1 MODIFYING CELL WALL COMPOSITION OF LIGNOCELLULOSIC SUGARCANE BIOMASS BY RNAI SUPPRESSION OF CAFFEIC ACID O METHYLTRANSFERASE TO ENHANCE BIOFUEL PRODUCTION By J E H YEONG J UNG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL O F THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Je Hyeong Jung
3 To my family mentors and friend s f or their love, encouragement and guidance
4 ACKNOWLEDGMENTS I would like to gratefully acknowledge my major advisor, Dr. Fredy Altpeter, for his guidance, encouragement and financial support. His enthusiasm kindness, and in depth knowledge of research have in spired my career and he has been a great role model I would also like to thank my committee members Special thanks to my Co A dvisor, Dr. Maria Gallo whose continuous motivation and positive thinking helped me accomplish my research goals. I am also gra teful to Dr. Wilfred Vermerris for helping me in building a solid foundation in this research I especially acknowledge Dr. James Preston for his continu al adv ice and commitment to discussions I sincerely thank Dr. Gary Peter for the thought provoking sug gestions. Dr s Wilfred Vermerris and Kevin Kenworthy provided useful advice while I was a T eaching A ssistan t in Genetics This class provided great opportunit ies to actively enga ge students during the semester Dr s John Erickson, Robert Gilbert, and Jim Boyer kindly provid ed the necessary field equipment to conduct my research. Heartfelt thanks to all my current and former lab members I especially thank Dr. Janice Zale for editing my manuscripts and dissertation I am indebted to Drs. Jae Yoon Kim an d Walid Fouad for their generous advice in research. I am appreciative for the friendships with Elizabeth Marco, Yang, Yuan, Yogesh, Hao and Baskaran. I sincerely thank Dr. Seo, Yong Weon for his continuous guidance and motivation during my research car eer. I am grateful to Dr s Lee, Ho Joung and Kim, Wook for helping me realize how enjoyable research can be. At last, my most sincere thanks go to my lovely family and wife for their love and support
5 TABLE OF CONTENT S page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIS T OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Sugarcane ................................ ................................ ................................ .............. 12 Sugarcane as S uperior F eedstock for B iofuel P roduction ................................ 12 Origin of S ugarcane H ybrids and G enome S tructure ................................ ....... 13 Genetic E ngineering in S ugarcane ................................ ................................ ... 15 P lant C ell W all ................................ ................................ ................................ ........ 17 Cell W all P olysaccharides ................................ ................................ ................ 18 Aromatic S ubstances ................................ ................................ ....................... 19 Structural P roteins ................................ ................................ ............................ 20 Lignin ................................ ................................ ................................ ...................... 21 Monolignol B iosynthesis ................................ ................................ ................... 21 Lignin P olymerization and D eposition in the S econdary C ell W a lls .................. 23 Forward and R everse G enetics in L ignin B iosynthesis and E ffects of M anipulating L ignin B iosynthetic G enes on the F ormation of L ignin and P lant D evelopment/ G rowth ................................ ................................ ........... 26 Phenylalanine ammonia lyase ( PAL ) ................................ ......................... 26 Cinnamate 4 hydroxylase (C4H) ................................ ................................ 27 4 coumarate:CoA ligase (4C L) ................................ ................................ ... 27 Hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) ................................ ................................ ................... 28 p ................................ ........................... 28 Caffeoyl CoA O methyltransferase (CCoAOMT) ................................ ........ 29 Cinnamoyl CoA reductase (CCR) ................................ .............................. 29 Ferulate 5 hydroxylase (F5H) ................................ ................................ .... 31 Caffeic acid O methyltransferase (COMT) ................................ ................. 31 Cinnamyl alcohol dehydrogenase (CAD) ................................ ................... 32 Lignocellulosic B iofuel ................................ ................................ ............................. 33 The P roduction P rocess of L ignocellulosic E thanol ................................ .......... 35 The I ntrinsic R ecalcitrance of L ignocellulosic B iomass for E thanol P roduction ................................ ................................ ................................ ..... 38 Improved Q uality of L ignocellulosic F eedstock through L ignin M odification ..... 39
6 RNA I nterference (RNAi) ................................ ................................ ........................ 41 2 ISOLATION AND EXPRESSIONAL CHARACTERIZATION OF CAFFEIC ACID O METHYLTRANSFERASE ( COMT ) IN SUGARCANE ................................ ......... 48 Introduction ................................ ................................ ................................ ............. 48 Materials and Methods ................................ ................................ ............................ 50 Isolation of COMT Gene from CP 88 1762 ................................ ........................ 50 cDNA L ibrary C onstruction and S creening ................................ ....................... 51 Sequence A nalysis ................................ ................................ ........................... 51 Sampling for E xpression A nalysis of the COMT G ene ................................ ..... 52 RT PCR and Qu antitative R eal T ime RT PCR ................................ ................. 52 Results ................................ ................................ ................................ .................... 53 Isolation of COMT G enes Through cDNA L ibrary S creening and PCR ............ 53 Sequence C omparison Between the I solated COMT and TCs ......................... 54 Deduced A mino A cid S equence C omparison of the I solated COMTa with Ot her M onocot COMTs ................................ ................................ ................. 55 Expression of the COMT in D ifferent T issues and D evelopmental S tages ....... 55 Discussion ................................ ................................ ................................ .............. 56 3 GENERATION OF COMT SUPPRESSED TRANSGENIC SUGARCANE AND EFFECTS OF COMT SUPPRESSION ON LIGNIN, PLANT GROWTH, AND SACCHARIFICATION PERFORMA NCE UNDER GREENHOUSE CONDITIONS ................................ ................................ ................................ ......... 66 Introduction ................................ ................................ ................................ ............. 66 Materials and Methods ................................ ................................ ............................ 69 Plant G rowth ................................ ................................ ................................ ..... 69 Vector C onstruction ................................ ................................ .......................... 70 Generation of T ransgenic S ugarcane ................................ ............................... 71 Evaluation of NPTII E xpression ................................ ................................ ........ 72 PCR Analysis ................................ ................................ ................................ ... 72 Southern B lot A nalysis ................................ ................................ ..................... 73 Small RNA N orthern B lot ................................ ................................ .................. 73 Quantitative R eal T ime RT PCR for Q uantification of COMT E xpression ........ 74 Microscopic and H istochemi cal A nalysis ................................ .......................... 75 Sample P reparation and D etermination of L ignin C ontent and C omposition .... 76 Dilute A cid P retreatment and E nzymatic H ydro lysis ................................ ......... 78 Statistical A nalysis ................................ ................................ ............................ 78 Results ................................ ................................ ................................ .................... 79 Generation and M olecular C har acterization of T ransgenic S ugarcane L ines ... 79 Lignin C ontent, C omposition, and E nzymatic S accharification ......................... 80 Plant P henotype and G rowt h ................................ ................................ ............ 81 Discussion ................................ ................................ ................................ .............. 83 4 FIELD EVALUATION OF COMT SUPPRESSED TRANSGENIC SUGARCANE ... 93
7 Introduction ................................ ................................ ................................ ............. 93 Materials and Methods ................................ ................................ ............................ 95 F ield D esign ................................ ................................ ................................ ..... 95 Evaluati on of P lant Performance ................................ ................................ ...... 96 Gene E xpression A nalysis ................................ ................................ ................ 97 Sample P reparation for the E valuation of C ell W all C omposition and S accharifica tion E fficiency ................................ ................................ ............. 97 Lignin C ontent and C omposition ................................ ................................ ...... 97 Analysis of H ydroxycinnamic A cids ................................ ................................ .. 98 Cell W all C arbohydrates and S tarch ................................ ................................ 99 Dilute A cid P retreatment and E nzymatic H ydrolysis ................................ ...... 100 Statistical Analysis ................................ ................................ .......................... 101 Results ................................ ................................ ................................ .................. 101 COMT G ene S uppression in T ransgenic Sugarcane L ines ............................ 101 Effec ts of COMT S uppression on L ignin C ontent and C omposition ............... 102 Effects of COMT S uppression on C ell W all C arbohydrates and C ell W all B ound H ydroxycinnamic A cids ................................ ................................ .... 102 Effect of L ignin R eduction on S accharification E fficiency ............................... 103 Growth P erformance of the T ransgenic S ugarcane G rown under F ield C onditions ................................ ................................ ................................ ... 104 Discussion ................................ ................................ ................................ ............ 105 5 CONCLUSION S ................................ ................................ ................................ ... 117 APPENDIX: LABORATORY PROTOCOLS ................................ ................................ 120 Generation of Transgenic S ugarcane using Particle Bombardment ..................... 120 Small RNA Northern Blot ................................ ................................ ...................... 123 Analysis of Cell Wall Components ................................ ................................ ........ 127 Evaluation of Saccharification Efficiency ................................ ............................... 130 LIST OF REFERENCES ................................ ................................ ............................. 133 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 151
8 LIST OF TABLES Table page 1 1 Improvement of fermentable sugar and/or ethanol yields in lignin reduced transgenic plants and mutants ................................ ................................ ............ 46 3 1 Lignin content, composition, and glucose yields after enzymatic saccharification ................................ ................................ ................................ ... 87 4 1 Lignin co ntent and composition in transgenic sugarcane ................................ 110 4 2 Composition of structural carbohydrates of the cell wall in control and transgenic sugarcane plants ................................ ................................ ............ 111 4 3 Recovery yields of cell wall bound p coumaric acid ( p CA) and ferulic acid (FA) after mild alkaline hydrolysis of the control and transgenic sugarcane ..... 112 4 4 Growth c haracteristics of the transgenic sugarcane lines under field conditions ................................ ................................ ................................ ......... 113
9 LIST OF FIGURES Figure page 1 1 Lignin biosynthesis pathway adapted from Boerjan et al. (2003) and Bonawitz and Chapple (2010) ................................ ................................ ............ 47 2 1 Nucleotide and amino acid comparisons among the isolated sugarcane COMTs ................................ ................................ ................................ ............... 61 2 2 Nucleotide s COMTa and TCs ...... 62 2 3 Nucleotide s equence comparison of coding region s spanning SAM binding residues between TCs and COMTa ................................ ................................ ... 63 2 4 Amino acid sequence comparison s between sugarcane COMTs and functionally characterized COMTs in monocot plants ................................ ......... 64 2 5 COMT expression patter ns in sugarcane ................................ ........................... 65 3 1 Generation and selection of transgenic sugarcane lines and investigation of transgene integration pattern in the transgenic sugarcane lines ......................... 88 3 2 Detection of COMT siRNA and evaluation of COMT expression levels in transgenic sugarcane ................................ ................................ ......................... 89 3 3 Phenotypic evaluation of transgenic sugarcane in comparison to wild type ....... 90 3 4 Microscopic and histochemical evaluation of transgenic sugarcane in comparison to wild type ................................ ................................ ...................... 91 3 5 Plant growth chara cteristics under greenhouse conditions ................................ 92 4 1 Real time RT PCR analysis of COMT expression level in transgenic sugarcane ................................ ................................ ................................ ......... 114 4 2 Field trial of the COMT suppressed transgenic sugarcane lines ....................... 115 4 3 Enzymatic saccharification performance in wild type (WT), transgenic control harboring npt II gene alone (TC), and transgenic sugar cane lines (T41 and T4) ................................ ................................ ................................ .................... 116
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MODIFYIN G CELL WALL COMPOSITION OF LIGNOCELLULOSIC SUGARCANE BIOMASS BY RNAI SUPPRESSION OF CAFFEIC ACID O METHYLTRANSFERASE TO ENHANCE BIOFUEL PRODUCTION By Je Hyeong Jung December 2012 Chair: Fredy Altpeter Co c hair: Maria Gallo Major: Agronomy Sugarcane ( Sac charum spp. h ybrid s ) is a prime bioethanol feedstock. Currently sugarcane ethanol is produced through the fermentation of sucrose which can easily be extracted from stem internodes. Processes for the production of biofuel from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per unit land area and unit biomass However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lig nocellulosic biomass to enzymatic hydrolysis The presence of lignin in the plant cell wall has been recognized as a major limitation to efficient bio conversion of lignocellulos ic biomass to biofuel I n this study, suppression of caffeic acid O methyltrans ferase ( COMT ) in transgenic sugarcane greatly enhances the bioconversion efficiency of lignocellulosic biomass into fermentable sugar s by reducing lignin content. COMT suppressed transgenic sugarcane lines have been successfully generated by deploying RNA interference Suppression of COMT decreases lignin content and alters lignin composition with reduced level s of s y r i ngyl unit incorporation while the accumulation of cell wall polysaccharides remains unaffected More
11 importantly, the s accharification effi ciency is improved up to 32% in transgenic sugarcane lines Furthermore, transgenic sugarcane lines require one third of the hydrolyzing time and 3 or 4 fold less enzyme to produce an equal or greater amount of fermentable sugar compared with the control l ines. P lant growth performance of transgenic sugarcane depended upon the extent of lignin reduction. A transgenic line with a 6 % reduction in lignin exhibit ed comparable growth performance to control plants under both greenhouse and field conditions, while lignin reduction between 8 % and 1 2 % result ed in impaired growth Reducing the recalcitrance of lignocellulosic sugarcane biomass by modifying lignin biosynthesis, without compromising plant growth performance is expected to improve the economic feasibili ty of lignocellulosic biofuel production. Transferring the reduced lignin trait to other high biomass varieties of sugarcane will further increase the value of sugarcane as a superior biofuel feedstock.
12 CHAPTER 1 LITERATURE REVIEW Sugarcane Sugarcane ( Sac charum spp. hybrid s ) is an economically important crop and is the main crop source for s ugar and bioethanol production. It is a highly productive perennial C 4 grass grown in tropical, semi tropical and sub tropical regions ( Tew and Cobill, 2008 ) Its perennial growth habit and C 4 p hotosynthetic pathway maximize carbon assimilation while minimizing water and nitrogen inputs ( Byrt et al., 2011 ) In 2010, more than 1.7 billion Mg of s ugarcane was produced using 2% of the total cultivated ar ea in over 90 countries (FAOSTAT, http://faostat3.fao.org/home/index.ht ml ) Its production quantity on a fresh weight basis is higher than any other crop in the world (FAOSTAT, http://faostat3.fao.org/home/index.ht ml ) The n et production value of sugarca ne worldwide was $ 54 billion in 2010 (FAOSTAT, http://faostat3.fao.org/home/index.ht ml ) Sugarcane as S uperior F eedstock for B iofuel P roduction Sugarcane is the most important crop for sugar production accounting for approximately 70% of the world s suga r supply ( Tew and Cobill, 200 8 ) It is also the second most important feedstock for bio ethanol production after maize. I n 2011, 21 billion liters of bioethanol were produced from sugarcane, accounting for 25% of the total bioethanol produced worldwide ( RFA, 2012 ) S ugarcane accumulates as much as 50% of its dry weight as sucrose in the internodes ( Waclawovsky et al., 2010 ) The production of s ugarc ane ethanol has been commercialized in Brazi l with low production costs, which are 24% less than corn ethanol in the US A ( Crago et al., 2010 ) Furthermore, it is considered an advanced biofuel due to its high environmental
13 sustainability ( Goldemberg, 2007 ; Schnepf and Yacobucci, 2012 ) The energy balance of sugarcane ethanol is about seven times higher than corn ethanol reflecting much less fossil fuel utilization during pro duction, similar to lignocellulosic ethanol ( Goldemberg, 2007 ) L and use efficiency in terms of ethanol productivity per unit area is 45% higher for sugarcane ethanol compared to corn ethanol ( Crago et al., 2010 ) T here has been a great deal of attention paid to sugarcane as a promisin g feedstock fo r lignocellulosic ethanol production due to its exceptional biomass productivity and the availability of a large amount of post harvest residues such as green tops leaves and bagasse. (39 Mg ha 1 ; stalk le aves and tops) is significantly higher than maize ( 17.6 Mg ha 1 ; grain and stover), switchgrass (10.4 Mg ha 1 ; biomass ) or Miscanthus (29.6 Mg ha 1 ; biomass ) ( Heaton et al., 2008 ; Waclawovsky et al., 2010 ) Post harves t residues and bagasse comprise approximately 55% of the total above ground biomass ( Somerville et al., 2010 ; Tew and Cobill, 2008 ) Together with sugar based bioethanol, the utilization of abundant lignocellulosic biomass further incr eases ethanol producti vity per unit land area or per unit biomass. Leite et al. (2009 ) suggested that assuming all of the post harvest residues and 50% of the bagasse are con verted to bioethanol, 33~38% less land is required to produce a n equal amount of sugar based bioethanol. Furthermore, Somerville et al. (2010 ) projected that bioconversion of all of the bagasse could increase total bioethanol yield by 59% Origin of S ugarcane H ybrid s and G enome S tructure Sugarcane b elongs to the genus Saccharum L. in the Poaceae family. S ix species have been traditionally classified as Saccharum gen era two of which are S robustum and S. spontaneum and four historically cultivated species, S
14 barberi S sinense and S edule Among the species, S barberi and S sinense are thought to be natural interspecific hybrid s between S spontaneum and S while the origin of S edule is un clear ( Piperidis et al., 2010 ) Current sugar cane cultivars are artificial interspecific hybrids between S as female and S spontaneum as male. A fter hybridization F 1 hybrids are backcrossed several times to S to recover high sugar yielding traits ( Piperidis et al., 2010 ) S is thought to have be en domesticated from S robustum and is ch aracterized by thick stalk s and high sugar content, but less tillering/ rat oo ning ability and tolerance to biotic and abiotic stresses. S spontaneum is a vigorous wild species which is genetically more diverse than S It develops relatively th in and tall stalk s and contains high fiber with low sugar content. S spontaneum has relatively higher biotic and abiotic tolerance than S ( Tew and Cobill, 2008 ) Both of the parental species are considered t o have an autopolyploid origin S spontaneum has a wide rang e of chromosome numbers and ploidy levels ranging from 2n=5x=40 to 2n=16x=128, while S has 2n=8x=80. The genome of the interspecific hybrid s is aneup lo idy with a high level of polyploidy (ave rage 12x) consisting of ~120 chromosomes Seventy eighty percent of the current sugarcane genome is derived from S 10 20% from S spontaneum and ~10% from recombination ( ; Piperidis et al., 2010 ) Allo polyploidization is a process of g enome duplication after interspecific hybridization and it often induces massive genome restructuring followed by gene silencing and the subsequent loss of duplicate d genes ( Adams and We ndel, 2005 )
15 G enes retained in the duplicated genome often undergo diver gence in gene function and expression ( Wang et al., 2012 ) In general, autopolyploids tend to experience less genome reshaping than allopolyploids ( Parisod et al., 2010 ; Wang et al., 2012 ) T he sugarcane genome resulting from interspecific hybridization between two parental autopolyploids is thought to be conserved without extensive gene loss or genome rearrangement ( Garsmeur et al., 2011 ; Jannoo et al., 2007 ; Wang et al., 2010 ) T he sugarcane genome is expected to have a high degree of functional genetic redundancy having a high level of co l linearity, structural conservation, and sequence similarity/identity among homo ( eo ) logous haplotypes ( Garsmeur et al., 2011 ; Jannoo et al., 2007 ) G enetic E ngineering in S ugarcane Conventional sugarcane breeding combined with better agronomic practices ha v e improved yields, disease resistance, and stress tolerance ( Jackson, 2005 ) However, conventional br eeding is challenging due to the lack of diversification within the sugarcane germplasm, the long breeding cycle s requiring more than ten years, its complex genome structure and limited genome information ( Dal Bianco et al., 2011 ; Jackson, 2005 ) T herefore, genetic engineering through transgenic approaches w ill play a cr itical role in overcoming the limitations of conventional breeding. Sugarcane is an attractive target for genetic transformation ( Altpeter and Oraby, 2010 ) Invitro culture of sugarcane has been advanced by techniques for propagation and the generation of disease free clones somaclonal variants, and transgenic plants ( Chengalrayan and Gallo Meagher, 2001 ; Gallo Meagher and Irvine, 1996 ; Lakshmanan et al., 200 6 ; Singh et al., 2008 ) Vegetative propagation of transgenic sugarcane may provide stable transgene expression over generations and prevent the
16 segregation of sta c ked transgenes ( Altpeter and Oraby, 2010 ) I n addition, poor pollen viability of sugarcane along with it s amenability to vegetative propagation will ensure a high level of transgene containment, which is a favorable characteristic in terms of bio safety ( Bonnett et al., 2008 ) Bower and Birch (1992 ) successfully generated the first stable tran sgenic sugarcane incorporating the npt II gene using microprojectile bombardment. Subsequently, a number of transgenic sugarcane lines have been generated through both p article bombardment (biolistics) and Agrobacterium mediated gene transfer mainly target ing h erbicide resistance, tolerance to abiotic or biotic stress increased sugar yields, and the production of value added metabolite s ( Altpeter and Oraby, 2010 ; Arencibia et al., 1998 ; Gallo Meagher and Irvine, 1996 ) Biolistic s is applicable to a wide variety of cell types and explants without genotype dep endency, and it is favorable for integrating multiple expressi on cassettes for gene stacking ( Altpeter et al., 2005 ) U tiliz ation of minimal expression cassette s in biolistics prevents host plant contamination with vector backbone s Furthermore, a lthough Agrobacterium mediated gene transfer tends to result in simpler transgene integration pattern s this also can be achieved by biolistic s utilizing a low concentration of minimal expression cassette s and optimizing the microprojectile s ize ( Jackson et al., 2012 ; Kim et al., 2012 ; Taparia et al., 2012b ) Somatic embryogenesis and regeneration are essential for the generation of transgenic plants S omatic embryos can be produced indirectly from callus induced from meristematic tissues or directly without an intervening callus phase ( Arnold, 2008 ) Immature leaf whorl s immature inflorescences, or basal shoot apical meristem s ha ve
17 been used as explants for somatic embryogenesis ( Altpeter and Oraby, 2010 ) Among the se the immature leaf whorl is ideal bec ause it is available year roun d ( Taparia et al., 2012a ) Indirect somatic embryogenesis has been routinely used for biolistic gene transfer in sugarcane ( Altpeter and Oraby, 2010 ) Sugarcane callus culture can be extended without compromising embryogenic potential. However, a prolonged callus phase / tissue culture period and delayed regeneration increase the rate of somaclonal variation often resulting in poor agronomic performance ( Basnayake et al., 2011 ; Hoy et al., 2003 ; Vickers et al., 2005 ) Direct somatic embryogenesis with rapi d regeneration could reduce the risk of undesirable somaclonal varia nts and the overall time require d for the generation of transgenic plants ( Lakshmanan et al., 2006 ) C ell fate is primarily determi ned by auxin to cytokinin ratio during invitro culture ( Arnold, 2008 ) Direct somatic embryogenesis from immature leaf whorl s of sugarcane cultivar CP88 1762 is achieved w ith p chlorophenoxyacetic acid : napthaleneacetic acid : 6 benzylaminopurine in the ratios of 1.86:1.86:0.09 mg L 1 in the tissue culture medium ( Taparia et al., 2012b ) The g eneration of transgenic sugarcane via direct somatic embryogenesis requires only 12 weeks, which is 12 24 weeks faster than those obtained through indirect somatic embryogenesis ( Taparia et al., 2012b ) P lant C ell W all P lant cell wall s have key roles in plant structural integrity plant morphogenesis, water/nutrient transport, plant defense, cell d ifferentiation, and cell comm unication ( Cosgrove, 2005 ) T hey also are the main so urce for food, feed, and bio based products which contain abundant organic carbon in the form of cell wall polysaccharides structural proteins and various aromatic compounds ( McCann and Rose, 2010 ; Vermerris, 2008 ) T he cell wall is highly complex and heterogeneous, and its chemical
18 composition and structure vary among different tissues and cell types reflecting diverse and differentiated functions of the cell walls ( Carpita and McCann, 2000 ) It is primarily classified into primary and secondary walls. T he primary wall formed during cell division and expansion is mostly composed of cellulose hemicelluloses, pectin, and a small amount of structural proteins ( Carpita and McCann, 2000 ) T he secondary cell is formed interior to the primary wall after cell growth ceases in the specialized cell types such as vessels and fibers ( Donaldson, 2001 ) One of the distinct characteristic s of these seco ndary cell walls is the massive imp regnation of lignin into the cell wall matrix ( Bonawitz and Chapple, 2010 ) C ell W all P olysaccharides T he most abundant polysaccharide in the cell wall is cellulose Fifteen to thirty percent of the primary cell wall consist s of cellulose on a dry w eight basis, and the amount is even higher in secondary cell walls ( Carpita and McCann, 2000 ) C ellulose is present in the form of microfibrils composed of an average of three dozen (1 4) D glucan chains interconnected by hydrogen bond s ( Vermerris, 2008 ) Cellulose has a crystalline structure and its hydrophobic surface limit s the accessibility of cellulolytic enzymes ( Himmel et al., 2007 ) In growing cell walls, cellulose is embedded and interlocked in a cell wall matrix of hemicelluloses and pectin s ( Srensen et al., 2010 ) Hemicelluloses ( cross linking glycans ) are heterogeneous polymer s of polysaccharides cross link ed with one another or cellulose microfi brils ( Carpita and McCann, 2000 ) G lucuronoarabinoxylan s (GA Xs) are the major hemicellulose in the cell wall of grasses and commelinoid monocots ( Carpita, 1996 ) GAXs have xylan backbone s with side chains of arabinose and glucuronic acid attached to the O 3 and O 2 positions of the backbone, respectively ( Carpita, 1996 ) L ess branched GAXs
19 confer more cell wall rigidity resulting from a relatively high degree of hydrogen bond formation with other GAXs and cellulose microfibrils ( Vermerris, 2008 ) Xyloglucans (XyGs) are predominant hemicelluloses in dicot s and non commelinoid monocots ( Carpita, 1996 ) XyGs have (1 4) D glucan chain s as backbone s with side chains of xylose. Some of the xylosyl units can be substituted with arabinose or galactose and galactosyl units are sometimes substituted with fucose ( Carpita and McCann, 2000 ) Pectins are a heterogeneous group of polysaccharides I n the primary wall of grasses and commelinoid monocots, the amount of pectin (2 10%) is relatively less than that of dicots and non commelinoid monocots (~35%) ( Mohnen, 2008 ) T wo main constituents of pectins are homogalacturonan (HG) and rhamnogalacturonan I (RG I) ( Mohnen, 200 8 ) HG has a backbone of (1 4) D galacturonic acid (GalA) It is often methyl esterifed at the C 6 position and structurally modified to xylogalacturonan (XGA) and rhamnogalacturonan II (RG II) XGAs have side chains of xylosyl units at the O 3 position of GalA. RG II is highly co mplex substituted HG with heterooligomeric s ide chains consis ting of several sugars. RG I has a backbone of the disaccharide unit s (1 2) L rhamnosyl (1 4) D GalA with side chains. Arabinans, galactans, and/or branched arabidnogalactans are attached to rhamnosyl units in RG I. These pectic polysaccharides are often cross linked to each other forming macromolecular pectin network ( Vincken et al., 2003 ) Non esterifed carboxyl resides of GalA are involved in calcium ion cross linking of HG s Borate diester bonding forms RGII dim er Furthermore transesterification could occur between p ectins and other cell wall polysaccharides. Aromatic S ubstances The cell wall s of grasses and commelinoid monocots contain considerable amount s of aromatic s ubstances, mostly hydroxycinnamic acids, such as p coumarate
20 and ferulate ( Carpita, 1996 ) F erulate is involved in the formation of cell wal l polysaccharide cross link s ( Grabber et al., 2000 ; Hatfield et al., 1999b ) Ferulate can be esterifed to the O 5 of arabinosyl units of arabinoxylans, and c ell wall polysaccharides are cross linked by radical coupling between esterified ferulates forming 8 O 4 8 5 8 8 or 5 5 linked diferulates. E xtensive lignin ferulate polysaccharide complex es are also formed by copolymeriz ation of f erulate monomer s or diferulate s with monolignols ( Grabber et al., 2002 ) F urthermore, during the early s tage s of lignification f erulates may play a role in providing a nucleation site for the attachment of monolignols ( Ralph et al., 1995 ) p C oumarate is mainly esterifed with lignin not with cel l wall polysaccharides and it is esterifed to the position of s i napyl alcohol forming sinapyl p coumarate conjugate ( Ralph et al., 1994 ) The incorporation of si napyl alcohol into the growing lignin polymer is thought to be facilitated by p c oumarate during the end wise polymerization of lig n i n ( Hatfield et al., 2008 ) Radical coupling of sinapyl alcohol alone is relatively slow, whereas radical form ation is rapid with the addition of si napyl p coumarate conjugate Structural P r otei ns T here are four major classes of structural proteins: hydroxy proline rich glycoproteins (HRGPs, commonly extensin s ), proline rich proteins (PRPs), gl ycine rich proteins (GRPs), and arabinogalactan proteins (AGPs) ( Carpita and McCann, 2000 ) In dicots and non commelinoid monocots, the cell wall carbohydrates are cross l inked with structural proteins. In contrast, grasses and commelinoid monocots have smaller amount s of structural proteins, but rathe r aromatic subst ances, mostly hydroxycinnamates have functions similar to structural proteins ( Carpita, 1996 )
21 Lignin L ignin is a major cell wall co mponent, mainly deposited in the secondary cell wall of vascular plants. Lignin plays important roles in plant growth and development by providing mechanical strength, defense, and a path for water transportation ( Boerjan et al., 2003 ) Lignin is a heterogeneous aromatic polymer consisting primarily of three monolignols, p coumaryl (4 hydroxycinnamyl) alcohol, coniferyl (3 methoxy 4 hydroxycinnamyl) alcohol, and sinapyl (3,5 dimethoxy 4 hydroxyc innamyl) alcohol. A fter incorporation of these monolignols into a lignin polymer, they are referred to as p hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively ( Bonawitz and Chapple, 2010 ) Monolignol B iosynthesis Monolignol biosynthesis has diverged from the general phenylpropanoid pathway involved in the biosynthesis of many metabolites such as (iso)flavonoids, anthocya nins, stilbenes, tannins, phenylpropens, and coumarins ( Vogt, 2010 ) T he biosynthesi s of monolignols commences with the deamination of phenylalanine and follows a series of enzymatic reactions on the monolignol precursors : hydroxylation at the C3, C4, or C5 position s of the aromatic ring; O methylation of the hydroxyl group at the C3 or C5 position of aromatic ring; two successive reductions at the side chain of the precursors from carboxylic acid to aldehyde and finally to alcohol ( Boerjan et al., 2 003 ; Bonawitz and Chapple, 2010 ) A s shown in Figure 1 1, and reviewed in Bonawitz and Chapple (2010 ) and Boerjan et al. (2003 ) m onolignol biosynthesis shares three initial enzymatic reactions with the general phenylpropanoid pathway catalyzed by p h enylalanine a mmonia l yase (PAL), c innamate 4 h ydroxylase (C4H), and 4 c oumarate: Co A l igase (4CL) The
22 deamination of phenylalanine to cinnamic acid catalyzed by PAL is the first step, followed by the hydroxylation at the C4 position of cinnamic acid which is catalyzed by C4H to form p coumaric acid. In grass species, tyrosine can be utilized by tyrosine a mmonia l yase (TAL) to yield p coumaric acid ( Rosler et a l., 1997 ) p C oumaric acid is further converted to p coumaroyl CoA by 4CL, which is a branch point metabolite between lignin and other phenylpr opanoid metabolites ( Vogt, 2010 ) In the traditional view of the monolignol biosynthesis pathway hydroxylation and O methylation in monolignol biosynthesis are thought to occur at the level of f ree hydroxycinnamic acids. However, invi vo biochemical stud ies and transgenic research demonstrate that this pathway is highly unlikely ( Dixon and Srinivasa Reddy, 2003 ) Instead, h ydroxylation at the C3 position of monolignol precursors occurs at shikimate or quinate ester of p coumaroyl CoA Firstly, h ydroxycinnamoyl CoA s hikimate/ q uinate h ydroxycinnamoyl transferase (HCT) converts p coumaroyl CoA to p coumaroyl shikimate or quinate while p coumaroyl CoA serves as a direct precursor for p coumaryl alcohol and ot her phenylpropanoids biosynthesis. H ydroxylation at the C3 position of p coumaroyl shikimate or quinate is then catalyzed by c oumarate 3 h ydroxylase (C3 H) to form caffeoyl shikimate or quinate ( Schoch et al., 2001 ) Although C3 H is active on both shikimate and quinate ester, the shikimate derivative is considered to be preferred substrate for C3 H ( Hoffmann et al., 2003 ) HCT is a reversible enzyme that converts caffeoyl shikimate to caffeoyl CoA. S ubsequently, O m ethylation at the C3 position o f caffeoyl CoA is catalyzed by c affeoyl CoA O m ethyltransferase (CCoAOMT) to produce feruloyl CoA
23 The first reduction occurs at the level of hydroxycinnamoyl CoA thioester. Cinnamoyl CoA r educt ase (CCR) catalyze s the reduction of p cou maroyl CoA to p coumaraldehyde However, considering the majority of G and S units in lignin CCR is thought to preferentially convert f eruloyl CoA to coniferaldehyde ( Bonawitz and Chapple, 2010 ) T he reduction of p coumaraldehyde and coniferaldehyde to the corresponding hydroxycinnamyl alcohol s, p coumaryl alcohol and coniferyl alcohol, respectively, is catalyzed by c innamyl a lcohol d ehydro genase (CAD) During the biosynthesis of sinapyl alcohol, h ydroxylation at the C5 position of c oniferaldehyde is carried out by f erulate 5 h ydroxylase (F5H) yielding 5 hydroxyconiferaldehyde F5H also can convert coniferyl alcohol to 5 hydroxyconife ryl alcohol ( Humphreys et al., 1999 ; Osakabe et al., 1999 ) S ubsequently, caffeic acid O methyltransferase (CO MT) catalyzes O m ethylation at the C5 position of 5 hydroxyconiferaldehyde and 5 hydroxyconiferyl alcohol, yielding sinapaldehyde and sinapyl alcohol, respectively Finally, enzymatic reduction occurs from sinapaldehyde to sin apyl alcohol by CAD Lignin P olymerization and D eposition in the S econdary C ell W alls F ollowing the monolignol biosynthesis, monolignols are exported from cytoplasm to the apoplast, although the mechanism of export and transport ha s not been fully elucidated ( Bonawitz and Chapple, 2010 ) S everal transportation models have been proposed including 4 O glucosylated monolignol translo cation, Golgi derived vehicles, transpor ter mediated, and free diffusion model s ( Vanholme et al., 2010 ) The l ignin polymer is formed through radical coupling of activated monolignols tha t are oxidized by wall bound peroxidases and/or laccases ( Ralph et al., 2004 ) I t has been challenging to elucidate which peroxidases and/or lacc ases catalyze enzymatic
24 oxidation because these enzymes belong to a large multigene family with overlapping activities ( Vanholme et al., 2010 ) Afte r the enzymatic oxidation, radical electron s on the oxygen atom at the C4 position of activated monolignols can be delocalized reversely to the C1, C3, C5, and side chain carbon ( Freudenberg, 1968 ) Monolignol radicals with a delocalized electron at the carbon of side ch ain are the most reactive with the highest electron density at this position ( Vermerris, 2008 ) As reviewed in Ralph et al. (2004 ) d uring the end wise polymerization, radical coupling occurs between the carbon of the incoming monolignol and the 4 O or C5 position of monolignol end unit resulting in O 4 or 5 linkage s 1 linkage s se em to be only form ed between a monolignol radical and O 4 linked end unit. linkage is only possible by dimerization of monolignols 4 O 5 and 5 5 linkage s are formed by cross coupling of oligonols and the frequency of these linkages is relatively mi nor 4 O 5 and 5 5 linkage s generate branched lignin structure, and since C5 position is not available for cross coupling in the oligonols ended with S unit, S rich lignin could be more linear than G rich lignin ( Bonawitz and Chapple, 2010 ) O 4 ether linkage is relatively liable t o ch emical degradation compared to biphe nyl and carbon carbon linkages ( Boerjan et al., 2003 ) P hysic ochemical characteristics of lignin are primarily determined by monolignol distribution incorporated into the polymer and following inter unit linkage patterns. The mechanism of lignin polymerization i s not fully understood. The traditional and most accepted model is that lignin polymerization is processed through random c oupling of monolignol radicals. The r elative abundance of the different monolignols and inter unit linkages in lignin polymer are det ermined by the spatio temporal regulation of monolignol biosynthetic genes and the availability of monolignol radicals with variable
25 oxidized sites at the site of polymerization ( Boerjan et al., 20 03 ) O n the other hand, a directed lignin polymerization model postulates that a lignin polymer is built through selective and regio specific coupling under rigid enzymatic reactions mediated by dirigent proteins ( Davin and Lewis, 2000 ) H owever, this biochemically controlled model has limitations on explain ing the racemic nature of lignin polymer, random sequence of units in lignin polymer and the flexibility of lignifications which is the incorporation of unusual monolignols into lignin in transgenic and mutant plants ( Boerjan et al., 2003 ; Ralph et al., 2004 ) Furthermore, genetic evidence is not enough to support dirigent proteins directing polymerization even in most of the model plants ( Bonawitz and Chapple, 2010 ; Vermerris, 2008 ) Lignin is deposited mainly in the seco ndary cell walls of speci alized cells such as vessels and fibers ( Donaldson, 2001 ) A fter cellulose and hemicellulose s are deposited in the secondary cell wall, lignification takes place in that region following the orientation of the cellulose microfibrils, and lignin impregnates the cell wall matrix ( Boerjan et al., 2003 ; Bonaw itz and Chapple, 2010 ) L ignin composition and structure are variable among and within species depending on developmental stages and cell types ( Boerjan et al., 2003 ) T he lignin of a ngiosperm s mainly consists of G and S units with trace amount s of H units in dicots and relatively more H units in monocots. The lignin of gymnosperms is mainly composed of G unit s with a lack of S unit s and t here is an intermediate amount of H units between dico ts and monocots. W ithin a species, G units predominat e in the earlier stage of development, and S units tend to be deposited later. T he secondary cell walls of vessels are enriched in G units, while structural fibers contain more S units.
26 Forward and R eve rse G enetics in L ignin B iosynthesis and E ffects of M anipulating L ignin B iosynthetic G enes on the F orm ation of L ignin and P lant D evelopment/ G rowth Phenyl alanine ammonia lyase ( PAL ) PAL is encoded by a multi gene family. There is functional redundancy among P AL family members, although each member has a distinctive role in the general phenylpropanoid pathway ( Huang et al., 2010 ; Rohde et al., 2004 ) In agreement with the role of PAL in the general phenylpropanoid pathway, T DNA insertional mutation s in PAL s : pal1 pal2 pal1/pal2 double or pal1/pal2/pal3/pal4 quadruple mutation in Arabidopsis affects the biosynthesis of lignin and other metabolites including flavonol glucosides, tannin, and anthocyaninn pigments, as well as SA ( Huang et al., 2010 ; Rohde et al., 2004 ) Furthermore transcript profiling in the mutants indicates that knock down of PAL not only changes expression patterns of other lignin biosynthetic genes but also affects the expression of genes related to amino acid and carbohydrate metabolism ( Rohde et al., 2004 ) S ingle mutant pal1 and pal2 have no obvious phenotypic difference s compar ed to wild type. However, a pal1/pal2 double mutation affects fertility depending on the genetic background of Arabidopsis (ecotype C24 vs. Col 0), and reduces tolerance to UV B irradiation ( Huang et al., 2010 ; Rohde et al., 2004 ) T he quadruple mutant shows dwarfism, male sterility, and reduced level s of SA and tolerance to pathogen s ( Huang et al., 2010 ) Down regulation of PAL induce s pleiotropic e ffects and the accumulation of other met abolites important for defense mechanism s For example, PAL suppressed tobacco is susceptible to fungal, bacterial, and viral pathogens ( Dixon and Srinivasa Reddy, 2003 )
27 Cinnamate 4 hydroxylase ( C4 H ) Arabidopsis reduced epidermal fluorescence 3 ( ref3 ) harboring a missense mutation in C4H shows redu ctions in total lignin content and i n several phenylpropanoid derivatives ( Ruegger and Chapple, 2001 ; Schilmiller et al., 2009 ) Reduced C4H a ctivity in the ref3 mutant primarily affects G unit biosynthesis with no changes in S unit s However, C4H down regulated tobacco and alfalfa show reduced S unit s ( S ewalt et al., 1997 ; Srinivasa Reddy et al., 2005 ) A series of ref3 mutants with different levels of reduced C4H activity indicate that severe loss of C4H activity results in dwarfism, collapse d xylem vessel s and male sterility ( Schilmiller et al., 2009 ) 4 coumarate:CoA ligase ( 4CL ) The brown midrib ( bmr2 ) mutant of s orghum carries a missense mutation of the 4CL 1 gene. B mr2 with r educed 4CL activity shows 20% reductions in total lignin content and in both G and S unit s ( Saballos et al., 2012 ) The d own regulation of 4CL in switchgrass and poplar results in a slight increase in H unit s and preferential reduction s in G over S unit s with reduced total lignin content ( Voelker et al., 2010 ; Xu et al., 2011 ) S everal 4CL isoforms encoded by a multigene family are found in planta and 4CL family genes are generally clustered into two grou ps in both monocots and dicots. Class I and II 4CLs are thought to be involved in lignin and flavonoid biosynthesis, respectively, with different substrate preferences, relative t ranscript abundance, and spatio temporal exp ression patterns ( Ehlting et al., 1999 ; Gui et al., 2011 ; Saballo s et al., 2012 ) At4CL1, Pv4CL 1 and Os4CL3 are identified as key 4CL enzymes involved in lignin biosynthesis in Arabidopsis switchgrass and rice, respectively ( Eh lting et al., 1999 ; Gui et al., 2011 ; Xu et al., 2011 )
28 Brown coloration in leaf midrib, basal stem, and/or roots is observed in 4CL suppressed mutant and trans genic plants ( Gui et al., 2011 ; Saballos et al., 2012 ; Voelker e t al., 2010 ) The bmr2 mutation has no adverse effect on plant phenotype and down regulation of Pv4CL1 in switchgrass does not affect biomass accumulation ( Saba llos et al., 2012 ; Xu et al., 2011 ) However, delayed plant growth and abnormal anther development are observed in transgenic rice and reduced biomass production is observed in transgenic poplar ( Gui et al., 2011 ; Voelker et al., 2010 ) Hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase ( HCT ) D own regulat ion of HCT in alfalfa and Arabidopsis reduces total lignin content with a strong reduction in G and S units and an increase in H unit s ( Hoffmann et al., 2004 ; Shadle et al., 2007 ) T herefore, the enzymatic reaction of HCT is a crucial initial step to drive the biosynthesis of coniferyl and sinapyl alcohols. HCT suppressed alfalfa and Arabidopsis exhibit dwarf phen otype s with reduced biomass ( Gallego Giraldo et al., 2011 ; Shadle et al., 2007 ) I mpaired plant growth i n HCT down regulated Arabidopsis appears to be associated with an increased level of SA derived from the shikimate pathway ( Gallego Giraldo et al., 2011 ) Involvement of shikimate during monolignol biosynthesis is required not only for supplying substrate for the 3 hydroxylation step, but also for regulating the flow of monolignol precu r sors ( Bonawitz and Chapple, 2010 ) p Coumarate 3 hydroxylase ( C 3 H ) The Arabidopsis ref8 mutant carrying a missense mutation in C3 H has increased level s of p coumarate esters and strongly reduced total lignin content ( Franke et al., 2002a ; Franke et al., 2002b ) Hydroxylation by C3 H is considered a committed
29 control step along with HCT for the biosynthesis of coniferyl and sinapyl alcohol The ref8 mutant has small amount s of G and S lignin, and the lignin almost entirely consists of H lignin ( Franke et al., 2002a ) C3 H down regulated alfalfa has strongl y reduced G and S units and increased H unit s s imilar to the ref8 mutant ( Ralph et al., 2006 ) Both the Arabidopsis mutant and the transgenic alfalfa show dwarf phenotype s Caffeoyl Co A O methyltransferase ( CCoAOMT ) Down regulation of CCoAOMT results in reduced total lig nin content with a preferential reduction in G unit s without a decrease in S unit s in transgenic tobacco and alfalfa plants ( Guo et al., 2001 ; Zhong et al., 1998 ) T hese results indicate that CCoAOMT is specifically involved in the 3 O methylation of caffeoyl CoA for the biosynthesis of coniferyl alcohol CCoAOMT suppressed alfalfa accumulates soluble caffeic acid D glucoside possibly indicating that excess caffeoyl CoA in the metabolite pool of monolignol biosynthesis is converted into caf feic acid and then modified with glucosyla tion ( Guo et a l., 2001 ) S imilar to transgenic alfalfa, soluble phenolic acids (vanillic, caffeic, and sinapic acids) glucosides are accumulate in CCoAOMT down regulated poplar ( Meyermans et al., 2000 ) T ransg enic poplar display s pink red color on its debarked stem and its overall morphology is similar to wil d type ( Meyermans et al., 2000 ) T here are contrasting results regarding the effects of CCoAOMT suppression on transgenic tobacco plants. Significantly d e creased stem length is shown in one study ( Pinon et al., 2001b ) whereas normal plant growth is shown in an other ( Zhong et al., 1998 ) Cinnamoyl CoA reductase ( CCR ) Down regulation of CCR in Arabidopsis tobacco, and p oplar results in ~50% reduction of total lignin content, wh ile the effects of CCR suppression on lignin
30 composition are variable ( Goujon et al., 2003 ; Lepl et al., 2007 ; Piquemal et al., 1998 ; Zhou et al., 2010b ) Lignin compositional variation in transgenic plants m ight depend on the species, developmental stages, culture conditions and the suppression of different CCR isoform s Zhou et al. (2010b ) suggest ed that two CCR isoforms in Medicago truncatula CCR1 and CCR2 provide non redundant independent routes for the biosynthesis of coniferyl and sinapyl alcohol. CCR1 preferentially ca talyzes reduction of feruloyl CoA, which is the product of the CCoAOMT reaction, to coniferaldehyde. CCR2 drives an alternative route via reduction of caffeoyl CoA to caffeyl aldehyde, and its subsequent conversion to coniferaldehyde by COM T ( Zhou et al., 2010b ) It is unclear whether the metabolite pool of coniferaldehyde resulting from C CoAOMT/CCR1 and CCR2/COMT route s is identical or separate. B ased on t he observation that CCoAOMT suppression results in reduced G units without a change in S unit s the CCR2/COMT route is thought to be involved in S unit formation at least in the absence of CCoAOMT ( Guo et al., 2001 ; Zhou et al., 2010b ) In addition, the Arabidopsis CCR1 knock out mutant deposit s increas ed level s of f eruloyl malate in the stem, and CCR down regulated poplar accumulates high level s of ferulic acid incorporated into lignin ( Lepl et al., 2007 ; Mir Derikvand et al., 2008 ) These results show a possible redirection of feruloyl CoA toward ferulic acid upon blocking CCR, and incorporation of intermediates into lignin polymer. T he CCR suppressed pla nts exhibit orange brown coloration around the xylem, decreased plant height, and collapsed vessel s with a less degree of secondary cell wall formation ( Goujon et al. 2003 ; Lepl et al., 2007 ; Piquemal et al., 1998 )
31 Ferulate 5 hydroxylase ( F 5H ) A F 5H deficient T DNA tagging mutant in Arabidopsis ( fah1 ) shows dra matic changes in lignin composition and structure. Consistent with the role of F5H in sinapyl alcohol biosynthesis, the lignin of fah1 contains only a trace amount of S units with increased occurrence s of 5 and 5 5 linkages ( Humphreys et al., 1999 ; Marita et al., 1999 ) In contrast over expression of F5H in Arabidopsis and poplar driven by the C4H promoter resu lts in lignin comprised of over 90% S unit s ( Marita et al., 1999 ; Stewart et al., 2009 ) The amount of O 4 linkage s are similar in the F5H over expressed poplar compared to control plants while linkage s are increased ( Stewart et al., 2009 ) In addition, t he lignin with t he elevated level of S unit contains no 5 linkages and less 5 5 linkage structure s D espite dramatic alteration s in relative abundance of G and S unit, the F5H over expressed transgenic poplar exhibits no differences in vessel development and cell wall t hickness ( Huntley et al., 2003 ) Caffeic acid O methyltransferase ( COMT ) COMT deficien cy in mutants (maize bm3 and sorghum bmr 12 ) and down regulati on of COMT in a wide variety of species, including alfalfa, tobacco, tall fescue, perennial ryegrass, switchgrass, and poplar consistently result in a strong reduction in S unit s indicating a primary role of COMT in sinapyl alcohol biosynthesis ( Chen et al., 2004 ; Fu et al., 2011a ; Guo et al., 2001 ; Marita et al., 2003 ; Palmer et al., 2008 ; Pinon et al., 2001a ; Tu et al., 2010 ; Van Doorsselaere et al., 1995 ) Furthermore COMT deficiency results in the accumulation of 5 hydroxyconiferaldehye which is generally the most p referential substrate for COMT. 5 hydroxyconiferaldeh ye is then reduced to 5 hydroxyconiferyl alcohol by CAD, and this atypical monolignol is incorporated into lignin
32 forming a novel benzodioxane structure referred to as the 5 hydroxyguaiacyl unit ( Palmer et al., 2008 ; Ralph et al., 2000 ; Van Doorsselaere et al., 1995 ) COMT is encoded by a multigene family belonging to the type 1 plant O methyltransferase s (OMT), and COMT generally has broad substrate permissiveness with different substrate preferences ( Louie et al., 20 10 ; Parvathi et al., 2001 ; Zubieta et al., 2002 ) Although COMT ha s the highest catalytic affinity toward 5 hydroxyconiferaldehye, 3 O or 5 O methyla tion could also occur at caffeoyl aldehyde caffeyl alcohol and /or 5 hydroxyconiferyl alcohol. Incomplete depletion of S unit s in COMT knock out mutant s may further support the idea that sinapyl alcohol can, at least in part, originat e from alternative ro utes driven by other OMT family members ( Barrire et al., 2004 ; Dixon et al., 2001 ; Parvathi et al., 2001 ) COMT defective mutants and transgenic plants show brown coloration in vascular tissues. COMT suppression generally ha s negligible effects on the plant growth of various transgenic plants, including alfalfa, maize, switchgrass, perennial ryegrass, and poplar ( Chen and Dixon, 2007 ; Fu et al., 2011a ; Pilate et al., 2002 ; Piquemal et al., 2002 ; Tu et al., 2010 ) Cinnamyl alcohol dehydrogenase ( CAD ) Deficiency of CAD in mutants such as t he maize bm1 and sorghum bmr6 and transgenic tobacco and poplar characteristically result in the incorporation of coniferaldehyde and/or sinapaldehyde into lignin polymer s which are typical substrates for CAD ( Chen et al., 2012 ; Lapierre et al., 2004 ; Marita et al., 2003 ; Ralph et al., 1998 ; Saballos et al., 2008 ) Consistent with the lack of S units in gymnosperms, only one CAD gene or a relatively small gene family is present in gymnosperms ( MacKay et al., 1997 ) On the ot her hand CAD i s encoded by a multigene family in a n giosperms. In
33 aspen, one of the CAD family members is reported to have a high s ubstrate affinity toward sinapaldehyde and is designated as sinapyl alcohol dehydrogenase (SAD) ( Li et al., 2001 ) While the presence of SAD in herbaceous angiosperm s ha s yet to be investigated, the enzymatic reduction of each hydroxycinnamaldehyde into corresponding alcohol seems to be governed rather by the combined action of each CAD family member in different developmental stages and tissues than by a highly specified CAD ( Sibout et al., 2005 ; Vermerris, 2008 ) CAD deficient mutant s and down regulated transgenic plants show brown col oration in lignified tissues due to the accumulation of conjugated aldehydes in the se tissues ( Baucher et al., 1996 ; Sattler et al., 2010 ) CAD down regulated poplar t rees, which have 15 47% of wild type CAD activity show normal growth performance under field conditions, while severe level s of C AD suppression result in impaired growth under greenhouse conditions ( Pilate et al., 2002 ) Lignocel lulosic B iofuel Given the environmental and socio economic problems caused by the hea vy dependence on fossil energy, it is expected that renewable biofuel s will replace a ce rtain amount of fossil fuel, particularly in the transportation sector A ccording to the U.S. Renewable Fuel Standard (RFS) with the enactment of the Energy Policy Act of 2007 the U.S. has the goal of produc ing 136.3 billion liters (36 billion U S gallon s ) of biofuel by 2022 ( Schnepf and Yacobucci 2012) T his amount of biofuel has t he potential to replace about 2 7 % of the gasoline by quantity in the current U.S. market (13 4 billion U S gallons consumed in 2011 U.S. Energy Information Administration, ht tp://www.eia.gov/ ). C urrent biofuel s especially bioethanol production mostly relies on starch and sugar feedstock s However, there have been concerns about the negative
34 impacts on the food and feed supply and relatively poor environmental sustainability particularly regarding starch based ethanol from corn In this respect, the production of corn ethanol cannot exceed more than 56.8 billion liters (15 billion U S gallons) from 2015 under the RFS ( Schnepf and Yacobucci 2012). Lignocellulosic biomass ha s been considered an abundant, inexpensive and sustainable energy resource that can provide bioethanol on a sufficient scale to replace a significant amount of gasoline. T he price of biomass is equivalent to 13 U S dollars per barrel of crude oil on the basis of energy value ( Yang and Wyman, 2008 ) In the U S A it is projected that 1.3 billion dry Mg of lignocellulosic biomass could be available annually by 2030, and it has the potential to replace 87 billion U S gallons of gasoline ( Carroll and Somerville, 2009 ) which is nearly 6 5 % of the gasoline currently consumed in the US A (13 4 billion U S gallons consumed in 2011 U.S. Energy Information Administration, http://www.eia.gov/ ) Furthermore the energy balance of li gnocellulosic ethanol has higher net benefits in terms of reductions in greenhouse gas emission s and energy input compared with fossil fuel s and also conventional ethanol from corn, wheat and sugar beet ( Goldemberg, 2007 ; Sims et al., 2010 ) Considering its a bu n dance and sustainability at least 60.6 billion liters (16 billion U S gallons) of ethanol are expect ed to be produced from lignocellulosic biomass by 2022 under the RFS ( Schnepf & Yacobucci 2012). D espite the exceptionally beneficial characteristics of lignocellulosic ethanol as a renewable alternative, full commercialization has been delayed due to eco nomic and technical limitations. T he production costs of lignocellulosic ethanol, which range from 0.6 1.3 U.S. dollars per liter are higher than gasoline and other conventional bioethanol
35 ( Goldemberg, 2007 ; Sims et al., 2010 ) In this respect, there have been tremendous efforts in developing efficient lignocellulosic feedstock plants and bioconversion procedures T he P roduction P rocess of L ignocellulosic E thanol T he process of ethanol production from lignocellulosic biomass includes the following steps: pretreatments of the feedstock, hydrolysis of cellulose and hemicelluloses into simple sugars, fermentation of sugars, and recovery and purification of ethanol ( Lu and Mosier, 2008 ) P retreatment is a process to disrupt the naturally resistant cell wall matrix and thereby increa se the acce ssibility of hydrolyzing enzymes to cellulose and hemicelluloses ( Mosier et al., 2005 ) A lthough pretreatment is the essential step for efficient bioconversion of lignocellulosic biomass into biofuel and other bio based products, it is considered the most expensive cost element because of intense chemical and energy requirements ( Yang and Wyman, 2008 ) O ver the years, various pretreatment techn ologies have been developed and evaluated targeting high recovery yields of fermentable sugars, limiting the release of undesirable compounds that inhibit fermentation, and minimizin g the chemical and energy demand ( Mosier et al., 2005 ) The e ffects of pretreatment on cell wall components vary among pretreatment methods I n general, pretreatments under alkaline conditions, such as a mmoni a fiber/freeze explosion (AFEX), a mmonia recycle percolation (ARP) and lime pretreatment have major effects on removing or depolymeriz ing lignin with minor effects on solubilizing hemicelluloses. On the other hand, acidic or neutral pretreatments such as diluted acid, steam explosion, and liquid hot water pretreatments effectively remove hemicelluloses rather than lignin ( Alvira et al., 2010 ) T h ere a re advantages and
36 disadvantages to all of the current pretreatment techn ologies that significantly increase the accessibility of enzymes and saccharification efficienc ies For example, acid based pretreatment s are applicable to a wide variety of lignocellulosic materials compared with AFEX, ARP, or lime pretreatment ( Mosier et al., 2005 ) There is a greater chance of generating toxic compounds tha t inhibit fermentation, especially in steam explosion and acid pretreatment, whereas, liquid hot water, alkaline, and AFEX pretreatments minimize the release of fermentation inhibitors ( Alvira et al., 2010 ) After pretreatment, t he enzyme hydrolysis step aims to more efficiently depolymerize polysaccharides in lignocellulosic biomass in to fermentable sugars, which then can be easil y converted to ethanol by ferment ation microorganisms. Hydrolysis of cellulose into glucose requires the synergistic action of three different types of cellulolytic enzymes, endo 1,4 glucanase (endoglucanase, EG), exo 1,4 glucanase (cellobiohydrolase, CBH), and glucosidase (cellobiase) ( Mansfield et al., 1999 ) EGs rando ml y cleave 1,4 glycosidic bonds of cellulose. CBHs remove cellobiose residues from the cellulose chain hydrolyzing not only non reducing ends, but also reducing ends of the chain. glucosidases finally hydrolyze cellobiose residues into glucose. The enzyme system for hydrolyzing hemicelluloses is more complex due to the heterogeneity of hemicelluloses ( Lu and Mosier, 2008 ; Shallom and Shoham, 2003 ) For hydrolyzing GAXs, which are the major hemicellulos ic compone nt in grass es endo 1,4 xylanase and exo 1,4 xylosidase are required to liberate xylose from the xylan backbone. T he side chains on GAXs can be removed by L a rabinofuranosidase and D g lucuronidases Hemicellulotytic esterases including acetyl xylan esterase and ferulo yl esterase improve xylose hydrolysis efficiency.
37 In the conventional process of lignocellulosic ethanol production, enzyme hydrolysi s and ethanol fermentation are conducted as separate steps or as one single step, referred to as separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF), respectively ( Lu and Mosier, 2008 ) If pentose sugars are fermented with hexose sugars, the SSF configurat ion is often classified as simultaneous saccharification and co fermentation (SSCF ) ( Margeot e t al., 2009 ) The integrated process configuration of SSF and SSCF reduce the capital cost of lignocellulosic ethanol production compared to SHF. A further integrated process, consolidated bio processing (CBP) has been suggested as a desired process conf iguration for more economical lignocellulosic ethanol production ( Galbe et al., 2007 ) In CBP, the simultaneous hydrolysis and fermentation are mediated by a single organism or a microbial consortium, while cellulolytic enzymes are produced in a separate system and added to saccharification /fermentation step in SSF and SSCF ( G albe et al., 2007 ) However, the integrated process is often operate d under compromised operational condition s due to the different optimal conditions for enzym atic hydrolysis and fermentation steps. In addition, the microorganism mediating both cellulos e hydrolysis and fermentation simultaneously in a satisfactory manner has not yet been developed ( Margeot et al., 2009 ) The bio conversion of fermentable sugars remain s suboptimal, especially for pentose s u gars in which fermentation is less efficient than hexose sugar fermentation. Pentose sugars comprise a significant portion in plant cell walls but the native strain of Saccharomyces cerevisiae a common choice of microorganism for ethanol production, is unable to convert pentose sugars into ethanol due to the lack of pentose sugar
38 metabolism ( Alper and Stephanopoulos, 2009 ) Therefore, efficient pentose sugar fermentation would improve the cost effective ness of lignocellulosic ethanol production. A dvances in metabolic engineering have signifi cantly i ncreased the efficienc y of pentose sugar fermentation by introducing heterologous pentose sugar metabolic pathways in S. cerevisiae and by improving ethanol productivity and ethanol tolerance of pentose utilizing organisms ( Van Vleet and Jeffries, 2009 ) T he I ntrinsic R ecalcitrance of L ignocellulosic B iomass for E thanol P roduction T he complexity and heterogeneity of the plant cell wall provides physical and ch emical resistance to hydrolysis into fermentable simple sugars ( Himmel et al., 2007 ) T he chain length of cellulose ( the degree of polymerization) and the ratio of crystalline to amorphous cellulose (crystallinity) are thought to affect the rate of enzymatic hydrolysis ( Man sfield et al., 1999 ) However, these characteristics alone do not directly explain the biomass recalcitrance to enzymatic hydrolysis ( Zhao et al., 2012 ) C rystalline cellulose itself can be hydrolyzed by the synergistic action of the fungal cellulase complexes ( Mansfield et al., 1999 ) The limited enzymatic hydrolysis of cellulose is associated with other factors, such as the surface area ava ilability of the substrate for cell u lolytic enzymes, pore size/volume, hemicellulose cross linking, and lignin impregnation ( Alvira et al. 2010 ; Zhao et al., 2012 ) T he d egree of substitution in hemicellulose determines pore size and volume in the cell wall matrix. I n grass species, the less branched GAXs result in a higher degree of self cross linking and with cellulose microfibrils, thus the cell walls containing less branched GAXs become more rigid and dense ( Vermerris, 2008 ) I n addition, the level of feruloylation of arabinoxylans affect enzymatic hydrolysis be cause ferulate cross link s with arabinoxylan and lignin interconnects the cell wall matrix ( Grabber et al., 1998 )
39 A ssociation of hemicelluloses in biomass recalcitrance is considered less important than that of lignin because hemicelluloses are rela tively liable to pretreatment and enzymatic hydrolysis ( Zhao et al., 2012 ) T he presence of lignin in the plant cell wall is a major obstacle to enzyma tic hydrolysis ( Jrgensen et al., 2007 ; Mansfield et al., 1999 ; Zhao et al., 2012 ) L ignin primarily limits the accessibility of cellulose to cellulolytic enzymes by shielding the cellulose chain. Lignin also corresponds to lower porosity of the cell wall, thus the surface area of substrate for the enzyme attack is highly limited in the presence of lignin. F urthermore, up to 60 70% of cellulolytic enzymes added will irreversibly adsorb to lignin, thereby overall enzyme activity is reduced ( Chernoglazov et al., 1988 ; Jrgensen et al., 2007 ) Improved Q uality of L ignocellulosic F eedstock through L ignin M odification The presence of lignin in the lignocellulosic biomass ha s long been recognized as a major obstacle in the manufactur ing of paper digestibility of forage and biochemical production including biofuel s For the efficient bioconversion of lignocellulosic biomass into ethanol, pretreatment is an essential operatin g process disrupting the cell wall matrix removing and relocating lignin ( Mosier et al., 2005 ) A lthough pretreatment is beneficial to increase ethanol production, it is an expensive and energy intensive process accounting for about 20% of the total production costs ( Yang and Wyman, 2008 ) Furthermore, depending on the pretreatment method, pretreatment could negatively impact subsequent steps causing degradation of sugars and the generation of undesirable compounds, such as acetic acid, furan, and phenolic derivatives, which are toxic to fermenting microorganisms ( Alvira et al., 2010 ; Jrgensen et al., 2007 ) T herefore, along with the efforts in developing advanced
40 pretreatment met hods, there has been tremendous interest in modifying lignin content and/or composition of lignocellulosic feedstock to improve the efficiency of ethanol production Chen and Dixon (2007 ) demonstrated that reducing lignin in lignocellulosic biomass significantly improved fermenta ble sugar yields up to 2.7 fold without pretreatment and up to 1.9 fold with dilute acid pretreatment. I n that study, six different lignin biosynthetic genes ( C4H HCT C3 H CCoAOMT F5H and COMT ) were independently down regulated in transgenic alfalfa. E ach transgenic line displayed different levels of lignin reduction and values of S to G unit molar ratio depending on the targeted lignin biosynthetic genes. In the set of transgenic lines, it was clearly shown that total lignin content was negatively correlated with fermentable sugar release after enzymatic hydrolysis with or without pretreatment (adjusted R 2 =0.91 or 0.79 with pretreatment or without pretreatment respec tively ). Although, the chemical and structural characteristics of the lignin polymer are mainly determined by the relative abundance of each monolignol, it was inconclusive whether there was a relationship between S/G ratio and saccharification efficiency. Improvement of saccharification efficienc ies and ethanol yields ha ve been consistently demonstrated in lignin reduced transgenic plants and mutants, demonstrating the negative impact of lignin on the bioconversion of lignocellulosic materials (Table 1 1 ). In the s orghum brown mid rib mutant s carrying mutations in 4CL COMT or CAD gene s ( Dien et al., 2009 ; Saballos et al., 2008 ) fermentable sugar yields were improved by 7 27% and ethanol yields w ere enhance d 22%, compared with the wild type counterpart. Furthermore, a double mutation in lignin biosynthetic genes
41 had an additive effect on sugar and ethanol yields ( Dien et al., 2009 ) L ignin modified transgenic s witchgrass es were generated by suppressing CAD COMT or 4CL ( Fu et al., 2011a ; Fu et al., 2011b ; Saathoff et al., 2011 ; Xu et al., 2011 ) Particularly, COMT suppressed transgenic switchgrass exhibited a 22 and 38% increase in saccharification efficiency and ethanol yields, respectively ( Fu et al., 2011a ) Moreover, it has been demonstrated that there is great potential to reduce pretreatment severity, i.e. pretreatment reaction time temperature and enzyme dosage for producing a comparable amount of ethanol from lignin modified transgenic feedstock s ( Fu et al., 2011a ) M anipulating lignin biosynthetic genes or identifying lignin modified mutant s could be a straightforward and viable strategy to increase the economic feasibility of lignocellulosic biofuel pro duction with diminished bioma ss recalcitrance and pretreatment requirement s. RNA I nterference (RNAi) RNAi is one of the cellular regulation processes of gene expression mediated by small silencing RNA (sRNA). sRNA can be primaril y categorized into two clas ses on the basis of biogenesis, namely microRNA (miRNA) and short interfering RNA (siRNA) ( Carthew and Sontheimer, 2009 ) miRNA originated from an endogenous non coding miRNA gene, and its expression is highly regulated in the genome. miRNA is processed from a miRNA gene transcript (pre miRNA) which forms a stem loop structured d ouble s tranded RNA (dsRNA) with i mperfect complementa rity On the other hand, siRNA is generated from perfectly complementary double strand ed RNA (dsRNA) precursor s derived from diverse sources, such as transgene s viral RNA s transposon s and gene/pseudogene duplex es
42 The mechanism of RNAi is reviewed in Carthew and Sontheimer (2009 ) In general, RNAi results in post transcriptional gene silencing (PTGS) and tran scriptional gene silencing (TGS). T he process begins with the formation of dsRNA o f the transcripts derived from exogenous sources or endogenous sources. dsRNA can be formed by either sequence complementa rity or by the action of an RNA dependent RNA polyme rase. dsRNA is then recognized and cleaved by Dicer into sRNAs. In plants, D icers are dsRNA specific RNAase III family ribonuclease s encoded by the small dicer like ( DCL ) gene family ( Hamilton et al., 2002 ; Xie et al., 2004 ) Each ha s distinctive and redundant roles in generating various types of s RNA with different length s In Arabidopsis DCL1/DCL4, DCL2, and DLC3 are kn own to produce ~21, ~22, and 24 ~ 26 nt long sRNA, respectively. 21~22 nt long sRNAs are mostly involved in PTGS, while 24~26 nt long sRNAs mainly trigger TGS ( Hamilton et al., 2002 ; Xie et al., 2004 ) Newly produced double stranded sRNAs are then loaded into one of the Argonaute (Ago) proteins in RNA induced silencing complex (RISC). D uring the assembly process, o ne of the strands of sRNA is removed or cleaved by Ago T he remaining guide strand of sRNA directs RISC and binds to the target RNA via Watson Crick base pairing F inally, PTGS is induced from the endonucleolytic cleavage of the target mRNA by the action of Ago, and then cellular exonucleases complete the degradation of the target mRNA. In few cases, during the cycle of PTGS mediated by siRNA or miRNA, cleaved target transcripts can be converted into dsRNAs by RNA dependent RNA polymerase and other cofactors. T his secondary dsRNAs can be further processed into secondary sRNA by Dicer in plan ts so that the silencing can be amplified and spread throughout the cell
43 The molecular mechanism of TGS is not fully understood compared to PTGS ( Carthew and Sontheimer, 2009 ) S pecialized RNA induced transcriptional silencing complex (RITC) is assemb led with sRNA which is homologous to the target chromosomal loci. sRNA within the complex appears to interact with RNA polymerase, transcribing the target region, and provid ing binding sites for the protein complex including DNA methyltransferase which med iates heterchromatin formation RNAi has been utilized as an excellent tool for the functional analysis of genes and in crop improvement. Sequence directed suppression of endogenous gene(s) is achieved by introducing the transgene which can be processed i nto siRNA in the host genome. RNAi has several advantages over other undirected methods such as physic ochemical and insertional mutagenesis. T hese conventional mutagenesis approaches require generating a large number of mutant lines to identify the desirab le mutation and the gene responsible for generating the phenotype ( Krysan et al., 1999 ; Wesley et al., 2001 ) Mor eover, particularly in a polyploid genome, these methods would be ineffective resulting in null phenotype s due to genetic redundancy ( Waterhouse and Helliwell, 2003 ) RNAi allows simultaneous suppression of homo(eo)logous gene copies, which may have functional redundancy and complementation ( Lawrence and Pikaard, 2003 ; Travella et al., 2006 ) S ilencing a specific gene or all members of a mu l tigene family is also possible by targeting unique and conse rved region s respectively ( Miki et al., 2005 ) RNAi is also favored to identify and characterize lethal alleles ( Waterhouse and Helliwell, 2003 ) with different levels of target gene suppression due to the variation of silencing efficiency in different
44 transgenic events. Furthermore RNAi inductio n can be moderated using spatio temporal specific or inducibl e promoter s ( Ossowski et al ., 2008 ) Off target silencing is considered a drawback to employing transgene induced RNAi. In general, contiguous sequence identity over at least 21 bp and/or overall homology over 88% between the transgene and endogenous sequences could induce RNAi ( Travella et al., 2006 ; Xu et al., 2006 ) Furthermore, a cleav a ge site recognized by Dicer is un known in dsRNAs deri ved from the transgene, thus the chance of off target silencing can be increased by the generation of various species of siRNAs with diverse sequences processed from different regions of dsRNAs ( Ossowski et al., 2008 ) It is challenging to select targeting sequences with p recise s pecificity given the limited sequence information for most of the crop species. Instead, the transgene construct c an be designed among closely related genes with short target sequences similar to miRNA precursors in order to reduce the number of siRNA sp ecies ( Ossowski et al., 200 8 ) T he efficiency of transgene induction med ia ted PTGS is affected by the transgene construct and the resulting dsRNA structure ( Smith et al., 2000 ; Wesley et al., 2001 ) The inverted repeat construct with the splicing intron generally shows 90~100% induction of PTGS in transgenic events, while the occurrence of PTGS was less than 70% with a linker, spacer, or non splicing intron. The c lassical sense or antisense silencing construct trigger s PTGS in less than 15% of transformants. The suppression level of the target gene differs among independent transgenic events. The effectiveness of target gene silencing is pri marily affected by the productivity of siRNA from the transgene and the accessibility of siRNA to the target ( Ossowski et al., 2008 ) Single or low copy number(s) of the transgene induce higher
45 level s of target gene suppression than multiple copies ( Kerschen et al., 2004 ) Transgene integration site in the genome would be also important in the generation of sufficient siRNA, since transgene expression can be repressively influenced by flanking host DNA sequences and/or u nfavorable chromosome regions ( Matzke and Matzke, 1998 )
46 Table 1 1 Improvement of fermentable sugar and/or ethanol yields in lignin reduced transgenic plants and mutants Plants Gene s affecte d Methods L ignin content a) F ermentable sugar yields b) E thanol yields c) Reference s Alfalfa C4H Antisense 29% 4 1 % n.d. Chen and Dixon (2007 ) Alfalfa HCT Antisense 52% 88 % n.d. Chen and Dixon (2007 ) Alfalfa C3 H Antisense 36% 59 % n.d. Chen and Dixon (2007 ) Alfalfa CCoAOMT Antisense 25% 35%* n.d. Chen and Dixon (2007 ) Alfalfa F5H Antisense 0% 0% n.d. Chen and Dixon (2007 ) Alfalfa COMT Antisense 22% 35 % n.d. Chen and Dixon (2007 ) Alfalfa CCR Antisense 31% 58% n.d. Ja ckson et al. (2008 ) Alfalfa CAD Antisense 13% 19% n.d. Jackson et al. (2008 ) Sorghum 4CL Mutation 1) 16% 17% n.d. Saballos et al. (2008 ) Sorghum u nknown Mutation 2) 17% 20% n.d. Saballos et al. (2008 ) Sorghum CAD Mutation 3) 22% 7% n.d. Saballos et al. (2008 ) Sorghum COMT Mutation 4) 13% 21% n.d. Saballos et al. (2008 ) Sorghum CAD Mutation 5) 13% 27% 22% Dien et al. (2009 ) Sorghum COMT Mutation 6) 15% 23% 21% Dien et al. (2009 ) Sorghum COMT+CAD Mutation 7) 27% 34% 43% Dien et al. (2009 ) Switchgrass CAD RNAi 24 % 44 % n.d. Fu et al. (2011b ) Switchgrass COMT RNAi 13% 22% 38% Fu et al. (2011a ) Switchgrass 4CL RNAi 22% 57% n.d. Xu et al ( 2012 ) a) Values are the percentage decrease of total lignin content in transgenic or mutant plants compared with corresponding control plants. b) Values are the p ercentage increase of glucose yields per unit biomass or per u nit cellulose in transgenic or mutant plants, compared with corresponding control plants. Only glucose yields from lignocellulosic m aterials pretreated with dilute sulfuric acid were used for this comparison. c) Values are the p ercentage increase of ethano l yields per unit biomass or per unit cellulose following simultaneous saccharification and fermentation with S. cerevisiae D5A in transgenic or mutant plants, compared with corresponding control plants. 1, 2, 3, and 4) Sorghum brown mid rib mutant bmr2 bmr3 bmr6 and bmr12 respectively. b mr2 was currently characterized harboring mutation in 4CL ( Saballos et al., 2012 ) The gene r esponsible for bmr3 is un known. 5, 6, and 7 ) Sorghum brown mid rib mutant bmr6 bmr 12 and bmr6 and bmr12 double mutant, respectively. T hese mutants were near isogenic lines with the genetic background of cultivar Atlas. V alues with an asterisk are approximately calculated based on the bar graph presented in the corresponding reference. n.d. Not determined.
47 (Iso) Flavonoids Stilbenes tannins A nthocyanins Coumarins etc Figure 1 1 Lignin biosynthesis pathway a dapted from Boerjan et al. (2003 ) and Bonawitz and Chapple (2010 ) Grey arrows indicate alternative pathways that are though t to function in the absence of par ticular enzymes, such as CCoAOMT and COMT. Abbreviations for enzymes: PAL, p henylalanine a mmonia l yase ; C4H, c innamate 4 h ydroxylase ; 4CL, 4 c oum arate: C oA l igase ; HCT, h ydroxycinnamoyl CoA s hikimate /q uinate h ydroxycinnamoyl transferase ; C3 H, c oumarate 3 h ydroxylase ; CCoAOMT, caffeoyl CoA O methyltransferase; CCR, c innamoyl CoA r educt ase, F5H, f erulate 5 h ydroxylase ; COMT, caffeic acid O methyltra nsferase; CAD, c innamyl a lcohol d ehydrogenase Lignin Transport to the cell wall and polymerization through radical coupling
48 CHAPTER 2 ISOLATION AND EXPRESSIONAL CHARACTERIZATION OF CAFFEIC ACID O METHYLTRANSFERASE ( COMT ) IN SUGARCANE Introduction T he monolignol biosynthetic pathway involves multiple O met h ylations of hydroxyl grou ps at the C3 and/or C5 positions of the monolignol precursors ( Boerjan et al., 2003 ) C affeic acid O methyltransferase (COMT) is one of two O methyltran s ferases with c affeoyl C oA 3 O methyltrans ferase (CCoAOMT) in the monolignol biosynthetic pathway In plants, COMTs are encoded by a multi gene family that belong s to the type 1 family of diverse S adenosyl L methionine (SAM) dependent O methyltran s ferases (OMTs) ( Louie et al., 2010 ) Unlike typ e 2 plant OMTs to which CCoAOMT belong s type 1 OMTs do not require divalent cations for catalytic activity In COMT mediated O methylation, the 3 or 5 hydroxyl group on the substrate is deprotonated by catalytic residues, and a reactive methyl group is transferred from SAM to the phenolate yielding S adenosyl L homocysteine (SAH) and the methyl ether derivatives as final produc ts ( Zubieta et al., 2002 ) I n monolignol biosynthesis COMT is primarily involved in sinapyl alcohol biosynthesis, catalyzing 5 O methylation at 5 hydroxyconiferyl aldehyde yielding sinapyl aldehyde COMT was historically known to catalyze 3 or 5 O methylation s at the level of free hydroxycinnamic acids including caffeic and 5 hydroxyfer ulic acid in monolignol biosynthesis H owever, COMT exhibits a strong k i netic preference for aldehydes (5 hydroxyconiferaldehyde and caffeoyl aldehyde) and slightly less for alcohols (5 hydroxiconiferyl alcohol and caffeoyl alcohol) favoring 5 O methylatio n over 3 O methylation ( Dixon et al., 2001 ; Li et al., 2000 ; Louie et al., 2010 ; Osakabe et al., 1999 ; Parvathi et al., 2001 ; Zubieta et al., 2002 ) F urthermore, COMT d eficient mutants and
49 down regulated transgenic plants showed a significant reduction in S unit s and the incorporation of 5 hydrox yconiferyl alcohol into lignin, which is derived from 5 hydroxyconiferyl aldehyde ( Guo et al., 2001 ; Palmer et al., 2008 ; Ralph et al., 2000 ) T he major route for sinapyl alcohol biosynthesis p asses through 5 O methylation at 5 hydroxyconiferyl aldehyde However, given the broad substrate permissiveness of COMT and presence of multiple family members 3 O or 5 O methylation could also occur a t the level of caffeoyl aldehyde caffeyl alcohol and 5 hydroxyconiferyl alcohol Residual S unit content and COMT activity in COMT knock out mutants such as maize bm3 and sorghum bmr12 suggest that multiple COMTs could be partially involved in monolignol biosynthesis depending on different developmental s tages and cell types ( Barrire et al., 2004 ; Dixon et al., 2001 ; Parvathi et al., 2001 ) Sugarcane has a highly polyploid genome with an average 12 haplotypes. D espite the high level of ploidy and the interspecific origin of commercial sugarcane cultivar s homo(eo)logous genes are considered functional and able to translate complete protein s ( Garsmeur et al., 2011 ; Jannoo et al., 2007 ) Considering the complexity of the sugar cane genome, ~31 different consensus COMT EST sequences are clustered within the genome ( Ramos et al., 2001 ) which could be potential homo(eo)logous genes and/or gene family members derived from segmental duplications O nl y one full length COMT gene has been isolated and identified in sugarcane ( GenBank a ccession no. AJ231133) ( Selman Housein et al., 1999 ) T he previously identified COMT shows preferential expression in stems and lignif ied tissues such as the epidermis, xylem, and sclerenchyma ( Ruelland et al., 2003 ; Selman Housein et al., 1999 )
50 In an attempt to modify lignin biosynthesis by suppressing COMT through an RNAi approach, COMT genes were isolated from a commercially important sugarcane cultivar CP88 1762 by cDNA library screening and PCR The expression patt erns of the COMT were investigate d in different tissues and at various developmental stages In addition, putative COMT homo(eo)logous gene sequences were retrieved from a public database and compared with the isolated full length COMT gene. Materials and Methods Isolation of COMT Gene from CP 88 1762 Tissue samples comprised of l ea ves intern ode s node s and immature leaf who rl s of sugarcane ( Saccharum spp. h ybrid s ) va r. CP88 1762 were obtained from the University of Florida Plant Science Research and Educa tion Unit, Citra, Florida Root s and newly emerging shoot s were collected from the plants grown in the Technaflora B.C. Grow hydroponic solution (1/1000 dilution) ( Technaflora Plant Products Ltd. Mission, British Columbia, Canada) Total RNA was extracte d from each tissue using Trizol (Invitrogen, Carlsbad, CA USA ), according to the manufacturer s instruction s Total RNA from each sample was mixed in equal proportion cDNAs were synthesized from 1 g of total RNA using the iScr ipt cDNA Synthesis Kit (Bio Rad, Hercules, CA, USA) Forward ( 5 GTCTCTCTCCTTGTATCCTCC TCTC 3 ) and reverse (5 ATTCGACAATTTAGAATCCAGAACAT 3 ) p rimers were designed from 5 and 3 UTR sequences of S officinarum caffeic acid 3 O methyltransferase (Gen Bank accession No. AJ231133). PCR was carried using a MyiQ cycler (Bio Rad) with iTaq polymerase (Bio Rad) with the following cycles : 15 min at 9 5 C followed by 35 cycles of 94 C for 1 min 52 C for 1 min and 72 C for 1 min and f inal elongation at 72 C for 10 min. PCR
51 products were cloned into pCRII TOPO (In vitrogen) and a total of six clones were sequenced cDNA L ibrary C onstruction and S creening The m ixture of the aforementioned total RNA was used for cDNA library construction. mRNA was purified from the total RNA using Oligotex mRNA Mini Kit ( Qiagen Valencia, CA, USA ). The cD NA library was con str ucted using the ZAP cDNA Synthesis Kit and ZAP cDNA Gigapack III Gold Clo ning Kit (Stratagene, Santa Clara, CA, USA) according to the s instruction s cDNAs synthesized from 5.9 g of mRNA were fractionized using Sepharose CL CB gel filtration column (Stratagene ). cDNAs over 500 bp were ligated into Uni ZAP XR, and recombinant lamda phage w ere p i cked and amplified. Total 4 10 5 pfu of the cDNA contain ing phage w ere primarily screen ed using a 3 46 bp sugarcane COMT probe spanning the SAM binding site S econdary screening was performed using the phage with positive signals from the primary screening. I nsert s containing pBluscript phagemids were excised from Uni ZAP XR and cDNAs of ap proximately 2.0 kb were sequenced Sequence A nalysis T o retrieve tentative consensus (TC) sequences for COMT a keyword search was performed against D ana F arber C ancer I nstitute (DFCI) Sugarcane Gene Index ( http://compbio.dfci.harvard.edu/cgi bin/tgi/gimai n.pl?gudb=s_officinarum ) N ucleotide sequence s and deduced amino acid sequence s were analyzed using ClustalW2 ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ) with the default parameter s C onserved regions o f COMT s were analyzed against structurally characteriz ed Lolium perenne COMT (LpCOMT, Gen B ank accession no. AAD10253 ) ( Louie et al., 2010 )
52 Sampling for E xpression A nalysis of t he COMT G ene Sugarcane tissues were collected from 8 month old greenhouse grown plants. S amples were immediately frozen in liquid nitrogen and stored at 80 C until further analysis. L eaf blade, mid rib, and sheath were collected from the top visible dewl ap (TVD) leaf (Leaf+1). A fter collecting a TVD leaf and leaf sheath, the remaining leaves and sheaths were removed from the stalk. L eaf whorl s shoot apical meristem s internodes, nodes, shoot s and roots were collected from the stalk. Internodes and nodes were numbered in order from shoot apical meristem to the bottom of the stalk. S hoot s were newly emerging tiller s and they were collected before leaf expansion Roots were collected from below the stem root junction, and washed with DEPC treated water befo re freezing. Epidermal, vascular, and ground tissues were collected from the fourth internode below the shoot apical meristem. A fter removing the leaf sheath, the internode was transversely and longitudinally sectioned and immediately frozen in liquid nit rogen. The sections were slowly thawed at 4 C. Vascular tissues were detached from ground tissues using forceps, and epidermal tissues were cut under a dissecting microscope. The tissues collected were immediately frozen in liquid nitrogen until RNA extrac tion s could be performed RT PCR and Qu antitative R eal T ime RT PCR Total RNA from each tissue was extracted using the Trizol reagent (Invitrogen) according to To prevent genomic DNA contamination, total RNA was treated with RNase Free RQ1 DNase (Promega Madison, WI, USA ). cDNA was synthesized from 500 ng of DNase treated total RNA using the iScript cDNA Synthesis Kit (Bio Rad). The primers (forward: 5 TAAATACGCACACCTGCTGCT 3
53 and reverse: 5 ATTCGACAATTTAGAATCCAGAACAT 3 ) were designed for amplification of a fragment of the 3 UTR region. Sugarcane GAPDH primers (forward: 5 CACGGCCACTGGAAGCA 3 and reverse: 5 TCCTCAGGGTTCCTGATGCC 3 ) were used to a mplify a fragment of the sugarcane GAPDH gene as a reference gene for normalization of transcripts as described by Iskandar et al. (2004 ) RT PCR was performed in the MyiQ cycler (Bio Rad) with Phire Hot Start DNA polym erase (New England Biolabs, Ipswich, MA, USA) with the following conditions: 30 s at 9 8 C followed by 26 cycles of 9 8 C for 5 s 60 C for 5 s and 72 C for 20 s and final elongation at 72 C for 1 min. PCR products were analyzed by 1 .2 % ag a rose gel electr ophoresis and visualized with ethidium bromide. Transcript abundance of COMT was quantified in epidermal, vascular, and ground tissues of the internode s. Quantitative r eal time RT PCR was performed in the MyiQ cycler (Bio Rad) with iQ SYBR Green Supermix ( Bio Rad) with the following conditions: 95C for 3 min denaturation, 40 cycles at 95C for 10 s and 55C for 45 s. Amplicon specificity was verified by melt curve analysis from 55C to 95C and by agarose gel electrophoresis. The relative expression of COM T in each tissue was calculated using 2 [ C t ( GAPDH) C t ( COMT) ] Results Isolation of COMT G enes Through cDNA L ibrary S cree n ing and PCR cDNA library screening was performed t o isolate putative COMT homo(eo)logs and other closely related genes within the COMT gene family of sugarcane. A total of six clones were sequenced out of 27 clones screened from the cDNA library derived from CP88 1762. Three different COMT genes were identified and designated as COMTa b and c The PCR products were identical to COM Ta and COMTb COMTc
54 was not detected in the amplified PCR product. T he length of their ORF s w ere 1089 bp and corresponded to 362 deduc ed amino acids in all of the isolated COMT s and the previously identified COMT ( Gen B ank accession No. AJ231133 ), designate d as COMTr. T he overall sequence identity including the UTR s was 98% among COMT s The sequence identities of the coding region s and the UTR s were 99% and 96%, respectively. Three different forms of the full length COMT genes had several nucleotide substi tutions and insertion s /deletion s (indel s ), compared with COMTr (Figure 2 1). The coding region of COMTa showed 100% sequence identity with COMTr while COMTb and c had 2 and 3 nucleotide substitut ions, respectively (Figure 2 1 B ). I ndels were observed in bo th 3 and 5 UTR s of isolated COMTs compared to COMTr (Figure 2 1, A and C ). In the deduced amino acid sequences, COMTb and COMTc had only one substitution Ile294Val compared to COMTr and COMTa ( Figure 2 1 D ). Sequence C omparison B etween the I solated COMT and TCs A t otal of 22 TC sequences for COMT were retrieved from the DFCI S. officinarum Gene Index database. Of the TCs, the overall coverage of the comp lete open reading frame s (ORFs) ranged from 19% to 100%. TC112705 show ed 100% coverage and was ide ntic al with COMTb with in the coding region but had several substitutions in the 3 UTRs A mong the 22 TCs, those containing 3 UTR sequences were selected, and their 3 UTR s were compared with that of COMTa The 3 UTR sequences of one group of TCs (TC112705, TC 114280, TC 127415, TC 138868, and TC 115978) showed relatively high sequence identity with COMTa rang ing from 95 to 99% (Figure 2 2 A ), similar to the sequence identity among the isolated COMT (96%). The 3 UTR sequences of the other
55 group s (TC 120283 and TC 121675) w ere relatively diverse compared to COMTa with 90 and 80% sequence i dentit ies, respectively (Figure 2 2 B ). To investigate whether the sequences were conserv ed in the coding region s among TCs and COMT s, six TCs containing the sequence encoding the SAM binding residues were selected and analyzed Sequence identity in th is r egion from +410 to +755 of COMTa among the selected TCs (TC112705, TC 116269, TC 121675, TC 149781, TC 120283 and TC 1337730) and COMTa ranged from 95% to 100% (Figure 2 3). I n particular, TC121675 and TC 120283, which had relatively diverse 3 UTR sequences compared with other TCs and COMTa showed 100 and 98% sequence identity with COMTa in th is region. Deduced A mino A cid S equence C omparison of the I solated COMT a with Ot her M onocot COMTs T he deduced amino acid sequence from sugarcane COMTa sh owed 94, 91, and 79% similarity with s orghum maize, and Lolium perenne COMT s respectivel y. A pairwise comparison between the sugarcane COMT and the previously characterized additional monocot COMTs showed that the substrate, the SAM binding site and the catalytic residues were perfectly conserved without any amino acid substitutions (Figure 2 4). Expression of the COMT in D ifferent T issues and D evelopmental S tages COMT transcripts were detected in all of the collected tissues but t he expression level wa s higher in the internodes, nodes, and shoots compared with the shoot apical meristem, lea ves or roots ( F igure 2 5 A ). In the internodes and the nodes, lower transcript abundance was apparent in relatively mature tissues, the ninth internode and node ( F igu re 2 5A ). Examining internode s COMT was highly expressed in the vascular
56 bundle and in the epidermal tissue s and significantly less in ground tissue (Figure 2 5, B and C ). Quantitative real time RT PCR results showed that expression level s of COMT in vas cular and epidermal tissue s were 2.5 and 2.2 fold higher than in ground tissue respectively (Figure 2 5 C ) Discussion T hree different forms of COMT were isolated from sugarcane cultivar, CP88 1762 through cDNA library screening and PCR. S tructural charac terization of COMT s in alfalfa and Lolium perenne elucidate d the catalytic resid u es responsible for methyl transfer and the binding domains for recogni tion of SAM and the monolignol precu r sors ( Louie et al., 2010 ; Zubieta et al., 2002 ) Maize and s orghum COMT s have been functionally characterized as the gene s responsible in lignin defective brown midrib mutants bm3 and bmr12 respectively ( Bout and Vermerris, 2003 ; Vignols et al., 1995 ) T he isolated sugarcane COMT has perfect ly conserved substrate and SAM binding domains and catalytic residues. T herefore, it is expected that the sugarcane COMT would have nearly identical catalytic properties toward monolignol precursors compared with the previously characterized lignin biosyn thetic COMT s The sugarcane COMT was preferentially expressed in developing and lignifying tissues, such as relatively immature internodes and nodes. H igher expression level s were evident from stem sections compared to leaves or roots which were similar t o the previously reported COMT expression patterns in sugarcan e and alfalfa ( Inoue et al., 1998 ; Selman House in et al., 1999 ) In the internode s COMT is highly expressed in the epidermal and vascular tissues where lignification take s place Consistent with th is result, the previously id entified sugarcane COMT protein accumulated in the vascular bundles, and ma ize COMT transcripts were preferentially expressed in differentiated
57 xylem cells and sclerenchyma cells under the epidermis ( Ruelland et al., 2003 ; Vignols et al., 1995 ) Thus, gene expression of the sugarcane COMT in lignifying tissues w ould support that the COMT is involved in monolignol biosynthesis. Although assignment of homo(eo)log s remain s to be clarified, a high level of sequence identity ( > 95%) in both the coding and UTRs among the isolated COMT s and most of the TC sequences might indicate these genes are homo(eo)log s In sugarcane, not only h omologous but also homoeo logous alleles display a high level o f col l inearity and sequence conservation ( Garsmeur et al., 2011 ; Jannoo et al., 2007 ) S equence identity among hom o(eo)logous genes was 95.9 and 87.5% in the exon and in tron region s respectively ( Garsmeur et al., 2011 ) Low copy numbers in the Southern blot s hybridized with the sugarcane COMT probe also suggest s a high level of sequence and structural conservation among COMT genes located in homo(eo)logous chromosomal regions ( Selman Housein et al., 1999 ) Moreover, other COMT family members originated by single gene duplications may have limited sequence identity and be below the limit of detection in Southern hybridizations Compared to gene copies derived from single duplications, such as dispersed and transposon based duplications, duplicated genes that have eme rged through polyploidization, especially autopolyploidization, tend to undergo relatively low rate s of sequence substitutions gene loss, and diversification in expression and function ( Parisod et al., 2010 ; Wang et al., 2012 ) Thus it could be anticipated that COMT homo(eo)logous genes are retaine d in the sugarcane genome with a smaller degree of divergence in exp ression and function.
58 Given the high degree of polyploidy and po tential functional redundancy among homo(eo)logous genes in the sugarcane genome, it would be challenging to isol ate and identify lignin defective mutants in sugarcane. One advantage in depl oying RNAi for metabolic engineering is that homo(eo)logous genes or gene family members can be simultaneously down regulated in the host genome targeting conserved regions among genes of interes t ( Lawrence and Pikaard, 2003 ; Travella et al., 2006 ) A mong the isolated COMT s and TCs coding regions spanning SAM binding domains show over 95% sequence identity. In ge neral, contiguous sequence identity greater than 21 bp and/or overall homology greater than 88% between transgene and endogenous sequences could induce RNAi ( Travel la et al., 2006 ; Xu et al., 2006 ) T herefore, this coding region would be a good target for the simultaneous suppression of functionally redundant COMT genes in sugarcane.
59 COMTr --------CCAACACTTCCCAAGC TCGCG CGCTGAGCTCCTCAAGCCCACCAGAAAAG 50 COMTa GGGCTGCAGGCAACACTTCCCAAGCTCGCGTCGCT C AGCTCCTCAAGCCCACCAGAAAAG 60 COMTb GGCACGAGGCCAA ---------GCTCGCGTCGCT C AGCTCCTCAAGCCCACCAGAAAAG 50 COMTc GGCACGAGGCCAACACTTCCCAAGCTCGCGTCGCT C AGCTC CTCAAGCCCACCAGAAAAG 60 COMTr GTCTCTCTCCTTGTATCCTCCTCTCCACCGGGCACCGGCCGGCCGTCGTCAGGCATGGGC 1 04 COMTa GTCTCTCTCCTTGTATCCTCCTCTCCACCGGGCACCGGCCGGCCGTC A TCAGGCATGGGC 1 14 COMTb GTCTCTCTCCTTGTATCCTCCTCTCCACCGGGCACCGGCC T GCCGTCGTCAGG CATGGGC 1 04 COMT c GTCTCTCTCCTTGTATCCTCCTCTCCACCGGGCACCGGCCGGCCGTCGTCAGGCATGGGC 1 14 COMTr ATG GGCTCGACCGCCGAGGACGTGGCCGCGGTGGCGGACGAGGAGGCGTGCATGTACGCG 60 COMTa ATG GGCTCGACCGCCGAGGACGTGGCCGCGGTGGCGGACGAGGAGGCGTGCATGTACGCG 60 C OMTb ATG GGCTCGACCGCCGAGGACGTGGCCGCGGTGGCGGACGAGGAGGCGTGCATGTACGCG 60 COMTc ATG GGCTCGACCGCCGAGGACGTGGCCGCGGTGGCGGACGAGGAGGCGTGCATGTACGCG 60 COMTr ATGCAGCTGGCGTCGGCGTCCATCCTGCCCATGACGCTGAAGAACGCGCTGGAGCTGGGC 120 COMTa ATGCAGCTGGCGTCGGCGTCCATCCTGCCCATGACGCTGAAGAACGCGCTGGAGCTGGGC 120 COMTb ATGCAGCTGGCGTCGGCGTCCATCCTGCCCATGACGCTGAAGAACGCGCTGGAGCTGGGC 120 COMTc ATGCAGCTGGCGTCGGCGTCCATCCTGCCCATGACGCTGAAGAACGCGCTGGAGCTGGGC 120 COMTr CTGCTGGAGGT GCTGCAGGCGGAGGCGCCCGCGGGGAAGGCGCTGGCGCCCGAGGAGGTG 180 COMTa CTGCTGGAGGTGCTGCAGGCGGAGGCGCCCGCGGGGAAGGCGCTGGCGCCCGAGGAGGTG 180 COMTb CTGCTGGAGGTGCTGCAGGCGGAGGCGCCCGCGGGGAAGGCGCTGGCGCCCGAGGAGGTG 180 COMTc CTGCTGGAGGTGCTGCAGGCGGAG GCGCC T GCGGGGAAGGCGCTGGCGCCCGAGGAGGTG 180 COMTr GTGGCGCGGCTGCCCGTGGCGCCCACCAACCCCGACGCGGCGGACATGGTGGACCGCATG 240 COMTa GTGGCGCGGCTGCCCGTGGCGCCCACCAACCCCGACGCGGCGGACATGGTGGACCGCATG 240 COMTb GTGGCGCGGCTGCCCGTGGCGCCCACCAACCCCGAC GCGGCGGACATGGTGGACCGCATG 240 COMTc GTGGCGCGGCTGCCCGTGGCGCCCACCAACCCCGACGCGGCGGACATGGTGGACCGCATG 240 COMTr CTCCGCCTCCTCGCCTCCTACGACGTCGTCAAGTGCCAGATGGAGGACAAGGACGGCAAG 300 COMTa CTCCGCCTCCTCGCCTCCTACGACGTCGTCAAGTGCCAGATGGAGGAC AAGGACGGCAAG 300 COMTb CTCCGCCTCCTCGCCTCCTACGACGTCGTCAAGTGCCAGATGGAGGACAAGGACGGCAAG 300 COMTc CTCCGCCTCCTCGCCTCCTACGACGTCGTCAAGTGCCAGATGGAGGACAAGGACGGCAAG 300 COMTr TACGAGCGGCGGTACTCCGCCGCCCCCGTCGGCAAGTGGCTCACCCCCAACGAGGACGGC 360 COMTa TACGAGCGGCGGTACTCCGCCGCCCCCGTCGGCAAGTGGCTCACCCCCAACGAGGA C GGC 360 COMTb TACGAGCGGCGGTACTCCGCCGCCCCCGTCGGCAAGTGGCTCACCCCCAACGAGGACGGC 360 COMTc TACGAGCGGCGGTACTCCGCCGCCCCCGTCGGCAAGTGGCTCACCCCCAACGAGGACGGC 360 COMTr GTCTCCATGGCCGCGCTCACGCTCATGAACCAGGACAAGGTCCTCATGGAGAGCTGGTAC 420 COMTa GTCTCCATGGCCGCGCT C ACGCTCATGAACCAGGACAAGGTCCTCATGGAGAGCTGGTAC 420 COMTb GTCTCCATGGCCGCGCTCACGCTCATGAACCAGGACAAGGTCCTCATGGAGAGCTGGTAC 420 COMTc GTCT CCATGGCCGCGCTCACGCTCATGAACCAGGACAAGGTCCTCATGGAGAGCTGGTAC 420 COMTr TACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGTACGGGATGACG 480 COMTa TACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGTACGG G ATGACG 480 COMTb TACCTCAAGGACGCGG TGCTTGACGGCGGCATCCCGTTCAACAAGGCGTACGGGATGACG 480 COMTc TACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGTACGGGATGACG 480 COMTr GCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGGGTGTTCAACGAGGGCATGAAG 540 COMTa GCGTTCGAGTACCACGGCACGGACCCGC GCTTCAACCG G GTGTTCAACGAGGGCATGAAG 540 COMTb GCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCG C GTGTTCAACGAGGGCATGAAG 540 COMTc GCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCG C GTGTTCAACGAGGGCATGAAG 540 COMTr AACCACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCT ACACGGGCTTCGAGGGCGTC 600 COMTa AACCACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCTTCGAGGGCGTC 600 COMTb AACCACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCTTCGAGGGCGTC 600 COMTc AACCACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCTTCGA GGGCGTC 600 COMTr TCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCATCACCTCGCAC 660 COMTa TCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCATCACCTCGCAC 660 COMTb TCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCATCACCTCGCAC 660 COMTc TCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCATCACCTCGCAC 660 A
60 COMTr CACCCGCAGATCAAGGGCATCAACTTCGACCTCCCCCACGTGATCTCCGAGGCGCCGCCG 720 COMTa CACCCGCAGATCAAGGGCATCAACTTCGACCTCCCCCACGTGATCTCCGAGGCGCCGCCG 720 COMTb CACCCGCAGATCAAGGGCATCAACTTCGACCTCCCCCACGTGATCTCCGAGGCGCCGCCG 720 COMTc CACCCGCAGATCAAGGGCATCAACTTCGACCTCCCCCACGTGATCTCCGAGGCGCCGCCG 720 COMTr TTCCCCGGCGTGCAGCACGTCGGCGGGGACATGTTCAAGTCGGTGCCGGCGGGCGACGCC 780 COMTa TTCCCCG GCGTGCAGCACGTCGGCGGGGACATGTTCAAGTCGGTGCCGGCGGGCGACGCC 780 COMTb TTCCCCGGCGTGCAGCACGTCGGCGGGGACATGTTCAAGTCGGTGCCGGCGGGCGACGCC 780 COMTc TTCCCCGGCGTGCAGCACGTCGGCGGGGACATGTTCAAGTCGGTGCCGGCGGGCGACGCC 780 COMTr ATCCTCATGAAGTGGATCC TCCACGACTGGAGCGACGCGCACTGCGCCACGCTGCTCAAG 840 COMTa ATCCTCATGAAGTGGATCCTCCACGACTGGAGCGACGCGCACTGCGCCACGCTGCTCAAG 840 COMTb ATCCTCATGAAGTGGATCCTCCACGACTGGAGCGACGCGCACTGCGCCACGCTGCTCAAG 840 COMTc ATCCTCATGAAGTGGATCCTCCACGACTGGAG CGACGCGCACTGCGCCACGCTGCTCAAG 840 COMTr AACTGCTACGACGCGCTGCCGGAGAACGGCAAGGTGATCATCGTCGAGTGCGTGCTGCCG 900 COMTa AACTGCTACGACGCGCTGCCGGAGAACGGCAAGGTGATCATCGTCGAGTGCGTGCTGCCG 900 COMTb AACTGCTACGACGCGCTGCCGGAGAACGGCAAGGTGATC G TCGT CGAGTGCGTGCTGCCG 900 COMTc AACTGCTACGACGCGCTGCCGGAGAACGGCAAGGTGATC G TCGTCGAGTGCGTGCTGCCG 900 COMTr GTGAACACCGAGGCCGTCCCGAAGGCGCAGGGCGTGTTCCACGTCGACATGATCATGCTC 960 COMTa GTGAACACCGAGGCCGTCCCGAAGGCGCAGGGCGTGTTCCACGTCGACATGATCAT GCTC 960 COMTb GTGAACACCGAGGCCGTCCCGAAGGCGCAGGGCGTGTTCCACGTCGACATGATCATGCTC 960 COMTc GTGAACACCGAGGCCGTCCCGAAGGCGCAGGGCGTGTTCCACGTCGACATGATCATGCTC 960 COMTr GCGCATAACCCCGGCGGCAGGGAGCGGTACGAGCGGGAGTTCCACGACCTCGCCAAGGGC 1020 CO MTa GCGCATAACCCCGGCGGCAGGGAGCGGTACGAGCGGGAGTTCCACGACCTCGCCAAGGGC 1020 COMTb GCGCATAACCCCGGCGGCAGGGAGCGGTACGAGCGGGAGTTCCACGACCTCGCCAAGGGC 1020 COMTc GCGCATAACCCCGGCGGCAGGGAGCGGTACGAGCGGGAGTTCCACGACCTCGCCAAGGGC 1020 COMTr GCCGGGTTCTCCGGGTTCAAGGCCACCTACATCTACGCCAACGCCTGGGCCATCGAGTTC 1080 COMTa GCCGGGTTCTCCGGGTTCAAGGCCACCTACATCTACGCCAACGCCTGGGCCATCGAGTTC 1080 COMTb GCCGGGTTCTCCGGGTTCAAGGCCACCTACATCTACGCCAACGCCTGGGCCATCGAGTTC 1080 COMTc GCCGG GTTCTCCGGGTTCAAGGCCACCTACATCTACGCCAACGCCTGGGCCATCGAGTTC 1080 COMTr ATCAAG TAA 1089 COMTa ATCAAG TAA 1089 COMTb ATCAAG TAA 1089 COMTc ATCAAG TAA 1089 COMTr ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGGTC CTCGTACAT 60 COMTa ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTG A TCCTCGTACAT 60 COMTb ATACGCACACCTGCTGCTGCCTGCGTCGAGCTG T GATGGCACATGGTGGTCCTCGTACAT 60 COMTc ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGGTCCTCGTACAT 60 CO MTr CGCCGTCGTCTTCTTCTTC -----AAGTTGTGTTGCTGCTGGTGCTCTCGCGCATGCAT 114 COMTa CGCCGTCGTCTTCTTCTTC -----AAGTTG C GTTGCTGCTGGTGCTCTCGCGCATGCAT 114 COMTb CGCCGTCGTCTTCTTCTTCTTCTTCAAGTTG C GTTGCTGCTGGTGCTCTCGCGCATGCAT 120 COMTc CGCCGTCGTCTTCTTCTTCTTC --AAGTTG C GTTGCTGCTGGTGCTCTCGCGCATGCAT 117 COMTr GATGTACTCTTTTTTGCTTATATTTTGCTTTCTTCATTCTGCAGTCTGGCCT -------166 COMTa GATGTACTCTTTTT GCTTATA C TTTGCTTTCTTCATTCTGCAGTCTGGCCTAGCCTGCC 173 COMTb GATGTACTCTT TTT GCTTATATTTTGCTTTCTTCATTCTGCAGTCTGGCCTAGCCTGCC 179 COMTc GATGTACTCTTTTT GCTTATATTTTGCTTTCTTCATTCTGCAGTCTGGCCTAGCCTGCC 176 COMTr ----------GGCGGCCATATCTTCGGTATGTCTTGTCGAGCTCTTGCATGTTCTGGAT 215 COMTa TCTATGGCGGTGGCGGCCATATCT TCGGTATGTCTTGTCGAGCTCTTGCATGTTCTGGAT 233 COMTb TCTATGGCGGTGGCGGCCATATCTTCGGTATGTCTTGTCGAGCTCTTGCATGTTCTGGAT 239 COMTc TCTATGGCGGTGGCGGCCATATCTTCGGTATGTCTTGTCGAGCTCTTGC G TGTTCTGGAT 236 COMTr TCTAAATTGTCGAATTGTCTCTGCCATGTACCAGTA ATAACAATCAATGATATACTTACT 275 COMTa TCT A AATTGTCGAATTGTCT C TGCCATGTACCAGTAATAACAATCAATGATATACTTACT 293 COMTb TCTAAATTGTCGAATTGTCTCTGCCATGTACCAGTAATAACAAT T AATGATATACTTACT 299 COMTc TCTAAATTGTCGAATTGTCTCTGCCATGTACCAGTAATAACAATCAATG ATATACTTACT 296 COMTr AT ------ACAAAAAAAAAAAAAA -----293 COMTa ATACAA --AAAAAAAAAAAAAAAAA ----316 COMTb ATACAAATGAAAAAAAAAAAAAAAAAAAAAA 330 COMTc A -------AAAAAAAAAAAAAAAAA ----314 B C
61 COMTr MGSTAEDVAAVADEEACMYAMQLASASILPMTLKNALELGLLEVLQAEAPAGKALAPEEV 60 COMTa MGSTAEDVAAVADEEACMYAMQLASASILPMTLKNALELGLLEVLQAEAPAGKALAPEEV 60 COMTb MGSTAEDVAAVADEEACMYAMQLASASILPMTLKNALELGLLEVLQAEAPAGKALAPEEV 60 COMTc MGSTAEDVAAVA DEEACMYAMQLASASILPMTLKNALELGLLEVLQAEAPAGKALAPEEV 60 COMTr VARLPVAPTNPDAADMVDR ML RLLASYDVVKCQMEDKDGKYERRYSAAPVGKWLTPNEDG 120 COMTa VARLPVAPTNPDAADMVDR ML RLLASYDVVKCQMEDKDGKYERRYSAAPVGKWLTPNEDG 120 COMTb VARLPVAPT NPDAADMVDR ML RLLASYDVVKCQMEDKDGKYERRYSAAPVGKWLTPNEDG 120 COMTc VARLPVAPTNPDAADMVDR ML RLLASYDVVKCQMEDKDGKYERRYSAAPVGKWLTPNEDG 120 COMTr VSMAALTLMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNRVFNEGMK 180 COMTa VSMAA LTLMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNRVFNEGMK 180 COMTb VSMAALTLMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNRVFNEGMK 180 COMTc VSMAALTLMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNRVFNEGMK 180 COMTr N HSVIITKKLLEFYTGFEGVSTLVDVGGGIGATLHAITSHHPQIKGINFDLPHVISEAPP 240 COMTa NHSVIITKKLLEFYTGFEGVSTLVDVGGGIGATLHAITSHHPQIKGINFDLPHVISEAPP 240 COMTb NHSVIITKKLLEFYTGFEGVSTLVDVGGGIGATLHAITSHHPQIKGINFDLPHVISEAPP 240 COMTc NHSVIITKKLLEFY TGFEGVSTLVDVGGGIGATLHAITSHHPQIKGINFDLPHVISEAPP 240 COMTr FPGVQHVGGDMFKSVPAGDAILMKWILHDWSDAHCATLLKNCYDALPENGKVIIVECVLP 300 COMTa FPGVQHVGGDMFKSVPAGDAILMKWILHDWSDAHCATLLKNCYDALPENGKVIIVECVLP 300 COMTb FPGVQHVGGD MFKSVPAGDAILMKWILHDWSDAHCATLLKNCYDALPENGKVI V VECVLP 300 COMTc FPGVQHVGGDMFKSVPAGDAILMKWILHDWSDAHCATLLKNCYDALPENGKVI V VECVLP 300 COMTr VNTEAVPKAQGVFHVDMI ML AHNPGGRERYEREFHDLAKGAGFSGFKATYIYANAWAIEF 360 COMTa VNTEAV PKAQGVFHVDMI ML AHNPGGRERYEREFHDLAKGAGFSGFKATYIYANAWAIEF 360 COMTb VNTEAVPKAQGVFHVDMI ML AHNPGGRERYEREFHDLAKGAGFSGFKATYIYANAWAIEF 360 COMTc VNTEAVPKAQGVFHVDMI ML AHNPGGRERYEREFHDLAKGAGFSGFKATYIYANAWAIEF 360 COMTr IK 362 COMTa IK 362 COMTb IK 362 COMTc IK 362 Figure 2 1 N ucleotide and amino acid c omparison s among the isolated sugarcane COMTs. A ) Nucleotide sequences of the 5 UTR. B) Nucleotide sequences in the coding region. Coding sequences for start and stop codon s are underlined. C) Nucleotide sequences of the 3 UTR. D) Deduced amino acid sequence comparison s among the isolated sugarcane COMTs. Underlined red letters ind icate sequence substitutions compared with COMTr D
62 COMTa TAA ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGATCCTCGTA TC115978 TAA ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGGTCCTCGTA TC127415 TAA ATACGCACACCTGCTGCTGCCTGCGTCGAG CTGCGATGGCACATGGTGGTCCTCGTA TC114280 TAA ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGGTCCTCGTA TC112705 TAA ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGGTCCTCGTA TC138868 TAA ATACGCACACCTGCTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGGTCC TCGTA *************************************************** ******** COMTa CATCGCCGTCGTCTTCTTCTTC --AAGTTGCGTTGCTGCTGGTGCTCTCGCGCATGCAT TC115978 CATCGCCGTCGTCTTCTTCTTCTTCAAGTTGCGTTGCTGCTGGTGCTCTCGCGCATGCAT TC127415 CATCGCCGTCGTCTTCTTCTTC --AAGTTGCGTTGCTGCTGGTGCTCTCGCGCATGCAT TC114280 CATCGCCGTCGTCTTCTTCTTC --AAGTTGCGTTGCTGCTGGTGCTCTCGCGCATGCAT TC112705 CATCGCCGTCGTCTTCTTCTTC --AAGTTGCGTTGCTGCTGGTGCTCTCGCGCATGCAT TC138868 CATCGCCGTCGTCTTCTTCTTC TTCAAGTTGCGTTGCTGCTGGTGCTCTCGCGCACGCAT ********************** ****************************** **** COMTa GATGTACTCTTTTT GCTTATACTTTGCTTTCTTCATTCTGCAGTCTGGCCTAGCCTGCC TC115978 GATGTACTCTTTTT GCTTATATTTTGCTTTCTTCATTCTGCAGT CTGGCCTAGCCTGCC TC127415 GATGTACTATTTTT GCTTATATTTTGCTTTCTTCATTCTGCAGTCTGGCCTAGCCTGCC TC114280 GATGTACTCTTTTT GCTTATATTTTGCTTTCTTCATTCTGCAGTCTGGCCTAGCCTGCC TC112705 GATGTACTCTTTTTTGCTTATATTTTGCTTTCTTCATTCTGCAGTCTGGCCT -------TC138 868 GATGTACTCTTTTTGCTTTATATTATGCTTTCTTCATTCTGCAGTCTGGACTAGCCTGCC ******** ***** ***** ************************ ** COMTa TCTATGGCGGTGGCGGCCATATCTTCGGTATGTCTTGTCGAGCTCTTGCATGTTCTGGAT TC115978 TCTATGGCGGTG GCGGCCATATCTTCGGTATGTCTTGTCGAGCTCTTGCGTGTTCTGGAT TC127415 TCTATGGCGGTGGCGGCCATATCTTCGGTATGTCTTGTCGAGCTCTTGCATGTTCTGGAT TC114280 TCTATGGCGGTGGCGGCCATATCTTCGGTATGTCTTGTCGAGCTCTTGCATGTTCTGGAT TC112705 ----------GGCGGCCATATCTTCGGTATGTC TTGTCGAGCTCTTGCATGTTCTGGAT TC138868 TCTATGGCGGGGGCGGACATATCTTCGGTATGTCTGGTCGAGCTCCTGCGTGTTCTGGAT ***** ****************** ********* *** ********** COMTa TCTGAATTGTCGAATTGTCT ATGCCATGTACCAGTAATA 275 TC115978 TCTAAATTGTCGAATTGTCT CTGCCATGTACCAGTAATA 278 TC127415 TCTAAATTGTCGAATTGTCT CTGCCATGTACCAGTAAAA 275 TC114280 TCTAAATTGTCGAATTGTCT CTGCCATGTACCAGTAATA 275 TC112705 TCTAAATTGTCGAATTGTCT CTGCCATGTACCAGTAATA 257 TC138868 TCTAAAT TGGCGAATTGTCTTCTGCCATGTACCAGTAATA 280 *** ***** ********** **************** COMTa TAA ATACGCACACCTGCT GCTGCCTGCGTCGAGCTGCGATGGCACATGGTGATCCTCGT TC121675 TAA TTAC CACACCTGCT GCTGGCTGGGTCGAGCTTGGAGGGCACATGGTGGTCCTTGG TC12 0283 TAA ATACGCACACCTGCTTGCTGCCTGCGTCGAGCTGCGATGGCACATGGTGGTCCTCGT *** *** ********** **** *** ******** ** *********** **** COMTa ACATCG CCGTCGTCTTCTTCTTC ---AAGTTGCGTTGCT GCTGGTGCTCTCGCGC A TC121675 ACATTGGCCG CGT TTTTTCTT ----AAAGTGCCGTGTTGGT -GTGCTTTCG GC A TC120283 ACATCGGCCGTCGTCTTCTTCTTCTTCAAAGTTGCGTTGCTTGCTGGTGCTCTCGCGCCA **** *** *** ** ***** ** *** ** ***** *** ** COMTa TGCAT GATGTAC TCTTTTT -GCTTA TACTT T -GCTTTC --TTCA TTCTGC -A TC121675 TGCAGGAACA AC -CTTTTT -GCTAA TATTTG -GCTTTC ----TAATTCCGA -A TC120283 TGCATTGATGTACCTCTTTTTTGGCTTAATAATTTTGGCTTTTCCTTTCAATTCTGGCAA **** ** ****** *** ** ** ***** *** COMTa GTCT -GGCCTAG -CCT -GCCT -CTATGG --CGGTGG -CGG --CCATA -TCTT TC121675 GCCT -GGCCTA --CCT -GCCT --TTTGG --CGG GG -CGG ---CATAA TTTT TC120283 GTCTTGGGCCTAAACCCTTGGCCTTCTTATTGGCCCGGTGGGGCGGGCCCCTTAATTCTT ** ****** *** **** *** ** *** ** ** COMTa C --GGTATG --TCTTG -TCGAGCTCTTGCATG --------TTCT -GGATTCT --TC121675 G -----ATG ---CTTG -CCAA -CCTTGCGGG ---------TC --GGATTT ---TC120283 TCCGGGAATGGTTCCTTGGTCCGA AAGCTTCCTTGGCCGTTGGTTTCTTGGGATTTTCTT *** **** *** ** ***** COMTa GAATT --GTC --GAATTG ---TCTATGCCA TG --TACCAG -TAATA ------TC121675 AAAT ---GGC ---AATTG ----CT TGCC -TGG -CCCCAA TACA ---------TC120283 AAAATTTGGGTCCGAAAATTGGCCTTCTTTGCCCCTGGGTTCCCAAGTTAATTTAACCCA *** ***** ** **** ** *** Figure 2 2. Nucleotide s equence comparison of the 3 UTR between COMTa and TCs. A) 3 UTR sequence of TCs showing over 95% identity with that of COMTa B) 3 UTR sequence of TCs showing less than 90% identity with that of COMTa Coding sequences for the stop codon are underlined Asterisk indicates identic al sequences among aligned sequences A B
63 COMTa AGAGCTGGTACTACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGT 60 TC112705 AGAGCTGGTACTACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGT 60 TC116269 AGAGCTGGTACTACCTCAAGGACGCGGTGCTTGACGGCGGC ATCCCGTTCAACAAGGCGT 60 TC121675 AGAGCTGGTACTACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGT 60 TC149781 AGAGCTGGTACTACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGT 60 TC120283 AGAGCTGGTACTACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGG CGT 60 TC133773 AGAGCTGGTACTACCTCAAGGACGCGGTGCTTGACGGCGGCATCCCGTTCAACAAGGCGT 60 COMTa ACGGGATGACGGCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGCGTGTTCAACG 120 TC112705 ACGGGATGACGGCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGCGTGTTCAACG 120 TC1162 69 ACGGGATGACGGCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGCGTGTTCAACG 120 TC121675 ACGGGATGACGGCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGCGTGTTCAACG 120 TC149781 ACGGGATGACGGCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGCGTGTTCAACG 120 TC120283 ACG GGATGACGGCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGCGTGTTCAACG 120 TC133773 ACGGGATGACGGCGTTCGAGTACCACGGCACGGACCCGCGCTTCAACCGCGTGTTCAACG 120 COMTa AGGGCATGAAGAACCACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCT 180 TC112705 AGGGCATGAAGAACC ACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCT 180 TC116269 AGGGCATGAAGAACCACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCT 180 TC121675 AGGGCATGAAGAACCACTCGGTCATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCT 180 TC149781 AGGGCATGAAGAACCACTCGGTCATCAT CACCAAGAAGCTCCTCGAGTTCTACACGGGCT 180 TC120283 AGGGCATGAAGAACCAC AG CGT G ATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCT 180 TC133773 AGGGCATGAAGAACCAC AG CGT G ATCATCACCAAGAAGCTCCTCGAGTTCTACACGGGCT 180 COMTa TCGAGGGCGTCTCCACGCTCGTCGACGTGGGCGGCGGCAT CGGCGCCACCCTGCACGCCA 240 TC112705 TCGAGGGCGTCTCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCA 240 TC116269 TCGAGGGCGTCTCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCA 240 TC121675 TCGAGGGCGTCTCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTG CACGCCA 240 TC149781 TCGAGGG T GTCTCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCA 240 TC120283 TCGA A GGCGTCTCCACGCTCGTCGACGTGGGCGGCGGCATCGGCGCCACCCTGCACGCCA 240 TC133773 TCGAGGGCGTCTCCACGCTCGTC A ACGTGGGCGGCGGCATCGGCGCCACCCTG G ACGCCA 240 COMTa TCACCTCGCACCACCCGCAGATCAAGGGCATC AACTT CGACCTCCCCCACGTGATCT 297 TC112705 TCACCTCGCACCACCCGCAGATCAAGGGCATC AACTT CGACCTCCCCCACGTGATCT 297 TC116269 TCACCTCGCACCACCCGCAGATCAAGGGCATC AACTT CGACCTCCCCCACGTGATCT 297 TC121675 TCACCTCGCACCACCCGCAGATCAAGGGCATC AACTT CGACCTCCCCCACGTGATCT 297 TC149781 TCACCTCGCACCACCCGCAGATCAAGGGCATC AACTT CGACCTCCCCCACGTGATCT 297 TC120283 TCACCTCGCACCACCCGCAGATCAAGGGCATC AACTT CGACCTCCCCCACGTGATCT 297 TC133773 T A ACCT T GCA CCACCCG G AGATCAAGGGC C TT T AACTT T CGACCTCCCCCACGTGAT T T T 300 COMTa CCGAGGCGCC GCCGTTCCCCGGCGT GCAGCACGTC -GGCGGGGAC ATGTT 346 TC112705 CCGAGGCGCC GCCGTTCCCCGGCGT GCAGCACGTC -GGCGGGGAC ATGTT 346 TC116269 CCGAGGCGCC GCCGTTCCCCGGCGT GCAGCAC GTC -GGCGGGGAC ATGTT 346 TC121675 CCGAGGCGCC GCCGTTCCCCGGCGT GCAGCACGTC -GGCGGGGAC ATGTT 346 TC149781 CCGAGGCGCC GCCGTTCCCCGGCGT GCAGCACGTC -GGCGGGGAC ATGTT 346 TC120283 CCGAGGCGCC GCCGTTCCCCGGCGT GCAGCACGTC -GGCGGGGAC ATGTT 346 TC1337 73 CCGA A GCGCC C GCCGTTCCCCGG G GT T GC CA CACGT TTG GGCGGGGAC C ATGTT 354 Figure 2 3 Nucleotide s equence comparison of coding region s spanning SAM binding residues between TCs and COMTa Underlined red letters indicate sequence substitutions compared with COM T a.
64 COMTa MGSTAEDVAAVADEEACMYAMQLASASILPMTLKNALELGLLEVLQAEAPAG KALAPEE 59 COMTb MGSTAEDVAAVADEEACMYAMQLASASILPMTLKNALELGLLEVLQAEAPAG KALAPEE 59 SbCOMT MGSTAEDVAAVADEEACMYAMQLASSSILPMTLKNALELGLLEVLQKDA -G KALAAEE 57 ZmCOMT MGSTAGDVAAVVDEEACMYAMQLASSSILPMTLKNAIELGLLEVLQKEAGGGKAALAPEE 60 LpCOMT MGSTAADMAASADEDACMFALQLASSSVLPMTLKNAIELGLLEILVAAG -G KSLTPTE 57 COMTa VVARLPVAPTNP DAADMVDR ML RLLASYDVVKCQMED KDGKYERRYSAAPVGKWLTPN 117 COMTb VVARLPVAPTNP DAADMVDR ML RLLASYDVVKCQMED KDGKYERRYSAAPVGKWLTPN 117 SbCOMT VVARLPVAPTNP AAADMVDR ML RLLASYDVVRCQMED KDGKYERRYSAAPVGKWLTPN 115 ZmCOMT VVARMPAAPSDPAAAAAMVDR ML RLLASYDVVRCQMED RDGRYERRYSAAPVCKWLTPN 119 LpCOMT VAAKL P SAANP EAPDMVDRILRLLASYNVVTCLVEEGKDGRLSRSYGAAPVCKFLTPN 115 COMTa EDGVSMAA L TL MN QDKV L MESWYYLKDAVLDGGIPFNKAYGMTA F EY H GTDPRFNRV F NE 177 COMTb EDGVSMAA L TL MN QDKV L MESWYYLKDAVLDGGIPFNKAYGMTA F EY H GTDPRFNRV F NE 177 SbCOMT EDGVSMAA L AL MN QDKV L MESWYYLKDAVLDGGIPFNKAYGMTA F EY H GTDPRFNRV F NE 175 ZmCOMT EDGVSMAA L AL MN QDKV L MESWYYLKDAVLDGGIPFNKAYGMTA F EY H GTDARFNRV F NE 179 LpCOMT EDGVSMAA L AL MN QDKV L MESWYYLKDAVLDGGIPFNKAYGMSA F EY H GTDPRFNRV F NE 175 COMTa G M KNH S VIITKKLLEFYTGFEG VSTLVDV GGG IGATLHAITSHHPQIKGIN FDL PHVIS 236 COMTb G M KNH S VIITKKLLEFYTGFEG VSTLVDV GGG IGATLHAITSHHPQIKGIN FDL PHVIS 236 SbCOMT G M KNH S VIITKKLLEFYTGFDESVSTLVDV GGG IGATLHAITSHHSHIRGIN FDL PHVIS 235 ZmCOMT G M KNH S VI ITKKLLDFYTGFEG VSTLVDV GGG VGATLHAITSRHPHISGVN FDL PHVIS 238 LpCOMT G M KNH S IIITKKLLELYHGFEG LGTLVDV GGG VGATVAAIAAHYPTIKGVN FDL PHVIS 234 COMTa EAPPFPGVQHVGG DMF KSVPAGDAILM K W IL H D W SDAHCATLLKNCYDALPENG KVI I V 295 COMTb EAP PFPGVQHVGG DMF KSVPAGDAILM K W IL H D W SDAHCATLLKNCYDALPENG KVI V V 295 SbCOMT EAPPFPGVQHVGG DMF KSVPAGDAILM K W IL H D W SDAHCATLLKNCYDALPEKGGKVI V V 295 ZmCOMT EAPPFPGVRHVGG DMF ASVPAGDAILM K W IL H D W SDAHCATLLKNCYDALPENG KVI V V 297 LpCOMT EAPQFPGVTHVGG DMF KEVPSGDTILM K W IL H D W SDQHCATLLKNCYDALPAHG KVV L V 293 COMTa ECVLPVNTEAVPKAQGVFH V DM I ML A HN PGGR E RYEREFHDLAKGAGFSGFKATYIYANA 355 COMTb ECVLPVNTEAVPKAQGVFH V DM I ML A HN PGGR E RYEREFHDLAKGAGFSGFKATYIYANA 355 SbCOMT ECVLPVTTDAV PKAQGVFH V DM I ML A HN PGGR E RYEREFRDLAKAAGFSGFKATYIYANA 355 ZmCOMT ECVLPVNTEATPKAQGVFH V DM I ML A HN PGGK E RYEREFRELAKGAGFSGFKATYIYANA 357 LpCOMT QCILPVNPEANPSSQGVFH V DM I ML A HN PGGR E RYEREFQALARGAGFTGVKSTYIYANA 353 COMTa WAIEFI K 362 COMTb WAIEFIK 362 SbCOMT WAIEFIK 362 ZmCOMT WAIEFIK 364 LpCOMT WAIEFTK 360 Figure 2 4 Amino acid sequence comparison s between sugarcane COMTs and functionally characterized COMT s in mon ocot plants SbCOMT ( Sorgh um bicolor Gen B ank accession no. AAO43609 ). ZmCOMT ( Zea Maize G en B ank accession no. Q06509 ). LpCOMT ( Lolium perenne Gen B ank accession no. AAD10253 ). Conserved regions among plant COMTs are presented according to the structurally characterized LpCOMT as described in Louie et al. (2010 ) Conserved residues involved in substrate and SAM binding are shaded yellow and red, respectively. C atalytic residues are underlined and marked with bold characters in His266, Asp267, and Glu326 (following the numbering in LpCOMT) R ed letters indicate a sequence substitution between sugarcane COMTs.
65 Relative COMT expression Figure 2 5 COMT expression patterns in sugarcane. A ) RT PCR of COMT expression in different tissues and at different developmental stages. SAM, shoo t apical meristemaitc tissue; Leaf whorl, immature leaf whorl above SAM; Leaf +1, leaf with top visible dewlap; Internode +1 ~ +9 and Node +3 ~ +9, the number of internode s or node s counted from SAM; Shoots, emerging tiller ; Root s roots collected from ste m root junction region. B ) and C ) RT PCR and qu a ntitative real time RT PCR of COMT expression in vascular, grou n d, and epidermal tissues collected from the internode Sugarcane GAPDH gene was used as an internal control. Leaf whorl SAM Blade M id rib Sheath +1 +3 +5 +9 +3 +5 +9 Shoot Root COMT GAPDH Nodes Internode s Leaf +1 COMT GAPDH Vascular Ground Epidermal A B C
66 CHAPTER 3 GENERATION OF COMT SUPP RESSED TRANSGENIC SUGARCANE AND EFFECTS OF COMT SUPPRESSION ON LIGNIN, PLANT GROWTH, AND SACCHARIFICATION PERFORMANCE UNDER GREENHOUSE CONDITIONS (Re printed from Jung et al. 20 12 with permission from Plant Biotechnology Journal 1 ) Introduction The environm ental, political and economic challenges associated with our dependence on fossil fuels, have sparked a tremendous interest in developing efficient biomass feedstock s to satisfy the increasing demand for sustainable energy sources ( Byrt et al., 2011 ; Hisano et al., 2009 ; Tew and Cobill, 2008 ) Sugarcane ( Saccharum spp. hybri ds) is a prime herbaceous biofuel feedstock. (39 Mg ha 1 ; stalk leaves and tops) is significantly higher than maize ( 17.6 Mg ha 1 ; grain and stover), switchgrass (10.4 Mg ha 1 ; biomass ) or Miscanthus (29.6 Mg ha 1 ; biomass ) ( Heaton et al., 2008 ; Waclawovsky et al., 2010 ) Its perennial growth habit and C 4 photosy nthetic pathway maximize carbon sequestration while minimizing light requirements, water, and nitrogen inputs ( Byrt et al., 2011 ; Somerville et al., 2010 ) Sucrose accumulates in the stalk internodes of sugarcane and is either utilized for sugar production or readily fermented to the transportation fuel ethanol. The abundant lignocellulosic sugarcane residues are cur rently underut ilized for bioenergy production ( Leite et al., 2009 ; Somerville et al., 2010 ) Including these re sidues for second generation biofuel production has the potential to boost biofuel yields per unit land area compared to current sucrose based conversion technologies. 1 Jung, J.H., Fouad, W.M., Vermerris, W., Gallo, M. and Altpeter, F. (2012) RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass. Plant B iotechnol. J. 10, 1067 1076.
67 Diminished recalcitrance to enzymatic hydrolysis is a desirable trait for lignocellulos ic feedstocks. The presence of lignin in the cell wall exacerbates biomass recalcitrance and limits bioconversion of lignocellulosic biomass into fermentable sugars ( Jrgensen et al., 2007 ; Mansfield et al., 1999 ; Weng et al., 2008 ) Energy intensive thermo chemical pretreatments are required to degrade the cell wal l matrix and to disrupt the crystalline structure of cellulose, thereby increasing binding sites for cellulolytic enzymes ( Mosier et al., 2005 ) Pretreatments are followed by saccharification, during which the cell wall polymers, primarily cellulose and hemicellulose, are enzymatically hydrolyzed into monomeric sugars for fermentation ( Lu and Mosier, 2008 ) Lignin is an aromatic, hydrophobic polymer primarily consisting of p hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, which are polymerized by radical coupli ng of three different monolignols: p coumaryl, coniferyl, and sinapyl alcohol, respectively ( Boerjan et al., 2003 ) During saccharification, cellulases can adsorb irreversibly to lignin thereby reducing the overall enzymatic activity ( Chernoglazov et al., 1988 ; Jrgensen et al., 2007 ) The manipula tion of lignin biosynthesis in feedstocks is a prime strategy to reduce biomass recalcitrance and improve fer mentable sugar yields Ten different enzymes catalyze a series of hydroxylation, methylation, and side chain reduction reactions of monolignol prec ursors ( Bonawitz and Chapple, 2010 ) Down regulation of monolignol biosynthetic genes in transgenic alfalfa reduced total lignin content a nd altered lignin subunit composition. Fermentable sugar yields, with or without pretreatment, were shown to be negatively correlated with total lignin content ( Chen and Dixon, 2007 ) Down regulation of enzyme s involved in the terminal steps of
68 the monolignol biosynthe tic pathway including COMT or CAD generally had little or no negative effects on plant growth ( Chen and Dixon, 2007 ; Fu et al., 2011a ; Jackson et al., 2008 ; Pilate et al., 2002 ) In grasses, biomass conversion is also influenced by lignin subunit composition based on studies in maize and sorghum brown midrib mutants ( Dien et al., 2009 ; Saballos et al., 2008 ; Vermerris et al., 2007 ) Recently, transgenic approaches for manipulat ing lignin biosynthesis h ave been successfully applied to switchgrass ( Fu et al., 2011a ; Saathoff et al., 2011 ; Xu et al., 2011 ) Caffeic acid O methyltransferase (COMT) functions late in the monolignol biosynthetic pathway and despite its name, methylates 5 hydroxyconiferyl aldehy de and 5 hydroxyconiferyl alcohol to form S unit precursors, sinapyl aldehyde and sinapyl alcohol, respectively ( Bout and Vermerris, 2003 ; Guo et al., 2001 ; Humphreys et al., 1999 ; Osakabe et al., 1999 ) In sugarcane ~31 different consensus EST sequences are clustered to COMT, which could be potential allelic variants or homo(eo)logous genes ( Ramos et al., 2001 ) The p r eviously identified full length COMT ( GenBank a ccession no. AJ231133) shows 91% amino acid similarity with maize lignin specific COMT, and i s preferentially expressed in stems and roots, and in lignif ying tissues such as epidermis, xylem, and sclerenchyma ( Ruelland et al., 2003 ; Selman Housein et al., 199 9 ) Conventional breeding and genetic engineering of sugarcane are challenging because of its highly polyploid genome derived from interspecific hybridization ( D al Bianco et al., 2011 ; Lakshmanan et al., 2005 ) Although most plants examined to date share many of the enzymatic reactions leading to the synthesis of lignin ( Xu et al., 2009 ) limited information is available for sugarcane and none of the lignin biosynthetic genes
69 have been functionally characterized through forward or reverse genetics ( Ramos et al., 2001 ; Ruelland et al., 2003 ; Selman Housein et al., 1999 ) Biolistic or A grobacterium mediat ed gene transfer into sugarcane has typically been achieved by using embryogenic callus as target tissue ( Arencibia et al., 1998 ; Bower and Birch, 1992 ; Gallo Meagher and Irvine, 1996 ) Recent improvements for biolistic gene transfer focused on the reduction of the tissue culture period by using direct embryoge ne sis and by delivering minimal expression cassette s instead of plasmids ( Kim et al., 2012 ; Taparia et al., 2012a ; Taparia et al., 2012b ) A number of transgenes have been introduced into sugarcane to incorporate traits including h erbicide resistance, tolerance to abiotic or biotic stress and production of sugar and value added metabolite s ( Altpeter and Oraby, 2010 ) However, li gnin modification or the application of RNAi for crop improvement has not been reported in sugarcane. Here we describe the generation of transgenic sugarcane with RNAi suppression of COMT and demonstrate that the resulting transgenics are more amenable to biomass conversion for the production of fuels and chemicals. Materials and Methods Plant G rowth Commercially important sugarcane cv. CP88 1762 stalks were collected from the Everglades Research and Education Center, University of Florida, Belle Glade, Fl orida, USA. Singl e node segments from these wild type plants were transplanted to 15 liter pots containing Fafard No. 2 mix (Conrad Fafard, Agawam, MA, US A) and grown to maturity under natural photoperiod in an air conditioned greenhouse set at 28C/22C ( day/night). Plants were irrigated once a day and fertilized biweekly with Miracle Gro Plant and Lawn Food (Scotts Miracle Gro, Marysville, OH, USA).
70 The primary tran sgenic lines and wild type plants were clonally propagated by single node segme nts of matu re plants and in 15 liter pots containing Fafard No. 2 mix. Plants were arranged in a randomized block design, with eight replications, and grown under the conditions as described above. A total of four stalks were maintained in each pot by removing juveni le tillers weekly. Above ground fresh weight was measured from the most mature tiller of the 7 month old plants. After removing leaves and leaf sheath, stalk diameter was measured in the middle of the stalk, and length was measured from soil surface to the apical meristem of the stalk. The basal internode of th e transgenic, control, and wild type plant were transversely or longitudinally cut, and photographed immediately without fixation and staining to display the brown coloration. Vector C onstruction The COMT RNAi vector was constructed using the pWFOsC4H::Bg4CLi vector ( Fouad et al., 2010 ) To amplify a 346 bp COMT fragment from the sugarcane cD NA, a pair of primers (forward: 5 AGAGCTGGTACTACCTCAAGGACG 3 reverse: 5 GTTTAAACATGTCCCCGCCGACGTG 3 ) was designed to the sugarcane COMT sequence (GenBank accession no. AJ231133 ) This fragment was located on the COMT coding region between nucleotide + 410 and + 75 5, spanning part of the highly conserved SAM binding pocket among plant COMTs ( Louie et al., 2010 ) PCR products were clone d into the pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA, USA), and confirmed by sequencing. Xba I and EcoR V restriction enzyme sites were introduced to the 5 end of the COMT fragment and cloned into the pCR2.1 TOPO vector. The inverted repeats of Bg4CL fra gment in the pWFOsC4H::Bg4CLi (Fouad et al., 2010) were replaced by the COMT fragment with two subsequent cloning steps. The resulting COMT RNAi cassette consisted of the 1994 bp Oryza sativa cinnamate 4 hydroxylase
71 ( C4H ) gene promoter (GenBank accession n o. AC136224), inverted repeats of the 346 bp sugarcane COMT fragment separated by the 94 bp of Paspalum notatum 4 coumarate : CoA ligase ( 4CL ) intron (Fouad et al., 2010) and the CaMV 35S 3 UTR (Figure 3 1, A ). The pJFNPTII vector ( Altpeter et al., 2000 ) provided the selectable marker and contained the neomycin phosphotransferase II ( npt II) gene under the transcriptional control of the Zea mays ubiquitin promoter with first i ntron and CaMV 35S 3 UTR. Generation of T ransgenic S ugarcane Embryogenic sugarcane calli were induced as described by Chengalra yan and Gallo Meagher (2001 ) Immature leaf whorls were cut transversely into 2~5 mm pieces and cultured on m odified MS basal medium (CI 3), supplemented with 20 g L 1 sucrose and 13.6 M 2,4 D, with the pH adjusted to 5.8 Tissues were sub cultured on C I 3 media every 2 weeks. Ten weeks after callus induction, particle bombardment was performed using the PDS 1000/He biolistic particle delivery system (Bio Rad, Hercules, CA, USA) as previously described ( Altpeter et al., 1996 ) For delivery of the minimum linear transgene cassette (MC), the COMT RNAi cassette and npt II expression cassettes were released by restriction enzyme digestion with Xmn I and I Sce I, separated by gel electrophoresis, and extracted using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA, USA). COMT and npt II MC DNAs were mixed in a 2:1 molar ratio, co precipitated onto 1 m diameter gold micro carriers (Bio Rad) and accelerated into target tissues as described earlie r ( Altpeter et al., 2010 ) 24 shots of bombardment were perform ed with the following conditions: 1100 psi rupture pressure, 2 7.5 Hg vacuum pressure, 6 cm distance from the rupture disk to call i Calli were transferred to CI 3 media containing geneticin (G 418, 30 mg L 1 ) 6 days after bombardment, for 3
72 biweekly subcul tures ( Kim et al., 2012 ) Calli that survived were regenerated into plants on media containing 50 mg L 1 thidiazuron (TDZ) for 2 weeks. For further selection, elongation, and rooting of shoots, regenerated plantlets were transferred to hormone free media containing 30 mg L 1 paromomycin for four biwe ekly subcultures. Regenerated plants with r oots were transplanted in 0.65 liter pots with Fafard No. 2 mix and placed in a growth room with 80% relative humidity with 16 h photoperiod and 500 mol m 2 s 1 light intensity. Evaluation of NPTII E xpression NP TII expression was evaluated by NPTII ELISA using a commercial kit (Agdia, Elkhart, IN, USA). Total protein was extracted from leaves and quantified with the Bradford assay ( Bradford, 1976 ) The ELISA assay was performed using 20 g of to tal transgenic plants was qualitatively evaluated by color development in comparison with the supplied NPTII standard and wi ld type protein extracts. PCR Analysis The presen ce of the COMT RNAi cassette in genomic DNA extracts of transgenic lines was confirmed by PCR. Genomic DNA was extracted from leaves using the CTAB method ( Murray and Thompson, 1980 ) and 75 ng was used per reaction as a template for amplification. Primers (forward: 5 CCTGCTAGTCTTCTCT CTCATTGTT 3 and reverse: 5 GTGATGATGACCGAGTGGTTCTT 3 ) designed to the C4H promoter region and annealing to the sense fragment of COMT (Figure 3 1 A ) were used with an expected amplification product of 550 bp. PCR was performed in the MyiQ cycler (Bio Rad ) with iTaq DNA Polymerase (Bio Rad) under the following conditions: 95C for 3
73 min denaturation, 35 cycles at 95C for 30 s 60C for 30 s 72C for 1 min, and final extension at 72C for 7 min. Southern B lot A nalysis High molecular weight genomic DNA w as extracted from leaves using the CTAB method ( Murray and Thompson, 1980 ) Twenty microgram of genomic DNA were digested to completion with EcoR I, separated by ele c trophoresis on 1.0% agarose gel, and transferred onto the Hybond (GE He althcare Biosciences, Pittsburgh, PA, USA). Probes were generated by PCR to the C4H promoter region of the COMT RNAi cassette and labeled with 32 P dCTP (Perkin Elmer, Waltham, MA, USA) using the Prime a Gene Labeling System (Promega, Mannheim, Germany). Hy bridization and washing were performed according to the ray film (Fisher Scientific, Atlanta, GA USA ) at 80C for 2 d. Small RNA N orthern B lot The third internode below the apical shoot m eristem was collected from primary transgenic or wild type plants, and total RNA was extracted from 3 g of internode tissue using the modified hot SDS/phenol method ( Shirzadegan et al., 1991 ) Briefly, 3 g of internode was ground under liquid nitrogen, and homogenized by the addition of pre warmed (65 C) 10 mL acidic phenol (pH 4.5) and 10 mL of extraction buffer containing 0.1 M LiCl, 0.1 M tris HCl (pH 8.0), 0.01M EDTA (pH 8.0), 1% SDS (w/v), and 0.1% PVP (w/v). Following 5 min incubation in a 65 C water bath, 10 mL of chloroform was added. T he homogenate was thoroughly mixed, and centrifuged at 1 3 ,000 g for 15 min at 4 C The upper aqueous phase was extracted once more with the equal volume of chloroform. Total RNA was precipitated by adding the equal volume of isopropanol, and
74 washed twice with 70% ethanol. Small RNA was separated from the total RNA using the method described in Lu et al. (2007 ) Briefly, high molecular weight RNA was precipitated by adding 1/10 volume of 50% (w/v) PEG (M.W. 8000) and 5 M NaCl. T he mixture was incubated on ice for 1 h, and centrifuged at 16 ,000 g for 15 min at 4 C The supernatant contain ing small RNA was transferred to a new tube and small RNA was precipitated with the equal volume of isopropanol for 16 h at 20 C. S mall RNA was r ecovered by centrifugation at 16 ,000 g for 30 min at 4 C and pellet was washed twice with 80% ethanol. Thirty g of small RNA was separated by electrophoresis in 15% polyacrylamide TBE/urea gel (Bio Rad) and transferred to the Hybond N+ membrane (GE Healthcare Biosciences) using a semi dry transfer cell (Bio Rad). The 346 bp COMT fragment, used for COMT RNAi vecto r construction, was labeled with 32 P dCTP (Perkin Elmer) using the Prime a Gene Labeling System (Promega) for use as a probe. Hybridization was carried out overnight at 38 ( Brown et al., 2001 ) Following hybridization, the membrane was briefly rin sed 2X SSC 0.2% SDS and then washed twice with 2X SSC 0.2% SDS at 50 C for 20 min each. The membrane was exposed to Kodak X ray film at 80 C for 2 d. Quantitative R eal T ime RT PCR for Q uantification of COMT E xpression The third internode below the ap ical shoot meristem was collected from the clonally produced progenies of transgenic and wild type plants. Total RNA was To prevent genomic DNA contamination, total RN A was treated with RNase Free RQ1 DNase (Promega). cDNA was synthesized from 500 ng of DNase treated RNA using iScript cDNA Synthesis Kit (Bio Rad). P rimers (forward: 5 TAAATACGCACACCTGCTGCT 3 and reverse: 5
75 ATTCGACAATTTAGAATCCAGAACAT 3 ) were designed for amplification of the 3 UTR region of the targeted COMT gene. Sugarcane GAPDH primers (forward: 5 CACGGCCACTGGAAGCA 3 and reverse: 5 TCCTCAGGGTTCCTGATGCC 3 ) were used to amplify a fragment of the sugarcane GAPDH gene as a reference gene for normali zation of transcripts as described by Iskandar et al. (2004 ) Quantitative real time PCR of the transcripts was performed in the MyiQ cycler (Bio Rad) with iQ SYBR Green Supermix (Bio Rad) under the following conditions : 95C for 3 min denaturation, 40 cycles at 95C for 10 s and 55C for 45 s Amplicon specificity was verified by melt curve analysis from 55C to 95C and by agarose gel electrophoresis. COMT expression levels in tra nsgenic plants relative to wild type pl ants were calculated using the 2 Ct method ( Livak and Schmittgen, 2001 ) Microscopic and H istochemical A nalysis The transverse stem sections were hand cut with a razor blade from the sixth internode below the sho ot apical meristem in both wild type and transgenic line T4. M ule and Wiesner staining were performed according to Vermerris and Nicholson (2006 ) For M ule staining, s ections were immersed in freshly prepared 1% (w/v) potassium permanganate for 30 min at room temperature, and then washed with water for 2 min. The stained sections were then treated with 6M HCl for 1 min and excessive HCl was removed using tissue paper. For Wiesner staining sect ions were immersed with 2% (w/v) phloroglucinol in 2:1 mixture of absolute ethanol and 12M HCl for 3 min at room temperature. Sections were observed using an Olympus BH 2 light microscope (Olympus, Tokyo, Japan). Images were taken and recorded in the Infin ity 1 camera and Infinity analyzer (Lumenera Corporation, Ottawa, Ontario, Canada).
76 Sample P reparation and D etermination of L ignin C ontent and C omposition Stalks with 12 internodes were collected from clonally propagated transgenic or wild type plants, and internodes 1 3 below the shoot apical meristem, all leaves and leaf sheaths were removed. The remaining mature portion of the stalks was dried at 45C, and ground using a Wiley mill (Thomas Scientific, Swedesboro, NJ, USA) with a 1.0 mm sieve. The ground samples were passed through 0.42 mm sieve to remove irregular particles. Following three successive extractions with 50% ethanol (v/v), under sonication at 45C for 30 min, samples were dried at 45C until constant weight. The modified acetyl bromide meth od was used to determine lignin content ( Foster et al., 2010 ; Hatfield et al., 1999a ) Two milligram of extract fr ee dried sample was placed in a 2 mL polypropylene tube, and 1 mL of freshly prepared 25% (w/w) acetyl bromide/glacial acetic acid solution was added. The tubes were incubated in a water bath at 50C for 4 h, and during the last hour, samples were thorough ly mixed at 15 min intervals, and placed on ice for 30 min. One hundred microliter of each reaction were transferred into a 2 mL polypropylene tube containing 200 L of 2 M NaOH and 1.7 mL glacial acetic acid. The absorbance of the solution was determined at 280 nm using an Evolution 300 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The lignin content was calculated by employing the molar extinction coefficient of 21.5 L g 1 cm 1 for milled sugarcane vascular bundle lignin ( He and Terash ima, 1990 ) Lignin composition w as determined by thioacidolysis method following Robinson and Mansfield (2009 ) One milliliter of thioacidolysis reagent (2.5% boron trifluoride etherate and 10% ethanethiol in distilled dioxane, v/v) was added to each reaction vial containi ng 10 mg of extract free sample. Each vial was flushed with N 2 and tightly sealed with Teflon lined screw cap. The sample was incubated at 100 C for 4 h with
77 manual agitation every hour. After the reaction was halted at 20 C for 5 min, 0.2 mL of tetracos ane (5 mg mL 1 methylene chloride) was added to each vial as an internal standard then the sample was acidif ied to pH between 3 and 4 with 0.3 mL of 0.4 M sodium biocarbonate. F or the extraction of reaction products 2 mL of distilled water and 1 mL of me thylene chloride were added and vortexed. After phase separation, organic phase was recovered, and simultaneously passed though the Pasteur pipette packed with 50 mg of anhydrous sodium sulfate. Extracted sample s were dried at 45 C under a stream of N 2 an d then resuspended in 1 mL of methylene chloride Twenty microliter of the sample was silylated by adding 20 L of pyridine and 100 L of BSTFA [ N O bis (trimethylsilyl) acetamide ] at room temperature for at least 2 h T hi oacidolysis derivatives for lignin monomers were analyzed using the Varian 3800 GC gas chromatograph coupled to the Varian 1200 MS mass spectrometer ( Varian, Walnut Creek, CA USA ). One microliter of the sample was injected onto a Factor 4 VF 5ht column (35 m, 0. 25 mm i.d.) with helium (1. 2 mL min 1 ) as carrier gas. GC conditions were as follows: Initial column temperature, 130 C held for 3 min, ramped at 3C min 1 to 250 C and held for 5 min; injector temperature, 250 C ; and 1:2 split ratio. The mass spectrometer was operated in electron impact mode at 70 eV. The detector was operated at 1.0 kV. M ass spectra were recorded every 0.2 s at a scanning range of 50 5 50 m / z and the date was registered and analyzed using Varian MS Workstation software Thio acidolysis monomers, G and S were quant ified by employing the response factors from internal control (IS) as follows: G versus IS, 0.47; and S versus IS, 0.53 ( Yue et al., 2012 )
78 Dilute A cid P retreatment and E nzymatic H ydrolysis Dilute acid pretreatment and enzymatic hydrolysis was performed according to published protocols ( Chen and Dixon, 2007 ; Selig et al., 2008 ) Extractive free samples (0.15 g) were so aked in 1 .35 mL of dilute sulfuric acid [ final concentration 1.3% (w/w) ] and autoclaved at 121C for 40 min. After autoclaving, the pretreated samples were washed twice with 25 mL distilled water. The pretreated or native biomass samples were placed in 50 mL polypropylene tubes, and suspended in 5 mL of 0.1 M sodium citrate buffer (pH 4.8). Autoclaved distilled water containing 100 L of 2% (w/w) sodium azide, 6 FPU of Kerry Biocellulase W (Kerry Bioscience, Cork, Ireland), and 6 .4 p NPGU glucosidase (Sigma, Saint Louis, MO, USA) were added to bring the total volume to 10 mL The hydrolysis was performed for 120 h in a shaking incubator at 50C and 250 rpm. After enzymatic hydrolysis, 1 mL of the each sample was colle cted, filtered through a 0.2 m syringe filter, and glucose yields were analyzed using a n YSI glucose analyzer (YSI Life Science, Yellow Springs, OH, USA). Statistical A nalysis ANOVA was performed using Proc GLM in SAS TM Version 9.3 ( SAS Institute Inc., Ca ry, NC USA ) Statistical significance among means for biomass, stalk diameter, and P < 0.05. T tests were performed to determine whether the means of total lignin content, S/G ratios, and glucose yields were statistically significant between the transgenic plants and the wild type, tissue culture or npt II only transgenic controls ( P < 0.05).
79 Results Generation and M olecular C haracterization of T ransgenic S ugarcane L ines The RNAi inducing transgen e cassette contained a highly conserved region of COMT This may suppress COMT expression from both targeted COMT and homo(eo)logous COMT shared 95 100% nucleotide sequence identity with five tentat ive consensus (TC) sequences, TC116269, TC120283, TC121675, TC133773, and TC149781, putative homo(eo)logs of sugarcane COMT (DFCI S. officinarum Gene Index; http://compbio.dfci.harvard.edu/cgi bin/tgi/geneprod _search.pl). The promoter of Oryza sativa C4H gene was used to induce expression of RNAi inducing transgene in lignifying tissues. Biolistic gene transfer was used to produce sugarcane transformants. Following 24 bombardments, regeneration of plants, and selection of putative transformants on genetic in and paromomycin, 43 lines tested positive for the NPTII enzyme as determined by ELISA (Figure 3 1 B ) This resulted in a transformation efficiency of 1.8 transgenic plants produced per bombardment. Thirty eight of these 43 transgenic lines tested positiv e for the presence of the COMT RNAi cassette as determined by PCR analysis (Figure 3 1 C ) resulting in a co transformation efficiency of 88% for the unlinked npt II and COMT minimal cassettes. To determine copy number of the COMT RNAi cassette in the transf ormants, genomic DNA was analyzed by Southern blot analysis (Figure 3 1 D ). A unique transgene integration pattern was detected for each transgenic line, confirming these were independent transformation events. Among the 12 transgenic lines analyzed, two li nes (T4 and T18) displayed a relatively simple transgene integration pattern with two
80 copies. Four lines had less than eight hybridization signals, and six lines showed a complex integration pattern with more than eight copies. Small RNA northern blot ana lysis was performed for 17 transgenic plants to examine whether siRNA was produced from the dsRNA precursor generated by the inverted repeats of the COMT fragment. Seven of the 17 transgenic plants (41%) produced COMT siRNA, while siR NA was not detected in the wild type plant (Figure 3 2 A ). The size marker indicated that siRNA from the transgenic plants was ~21 nt long, whereas another siRNA class of ~24 nt was not detected in the transgenic plants (Fi gure 3 2 A ). Clones of primary transformants were vegeta tively propagated and grown in the greenhouse, and quantitative real time RT PCR of the transcripts was performed in order to investigate siRNA induced suppression of COMT The expression level was significantly reduced by 67, 97, and 97% in lines T41, T23 and T4, res pectively, compared to the wild type controls (Figure 3 2 B ) Lignin C ontent, C omposition, and E nzymatic S accharification Total lignin content and subunit composition were determined to examine the effect of COMT suppression on lignin biosynth esis in the vegetative progenies of the tran sgenic plants. Compared to wild type, total lignin was reduced by 3.9, 8.4, and 13.7% in lines T41, T23, and T4, respectively (Table 3 1 ). Lignin of transgenic plants was composed of significantly less S unit and similar a mount of G unit compared to wil d type (Table 3 1 ). Transgenic lines had lower S/G ratios ranging from 1 .27 to 0.79, while that of wild type was 1.47. There was no difference in total lignin content or lignin monomer composition of the transgenic control plants transfor med with npt II compared to wild type plants (Table 3 1 ).
81 To obtain an estimate of recalcitrance to enzymatic saccharification, enzymatic hydrolysis of lignocellulosic biomass from all samples was performed, with and without dilute acid pretreatments. Transgenic plants with reduced total lignin content and reduced S/G ratios, showed improvements in enzymatic digestibility of t he biomass compared to the wild type controls (Table 3 1 ). Without pretreatment, the biomass generated from t ransgenic line T4 yielded 41 % more glucose than the wild type plants, while T41 and T23 showed sim ilar glucose yields as the wild type plant. With dilute acid pretreatment, significant increases were observed for all of the lines T41, T23, and T4, which yi elded 25 %, 2 6 %, and 51 % more glucose, respectively. Pretreatment enhanced the saccharification efficiencies of the transgenics 2.1 to 2.5 fold compared to 1.9 fold for wild type. No significant differences were detected in glucose yields between the npt II only and wild type controls. Plant P henotype and G rowth Clonally propagated progeny of transgenic sugarcane displayed phenotypes similar to wild type under greenhouse conditions, without lodging or excessive tillering (Figure 3 3 A ). Microscopic evalu ation suggested that vascular bundle tissues and sclerenchyma fiber cells were intact in transgenic sugarcane with reduce d total lignin content (Figure 3 4 ). In contrast to wild type or npt II only control plants, transverse stem sections in the internode r egion of the transgenic lines revealed a deep brown color (Figure 3 3 C ). The intensity of brown color appeared to be correlated to the level of lignin reduction. Transgenic line T4, with the highest reduction in total lignin content, displayed the darkest brown color of all transgenic lines extending all the way from the basal node to the 4 th node below the apical meristem. T41 and T23 only showed brown
82 color in the basal internodes. The midrib of leaves from transgenic plants did not display brown colorati on (data not shown). Microscopic evaluation revealed a reddish brown color of vascular tissues and surrounding sclerenchyma cells of stem tissues fro m transgenic sugarcane (Figure 3 4 A and B ). Following histochemical analysis with Mule reagent vascular bundles displayed a red color in both wild type and transgenic sugarcane. However, the sclerenchyma fiber cells surrounding the vascular bundles displayed a yellow color in transgenic sugarcane, indicating a reduction of S units of lignin (Figure 3 4 C a nd D ). Staining with Wiesner reagent produced similar results in both wild type and transgenic sugarcane, indicating no changes in hydroxycinnamaldehyd e end groups of lignin (Figure 3 4 E and F ). The effect of COMT suppression on plant growth was investi gated using clonally propagated progenies grown in randomized, complete blocks and analyzed by ANOVA. Biomass production and stalk diameters of transgenic lines T41 and T23, with moderate reductions in lignin (3.9 and 8.4%, respectively), were similar to t hose of the non tran s genic tissue culture control plants (TC1) and npt II only transgenic control plants (TC2) (Figures 3 5, A and B ). Transgenic line T4, with a 13.7% reduction in lignin, produced less biomass and had thinner stalks compared with the contr ols. The tissue culture control (TC1) and the npt II only transgenic control (TC2) plants produced 12% and 10% less biomass than the wild type plants, respectively. Transgenic lines T41, T23, and T4 accumulated 17%, 13%, and 35% less biomass than the wild type plants, respectively. Since the controls and transgenic sugarcane exhibited similar or slightly taller stalk lengths compared with the wild type sugarcane, the reduced biomass
8 3 production was mostly due to decreased stalk diameters (F igure 3 5 B ). Trans genic plant T4 with the most severe lignin reduction flowered in the greenhouse under natural photoperiod and di d not display lodging (Figure 3 3 B ). Discussion Genetic improvement of sugarcane through breeding or biotechnology is challenging due to the hig hly polyploid genome of this interspecific hybrid. To our knowledge this is the first report of sugarcane improvement via an RNAi approach. Transgenic sugarcane plants with RNAi suppression of COMT had significantly lower lignin contents, ranging from 3.9 13.7% reduction relative to wild type Yields of fermentable glucose were significantly increased up to 41 % even w ithout pretreatment and up to 51 % with dilute acid pretreatment. These observations are consistent with what was observed for transgenic swi tchgrass in which COMT had been down regulated, leading to significantly reduced total lignin content by 11.4 13.4% and improved saccharification efficiencies by 16.5 21.5% following mild pretreatment ( Fu et al., 2011a ) as well as maize ( Vermerris et al., 2007 ) and sorghum ( Dien et al., 2009 ; Saballos et al., 2008 ) brown midrib mutants with re duced COMT activity. The reduction in lignin content combined with the lower S/G ratio in the lignin of these plants results in more efficient enzymatic saccharification, possibly due to altered physico chemical properties of the lignin, so that a smaller proportion of the cellulases irreversible adsorb on the lignin. The brown coloration of the stems is also consistent with the phenotype observed in other plants with reduced COMT activity ( Fu et al., 2011a ; Piquemal et al., 2002 ) The lack of brown midribs in the transgenic sugarcane plants is consistent with the findings in transgenic switchgrass with RNAi suppression o f COMT ( Fu et al., 2011a )
84 Lignin plays an important role in plant growth and development and serves to protect plants from abiotic and biotic stress ( Boerjan et al., 2003 ; Dixon and Paiva, 1995 ) Therefore, reducing lignin content could compromise plant performance, stress tolerance, or defense mechanisms. Our results indica te that in vitro generated transgenic sugarcane lines, with a moderate reduction in total lignin ranging from 3.9% to 8.4%, showed comparable biomass production to the tissue culture or transgenic control plants. Similarly, partial suppression of COMT thro ugh RNAi or antisense strategies in transgenic switchgrass and maize resulted in normal phenotypes under controlled environment conditions ( Fu et al., 2011a ; Piquemal et al., 2002 ) Suppression of COMT may adversely affect plant growth depending on the impact on lignin content and the genetic background. Transgenic sugarcane line T4, with 97% reduction of COMT transcript s and 13.7% reduction in total lignin content, displayed significantly reduced stalk diameter and biomass production compared with the control and wild type plants. Reduced COMT activity in brown midrib mutants of maize ( bm3 ) and sorghum ( bmr12 ) resulted i n ~10% reduction in stover and dry matter yields, respectively ( Miller et al., 1983 ; Oliver et al., 2005a ) A comparison of bmr12 near isogenic lines (NILs) in diffe rent inbred backgrounds showed decreases in dry matter yields ranging from 6 22%. However, the bmr12 NIL in the genetic background of Early Hegari Sart showed the greatest reduction in acid detergent lignin, but was able to produce the same amount of dry matter compared to its wild type counterpart ( Oliver et al., 2005a ) This suggested that there is an interaction between the genetic background and tolerance to reduced lignin content for bioma ss production. This was corroborated by analyses of soluble and cell wall bound aromatics in sorghum bmr6 bmr12 and bmr6 bmr12
85 mutants in different genetic backgrounds ( Palmer et al., 2008 ) Transgenic sugarcane line T4, with the most severe lignin reduction, was still able to mature and produce flowers. Line T4 displays a simple transgene integration pattern with two copies of the COMT RNAi cassette and CP 88 1762 is a fertile cultivar. Transferring the reduced lignin trait into energycane, a high fiber, high biomass variant of sugarcane ( Tew and Cobill, 2008 ) grown primarily for biomass production is ther efore expected to be feasible. While not affecting lignin content or composition, reduced stalk diameters and biomass production were observed in tissue culture and transgenic control plants when compared with wild type CP 88 1762. Somaclonal variation re sulting in less than optimal field performance has been reported for sugarcane ( Gilbert et al., 2005 ; Taparia et al ., 2012a ) A single backcross has the potential to eliminate tissue culture derived mutations ( Bregitzer et al., 2008 ) Sugarcane has a high level of genetic redundancy with an average of 12 homo(eo)logous haplotypes ( Le Cunff et al., 2008 ) and most of these homo(eo)logs are considered to be functional ( Garsmeur et al., 2011 ) Because of this functional redundancy, identification of mutant sugarcane plants with substantial changes in lignin content and/or lignin subunit composition is highly unlikely. RNAi mediated gene silencing allows simultaneous suppression of homo(eo)logs in a high ploidy genome within members of a gene family ( Lawrence and Pikaard, 2003 ; Miki et al., 2005 ) In this study, the hairpin structure of COMT transgene successfully trigger ed generation of siRNA and induced suppression of the targeted endogenous COMT gene expression. The reduction in total lignin content and altered S/G ratios suggest that the conserved
86 sequence that was used to suppress COMT may have supported co suppressio n of related COMT homo(eo)logous genes. RNAi mediated gene suppression is a useful tool for elucidation of gene function ( Miki et al., 2005 ; Travella et al., 2006 ) This study confirms that RNAi is an effective method for suppression of target genes in sugarcane. This finding is consistent with an earlier report on RNAi suppression of the phytoene desaturase in sugarcane ( Osabe et al., 2009 ) The sugarcane COMT gene was previously annotated and characterized based on sequence identity, transcript expression pattern, and tissue/cellular localization ( Ruelland et al., 2003 ; Selman Housein et al., 1999 ) However, COMT belongs to a large S adenosyl L methionine (SAM) dependent O methyltransferase (OM T) family including lignin specific OMT and other phenylpropanoid specific OMT genes. Therefore, additional evidence for confirming the correct annotation was needed ( N oel et al., 2003 ; Zhou et al., 2010a ) The transgenic evidence provided by this study verifies the function of COMT participating in lignin biosynthesis, particularly in S unit formation. Transgenic sugarc ane plants displayed a significant reduction of S units without changing quantities of G units, thereby causing a lower S/G ratio compared to wild type Similarly RNAi suppression of COMT in switchgrass also resulted in reduced S/G ratio ( Fu et al., 2011a ) Reducing the recalcitrance of lignocellulosic sugarcane biomass to enzymatic hydrolysis is expected to enhance the value of this prime biofuel feedstock. Follow up field evaluations will elucidate the influe nce of the genetic background of the transgenic sugarcane lines on biofuel yield per land area.
87 Table 3 1 Lignin content composition, and glucose yields after enzymatic saccharification Line s a) Total lignin b) G unit c) S unit d) S/G molar ratio Glucose y ield (mg g 1 biomass) (mg g 1 ) ( mol g 1 lignin) Pretreatment No pretreatment WT 181.4 2.2 158.4 2.7 233.4 2.0 1.47 190.9 4.9 96.1 3.0 TC2 182.0 2.0 154.0 0.9 230.0 7.9 1.49 196.9 6.6 95.6 3.7 T41 174.3 4.6* 150.4 1.6 191 .6 7.6* 1.27* 238.7 3.5* 95.2 3.9 T23 166.1 1.1* 163.6 4.7 179.8 5.0* 1.10* 241.3 1.8* 94.4 2.3 T4 156.6 1.9* 165.8 6.4 131.5 2.3* 0.79* 288.0 1.2* 135.6 2.8* a) L ine s included WT: Wild type sugarcane; TC2: Transgenic control h arboring npt II gene alone; T41, T23, and T4: Transgenic sugarcane b) Total lignin content was analyzed using the acetyl bromide (AcBr) method. c) G: Guaiacyl subunit. d) S: Syringyl subunit. Values are means standard errors of the mean ( n =3 for total l ignin n =2 for lignin composition and n =3 for glucose yields) *Significantly different from the wild type plants at P < 0.05 in t test.
88 Figure 3 1 Generation and selection of transgenic sugarcane lines and investigation of transgene integration pattern in the transgenic sugarcane lines A ) COMT RNAi cassette consists of inverted repeats of the 346 bp COMT gene fragment separated by 94 bp Paspalum notatum 4CL intron under control of 1994 bp Oryza sativa C4H promoter and CaMV indicate the primer binding site for PCR analysis to confirm transgenic events. The probe indicates the complementary binding site for Southern blot analysis B ) NPTII ELISA assay. Wells A1, B1, and C1 were negative control without pro tein, wild type and positive control with NPTII standard, respectively. The rest of wells were transgenic lines. Yellow coloration indicated NPTII expression in transgenic plants, and no coloration indicated little or no expression of NPTII. C ) PCR analys is of COMT RNAi cassette integration. 550 bp PCR products indicated the presence of COMT RNAi cassette in the transgenic sugarcane T1 ~ T21: Analyzed putative transgenic sugarcane. NC: Negative control without DNA template. PC: COMT RNAi c assette. WT: Wil d type sugarcane M: 2 kb DNA ladder. D ) Southern blot analysis of transgenic sugarcane plants. PC: 9.7 kb linearized COMT RNAi vector; WT: Wild type sugarcane; T1 T47: T ransgenic sugarcane. The displayed image was generated from a single Southern blot. Af ter exposure to x ray film for different periods of time lanes with best resolution were merged to minimize overexposure of individual signals WT T31 T41 T4 T23 T47 T17 T1 T38 T18 T33 T24 T26 PC T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 M NC T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 PC M WT A 550 bp D B C D E A 1 2 3 4 5 F B C P robe 4CL intron 35S 3 UTR COMT RNAi
89 Figure 3 2 Detection of COMT siRNA and evaluation of COMT expression le vel s in transgenic sugarcane. A ) Small RNA northern blot. The hybridization signal indicates the generation of ~21 nt long COMT siRNA by cleavage of the hairpin structure dsRNA. Ethidium bromide stained 5S rRNA and tRNA were used as a loading control to verif y the amount and integrity of the small RNA. WT1 and WT2: Wild type sugarcane plants; NC: buffer; SM: Size marker corresponding to 21 nt long RNA; T : Transgenic sugarcane plants; PC: T17 transgenic plant used as a positive co ntrol on a separate membrane. B ) Quantitative RT PCR analysis of COMT expression level in the transgenic sugarcane. Error bars represent the 95% confidence intervals of the 2 value of the wild type vs. transgenic plants. Significant differences in COMT expression between the wild type and the transgenic sugarcane are indicated by an asterisk ( n =3 P < 0.05 in t test). WT2 NC SM T4 1 T38 T1 T 1 7 T13 T29 T37 T44 WT1 PC T18 T3 1 T33 T21 T26 T47 T24 WT1 T23 T4 WT1 COMT siRNA COMT siRNA 5S/tRNA 5S/tRNA A B
90 Figure 3 3 Phenotypic evaluation of transgenic sugarcane in comparison to wild type WT: Wild type sugarcane; TC1: non transgenic plant co re generated with transgenic plants from tissue culture; TC2: Transgenic control plant harboring npt II alone; T41, T23, and T4: Transgenic sugarcane plants. A ) Greenhouse grown wild type (WT) and transgenic sugarcane plants (T4 T41). B ) Flowering of the trans genic sugarcane plant (T4) under greenhouse conditions with natural photoperiod C ) Coloration of internodes of transgenic and non transgenic plants. The basal internode of the transgenic, control, and wild type plant were transversely or longitudinally se ctioned, and the picture was taken immediately without fixation and staining. Scale bar indicate s 2 c m WT T 41 TC1 T 23 TC2 T 4 WT T 41 TC1 T 23 TC2 T 4 Transverse section WT T 4 1 1 T 23 T 4 C A B Longitudinal section
91 Figure 3 4 Microscopic and histochemical evaluation of transgenic sugarcane in comparison to wild type Microscopic observations o f transverse sections of th e internode, without staining ( A and B ), stained with M ule reagent ( C and D ), or stained with Wiesner reagent ( E and F ). Note the differences in color of vascul ar tissues between wild type (A) and T4 transgenic line ( B ) in the a bsence of staining and arrows indicating yellow coloratio n in the sclerenchyma fibe r cells in T4 transgenic line ( D ) compar ed with that in the wild type ( C ). Ph: Phloem; X: Xylem; S: Sclerenchyma; P: Parenchyma. Scale bars indicate 250 m. WT T4 A B C D E F Ph X S P
92 Figure 3 5 Plant growth characteristics under greenhouse conditions. A ) Biomass production. Values represent means of 8 clonally propagated plants, grown in an air conditioned greenhouse following randomization. Error bars represent standard e rror. Means with different letter are significantly different at P < 0.05 the most matur e stalk of 7 month old plants. B ) Stalk diameter (grey bar) and length (black bar) from the se t of plants described under ( A ) Stalk diameter was measured in the middle of the stalk, and length was measured from the soil surface to the apical meristem of the stalk. Means of stalk diameter (a, b, c) or means of stalk length (x, y) with different let ter are significantly different at P Diameter Length A a b b b b c B
93 CHAPTER 4 FIELD EVALUATION OF COMT SUPPRESSED TRANSGENIC SUGARCANE Introduction Sugarcane ( Saccharum spp. hybrids) is a highly productive perennial C 4 grass and a majo r feedstock for global bio ethanol and sugar production ( Tew and Cobill, 2008 ) In 2010, more than 1.7 billion tonnes of s ugarcane were produced on 23.9 million hectares worldwide, and i ts production on a dry weight basis, is higher than any other crop in the world (FAOSTAT ; http:// faostat3.fao.org/home/index.html#DOWNLOAD ) S ugarcane accumulates up to 50% of its dry weight as sucrose in the internodes ( Waclawovsky et al., 2010 ) Sucrose derived ethanol production from sugarcane has been successfully commer cialized in Brazil, with high environmental sustainability and average production costs that are 24% lower than the cost of ethanol production from maize in the USA ( C rago et al., 2010 ; Goldemberg, 2007 ) Sugarcane stalk harvest follow ed by s ucrose extraction generates a large amount of lignocellulosic residue (green tops, leaf litter, and bagasse) compris ing 55% o f total aboveground biomass ( Somerville et al., 2010 ; Tew and Cobill, 2008 ; Vermerris, 2011 ) Including both sucrose and lignocellulosic biomass from sugarcane as feedstocks for ethanol production will boost the ethanol yield per land area while increasing sustainability and adding environmental benefits ( Goldemberg, 2007 ; Leite et al., 2009 ; Somerville et al., 2010 ) T he presence of lignin in the cell wall is a major problem for cellulosic ethanol production because it limits the accessibility of cellulose and hemicellulose and reduces the a ctivity of cellulolytic enzymes ( Jrgensen et al., 2007 ; Mansfield et al., 1999 ) Therefore, an energy intensive pretreatment of the biomass is typically required for disrupti on of the cell wall matrix and degradation of lignin. Such pretreatment s may
94 adversely affect down stream ethanol pro duction by degrading sugars and generating inhibitory molecul es ( Alvira et al., 2010 ; Yang and Wyman, 2008 ) T herefore, reduction of lignin content or modif ications of its structure are attractive target s to enhance the production of cellulosic biofuel Lignin is form ed from the polymerization of three monolignols, p coumaryl, coniferyl, and sinapyl alcohol After incorporation of these monolignols into the lignin polymer, they are referred to as p hydroxyphenyl (H) guaiacyl (G), and syringyl (S) unit s respectively ( Bonawitz and Chapple, 2010 ) G rass lignin also contains considerable amount s of the hydroxycinnamic acids p coumar ate and ferul ate. Ferula te plays a role in interconnect ing hemicellulosic cell wall polysaccharide s and lignin ( Grabber, 2005 ; Grabber et al., 1996 ) whereas p coumarate, esterified primarily to the gamma carbon of sinapyl alcohol ( Ralph et al., 199 4 ), enhances the incorporation of this monolignol in the growing lignin polymer ( Hatfield et al., 2008 ) Among monolignol biosynthetic genes, caffeic acid O methyltransferase ( COMT ) encodes the enzyme which catalyzes O methylation at the C5 position of 5 hydroxyconiferaldehyde and 5 hydroxyconiferyl alcohol, yield ing sinapaldehyde and sinapyl alcohol, respectively ( Bout and Vermerris, 2003 ; Humphreys et al., 1999 ; Louie et al., 2010 ; Osakabe et al., 1999 ) C OMT deficiency or suppression in brown midrib mutant s (maize bm3 and sorghum bmr1 2 ) or transgenic plants reduc es S unit s in addition to total lignin content, c onsistent with the role of COMT in sinapyl alcohol biosynthesis ( Barrire et al., 2004 ; Fu et al., 2011a ; Guo et al., 2001 ; Marita et al., 2003 ; Palmer et al., 2008 ) D own regulati on of lignin biosynthetic gene(s) or the identif ication of mutants with reduced lignin content is a viable strategy to improve saccharification efficienc ies and /or
95 cellulosic ethanol yields as demonstrated with a number of crops ( Chen and Dixon, 2007 ; Dien et al., 2009 ; Saballos et al., 2008 ) including transgenic switchgrass ( Fu et al., 2011a ; Saathoff et al., 2011 ; Xu et al., 2011 ) and sugarcan e ( Jung et al., 2012 ) COMT suppressed transgenic suga rcane lines were previously generated in our laborator y by RNA interference (RNAi) and they showed significant reductions in total lignin and S subunit content (Jung et al., 2012). Fermentable glucose yields were significantly increased in the transgenic sugarcane lines following enzymatic hydrolysis of the lignocellulosic biomass A moderate reduction of l ignin ( up to 8% ) did not compromise biomass production under greenhouse conditions ( Jung et al., 2012 ) T o our knowledge, data on field performance of transgenic C 4 grass species with modified lignin conte nt or composition have not been reported. Such data are critically important to evaluate the growth and feedstock performance following production under realistic growing conditions. In this study, cell wall characteristics, saccharification efficienc ies and plant growth performance of COMT suppressed sugarcane lines were evaluated in a field trial under USDA APHIS permit 11 040 120n Materials and Methods F ield D esign COMT suppressed t ransgenic sugarcane RNAi lines were generated using the commercially i mportant cultivar CP88 1762 as previously described ( Ju ng et al., 2012 ) Rootstocks of clonal propagules of the V 1 (the first generation of vegetativ e progeny from transgenic plants) tissue culture control, transgenic control, and the original CP88 1762 ( wild type ) plants were transplanted on 30 March 2011 at the University of Florida, Plant Science Research and Education Unit, Citra Florida, USA under USDA/APHIS permit 11 040 120n. The f ield design was a randomized block design with three
96 replications. Four plots of transgenic lines representing four indep endent transformation events, and three plots of wild type, non transgenic control s derived from tissue culture, and the transgenic control harboring the npt II gene were included in each replicate. E ach plot consisted of one row with f ive clonal propagule s planted per plot. Spacing between plants rows and blocks was 90, 150 and 450 cm, respectively. Each block was surrounded by one row of wild type sugarcane plants Weeds were removed manually during the establishment phase, and Orthene was applied in A ugust 2011 for insect control P l ots w ere fertilized with 40 kg ha 1 N 15 kg ha 1 P and 60 kg ha 1 K at planting and with 70 kg ha 1 N 15 kg ha 1 P and 60 kg ha 1 K on 9 Ma y 2011. Plots were irrigated once or twice a week with a rate of 10 mm depending on rainfall and harvested on 26 October 2011 for determination of biomass and compositional analysis E valuation of P lant P erformance The f resh weight of the harvested biomass w as determined on site immediately after the plot harvest T he number of stal ks was counted for each plant, and f ive stalks were collected for milling The leaves and leaf sheaths were removed from the stalks the number of internodes was counted, and stalk height from the shoot apical meristem to the soil surface was measured S talk diameter was measured at the center of the 8 th internode from the b ase of the plant I nternodes 1 4 below the shoot apical meristem were removed and the remaining portion of the stalk w as crushed to extract juice using a custom made juice extractor. P ercentage soluble solid in the extracted juice ( Brix) was measured usin g a PAL 1 portable refractometer ( ATAGO U.S.A., Inc., Bellevue, WA, USA ) Crushed stalks were dried at 45 C and stored for further analysis. Pests and diseases [ e.g. s ugarcane brown l eaf rust ( Puccinia melanocephala ) orange leaf rust ( P. kuehnii ), and pink sugarcane mealybug ( Saccharicoccus sacchari )]
97 w ere monitored in monthly intervals Leaf rust was rated using a 0 4 scale as described in ( Comstock et al., 2010 ) For evaluating the rate of mealybug infestation, six stalks per plant were randomly selected and the number of infested internodes was recorded T he rate of infestation was calculated by the number o f infested internodes over the total number of internodes. T he lodging incidence was monitored every month. T he lodging susceptibility was scored in every plant using a 0 7 scale with 0 being no lodging (0 20 deviation from erect) and the score increasin g with every 10 lodging, until 7 which equated to complete lodging. Gene E xpression A nalysis The third internode below the shoot apical meristem was collected from one of the millable stalks in each plot. RNA extraction, cDNA synthesis and quantitative r eal time RT PCR were performed as previously described ( Jung et al., 2012 ) Sample P reparation for the E valuation of C ell W all C omposition and S accharification E fficiency Crushed stalks dried at 45 C as described above were ground using a Wiley mill (Thomas Scientific, Swedesboro, NJ, USA) with a 1.0 mm si eve and the samples were further p a ssed through a 0.42 mm sieve Soluble extract was removed from the samples by three successive extractions with 50% ethanol (v/v) and sonication at 45C for 30 min Extract free samples were dried at 45C until constant weight was achieved in order to analy ze lignin, hydr o xycinnamic acids, and cell wall polysaccharides, and the evaluation of saccharification efficienc ies Lignin C ontent and C omposition Total lignin content was measured using the acetyl bromide (AcBr) met hod as previously described ( Jung et al., 2012 ) Ligni n composition was determined by
98 thioacidolysis ( Robinson and Mansfield, 2009 ) T hioacidolysis derivatives for lignin monomers were analyzed using a Varian 3800 gas chromatograph ( GC ) coupled to the Varian 1200 mass spectrometer ( MS ) ( Varian, Walnut Creek, CA, USA ). One microliter of the sa mple was injected onto a Factor 4 VF 5ht column (35 m, 0.25 mm i.d.) (Varian) with helium (1.2 ml min 1 ) as carrier gas. GC conditions were as follows: i nitial column temperature 130 C held for 3 min, ramped at 3C min 1 to 250 C and held for 5 min; inje ctor temperature, 250 C ; and 1:2 split ratio. The mass spectrometer was operated in electron impact mode at 70 eV. The detector was operated at 1.0 kV. M ass spectra were recorded every 0.2 s at a scanning range of m/ z 50 550, and the dat a w ere re corded and analyzed using Varian MS Workstation software Thioacidolysis monomers, G and S were quantified by integration of the respective peak areas, and employing the response factors of G and S against an internal standard, tetracosane (IS) as follows: G versus IS, 0.47; and S versus IS, 0.53 ( Yue et al., 2012 ) Analysis of H ydroxycinnamic A cids Ester linked cell wall bound p coumaric acid ( p CA) and ferulic acid (FA) were determined following Hatfield et al. (1999c ) with modifications. Briefly, 25 mg of extract free sample was saponified with 1.7 ml of 2 M NaOH and 20 l 2 hydroxycinnamic ac id (1 mg ml 1 in 2 M NaOH) ( Sigma Aldrich, Saint Louis, MO, USA ) as an internal standard. The reaction was carried out at room temperature for 20 h in the dark, then acidified with 0.3 ml 12 M HCl. Samples were extracted three times with an equal volume of diethyl ether, and supernatants were combined and dried under a stream of N 2 Derivatization was performed by adding 40 l methoxyamine hydrochloride (20 mg ml 1 in pyridine ) ( Sigma Aldrich ) and incubating at 37 C for 90 min. The sample was trimethylsilyl ated by adding 60 MSTFA [ N methyl N ( trimethylsilyl )
99 trifluoroacetamide ] (Sigma Aldrich ), and incubating at 37 C for 30 min. p CA and FA were identified and quantified using GC MS with a Factor 4 VF 5ht column (35 m, 0.25 mm i.d.) (Varian). One microl iter of the sample was injected and helium was used as a carrier gas (1.2 ml min 1 ). The injector temperature was 2 2 0C; the split ratio was 1:10. The oven temperature was held for 4 min at 70 C, and increased to 250C at 10 C per min without holding, then ramped at 40 C per min and held constant for 2 min. MS operating procedure was the same as that for lignin compositional analysis except for a scanning range of m/ z 45 650. Peaks from derivatized p CA, FA, and internal standard were identified by characte ristic mass spectrum ions obtained from the reference compounds. Concentration s of p CA, FA, and internal standard w ere determined using the standard curve. T he concentration s of p CA and FA w ere normalized with the concentration of internal standard among t he sample s Cell W all C arbohydrates and S tarch Cell wall carbohydrates in the stalk sample s were determined according to the National Renewable Energy Laboratory (NREL) protocol by Sluiter et al. (2008 ) Three hundred milligram of e xtract free sample w ere hydrolyzed with 72% H 2 SO 4 at 30 C for 1 h and then treated with 4% H 2 SO 4 at 121 C for 1 h. Liberated monomeric sugars were identified and quantified with an Agilent/HP 1090 HPLC equipped with an RI detector ( Agilent Technologies, Santa Clara, CA USA). The HPLC an al ysis was carried out using a HPX 87H column ( Bio Rad, Hercules, CA, USA ), operating at 50 C with a 0.004 M H 2 SO 4 mobile phase at a flow rate of 0. 4 m l min 1 Starch content in the sample was determined by the NREL procedure by Ehrman (1996 ) Glucose yield w as measured using a YSI glucose analyzer ( YSI Life Sc ience, Yellow Springs, OH, USA) followin g
100 enzymatic digestion of the extracted starch, and its value was adju sted by multiplying 0.9 for the weight gained by hydration. Dilute A cid P retreatment and E nzymatic H ydrolysis Extract free sample s possessing 0.1 g of cellulose w ere pretreated as previously described ( Chen and Dixon, 2007 ) T he amount of cellulose was defined as the amount of glucose from cell wall carbohydrate corrected by subtractin g the amount of glucose from starch ( Selig et al., 2008 ) Five sets of pretreated sample s were prepared per sugarcane line to evaluate saccharification performance at different hydroly sis tim e s and enzyme dosage s The enzymatic hydrolysis was performed according to the NREL protocol by Selig et al. (2008) with modification s T he pretreated sample was suspended in 8 ml distilled water, and placed in a pre weighed 50 ml polypropylene tube Te n milliliter of 0.1 M sodium citrate buffer (pH 4.8) and 200 l of 2% (w/w) sodium azide (Sigma Aldrich ) were added to each tube The en zymatic hydrolysis was performed by adding the appropriate volume of Kerry Biocellulase W (Kerry Bioscience, Cork, Irela nd) and glucosidase ( Sigma Aldrich ) O ne set of the pretreated sample s was used as the no enzyme blank (no addition of cellulolytic enzymes ) The appropriate volume of distilled water was added to each tube to bring the total mass of each sa mple to 20 g, assuming all solutions and the biomass sample have 1.000 g ml 1 specific gravity. T he hydrolysis was carried out for 168 h in a shaking incubator at 50C and 250 rpm. The time course of enzymatic hydrolysis with 60 FPU g 1 cellulose of Kerry Biocellulase W and 64 p NPGU g 1 cellulose of Novozyme 188 glucosidase was monitored by measuring glucose yields at the time poi nt s of 0, 3, 6, 24, 48, and 72 h. The effect of enzyme loading on saccharification was evaluated with Kerry Biocellulase
101 W loading s of 5, 20, or 60 FPU g 1 cellulose. glu cosidase loading was constant as 64 p NPGU g 1 cellulose. One milliliter of the hydrolyzed sample was collected for each treatment and time point, and filtered through a 0.2 m syringe filter Glucose yields were analyzed using the YSI glucose analyzer (YSI Life Science). Saccharification efficiency was calculated as the ratio of glucose released following the enzymatic hydrolysis to the amount of glucose present in the cell wall before the hydrolysis. The glucose amount derived from starch in the stalk was subtracted in order to calculat e the glucose amount in the cell wall. Glucose yields were adjusted by multiplying 0.9 for the weight gained by hydration. Statistical A nalysis ANOVA was performed using Proc GLM in SAS TM Version 9.3 ( SAS Institute Inc., Cary NC USA) Statistical significance among the means for plant growth performance was determined using P < 0.05. Significant differences ( P < 0.05 ) of means for the content of cell wall components among the samples were deter mined using test. For the gene expression analysis the t test was used to determine whether the means of Ct were significantly different between wild type and a transgenic line ( P < 0.05 ). Results COMT G ene S uppression in T ransgenic Sugarcane L in es COMT gene suppression through RNAi was evaluated six month s after initiation of the field trial. The transcript abundance of COMT was significantly reduced by 80, 89, 92, and 91% in the transgenic lines, T41, T23, T31, and T4, respectively compared to wild type (WT) sugarcane (Figure 4 1 ).
102 Effects of COMT S uppression on L ignin C ontent and C omposition T otal lignin content in stalk sample s of transgenic and control plants grown in the field was determined using the acetyl bromide (AcBr) method. T here w a s no significant difference in total lignin content of the WT, the non transgenic tissue culture control (NT), or the npt II transgenic control (TC) (Table 4 1). However, t he transgenic lines had 4.5 11.1% lower total lignin content compared to WT. In compa rison to the corresponding TC, the level of total lignin reduction was 5.5, 7.5, 11.2, and 12.0% in the transgenic lines T41, T23, T31, and T4, respectively (Table 4 1). The S subunit content was significantly reduced by 16 % and 49% in T41 and T4, respecti vely, compared to the TC control (Table 4 1). There were no significant differences in G subunit content among the transgenic lines, WT or the TC line (Table 4 1). Due to a reduction in S subunit s T41 and T4 transgenic lines had significantly lower S/G m olar ratio s of 1.17 and 0.72, respectively, compared to 1.47 for the TC and 1.48 for the WT. Effects of COMT S uppression on C ell W all C arbohydrates and C ell W all B ound H ydroxycinnamic A cids The consequences of COMT suppression on other cell wall compone nts, such as cellulose, hemicellulose, and cell wall bound hydroxycinnamic acids, were investigated in the two transgenic lines, T41 and T4 which represented the lines with the least and most reduction in lignin content respectively versus the WT and th e TC (Table 4 2). T he amount s of glucose mostly derived from cellulose did not differ significantly between the transgenic and control plants The amount of xylose, major hemicellulose component, was significantly increased in T4 compared to T41 or control plants, while
103 the amount of arabinose was not different among the lines. The t otal amount of cell wall sugars did not differ significantly between the transgenic and control plants T he content of cell wall esterified p coumaric acid ( p CA) and ferulic ac id (FA) were evaluated following mild alkaline hydrolysis. The e sterified p CA content was significantly decreased by 8% and 32% in T41 and T4, respectively, compared to the WT (Table 4 3) Esterified FA did not differ significantly between the transgenic a nd control plants Effect of L ignin R eduction on S accharification E fficienc y Enzymatic hydrolysis following dilute acid pretreatment was performed to evaluate the bioconversion efficiency of lignocellulosic biomass from field grown transgenic sugarcane in to directly fermentable sugar s Th e time course of saccharification describes elevated saccharification efficiencies of the transgenic biomass compared to WT and TC during the entire 72 h period of enzymatic hydrolysis (Figure 4 3 A ) A maximum saccharifica tion rate was reached at 72 h of enzymatic hydrolysis and saccharification efficienc ies for T41 and T4 w ere 23.2 and 32.4% higher than for the TC control C ellulose in the transgenic lines was converted to glucose more rapidly compared to WT or the TC con trol Transgenic lines T41 and T4 had saccharification efficienc ies of 49.2 and 54.8%, respectively, a fter only 24 h of enzymatic hydrolysis values which exceeded the 48.4 and 46.7% conversion for WT or TC at 72 h of enzymatic hydrolysis respectively. T o evaluate the effect of enzyme dosage on the saccharification of transgenic and control sugarcane plants, hydrolysis was performed with different cellulase loadings of 5, 20, and 60 filter paper unit (FPU) per gram of cellulose. A ll lines showed the highe st saccharification efficienc ies at the highest cellulase loading (60 FPU) (Figure 4
104 3 B ) However, l ignocellulosic biomass from transgenic sugarcane plant s w as more effectively converted to glucose than that of WT or the TC control regardless of enzyme do sage. For T41 and T4, s acch a rification efficienc ies at 5 FPU w ere similar to those at 20 FPU for WT or the TC cont r ol Furthermore, at 20 FPU, saccharification efficienc ies of 49.9 and 55.9% in T41 and T4 w ere significantly higher than those obtained with 60 FPU in the WT or TC control, respectively G rowth P erformance of the T ransgenic S ugarcane G rown under F ield C onditions Clonally produced r ootstocks derived from the vegetative progeny (V1) of transgenic events and control plants established well and w ere grown for seven months under field conditions in Citra, FL (Figure 4 2 A and B ). The transgenic line, T41, with 6% lignin reduction displayed no significant difference in b iomass production compared with the TC and NT lines (Table 4 4 ). Stalk length and diameter were also not significantly different between T41, TC and NT lines However lines of T23 T31 and T4 with 8, 1 1, and 12% reduction in lignin, respectively, displayed a 21 64 and 65% reduction in biomass, respectively, compared to TC lines T his reduction in biomass can primarily be attributed to reduced stalk diameter compared to the controls In comparison to the original CP88 1762 WT plants, both the NT tissue culture controls and the TC control lines had an 18% reduction in biomass. For th e transgenic lines, t he number of internodes per stalk w as not statistically different from WT or the control lines indicating that the developmental stage was similar among the lines at the time of harvest (Table 4 4). T he number of stalks per plant was n ot different among the lines except in transgenic line T31, which had fewer stalks per plant (Table 4 4). T he amount of soluble solids in the stalk did not differ significantly between the transgenic lines T41 and T23 and control lines However
105 transgeni c lines T31 and T4 displayed significantly reduced concentrations of soluble solids in their stalks compared to the control lines (Table 4 4). D isease s, pests and lodging w ere monitored monthly A m inor occurrence of orange rust ( Puccinia kuehnii ) was obse rved during the main growth period and was rated between 0 1 in all of the lines with no significant differences between the lines A pink sugarcane mealybug infestation was observed in August and the pest was eliminated by pesticide application after sco ring. The rate of mealybug infestation did not significantly differ between the transgenic and control plants ranging from 10 to 37% of the internodes being infested T he transgenic and control plants did not show any l odging throughout the growing season until an isolated thunderstorm hit the field on 10 October 2011 with a maximum wind speed of 68 km/h. After the storm, lodging was most severe in the part of the field facing the prevailing wind direction during the severe thunderstorm, with no significan t difference between transgenic lines and WT or TC controls (Figure 4 2). Discussion To our knowledge, t his is the first report on the field performance of a transgenic C 4 grass with modified cell wall composition The field grown COMT suppressed transgeni c sugarcane lines showed improve ment in bioconversion efficiency of mature lignocellulosic biomass to fermentable glucose. S uppression of COMT transcripts ranging from 80% to 91% resulted in a reduction of lignin content by 6 % to 12% in different transgeni c lines and an improvement of saccharification efficiency by 19% to 32% compared to non transgenic and transgenic ( npt II only) control s. The transgenic lines required one third of the hydrolysis time and 3 to 4 fold less enzyme to produce an equal or high er amount of glucose than control plants. These findings are consistent
106 with earlier reports on greenhouse grown COMT suppressed transgenic switchgrass ( Fu et al., 2011 a ) or sugarcane (Jung et al. 2012). COMT s uppressed switchgrass increased ethanol production by 38% or enabled reduced pretreatments and 3 to 4 fold less enzyme loading to produce an equal amount of ethanol compared to the control. Elevated biofuel production from lignocellulosic sugarcane biomas s will boost the sucrose derived biofuel yields for this prime biofuel crop. Accelerated rates of bioconversion less severe pretreatment conditions and/or lower enzyme loadings to achieve yields of fermentable sugars equal to or higher than with wild type biomass are expected to significantly reduce biofuel production costs T he RNAi induced level of COMT suppression in the different transgenic lines that was observed earlier under greenhouse conditions (Jung et al. 2012) was stably maintained under fiel d conditions Reductions of total lignin and S subunit content in the field grown transgenic lines were consistent with those previously reported for greenhouse grown plants S uppression of COMT in transgenic sugarcane specifically resulted in a significan t reduction of S subunit content without affecting G subunit content similar to COMT deficient maize brown midrib ( bm3 ) and sorghum bmr12 mutants ( Barrire et al., 2004 ; Palmer et al., 2008 ) The transgenic sugarcane lines displayed reduced p coumaric acid ( p CA) levels p CA is primarily esterif i ed to S lignin subunit s i n grasses ( Grabber et al., 1996 ; Ralph et al., 1994 ) The r educed p CA content most likely result ed from a reduction of S subunit s in the COMT suppressed tran sgenic sugarcane plants similar to maize bm3 and sorghum bmr12 mutants ( Marita et al., 2003 ; Palmer et al., 2008 ; Piquemal et al., 2002 ) ) and COMT suppressed transgenic maize ( Piquemal et al., 2002 ) Ferulic acid
107 ( FA ) cross linking of the cell wall in grass speci es negatively affects enzymatic hydrolysis ( Grabber et al., 1998 ) FA is esterifi e d to arabinoxylans, and xylans are interconnected by radical coupling of esterif i ed FA into FA dimers or trimers. Furthermore, esterif i ed FA is incorporated into lignin forming an extensive lignin ferulate polysaccharide complex ( Grabber, 2005 ; Ralph, 2010 ) Elevated level s of FA w ere not observed in the sugarcane lines with COMT suppression In this study, there was no differenc e in the amount of glucose in the transgenic sugarcane lines analyzed, while xylose was significantly increased in the transgenic line T4 that displayed the greatest lignin reduction. Similarly, xylose amounts in the stem of COMT suppressed transgenic swit chgrass were increased in both T0 and T1 plants ( Fu et al., 2011a ) However, it is unclear whether there is a compensatory increase in cell wall carbohydrates for lignin reduction as proposed in 4 coumarate:CoA ligase (4CL) suppressed transgenic poplar ( Hu et al., 1999 ) The m aize bm3 mutant, cinnamyl alcohol dehydrogenase (CAD) or 4CL suppressed transgenic switchgrass did not exhibit an increase in cell wall carbohydrates ( Marita et al., 2003 ; Saathoff et al., 2011 ; Xu et al., 2011 ) Furthermore, no increase in the amount of cellulo se or the transcription level of cellulose biosynthetic genes was observed in a set of Arabidopsis lignin mutant s ( Vanholme et al., 2012 ) Plant biomas s yield is one of the most important factors for determin ing the economic viability of a lignocellulosic feedstock. Despite the beneficial effects of reducing lignin for ethanol conversion, blocking the monolignol biosynthetic pathway may be associated wit h impaired growth and development of the transgenic plants. Particularly, suppression of h ydroxycinnamoyl CoA s hikimate/ q uinate h ydroxycinnamoyl
108 transferase (HCT), p c oumarate 3 h ydroxylase (C3 H) or c innamoyl CoA r educt ase (CCR) frequently leads to developmental arrest, dwarfism, collapsed xylem vessel s and/or abnormal flowering ( Gallego Giraldo et al., 2011 ; Goujon et al., 2003 ; Piquemal et al., 1998 ; Ralph et al., 2006 ; Shadle et al., 2007 ; Srinivasa Reddy et al., 2005 ) These negative effects may result from disruption of non lignin metabolite biosynthesis, such as c oniferaldehyde derivatives and/or shikimate derivatives, which influence cell growth and defense mechanism s ( Bonawitz and Chapple, 2010 ; Gallego Giraldo et al., 2011 ) Interestingly, COMT suppress ed plants were reported to display normal plant growth in a variety of species including tobacco, alfalfa, maize, and switchgrass under greenhouse conditions, and p oplar under field conditions ( Chen and Dixon, 2007 ; Fu et al., 2011a ; Pila te et al., 2002 ; Pinon et al., 2001b ; Piquemal et al., 2002 ) H owever, c omplete knock out of COMT in maize and sorghum brown midrib mutants wa s genera lly associated with lower biomass yields ( Lee and Brewbaker, 1984 ; Miller et al., 1983 ; Oliver et al., 2005a ; Oliver et al., 2005b ; Sattler et al., 2010 ) In our results, 80% suppression of COMT and 6% reduction of lign in did not cause significant reduction of biomass production or related traits, such as stem diameter, height, tillering, and accumulated sugars compared to non transgenic tissue culture derived control plants. However, COMT suppression of 91% and a 12% re duction in lignin compromised biomass production under both greenhouse (Jung et al. 2012) and field conditions T hese results indicate that adverse effects on plant growth c an be avoid ed by target ed suppression of specific lignin biosynthetic gene(s) whi ch do not cause pleiotropic effects and by determining the level of lignin modification that allows high biomass yield
109 along with improved conversion performance. This strategy is facilitated by the range of gene expression/suppression that is typically f ound among different transgenic events. The tolerance to reduced lignin may also differ among different species and even within a species. In brown midrib mutants agronomic traits in the mutant cultivar or hybrid are influenced by the interaction betwe en the specific gene and the genetic background ( Pedersen et al., 2005 ; Sattler et al., 2010 ) F o r example, a sorghum COMT deficient bmr12 near isogenic line in the genetic background of Early Hegari Sart exhibited similar plant height and dry matter yield as the wild type counterpart unlike what was observed for the comparison in the background of Atlas, Kansas Collier, or Rox Orange ( Oliver et al., 2005a ) A lthough line T4 produced the least amount of biomass, it displayed the high est saccharification efficiency and the lowest lignin content among t he transgenic sugarcane lines This would suggest its use as a parent in future breeding efforts to explore if further improvements can be achieved by crossing COMT down regulated sugarcane lines (e.g. T4) to genetically div erse accessions (e.g. high bioma ss type energycanes) Crossing would also eliminate tissue culture derived mutations ( Bregitzer et al., 2008 ) Among the transgenic sugarcane lin es evaluated under both greenhouse and field conditions, T41 has the greatest promise for crop improvement since its field performance was comparable to control lines but with a 19 to 23% increase in saccharification efficiency Improving the saccharific ation efficiency o f lignocellulosic sugarcane biomass by modifying lignin biosynthesis will greatly benefit the biofuels industry
110 Table 4 1 Lignin content and composition in transgenic sugarcane Line s a) AcBr lignin b) G units c) S units d) S/G molar ratio mg g 1 DW e) mol g 1 AcBr lignin WT 192.2 1.9 a 97.4 5.4 a 143.9 3.5 a 1.48 a NT 193.3 2.1 a n a f) na na TC 194.3 1.7 a 102.4 4.0 a 150.0 4.8 a 1.47 a T41 183.6 0.7 b 106.8 1.6 a 125.4 2.6 b 1.17 b T23 179.8 1.2 b na na na T31 172.5 0.8 c na na na T4 170.9 1.3 c 107.2 5.7 a 77.1 1.0 c 0.72 c a) Lines included W T: W ild type sugarcane NT: Non transgenic control derived from tissue culture, TC: Transgenic control harboring npt II gene and T41, T23, T31, and T4: Transgenic sugarcane line s. b) Total lignin content was analyzed using the acetyl bromide (AcBr) method. c) G: Guaiacyl subunit. d) S: Syringyl subunit. e) DW: Dry weight f) na: not analyzed Values are means standard errors of the mean ; different letters with in the same colu mn indicate significant differences among means ( n =3, P < 0.05 ) as determined by Tukey test
111 Table 4 2 Composition of structural carbohydrate s of the c ell wall in control and transgenic sugarcane plants Line s a) Glucose Xylose Arabinose Total sugar mg g 1 D W b) WT 46 2 .0 1 7 .5 a 234.9 6.2 b 7.7 0.3 a 704.6 23.6 a TC 446.1 2.6 a 228.6 1.1 b 7.5 1.0 a 682.3 4.5 a T41 429.0 2.7 a 223.6 0.6 b 9.5 0.1 a 662.1 3.1 a T4 441.8 5.0 a 250.2 0.8 a 9.2 0.1 a 701.2 5.4 a a) Lines included W T : W ild type sugarcane TC: Transgenic control harboring npt II gene and T41 and T4: Transgenic sugarcane lines b) DW: Dry weight Values were means s tandard errors of the mean ; different letters in the same column indicated significant differences amo ng means ( n =3, P < 0.05 ) as determined by Tukey test
112 Table 4 3 Recovery yields of cell wall bound p coumaric acid ( p CA) and ferulic acid (FA) after mild alkaline hydrolysis of the control and transgenic sugarcane Line s a) p CA FA mg g 1 DW b) WT 1 4.4 0.3 a 2.2 0.2 a TC 14.5 0.3 a 2.3 0.1 a T41 13.3 0.3 b 2.4 0.2 a T4 9.9 0.1 c 2.7 0.1 a a) Lines included W T: W ild type sugarcane TC: Transgenic control harboring npt II gene and T41 and T4: Transgenic sugarcane lines b) DW: Dry weight V alues were means standard errors of the mean ; different letters in the same column in dicated significant differences among means ( n =3, P < 0.05 ) as determined by Tukey test
113 Table 4 4 Growth characteristics of the transgenic sugarcane lines under f ield conditions Line s a) No. of Internode s Biomass (kg plant 1 ) Stalk h eight (cm) Stalk d iameter (mm) No. of Stalks Soluble solids (% Brix) WT 18.5 a 18.5 a 204 a 28.2 a 20.5 a 20.0 a NT 17.6 a 15.2 b 180 bc 27.5 a 18.7 a 19.9 a TC 18.3 a 15.7 b 185 b 26.5 ab 19.8 a 20.4 a T41 18.2 a 15.7 b 182 b 25.1 bc 22.8 a 20.3 a T23 18.9 a 12.4 c 191 ab 23.7 cd 23.0 a 20.1 a T31 17.9 a 5.5 d 161 dc 21.6 de 14.0 b 17.5 b T4 18.7 a 5.3 d 155 d 19.8 e 19.0 a 17.2 b a) Lines included W T: W ild type sugarcane NT: Non transgenic control derived from tiss ue culture, TC: Transgenic control harboring npt II gene and T41, T23, T31, and T4: Transgenic sugarcane lines. Values are means D ifferent letters in the same column indicate significant differences among means ( n =3, P < 0.05) east Signifi cant Differences
114 Figure 4 1. Real time RT PCR analysis of COMT expression level in transgenic sugarcane W T: wild type sugarcane ; T41, T23, T31, and T4: Transgenic sugarcane lines. Error bars represent the 95% confidence interv als of the 2 value ( n =3)
115 Figure 4 2. Field trial of the COMT suppressed transgenic sugarcane lines. A) The field trial of transgenic sugarcane 6 months after establishment October 1 st 2011 B ) Sugarcane growth in block 1 at the time of harve st. October 26 th 2011. W T: wild type sugarcane ; TC; transgenic control harboring npt II ; T41, T23, T31, and T4: Transgenic sugarcane lines. Border plants were removed before harvest. A B T41 WT T23 T4 TC T31
116 Figure 4 3. Enzymatic saccharification performance in wild type (WT), t ransgenic control harboring npt II gene alone (TC), and transgenic sugarcane lines (T41 and T4). A ) Time course of enzymatic hydrolysis of ground extract free stalk sample with 60 FPU g 1 cell ulose following dilut e acid pretreatment. B ) Saccharification efficiencies at different cellulase dosage s (5, 20, and 60 FPU g 1 cellulose). Error bars represent standard errors of the mean ( n =3). A B
117 CHAPTER 5 CONCLUSION S Li gnin reduced transgenic sugarcane has been succes sfully developed by suppressing c affeic acid O methyltransferase ( COMT ) The suppression level of the gene, c ell wall characteristics, enzymatic saccharification efficiency and agronomic performance o f transgenic sugarcane plants were evalu ated under both greenhouse and replicated field conditions Despite the high level of ploidy and genetic redundancy in the sugarcane genome, transgene induced RNAi targeting the highly conserved sequences among the genetically redundant COMT genes, effecti vely suppressed the expression of COMT by 80 91%. Corresponding to the level of COMT suppression, total lignin content was reduced by 6 12% in different transgenic lines T he suppression of COMT also altered lignin composition. L ignin in the t ransgenic s c ontained 49% fewer S unit s without chang ing the G unit content compared to that of control plants The r educed S unit content in the lignin polymer subsequently resulted in a reduced level of p coumarate incorporation into lignin. No impact of COMT suppres sion was observed on the accumulation of total cell wall carbohydrates A reduction in total lignin content was associated with diminished recalcitrance of lignocellulosic biomass to enzymatic hydrolysis. T he s accharification efficienc y of lignin reduced t ransgenic lines w as 32 % higher than in control lines. Cellulose in the transgenic lignocellulosic material was converted to sugars more effectively at a ny given hydrolyzing time and enzyme dosage.
118 A moderate reduct ion of total lignin by up to 6 % had negli gible impacts on biomass yield, plant height, stalk diameter, tillering ability, and soluble solid accumulation compared with control lines under both greenhouse and field conditions However, 8 12 % lignin reduction had negative effects on plant growth per formance under field conditions. Future research should focus on the transfer of t hese trait s for reduced lignin and biomass recalcitrance into other high biomass yielding sugarcane or energycane. Given the association b etween the genetic background and t olerance to reduced lignin cross es with transgenic sugarcane from diverse genetic background s will further improve biomass production under optimal co nditions It is also expected that somaclonal variation observed in the transgenic sugarcane w ill be elim inated during the ( back ) crossing proce dure. Finally, it will be critical to investigate the ability of these transgenic sugarcane lines with reduced lignin to maintain agronomic fitness after extensive field testing across multiple years and through succe ssive vegetative progenies. In conclusion the improvement of feedstock quality has been an important task for efficient and cost competitive production of biofuel. Sugarcane can be considered a superior feedstock compared to conventional starch feedstock s or other dedicated biofuel crops. T he economic feasibility and productivity of sugarcane ethanol can be significantly increased by the co utilization of readily extractable sugar and abundant lignocellulosic biomass. Reducing lignin content by suppressin g lignin biosynthetic gene s has been established as a straightforward strategy to reduce biomass
119 recalcitrance, thereby improv ing the bioconversion efficiency of lignocellulosic sugarcane biomass.
120 APPENDIX LABORATORY PROTOCOLS Generation of Transgenic S ug arcane using Particle Bombardment Callu s Induction from Immature Leaf Whorl s 1. Harvest sugarcane tops with two or three internodes below the shoot apical meristem. 2. Wipe the outermost leaf sheath with 70% ethanol, and remove a few additional layers of oute r leaves under aseptic conditions. 3. Transversely cut immature leaf whorl in 2 5 mm thick sections from the region above shoot apical meristem 4. Place explants on a callus induction media (CI 3 ; see media preparation below) and subcultur e to the new medium o f the same composition biweekly. 5. Maintain cultures under low light intensity (30 mol m 2 s 1 light) at 28C and 16 h /8 h (light/ dark) photoperiod in an incubator. 6. Continue the callus induction phase for 10 weeks. Calli are bombarded 10 weeks after culture initiation. Preparation of Gold Microparticle Stock (60mg mL 1 ) 1. Weigh 60 mg of 1.0 m gold particles and place in a sterile 1.5 mL tube. 2. Add 1 mL of 70% ethanol and v ortex for 3 5 min 3. Centrifuge briefly (5 s ) to pellet the microparticles. 4. Discard the s upernatant follow ed by three washes with 1 mL autoclaved ddH 2 0. 5. Vortex for 1 min. 6. Centrifuge briefly (3 5 s) and remove the supernatant. 7. Add 1 mL sterile 50% ( v/v ) glycerol. 8. Store the gold stock at 20C. Preparation of DNA Coated Microparticles 1. V ortex g old stock suspension and transfer 30 L in to a sterile 1.5 mL tube. 2. Add the expression cassette s of the target gene and selectable marker gene as 2:1 molar ratio, respectively, and add sterilized ddH 2 O to a final volume of 60 L and vortex at low speed for 30 s 3. Place 20 L of 0.1 M freshly prepared spermidine and 50 L of 2.5 M CaCl 2 on the lid of the 1.5 mL tube. 4. Mix all components by closing the lid and vortex for 1 min. 5. Centrifuge briefly (3 5 s) to pellet the gold. 6. Discard the supernatant without dis turbing the pellet and add 250 L of absolute ethanol as a wash. 7. Centrifuge briefly (3 5 s ) and discard the supernatant. 8. Repeat the previous wash with ethanol once more. 9. Re suspend the pellet in 90 L absolute ethanol by sonication for 1 s.
121 10. Keep the DNA c oated microparticles on ice. Biolistic Bombardment using on PDS 1000/He Particle Delivery System 1. Place callus on CI 3 media supplemented with 0.4 M sorbitol 4 6 h prior to bombardment. 2. Turn on PDS 1000/He Particle Delivery System and vacuum pump E nsu re the helium supply is at least 200 psi above the desired pressure optimum. 3. Place the rupture disk in the centre of the rupture disk holder and secure it properly inside the chamber. 4. Place macrocarriers into holders with forceps and push down with a ster ile blunt object to secure it in holders. 5. R e suspend the DNA coated microparticles by vortexing briefly 6. Apply 5 l of the suspension of DNA coated microparticles into the center (inner 5 m m diameter) of the macrocarrier A llow for complete evaporation of ethanol before bombardment 7. Place stopping screen into the macrocarrier plate and insert the inverted macrocarrier assembly on top. Secure the lid on top of the shelf assembly. 8. Place macrocarrier plate containing the macrocarrier at the highest level of the inner chamber. 9. Place tissue culture plate on the second shelf below the macrocarrier plate ( 6 cm below). 10. Initiate a vacuum to 27.5 Hg press and hold the fire button until the disc ruptures at 1100 psi. 11. Vent the vacuum and remove bombarded calli 12. Dism antle the assembly and prepare for the next shot. Selection and Regeneration Protocol 1. Sixteen h ours (overnight) after bombardment, transfer the bombarded calli to CI 3 media and maintain the culture for 6 d under 30 mol m 2 s 1 light intensity at 28C a nd 16 h/8 h (light/ dark) photoperiod in an incubator 2. Transfer the calli onto selection medium which consists of C I 3 with 30 mg L 1 geneticin (G 418) Phytagel is replaced by agarose (Type I, Sigma). Track the identity of independent callus lines through the selection and regeneration procedure to rec over independent events Subculture calli to new selection mediu m 3 X biweekl y. 3. T ransfer the selected calli to regeneration CI 3 medium supplement with 50 mg L 1 thidiazuron (TDZ) and culture for 2 weeks unde r 100 mol m 2 s 1 light intensity at 28C and 16 h/8 h (light / dark) photoperiod in an incubator. 4. Transfer calli with shoots to selection rooting CI 3 media without 2,4 D containing 30 mg L 1 parom omycin Subculture 4 X biweekly under 100 mol m 2 s 1 light intensity at 28C and 16 h/8 h (light/ dark) photoperiod in an incubator. If the plantlet reach es the lid, use a deep Petri dish. 5. Transplant elongated shoots with roots into soil and maintain in a growth chamber with 80% relative humidity under 500 mol m 2 s 1 light intensity at 28C and 16 h/8 h
122 (light/ dark) photoperiod. Keep the regenerated plants covered during the first 4 6 d with a transparent container to maintain humidity. 6. After 14 d of acclimatization in the growth chamber move the transgenic plant s to glasshouse maintained at 28C day and 22C night, under natural photoperiod. Stock Solution and Media Preparation Macro stock solution dd H 2 O 800 mL Ammonium Nitrate 16.5 g Potassium Nitrate 19.0 g Calcium Chloride dehydrate 4. 4 g Magnesium Sulfate heptahydrate 3.7 g Potassium Phosphate, monobasic 1.7 g dd H 2 O Fill up to 1000 mL Mix all ingredients under constant stirri ng. S tore in bottle at 4 C. Micro stock solution dd H 2 O 400 mL Potassium Iodide 0.04 150 g Boric Acid 0.31000 g Manganese Sulfate 0.64000 g Zinc Sulfate heptahydrate 0.43000 g Sodium Molybdate dihydrate 0.01250 g Cupric Sulfate pentahydrate 0.00125 g Cobalt Chloride hexahydrate 0.00125 g dd H 2 O Fill up to 500 mL Mix all ingredients under constant stirring. S tore in bottle at 4 C. Fe stock solution dd H 2 O 4 00 mL Na 2 EDTA 0.93 g FeSO 4 7H 2 O 0.65 g dd H 2 O Fill up to 500 mL Heat 400 mL dd H 2 O in beaker, but do not boil water. Add Na 2 EDTA to hot water under constant stirring. Once it dissolves, remove from heat before adding FeSO 4 but continue stirring. S tore in light protectiv e bottle at 4 C 2,4 Dichlorophenoxyacetic acid (3 mg mL 1 ) 0.15 g powder dissolved in 500 L 1N NaOH. Make up to 50 mL with ddH 2 O. Store in aliquots at 20C. Use 1 mL L 1 media. CuSO 4 stock (12.45 mg mL 1 ) 0.6225g of CuSO 4 5H 2 O dissolved in 50 mL ddH 2 O. Store at 20C. Use 100 L L 1 media.
123 B5G Vitamin S tock solution/filter sterilized dd H 2 O 90 mL Nicotinic Acid 0.10 g Thiamine HCl 1.00 g Pyridoxine HCl 0.10 g Glycine 0.20 g Myo Inositol 10.0 g dd H 2 O Fi ll up to 1 00 mL Bring to clean bench and filt e r the solution through 0.2 m syringe filter and place into prepared sterile 1.5 mL tubes. Store tubes at 20C and only open them in clean bench when preparing media. Before addin g to media thaw it completely and vortex. Geneticin sulfate for callus selection media (30 mg mL 1 ) Dissolve 0.3 g Geneticin G418 in 10 mL ddH 2 O. Filter sterilize and store in aliquots at 20C. Use 1 mL L 1 CI 3 media. Thidiazuron (TDZ) for shoot regeneration media ( 5 0 mg mL 1 ) 0.5 g TDZ dissolved in 500 L 1N NaOH. Make up to 10 mL with ddH 2 O. Store in aliquots at 20C. Use 1 mL L 1 CI 3 media. Paromomycin sulfate for selection rooting media (30 mg mL 1 ) Dissolve 0.3 g paromomycin sulphate in 10 mL ddH 2 O. Filter sterilize and store in aliquots at 20C. Use 1 mL L 1 CI 3 media without 2,4 D Callus induction CI 3 m edia Pre Autoclave ddH 2 O 400 mL Sucrose 10 g Macro stock 50 mL Micro stock 5 mL Fe stock 3) 10 mL 2,4 Dichlorophenoxyacetic acid stock 500 L CuSO 4 stoc k 50 L ddH 2 O Fill up to 500 mL pH 5.8 for each bottle Phytagel (Gelrite) 1.5 g per bottle Post Autoclave B5G Vitamin stock 500 L per bottle Small RNA Northern B lot Total RNA Extraction 1. Prepare 10 mL total RNA extraction bu ffer and 10 mL phenol (pH 4.5) fo r each sample and warm to 55C. 2. Grind 3 g of internode tissue under liquid nitrogen and place the sample in a 50 mL polypropylene tube. Do not let the sample thaw
124 3. Add 10 mL extraction buffer and 10 mL phenol, and mix tho roughly by vo r texing I ncubate at 55 C for 5 min. 4. Cool to room temperature with shaking for 5 min. 5. Add 10 mL of chloroform and shake at room temperature for 20 min. 6. Centrifuge 13,000 g at 4C for 15 min. 7. Transfer supernatant to a fresh 50 mL polypropylene tube. 8. Add an equal volume of chloroform to the tube and shake at room temperature for 15 min. 9. Centrifuge 13,000 g at 4C for 10 min. 10. Transfer the supernatant to a fresh 50 mL polypropylene tube. 11. Add an equal volume of isopropanol and i ncubate at 20C for 16 h 12. Centrifuge 13,000 g at 4C for 20 min to precipitate total RNA. 13. Discard the supernatant, add 10 mL of cold 80% ethanol, and centrifuge 13,000 g at 4C for 5 min 14. Repeat step 13 15. Remove supernatant and let RNA pellet dry at room temperature for 5 min Do not completely dry the pellet. 16. Add 250 L of DEPC treated water and dissolve RNA. Take 10 L of total RNA and check RNA quality using formaldehyde gel electrophoresis. Small RNA S eparation from T otal RNA 1. Take 240 L of total RNA and add 30 L of 50% (w/v) PEG (M W 8 000) and 30 L of 5 M NaCl The f inal concentration s of PEG and NaCl are 5% and 0.5M respectively. 2. Mix thoroughly and incubate o n ice for at least 30 min. 3. Centrifuge at 1 6 000 g for 10 min at 4 C to pellet the high molecular weigh t RNA. 4. Transfer supernatant to a fresh 1.5 mL tube 5. Add an equal volume of cold isopropanol. Mix well and place at 20 C for at least 2 h. 6. Centrifuge at 1 6 000 g for 30 min at 4 C to pellet the small RNA. 7. Carefully remove the supernata nt add 1 mL of cold 80% ethanol and c entrifuge at 1 6 ,000 g at 4 C for 5 min. 8. Air dry for 5 min at room temperature and dissolve small RNA with 10 L of DEPC treated water. 9. Check RNA quantity and quality using the Nano Drop and 1.5% agarose gel electrophoresis Membrane Transfer 1. Assemble M ini PROTEAN system (Bio rad) with 1TBE buffer, 15% polyacrylamide/urea ready gel (Cat No. 161 1189, 12 well, Bio rad), and fill the outside of the tank with ice or set the assembly at 4C 2. After the system assembly, w ash the slots with 1x TBE using a 1 mL syringe. 3. Pre run a gel for 45 min at 180 V /400 mA. 4. Prepare 20 L of RNA loading small RN A in 10 L DEPC treated water and 10 L of loading buffer Prepare 100 fmole of 23 mer DNA oligo in
125 20 L of loading sample as a positive control and a size marker. For the controls, 21 and 24 mer RNA oligo s can be used. 5. Denature the samples and the contr ols at 65 C for 10 min, and put on ice immediately. 6. Wash slots again with 1x TBE using 1 mL syringe. 7. Load samples, and run at 180 V/400 mA for about 75 min until bromphenol blue dye reaches the bottom of the gel. 8. Take a gel out of the running chamber and stain the gel with EtBr mL 1 1TBE buffer) for 5 min. 9. Verify small RNA quality with a p hoto and de stain EtBr with 1TBE until the membran e transfer 10. Soak Hybond N+ membrane (GE Healthcare Biosciences) with 1TBE buffer for 5 min. 11. Prepare Semi Dry Transfer Cell (B io R ad) in the follow ing order s : Top 12. Perform membrane transfer at 10 V/400 mA for 35 min. Do not exceed 35 min. 13. W ash membrane wi th 1TBE for 1 min 14. Let the membrane dry briefly at room temperature 15. P erform UV cross linking for 1min. 16. Bake membrane for at least 1 h at 80 C. 17. Store membrane at 4 C until use. Probe Labeling, Hybridization and Detection 1. C heck the radioactive working area for previous radioisotope contamination before commencing work 2. Thaw Prime a Gene Labeling System (Promega), and place 32 P dCTP (Perkin Elmer) behind the plexiglass shield to thaw. 3. Warm the Church's Hybridization buffer (see buffer preparation below) to 38C 4. Boil 25 ng of probe in 30 L ddH 2 O for 5 min, and place th e denatured probe in ice for 5 min. 5. S Add the following to the denatured probe 5x labelling buffer 10 L Unlabeled dNTP mix 2 L BSA 2 L Klenow (5U/ L ) 1 L Total volume 45 L 6. Move the mixture behind the plexiglass shield, add 5 L of 32 P dCTP, and mix well by pipetting. The f inal volume of the reaction is 50 L 7. Incubate the mixture behind the plexiglass shield at room temperature for 4 h. 8. Durin g the probe incubation time, place the membrane inside a hybridization tube, and per form pre hybridization in the hybridization oven for 4 h at 38C with 1 mL of Church's Hybridization buffer per 10 cm 2 of membrane. Do not add blocking DNA, i.e. sheared sa lmon sperm DNA.
126 9. Just before performing hybridization, boil the probe for 5 min behind the plexiglass shield. Do not add blocking DNA 10. Discard the pre hybridization buffer and add new hybridization buffer into the tube 11. Immediately after probe boiling, ad d probe mixture into the tube behind the plexiglass shield. 12. Perform hybridization at 38C for 16 h in the hybridization oven. 13. After hybridization, prepare washing solution, 2SSC 0.2% SDS, and warm to 50C. 14. Working behind the plexiglass shield, dispo se of the hybridization solution into the hazardous waste container using a funnel taking care to avoid any spills. 15. W ash the membrane three times with washing solution for 20 min at 50C. 16. Remove the tubes from the oven and place them behind the plexigla ss shield. Dispose of the wash solution into the hazardous waste container and wrap the membrane in Saran wrap. 17. Check for radioactivity on the membrane using a survey meter In addition, check the working area for any radioactive contamination. 18. If there is radioactivity on a blank lane, wash the membrane once more with 1SSC 0.1% SDS for 20 min at 50C 19. Place the membrane with an X ray film (Kodak) in an autoradiography cassette and allow 16 18 h for exposure. Place the cassette at 80C during expos ure. Buffer Preparation DEPC treated water (0.1%, v/v) Dissolve 1 mL DEPC in 1 L ddH 2 O under constant stirring for 16 h. Autoclave the solution. Total RNA extraction buffer 0.1M LiCl 0.1M Tris HCl, pH 8.0 0.01M EDTA, pH 8.0 1% (w/v) SDS 0.1% (w/v) PV P (F W 40,000) Store at room temperature Loading buffer 98% (v/v) deionized formamide 10 mM EDTA pH 8.0 0.025% (w/v) xylene cyanol 0.025% (w/v) bromphenol blue Store at 20 C 0.5 M sodium phosphate buffer, pH 7.2 1 mM EDTA, pH 8.0 7% (w/v) SDS
127 Store up to 1 year at room temperatur e 20X SSC 3 M s odium chloride 0.3 M sodium citrate Analysis of Cell Wall Component s Determinatio n of Dry W eight (DW) of the Extract Free S ample 1. Dry crucibles at 105 C for at least 4 h and cool down in a desiccator. Weigh crucibles to the nearest 0.1 mg. 2. Weigh 100 10 mg extract free sample in a crucible and record the weight to the nearest 0.1 mg 3. D ry the crucible with the sample at 105 C for at least 4 h and cool down in a desiccator. Weigh to the nearest 0.1 mg. 4. Place the sample back into the dry oven at 105 C and dry to the constant weight, which is defined as 0.1% change in the weight percent s olids upon 1 h of re drying the sample. Determination of Total Lignin Content using Acetyl Bromide 1. Weigh 2 0.1 mg of extract free sample, and place 2 mL of polypropylene tube with screw cap. 2. Add 1 mL of freshly prepared 25% (w/w) acetyl bromide in gl acial acetic acid into the tube. Prepare the blank solution without the sample. Perform the reaction under the fume hood. Do not use molecular grade acetic acid. 3. Incubate the tube in a water bath at 50C for 4 h, and during the last hour, thoroughly mix t he sample at 15 min intervals. 4. Take the tube out of the water bath, and incubate the tube o n ice for 30 min. 5. Prepare a new tube containing 200 L of 2 M NaOH and 1.7 mL of glacial acetic acid. 6. Take 100 L from the reaction mixture and transfer to the new t ube with NaOH and acetic acid. Prepare three reactions per sample given inaccuracies due to pipetting errors 7. Mix thoroughly and transfer all of the mixture to a UV quartz cuvette. Do not use a plastic disposable cuvette. 8. Measure the absorbance at 280 n m. 9. The lignin content can be calculated by employing the molar extinction coefficient of 21.5 L g 1 cm 1 for milled sugarcane vascular bundle lignin. Determination of Lignin Composition using T hioacidolysis following GC/MS 1. Weigh 10 0.1 mg of extract fr ee sample and place into a 5 mL glass reaction vial with Teflon lined screw cap.
128 2. Add 1 mL of freshly prepared reaction mixture and blank with nitrogen gas prior to sealing. Reaction mixture: 2.5% (v/v) boron trifluoride etherate and 10% (v/v) ethanethiol in recently distilled dioxane. Prepare reaction mixture under the fume hood wearing a respiratory protection mask. 3. Incubate at 100 C for 4 h with manual agitation hourly. 4. Stop the reaction at 20 C for 5 min. 5. Add 0.2 mL of internal standard (tetracosan e, 5 mg mL 1 methylene chloride) to each vial 6. Add 0.3 mL of 0.4 M sodium bicarbonate enough to bring the reaction pH to between 3 and 4, check using pH paper. 7. Add 2 mL of water 1 mL of methylene chloride, vo r tex and let settle to separate the phase s 8. Prepare a Pasteur pipette packed with a small piece of Kimwipes and ~ 50 mg of granular anhydrous sodium sulfate 9. Remove a n ali quot (1.5 mL ) of organic lower phase using an autopipette from the reaction vial a nd simultaneously pass through the Pasteur pip ette and transfer directly into a 2 mL polypropylene tube. 10. Dry the sample under a stream of nitrogen at 45 C for 90 min and re suspend sample in 1 mL of methylene chloride. 11. Prepare 20 L of pyridine and 100 L of N, O bis(trimethylsilyl)acetamide (BST FA) in a GC glass vial. 12. Take 20 L of resuspended sample and add into the vial. 13. Incubate reaction mixture for at least 2 h at room temperature. 14. Prepare tetracosane standard ranging from 100 g to 1000 g mL 1 methylene chloride. 15. Set up the GC (Varian 3800 ) conditions as follows; Carrier: helium 1.2 mL min 1 Injector temperature: 250 C Oven temperature: hold at 130 C for 3 min, increase to 250 C at 3C min 1 and hold constant for 5 min. 16. Set up the MS (Varian 1200) operational conditions as follow s; Electron impact mode at 70 eV Detector operation at 1.2 kV Mass range m/z 50 550, and scan every 0.2 s 17. After setting the GC/MS, inject 1 L of sample and standard into Factor 4 VF 5ht column (35 m, 0.25 mm i.d.) with 1:10 split ratio. 18. Analyze the data with MS Workstation software. Peaks from thioacidolysis derivatives for H, G, and S units can be identified by characteristic mass spectrum ions of m/z 239, 269, and 299, respectively. The p eak from IS can be identified based on the peak from tetracosane standard. 19. Retrieve peak area corresponding to H, G, and S unit, and internal standard (IS). 20. Calculate the recovery rate based on the peak area of IS and tetracosane standard curve and normalize the lignin monomer peak area with the recovery rate. 21. Calc ulate the quantity of l ignin monomer s using the response factors of each monomer against IS as follows: H versus IS, 0.42, G versus IS, 0.47, and S versus IS, 0.53.
129 22. Calculate the molar concent ration of each monomer. Molecular weight s for H, G, and S are 3 88, 418, and 448, respectively. Determination of Cell Wall Carbohydrates Acid Ins oluble/Soluble Lignin, and Ash C ontent 1. Burn filtering crucibles at 575 C for 24 h, and cool down in a desiccator, and record the weight to the nearest 0.1 mg 2. Weigh 300 0.1 mg of extract free sample, and place into a glass pressure tube (38 203 mm) with Teflon front seal plugs 3. Add 3 mL of 72% (w/w) sulfuric acid to each tube a nd mix thoroughly to wet the sample. 4. Incubate the tube in a shaking water bath at 30 C for 1 h. 5. Add 84 mL of ddH 2 O to dilute sulfuric acid to make 4% solution and mix sample s thoroughly by inverting to eliminate the phase separation. 6. Perform autoclave at 121 C for 1 h. 7. Vacuum filter the autoclaved hydrolysis solution through previously weighed fi ltering crucibles. 8. Transfer 20 mL of a n aliquot to a sample storage bottle for the determination of acid soluble lignin. 9. For acid soluble lignin, measure the absorbance of the aliquot at 240 nm blanked with 4% sulfuric acid. Use molar extinction coeffici ent of 2 5.0 L g 1 cm 1 for the calculation of acid soluble lignin. 10. Transfer and fil er all remaining solids in the pressure tube using ddH 2 O. 11. Dry crucible and acid insoluble residues at 105 C until a constant weight is achieved 12. Cool down the crucible in a desiccator and record the weight to the nearest 0.1 mg. 13. Burn the crucible at 575 C for 24 h for the determination of ash content C ool down the crucible in a desiccator and record the weight to the nearest 0.1 mg. 14. T ransfer 20 mL of an aliquot after step 1 0 into a 50 mL Erlenmeyer flask. 15. Add calcium carbonate to bring the sample pH 5 6. Manually agitate the sample frequently and monitor the pH while adding calcium carbonate using pH paper After reaching pH to 5 6, stop adding calcium carbonate, and allow t he sample to settle. 16. Decant the supernatant and filtrate through a 0.2 m syringe filter. Collect 1 mL of filtrate into a HPLC glass vial. 17. Analyze the calibration standard (D glucose, D xylose, L arabinose) internal standard, and samples by HPLC coupled with RI detector using a Bio rad Aminex HPX 87H column and oper ating at 65C with 4 m M H 2 SO 4 mobile phase at a flow rate of 0.6 mL min 1 Determination of Ester Linked p Coumarate and Ferulate C ontent 1. Weigh 25 0.1 mg of extract free sample and place in to a 2 mL polypropylene tube with a screw cap. 2. Add 1.7 mL of 2 M NaOH containing 20 L of 2 hydroxycinnamic acid (1 mg mL 1 2M NaOH) as an internal control, and blank with nitrogen gas prior to sealing. 3. Incubate at room temperature for 20 h under dark cond itions with shacking.
130 4. Add 0.3 mL of 12 M HCl to acidify the sample. 5. Take 1 mL of reaction mixture a nd transfer into a new 2 mL centrifuge tube. 6. Add 1 mL of diethyl ether vo r tex for 5 s and centrifuge 16,000 g for 5 min. 7. Take the supernatant and add the e qual volume of diethyl ether, and repeat step 6. 8. Repeat step 7. 9. Combine the supernatant s and dry under a stream of nitrogen gas. 10. Add 40 L of methoxyamine hydrochloride (20 mg mL 1 pyridine) and incubat e at 37C for 90 min 11. A dding 60 of MSTFA [ N methyl N (trimethylsilyl) trifluoroacetamide], and incubat e at 37C for 30 min. 12. Prepare the standard reference for p coumarate, ferulate, and 2 hydrox ycinnamic acid 13. Set up the GC (Varian 3800) conditions as follows; Carrier: helium 1.2 mL m in 1 Injector temperature: 220 C Oven temperature: hold at 70 C for 4 min, increase to 250 C at 10C min 1 and hold constant for 2 min. 14. Set up the MS (Varian 1200) operational conditions as follows; Electron impact mode at 70 eV D etector operation at 1.0 kV M ass range m/z 45 6 50, and scan every 0.2 s 15. After setting the GC/MS, inject 1 L of sample and standard into Factor 4 VF 5ht column (35 m, 0.25 mm i.d.) with 1:10 split ratio. 16. Analyze data with MS Workstation software. 17. Peaks from derivatized p co umarate, ferulate, and internal standard can be identified by their characteristic mass spectrum ions obtained from the reference compounds. The c oncentration of p coumarate, ferulate, and internal standard s can be determined using the standard curve. The concentration of p coumarate and ferulate must be normalized with the concentration of internal standard among the sample s Evaluation of Saccharification Efficiency Diluted Sulfuric Acid Pretreatment 1. Weigh a sample equivalent to 0.1 g of cellulose and place into a 50 mL glass tube The c ellulose content is determined as glucose minus the contribution of any starch present in the sample. Prepare 0.1 g of No.1 Watmann filter paper for the positive control and check the saccharification efficiency. 2. Mix t he sample at a solid loading of 10% (w/w) with dilute sulfuric acid (final concentration of 1.3%, w/w). 3. Incubate the sample at room temperature for 1 h. 4. Autoclave the sample at 121 C for 40 min. 5. Wash the sample with 50 mL of ddH 2 O and drain the liquid th rough nylon vacuum filter.
131 6. Collect the all remaining pre treated residues with 50 mL of ddH 2 O and repeat step 5 twice. 7. Collect the all remaining pre treated residues with 8 mL of ddH 2 O and place it into a pre weighed 50 mL polypropylene tube. Do not let th e sample dry. Enzymatic S accharification and Determination of Glucose Y ields 1. Add 10 mL of 0.1 M sodium citrate buffer (pH 4.8) and 200 L of 2% (w/w) sodium azide into the tube with pre treated sample. 2. Add the appropriate volume of cellulases which is d epend ent on the activities of each enzyme and the enzyme dosage per unit biomass Prepare the sample without adding cellulases for the enzyme blank control. 3. Add the appropriate volume of ddH 2 O to bring the final mass of the sample to 20 g, assuming all so lutions and the biomass sample have 1.000 g mL 1 specific gravity 4. Incubate the sample in a shacking incubator at 50 C and 250 rpm for 72 h. 5. T ake 1 mL of the hydrolyzed sample while shaking the sample, and filt e r it through a 0.2 m syringe filter. 6. Measur e the glucose amount from the filtrate using the YSI glucose analyzer. 7. Correct the YSI reading value for hydration by multiplying with 0.9. De termination of Starch C ontent 1. Weigh 100 0.1 mg of sample and place into a 50 mL Erlenmeyer flask. Use 100 0. 1 mg of amylopectin as a standard reference material which is run in parallel to a batch of samples. 2. Add 5 mL of ddH 2 0 and mix thoroughly to wet the sample. 3. Add 2 mL of 2N NaOH and incubate at 90C for 20 min. Swirl to mix every 5 min. 4. Add 2 mL of 2N HC l and swirl to mix. Cool the sample to below 50C. 5. Add 2 mL of 1M sodium acetate buffer (pH 4.2) and swirl to mix. 6. Add 1 mL of amyloglucosidase (60 U mL 1 ) mix well, and incubate the sample in a shaking incubator at 40 C and 200 rpm for 60 min. 7. Take the sample out of the incubator and add 1 mL of 25% (w/v) trichloroacetic acid to stop the enzyme reaction. 8. Cool down the sample to room temperature and transfer it to a 20 mL of volumetric flask. Rinse out all of the remaining residues using 0.4 M sodium phos phate buffer (pH 6.2) and transfer to the flask. 9. Add additional sodium phosphate buffer to the final volume of 20 mL 10. Mix well take 1 mL of the sample and filt er through a 0.2 m syringe filter. 11. Measure the glucose amount from the filtrate using the YSI g lucose analyzer. 12. Measure the free glucose amount in the enzyme, and subtract it from the value of samples. 13. Correct the YSI reading value for hydration by multiplying 0.9 14. Correct the value with the digestion efficiency of standard reference material with a given enzyme.
132 Buffer Preparation 0.1 M sodium citrate buffer (pH 4.8) ddH 2 O 400 mL Citric acid monohydrate 4.4 g Trisodium citrate dihydrate 8.5 g ddH 2 O Fill up to 500 mL 1 M sodium acetate buffer (pH 4.2 ) ddH 2 O 400 mL Acetic acid (glacial) 23.4 g Sodium acetate anhydrous 9.1 g dd H 2 O Fill up to 500 mL 0.4 M sodium phosphate buffer (pH 6.2) 0.4 M sodium phosphate monobasic 815 mL 0.4 M sodium phosphate dibasic 195 mL Total 1000 mL
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151 BIOGRAPHICAL SKETCH Je Hyeong Jung was born in Seoul, Kor ea. He went to the Korea n University, Seoul, Korea, in 1998. During his undergraduate degree he was quite active in both academic and extra curricular activities He was awarded the Academic Excellence Scholarship for three semesters. He was the P resident of the U ndergraduate S tudent A ssociation in the D epartment of C rop S cience. He also served 26 months in the army, and after finishing military service, he travelled to Canada for 6 months. A fter he graduated with a B.S. ( c rop s cience) in 2005, he joine d the G raduate S chool at the Korea n U niversity and pursued a n M.S. in l ife s cience s and b iotechnology under the guidance of Dr. Yong Weon Seo. A fter obtaining a n M.S. degree in 2007, he got an offer from KT&G Central Research Institute, Daejeon, Korea, and participated in several research programs as an A ssistant Researcher until he came to the U S to pursue a Ph.D. In 2009, h e joined Dr. Fredy Altpeter s lab at University of Florida. He was a T eaching A ssistant in Genetics from 2009 to 2012. H e was awar ded The Paul Robin Harris Memorial Scholarship for three consecutive years from the Agronomy Department, University of Florida. He won the Wilton Earle Award at the International Plant Biotechnology Conference in 2010 and The Philip White Memorial Award at the Society for In Vitro Biology in 2011.