Citation
The control of the tomato fruitworm, the tomato pinworm, the tomato russet mite, and hornworms

Material Information

Title:
The control of the tomato fruitworm, the tomato pinworm, the tomato russet mite, and hornworms
Creator:
Wilcox, Joseph, 1901-1982
Elmore, J. C ( John Clifford ), b.1896
United States -- Bureau of Entomology and Plant Quarantine
Place of Publication:
[Washington, D.C
Publisher:
Bureau of Entomology and Plant Quarantine
Publication Date:
Language:
English
Physical Description:
7 p. : ; 26 cm.

Subjects

Subjects / Keywords:
TomatoDiseases and pests ( lcsh )
Heliothis zea -- Control ( lcsh )
Tomato pinworm -- Control ( lcsh )
Sphingidae -- Larvae -- Control ( lcsh )
Genre:
federal government publication ( marcgt )
non-fiction ( marcgt )

Notes

General Note:
Caption title.
General Note:
Typescript.
General Note:
"April 1943 ; E-589."
General Note:
Supersedes Circular E-489, Suggestions for the control of the tomato fruitworm, issued in September 1939.
Statement of Responsibility:
by Joseph Wilcox and J.C. Elmore.

Record Information

Source Institution:
University of Florida
Rights Management:
This item is a work of the U.S. federal government and not subject to copyright pursuant to 17 U.S.C. §105.
Resource Identifier:
030285031 ( ALEPH )
82365119 ( OCLC )
Classification:
635.6429 ( ddc )

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STATE PLANT BOARD
April 1943 E- 39

United States Department of Agriculture Agricultural Research Administration Bureau of Entomology and Plant Quarantine


THE CONTROL OF THE TOMATO FRUITWOPR, 1/THE TOT"ATO PIMWOR", THE T0"ATO RUSSET 77TE, AD TTOEWOPUS

By Joseph Wilcox and J. C. Elmore, Division of Truck Crop Insect Investigations



Introduction

Tomato plants and fruits are attacked commonly by several species of
insects, and important among these are the tomato fruitworm, the tomato pinworm, hornworms, and a new pest of tomatoes in California known as the tomato
russet mite. It is the purpose of this circular to set forth methods of
controlling these pests which have developed during recent years.

The tomato fruitworm (Helinthis armigera(Hbn.)), also known as the corn
earworm on corn and as the bollworm on cotton, is a pest of tomatoes, regularly or occasionally, in practically all sections of the United States where
tomatoes are grown. Its principal damage to tomatoes is through the insects
feeding on and in the tomato fruits, rendering them, unmarketable.

The tomato pinworm (Keiferia lycopersicella (Busck)) is a pest of
tomatoes in southern California. It is known to occur in Arizona, Niew Mexico,
Missouri, Florida, Mississippi, Virginia, Delaware, and Pennsylvania. In
addition to tomatoess this insect also occasionally attacks potato and eggplant. In California, Arizona, 1ew Mexico, and Florida the tomato pinworm has been primarily a pest of field-grown tomatoes, whereas thus far in Mississippi,
Virginia, Delaware, Pennsylvania, and Missouri it has been present only as a pest of tomatoes grown in greenhouses and in tomato fields near the infested
greenhouses.

The tomato russet mite (Phyllocoptes destructor Keifer) was first found
in Modesto, Calif., in 1940. By 1941 it had spread over most of the San Joaquin and Sacramento valleys and in 1942 it was widespread in southern
California. Besides tomato, other host plants recorded by the University of California are petunia, nightshade, Datura, potato, and morning-glory. This
mite is minute in size, and its presence on the plant can be detected only by
a microscopic examination. However, the injury caused by its feeding is readily recognizable. The plants are first attacked at the base and the
leaves develop a bronze appearance before they die. During warm weather the
mites increase very rapidly and can kill a vine or a whole field in a few
weeks if control measures are not applied promptly.

1/ This supersedes Circular E-489, Suggestions for the Control of the
Tomato Fruitworm, which was issued in Seotember 1939.





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There are two species of hornworms that attack tomatoes--the tomato hornwmrm (Protoparce uinquemaculata (Haw.)) and the tobacco hornworm (P. sexta (Johanj), which are widely distributed over this country. In certain sections they are, unless controlled, very destructive. They feed on the foliage of the plant, first attacking the leaves, oftentimes stripping the plants to thepoint where pnly the main stalk or stems remain.

Control

The problem of controlling these several pests when they occur in the
same field is somewhat involved, as a remedy for one may not be suitable for the other. In California the tomato fields may be infested at some period during the same season with the tomato fruitworm, the tomato pinworm, hornworms, the tomato russet mite, Putworms, and the beet armyworm.

In consideration of these factors, the determination of the proper remedy or remedies will depend on the kind of insects present in the field. The discussion on the control of these several pests and their joint control follows.

Tomato Fruitworm Control

Cryolite and calcium arsenste dusts ard a bait prepared by mixing corn
meal with cryolite are all about equal in effectiveness for the control of the fruitworm. The choice between these materials is discussed in more detail in the following paragraphs.

Cryolite.--Extensive experiments have demonstrated that this insecticide is one mf the best and safest materials to use on tomatoes for the control of the fruitworm, and is superior in the control of the pinworm. The active ingredient in erynlite is sodium fluoaluminate. Tests with the several brands and with different strengths of cryolite have not shown any great degree of difference in control when they were used at the same sodium fluoaluminate content and at the same rate per acre. For example, the same control can be expected from the use of a total of 180 pounds per acre of cryolite dust containing 35 percent of sodium fluoaluminate as can be expected from the use of a total nf 90 pounds per acre mf a cryolite dust containing 70 percent of sodium flunaluminate. As it is cheaper ti mix and apply 90 pounds of a dust containing 70 percent of sodium fluoaluminate per acre than it is to apply 180 pounds of the weaker dust, a 70-percent cryolite dust is recommended for general use.

Calcium arsenate.--Extensive experiments have demonstrated that calcium arsenae (undilute is as effective as a cryolite dust containing 70 percent of sodium fluoaluminate when both materials ate used at the same rate per acre. Calcium arsenate also has the added advantage of being cheaper than oryolite and in being more effective than cryolite in the control of hornworms and the best armyworm. CalciLun arsenate has the disadvantage when compared with cryolite of not being so effective in the.control of the pinworm, and in humid climates it may seriously burn the foliage and fruit. Calcium arsenate is preferred for use in localities where there is no chance of a pinworm infestation and in localities where it can be used without burning the plants and fruit.







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Corn meal bait.--This bait is prepared by mixing 9 pounds of corn meal (as finely ground as for pout ry feed) with 1 pound of cryolite containing 90 percent of sodium fluoaluminate. A total of 180 pounds per acre of this mixture has proved to be about as effective in the control of the fruitworm as a total of 90 pounds per acre of.a cryolite dust containing 70 percent of sodium flucaluminate, or as undiluted calcium arsenate. This corn meal bait mixture is cheaper than either of the .dusts and is scattered over the leaves of the plants by hand, no sppciail equipment being necessary, but it has the disadvantage when compared with the dusts of being ineffective against the pinworm, hornworm, beet armyworm, and russet mite.

When to treat.--The first application of either dust or bait for fruitworm control should be made as soon as the fruits of the main crop begin to set. At this time, depending on weather and other growing conditions, the plants may vary from 1 .to 3 feet in diameter but there will be an average of about 7 tomatoes per vine, 5 of which should be 1/2 inch in diameter or less. The timing of the first application is very important, as under southern California conditions it has been demonstrated that a delay of 1 week in making the first application results in a marked increase in the number of fruits damaged by the fruitworm.

Number of applications.--Three applications of either the bait or the
dusts are made at 2-week intervals.

Poundage of material per acre.--Experiments in southern California have
shown that 90 pounds of the cryolite dust mixture containing 70 percent of sodium fluoaluminate, or of the undiluted calcium arsenate, is the most profitable quantity to use in this area. An application of a. total of 150 pounds per acre for three applications proved superior to the 90-pound rate, but under the conditions of this experiment the added degree of control did not justify the expense of the additional material. Under war conditions the 150-pound rate is justified in many fields.

The experiments indicate that a cryolite dust mixture containing 70 percent of sodium fluoaluminate is preferable to one of lower strength. The three applications at 2-week intervals of 70-percent cryolite or undiluted calcium arsenate are made at 20, 30, and 40 pounds per acre, respectively, for a total of 90 pounds per acre, and at 25, 50, and 75 pounds per acre, respectively, for a total of 150 pounds per acre. If the cryolite is diluted to contain less than 70 percent of sodium fluoaluminate, the quantity of insecticide applied per acre should be increased accordingly. FMr example, when a cryolite dust mixture containing 50 percent of sodium flucaluminate is used, it should be applied at a total rate of 125 pounds per acre to be equivalent ti 90 pounds of a 70-percent cryolite. Likewise, if calcium arsenate is diluted with 25 percent of sulfur for tomato russet mite control, it will be necessary to use a total of 120 pounds per acre to get the equivalent of 90 pounds of undiluted calcium arsenate.

ThrP applications Af corn meal bait should be made at 2-week intervals, using 40, 60, and 80 pounds per acre, respectively, making a total of 180
pounds for the season.





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Tomato Pinrworm Control

Control of the.tomato pinworm may be obtained by using a cryolite dust
mixture containing 7Q percent of sodium fluoaluminate, with talc as a diluent.

When to treat,.--In the regular .treatment schedule, the first application of cryolite dust.mixture. should be made when the first fruits on the tomato plant are approximately 1 inch.in diameter. Under unusual conditions or in areas where the pinworm has been causing heavy losses, a careful watch should be made of the plant bed and ef the newly set transplants, and if a noticeable infestation occurs, an application eof cryolite should be made at once.

Number of applications.--Whether the plant bed or transplants have been
treated or not, the.regular. treatment schedule, beginning as indicated, should consist-of four applications at 10-day intervals. It is-important, however, that the fourth application be made about the time of the first picking-of. tomato fruits for the cannery, or after the first picking for market, and therefore it may be necessary to allow 2 or 3 weeks between the third and f6urth applications, depending upon weather conditions.

Poundage of material per acre.--On the basis of 1,000 tomato plants per acre, which is the prevailing planting rate in southern California, the most economical results have been obtained by using a range of from 20 to 25 pounds of the cryolite dust mixture per acre for each application, depending on the size of the plants, or a total of approximately 90 pounds for the four applications. In any event it is important that the plants be covered thoroughly by the insecticide at each application.

Although satisfactory control of the pinworm has been obtaine-d by using
a cryolite dust mixture containing 50 percent of sodium fluoaluminate, experiments indicate that better results can be obtained with the higher strength unless the quantityof the insecticide applied at the 50-percent strength is increased so that the same number of pounds of the active ingredient, sodium fluoaluminate, is applied per acre. If the 50-percent strength is used, it is suggested that the rate of application range from 25 to 35 pounds per acre per application, or a total of.125 pounds for the four applications.

Tomato Russet Mite..Control

Sulfur is used for the control of this mite. On canning tomatoes, not
more than 25 percent of sulfur should be used when there is fruit on the vines, as excess amounts of sulfur might damage the canned product. Undiluted sulfur can be used on small plants before the fruit has set and on larger vines if the fruit is not to be canned.

When to treat.--When there is danger of -early infestations (i.e., in later fields and in fields set out with plants grown in or near old vines or fields), applications of undiluted' sulfur .should be made at or about the third and sixth weeks after transplanting. During the fruiting period of the plhts, applications of 25-percent sulfur dust are made at the same time that the fruitworm or pinworm applications are made. If the frtit-is not to be used for canning 'and insecticides are not necessary for fruitworm or pinworm control, undiluted sulfur can be' used for the later applications after the fruit sets.









Poundage of material per acre.--From 5 to 10 pounds of undiluted sulfur per acr per application should be used when the plants are small. From 20 to 50 pounds of 25-percent sulfur dust should be used per acre per application during the fruiting period of the plants when the regular fruitworm or pinvorm applications are being made.

Number of applications.--In fields that are transplanted before June,
3 or 4 applications of 25-percent sulfur dust made at the time of the fruitworm or pinworm applications should be sufficient. In fields transplanted in June or later, it is desirable to make 2 additional applications of undiluted sulfur to the small plants at about the third and sixth weeks after transplanting.

Ordinarily it is not necessary to make special or extra applications of sulfur for mite control, as 25 percent of sulfur can be incorporated in the regular dust applications for fruitworm or pinworm control. Previous to these
regular dust applications, cutworms, pinworms, flea beetles, or army-wuorms frequently attack the small plants. In case of such attacks, 25 or 50 percent of sulfur can be incorporated in either calcium arsenate or cryolite used for their control.

Joint Control of the Tomato Fruitworm, the Tomato Pinworm, and the Tomato Russet Mite

In the event that both the tomato fruitworm and the tomato pinworm are present in injurious numbers in the same field, which is likely to be the case in many fields, a cryolite dust mixture containing 70 percent of sodium flunaluminate should be used to control these two species of insects. If the tomato russet mite is also present, sulfur should be used as the diluent for cryolite in these applications. Four dustinrs of this 1m.ixture should be applied. The first three applications should be made at 2-week intervals, beginning as soon as the fruits of the main crop begin to set, 20, 30, and. 40 pounds being used per acre per application. The fourth application should be made shortly after the first picking of the fruits for canning, or ordinarily about a week or 10 days after the third application. Pwenty-five pounds per acre of the cryolite dust mixture containing 70 percent of sodium fluoaluminate should be applied for this fourth application. The plants should be covered thoroughly by the insecticide at each application.

If the pinworm is not a problem and calcitur arsenate can be used in your locality without danger of burning the plants, then a dust containing 75 oercent of calcium arsenate and 25 percent of sulfur can be used. To obtain control of the fruitworm equivalent to the control obtainable with a cryolite dust containing 70 percent of sodium flucaltminate, 30, 40, and 50 pounds per acre of the calcium arsenate mixture should be used for the three applications, respectively.

A bait prepared by mixing 9 pounds of corn meal with 1 pound of cryolite containing 90 percent of sodium fluoaluminate is effective in the control of the fruitworm and cutworms. This material is somewhat cheaper than dusting and would be the preferred treatment in localities where the fruitworm is the main pest to be controlled, and in localities where the margin of profit is small and the cost of treatment is a major consideration. (For rate of







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application see page 3). This material is not effective against the pinworm, russet mite, hornworms, or the beet armyworm, and should not be used in localities where these pests usually cause damage.

Hornworms.--The regular applications of cryolite or calcium arsenate for fruitworm and pinworm control ordinarily give satisfactory control of these insects. In cases of severe infestation it may be necessary to make one or more supplementary applications of unrliluted calcium arsenate for the control of these pests. Calcium arsenate has proved superior to cryolite for the control of these insects, and, if a regular cryolite schedule is being followed for pinworm control, calcium arsenate should be applied in the interval between the regular applications. If the pinworm is not a serious menace, then calcium arsenate can be used for one or more of the regular fruitworm applications.

Other Insects

Cutworms and the boot armyworm may also be present in the same field
with the tomato fruitworm, pinworm, hornworms, and russet mite. Ordinarily the regular applications for the control of either the fruitworm or the pinworm will adequately control those insects, but in epidemic years, or in unusual infestations, special treatments may be necessary as outlined below.

Beeoot armyworm.--This insect usually attacks only tomatoes which mature
in the fall, and in ordinary years the regular "worm" applications give satisfactory control of the booeet armyworm. In epidemic years of this insect, it may be necessary to make extra applications of undiluted calcium arsenate to the plants when they are small and previous to the r-gular frinitworm or pinworm applications, and if cryolite is being used in the regular fruitworm or pinworm applications, calcium arsenate should be substituted for cryolite in one or more of these applications, or else an additional application of calcium arsenate for armyworm control should be made. In our experiments it has been demonstrated that calcium arsenate is superior to cryolite in the
control of the boeet armyworm.

Cutworms.--In southern California the rcgulo. dusting program for the tomato fruitworm has given rl:collent control of cutworms 1hen they have attacked the plants during the fruiting period. But at times severe infestations will occur on young plants before the time for the regular fruitworm treatment schedule begins. Such infestations must be handled immediately, or many plants will be killed outright. Control can be accomplished by either dusting the plants with 70-percent cryolite or undiluted calcium arsenate, or by propering a bait composed of 4 pounds of paris green, 100 pounds of wheat bran, and sufficient water to make a crumbly mash. Ten pounds of the dust or bran bait is sufficient to cover an acre. The dust should be applied primarily to the stem of thle plant, and the bait should be scttered around the base of the plant, preferably in the evening. Sodium fluosilicate may be substituted for paris green in the bait mixture.

Warn ing

All the materials recommended in this circular arc likely to leave
insecticidal residues on the fruit if they are applied within 2 weeks of the time the fruits are harvested. In cases where excess residue is present, the





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indications are that the washing process in the canneries and the process of wiping the fruit for market with a cloth will reduce the residue to a quantity well below the danger point. If any insecticidal materials are visible on the fruit when pi cked, such residue must be removed b. wiping or washing before the fruit is offered for sale.

CAUTION.--In handling mixin, and nplin7 poisonous irsecticides care
should be taken not to inhale excessive qu -,rtitics at ny time. .Well-desi ned respirators affording protection to the entire face are available and should
be used when such danger exists. After working with insecticides the hands or any exposed parts of the bodg should be washed thoroughly.

Where to Obtain Insecticides

Information regarding the purchase of the insecticide materials mentioned in this circular may be obtained usually through local dealers in agricultural supplies, seedsmen, general stores, or through the county agricultural agent, State agricultural experiment station, State department of agriculture, or the Bureau of Entomology and Plant Quarantine, Agricultural Research Administration, United States Department of Agriculture.






UNIVERSITY OF FLORIDA 3 1262 09224 6916




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PAGE 1

IDENTIFICATION AND CHARACTERIZATION OF COLD-REGULATED GENES IN COLD-HARDY CITRUS RELATIVE Poncirus trifoliata (L.) Raf. By MEHTAP SAHIN-CEVIK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by MEHTAP $AHIN-CEVIK

PAGE 3

To my husband Bayram.

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ACKNOWLEDGMENTS I would like to express my sincere gratitude, thanks, and appreciation to Dr. Gloria Moore, chair of my supervisory committee, for her advice, support, encouragement, and understanding throughout this study and the preparation of this manuscript. I could not have reached my goals without her guidance and help. I want to extend my gratitude to Dr. Maria Gallo-Meagher, Dr. Charlie Guy, and Dr. Wayne Sherman for kindly serving on my committee; and for their helpful suggestions and comments and for reviewing this dissertation. I want to thank Kim Ruesch for her help and support; that made this work easier. I also want to thank Dr. Sue Moyer and her lab for their help and support; and for providing the phosphoimager and hybridization bath for my experiments. I extend my thanks and appreciation to Dr. Morse for her help in analysis of expression data; and to Dr. Folta for letting me use his lab facilities. I would also like to thank members of Dr. Cline's lab for their help and support; and for sharing their lab equipment. I thank all of my friends in Gainesville and around the world (especially to Basma) for the support and friendship they provided in the last four years. My deepest thanks and appreciation go to my mother, Sultan, for her constant support and unconditional love throughout my life. I appreciate the endless love and support I receive from my sisters Ayten, Ezmehan, and Nesrin; and from my brothers Mehmet and Talat and his family. I also want give my thanks to my in-laws for their support and encouragement. IV

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I give my special thanks and love to my wonderful husband Bayram £evik to whom this dissertation is dedicated, for his constant love, help, support, understanding, and encouragement since the day we met. Last, but not least, I would like to give my most special thanks to my precious little girl, Beste Selena, for being with me and bringing me the joy and happiness that made finishing up my research and writing my dissertation more enjoyable. Finally, I thank the Turkish Ministry of Education for their generous financial support during my graduate studies at the University of Florida. v

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TABLES OF CONTENTS Page ACKNOWLEDGMENTS iv TABLES OF CONTENTS vi LIST OF TABLES viii LIST OF FIGURS ix ABSTRACT x CHAPTER 1 INNTRODUCTION 1 2 LITERATURE REVIEW 4 Environmental Stresses 4 Temperature Stress 4 Chilling Injury 5 Freezing Injury 6 Plant Responses and Adaptation to Low Temperatures 7 Supercooling 7 Cold Acclimation 8 Accumulation of Compatible Solutes 9 Alterations of Lipid Composition and Membrane Structure 12 Changes in ABA Level 14 Changes in Antioxidant Activity 15 Changes in Gene Expression 17 Methods Used for Identification of Cold Regulated Genes 25 Subtractive Hybridization 27 Array Analysis of Gene Expression 28 Cold Response in Citrus 30 3 IDENTIFICATION OF COLD REGULATED GENES FROM Poncirus trifoliata (L.) Raf. USING SUBTRACTIVE cDNA LIBRARIES 36 Introduction 36 vi

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Materials and Methods 39 Plant Materials 39 Environmental Stress Treatments for Gene Expression Study 39 Isolation of RNA 40 Construction of the Subtractive Library 42 Sequence analysis 42 Reverse Northern Blot Analysis of Subtracted cDNAs 42 Data Analysis 43 Northern Blot Hybridization 44 Results 45 Discussion 56 4 ISOLATION and CHARACTERIZATION OF COLD INDUCED PUTATIVE TRANSCRIPTION FACTORS IN Poncirus trifoliata (L.) Raf. 63 Introduction 63 Materials and Methods 67 Plant Materials 67 Environmental Stress Treatments 67 Isolation of RNA 68 Rapid Amplification of cDNA Ends (RACE) 69 Sequence Analysis 69 Northern Blot Hybridizations 69 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 70 Results 71 Cold-Induced PI-B05 cDNA from Poncirus Encodes an AP2 Domain Containing Protein 71 Cold-Induced PI-C10 cDNA from Poncirus Encodes a RAV-like Protein with two Different DNA-binding Domains 75 PII-C02 cDNA from Poncirus Encodes a RING Zinc Finger Protein and is Induced in Response to Cold and Drought 81 Discussion 86 5 SUB-CELLULAR LOCALIZATION OF COLD REGULATED GENES COR11 andCOrl9 94 Introduction 94 Materials and Methods 97 Results 100 Discussion 103 6 SUMMARY AND CONCLUSIONS 1 06 LIST OF REFERENCES 110 BIOGRAPHICAL SKETCH 125 vii

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LIST OF TABLES Table Page 3-1. Summary of sequence and expression analysis of cold-regulated genes identified from cold-acclimated Poncirus cDNA libraries 48 viii

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LIST OF FIGURES Figure Page 3-1. Construction of cold-regulated cDNA libraries using a subtractive hybridization method 41 3-2. Differential screening of forward and reverse subtracted cDNA libraries of cold acclimated Poncirus by reverse northern analysis 46 3-3. Northern blot analysis of selected cDNAs from the cold-induced library 52 34. Northern blot analysis of C12 expression in response to environmental stresses 55 41. Full-length sequence of Poncirus cDNA PI-B05 72 4-2. Multiple alignment of predicted amino acid sequences PI-B05 with other plant proteins in GenBank showing homology with PI-B05 73 4-3. Analysis of amino acid sequences of PI-B05 cDNA 74 4-4. Northern blot analysis of expression of PI-B05 cDNA in response to environmental stresses 76 4-5. Full-length sequence of Poncirus cDNA PI-C10 78 4-6. Multiple sequence alignment of predicted amino acid sequences of Poncirus PI-C10 cDNA with proteins in GenBank showing the highest homology with PI-C10 79 4-7. Conserved DNA-binding domains of PI-C10 80 4-8. Expression of PI-C10 cDNA in response to environmental stresses 82 4-9. Full-length sequence of Poncirus cDNA PII-C02 84 4-10. Analysis of predicted amino acid sequences of Poncirus PII-C02 cDNA 85 41 1. Northern blot analysis of expression of PII-C02 cDNA in response to environmental stresses g7 51. Sequence alignment and cloning of COR genes for sub-cellular localization study..98 5-2. Sub-cellular localization of COR genes in onion epidermal cells 101 ix

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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 IDENTIFICATION AND CHARACTERIZATION OF COLD-REGULATED GENES IN COLD-HARDY CITRUS RELATIVE Poncirus trifoliata (L.) Raf. By MEHTAP SAHIN-CEVIK December 2003 Chair: Gloria A. Moore Major Department: Horticultural Sciences Citrus is a cold-sensitive plant and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata is an interfertile Citrus relative that can tolerate temperatures as low as -26°C when cold-acclimated. To identify genes involved in cold tolerance in P. trifoliata, cDNA libraries were constructed from 2day cold-acclimated and nonacclimated Poncirus seedlings using a subtractive hybridization method. A total of 192 randomly picked clones, 136 from the cold induced library and 56 from the cold repressed library, were sequenced. Most of these clones showed sequence homology to previously characterized cold-induced or environmental stress-regulated genes from other plants that were deposited in GenBank. Expression of these cDNAs was analyzed by reverse northern blot hybridization with cold-acclimated and nonacclimated probes. Analysis of expression data showed that expression of 96 cDNAs was increased 2to 49-fold during cold acclimation. On the other hand, expression of only a few genes was repressed in response to cold. Three partial cDNAs (PI-B05, PI-C10, and PII-C02) showing homology to previously x

PAGE 11

characterized transcription factors were selected for further characterization. The fulllength cDNA sequences of these genes were obtained by 5' and 3' rapid amplification of cDNA ends (RACE). Sequence analysis revealed that PI-B05 contains the AP2 domain, PI-C10 contains the AP2 and B3 DNA binding domains, and PII-C02 has a RING zinc finger domain conserved in transcription factors involved in environmental stressinduced gene expression. Expression patterns of these genes in cold-tolerant P. trifoliata and cold-sensitive pummelo {Citrus grandis) in response to cold and drought were analyzed by northern blot hybridization at different time points. Expression analysis showed that PI-B05 and PI-C10 were induced only in response to cold in Poncirus. Expression of PII-C02 was induced by both cold and drought in Poncirus and pummelo. In addition, sub-cellular localization of two previously identified cold-regulated genes, COR1 1 and COR19, was studied in onion epidermal cells using COR::GUS fusion constructs. Transient expression assay followed by 4',6-Diamidino-2-phenylindole (DAPI) staining showed that these proteins are localized in the nucleus and cytoplasm, but accumulated predominantly in the nucleus. Deletion of the N-terminal half of COR 11 demonstrated that C-terminal half conserved in both genes was sufficient for nuclear localization. xi

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CHAPTER 1 INTRODUCTION Citrus originated in tropical regions of Southeast Asia, and have been cultivated for about 4,000 years. With annual production of more than 100 million metric tons, citrus has become one of the world's most important fruit crops. It is distributed and commercially grown widely in tropical and subtropical regions of the world. Citrus production is mostly limited by low temperatures outside of this region. Since citrus is a cold-sensitive plant, low temperatures and freezes result in significant damage and economic losses in subtropical citrus growing regions, including Florida. Although mild freezes occur relatively frequently in citrus growing regions, a number of severe destructive freezes have affected citrus production in the last century. Beside the economic losses due to destruction of citrus trees and fruits, these freezes have forced relocation of citrus production to warmer regions. Environmental conditions often are limiting factors for plant growth and development. To cope with changes in their environment, plants have developed different mechanisms for adapting to new environments. Since agricultural production of commercially important crops is adversely affected by unfavorable environmental conditions, understanding of plants' response to environmental stresses has been an important research area. The study of environmental stress and adaptation not only provides an understanding of the environmental stress mechanisms of plants, but also allows the development of strategies for the improvement of stress tolerance in agricultural crops. 1

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2 Temperature is one of the variable environmental conditions showing daily and seasonal changes. Therefore, exposure to low temperature is one of the most common environmental stresses on plants. Low temperatures can have a negative affect on plant growth and development. Thus, some plants have developed adaptive mechanisms, such as cold acclimation, to survive in low temperature conditions. Cold acclimation is a process by which exposure to low non-freezing temperatures increases a plant's tolerance to subsequent freezing temperatures. During this process, a series of measurable physiological and biochemical changes is induced by the expression of specific genes. Gene-expression analysis of cold-acclimated plants showed that several hundred genes are induced during low-temperature exposure of plants, including the model plant Arabidopsis thaliana L. and economically important agricultural crops. Isolation and characterization of genes induced during cold acclimation revealed that they were involved in a variety of cellular functions. Identifying these genes gives a better understanding of the mechanisms of cold tolerance; and allows the development of more cold-tolerant plants using genetic engineering. Since it is a cold-sensitive and economically important crop, the development of a cold-tolerant citrus plant is an important objective for citrus industries in subtropical regions affected by freezing. In the last century, breeders discovered the existence of major cold hardiness in the cross-compatible trifoliate orange, Poncirus trifoliata. Since then, a number of crosses between Poncirus and Citrus have been made to integrate the cold hardiness trait into commercial citrus varieties. However, even though breeding programs generated a few hybrid cultivars for rootstocks, the development of cold-

PAGE 14

3 tolerant citrus scion cultivars with acceptable horticultural characteristics has yet to be successful to date. Recent developments in molecular biology have enabled the identification of key genes involved in cold acclimation and cold tolerance in plants. It has been shown in a number of different plants that cold tolerance can be improved by manipulating the expression of these genes in transgenic plants. This approach has the potential to improve cold tolerance in citrus as well. Efficient genetic transformation methods are in place for citrus. However, there is insufficient information about regulation of cold tolerance in Poncirus and also a lack of genes that can be used for improving cold tolerance by genetic engineering in Citrus. Therefore, studying gene expression during cold acclimation of Poncirus is needed to identify genes involved in cold tolerance and to develop cold-hardy citrus cultivars. The overall objective of this study was to identify and characterize genes involved in cold tolerance in Poncirus. The specific objectives were (1) to construct subtractive cDNA libraries for identification of cold-regulated genes from cold-acclimated Poncirus trifoliate; (2) to analyze expression of cold-regulated genes from the cDNA libraries; (3) to isolate and clone cold-induced genes from cold-acclimated Poncirus; (4) to study expression of selected cold-induced genes under cold and drought stresses; and (5) to demonstrate nuclear localization of two previously identified cold-induced genes (COR11 and COR19).

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CHAPTER 2 LITERATURE REVIEW Environmental Stresses Plant species are distributed all over the world and are grown in different climates and environmental conditions. Like other organisms, plants are exposed to abiotic and biotic stresses in their environment. Thus, plant growth and development as well as crop production are highly influenced or even limited by environmental conditions such as soil and nutrition, light, temperature, and water. To survive extreme environmental conditions, plants evolved to adapt to their environment and develop mechanisms to cope with the stresses induced by these conditions. Photoperiodism, seed and bud dormancy, and low-temperature response are some examples of adaptive mechanisms that plants develop through their growth period. Many dicots and grasses promote or delay flowering in response to day length by using photoperiodism. Seeds delay developmental processes until the conditions required for germination are met using a mechanism called seed dormancy. Similarly, many temperate-zone trees stop bud growth in response to low temperatures (Taiz and Zeiger 1991), which is called endo-dormancy. Temperature Stress Temperature is one of the most common environmental conditions causing abiotic stress on plants. It has a significant role in the plant life cycle and is important for both developmental and physiological aspects of plants. Therefore, exposure of plants to high, low and freezing temperatures imposes significant stress to them and adversely affects their growth and development. Although high-temperature-induced stress is as important 4

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5 as low-temperature-induced stress, low-temperature-induced stress is the focus of this review. When plants are exposed to low temperatures, it causes two types of injury: chilling and freezing (Stushnoff et al. 1984). Chilling Injury Plant exposure to low but non-freezing temperatures is called chilling. Depending on the evolutionary origin of plants, chilling injury may occur at temperatures between 0 and 15 C. While tropical and subtropical plants show chilling injury at 8°C and 12C, respectively, most temperate plants show chilling injury at temperatures between 0°C and 4 C (Nishida and Murata 1996; Fowler and Limin 2002). Severity of injury during chilling is species-dependent; and plants can be divided into three different classes according to their chilling sensitivity. The first group is extreme chilling-sensitive plants, generally tropical originating plants, including Ephedra vulgaris (Richt.), Gossypium hirsutum L., and Vigna radiata L. In this group, chilling injury is rapid and irreversible. A second group showing delayed response to chilling is called chilling-sensitive plants, such as Cucumis sativum L., Glycine max (L.) Merr., Lycopersicon esculentum L., and Nicotiana tabacum L. The third group is chilling-resistant plants including Arabidopsis thaliana L., Brassica oleracea L., Hordeum vulgare L., and Triticum aestivum L. This group shows no chilling injury unless plants are exposed to another stress factor (Kratsch and Wise 2000). Symptoms of chilling injuries are generally similar across species and it can cause many physiological disruptions in plants including swelling and disorganization of both chloroplasts and mitochondria, reduced size and number of starch granules, dilation of thylakoids and unstacking of grana, lipid droplet accumulation in choloroplasts, and condensation of chromatin in the nucleus ( Kratsch and Wise 2000). Severity of chilling

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6 injury depends upon different factors including light, duration of chilling, relative humidity, acclimation, and certain stages of plants (Nishida and Murata 1996; Kratsch and Wise 2000). When Selaginella spp. were evaluated, no chilling injury was observed in the darkness. However, symptoms of chilling injury such as bleaching of chlorophyll, accumulation of lipid droplets, and degeneration of thylakoids was observed during chilling in the light (Jagels 1970). Although short exposure to chilling temperatures may not induce any injury to certain plants, longer exposure to the same chilling temperature can cause irreversible injuries (Kratsch and Wise 2000). Similarly, it was observed in both cotton and bean that high relative humidity acts as a protective factor for chloroplasts; and reduces the risk of injury during chilling (Wise et al. 1983). When different developmental stages were evaluated, it was found that seedlings were more susceptible than mature plants; and the pollen developmental stage was the most sensitive to chilling temperatures (Nishida and Murata 1996). When injury occurs during chilling, cells can die in two different ways. The first one is called necrotic cell death, which involves swelling of the cell and then lysis resulting in leakage of cellular contents. The other proposed mechanism is programmed cell death (PCD). This involves generation of reactive oxygen species that in turn trigger an increase in free Ca 2+ ions that act as second messenger, and stimulate a cascade of proteolytic enzymes involved in the cleavage of key proteins that finally results in the systematic death of the cell (Kratsch and Wise 2000). Freezing Injury The second type of injury caused by low temperatures is freezing injury, which occurs when the temperature drops below the freezing point of water. Exposure to low freezing temperatures may result in intracellular or extracellular freezing. Intracellular

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7 freezing occurs when ice crystals form within the cells either by internal nucleation in the cell or by penetration of an external ice crystal into the cell. This type of freezing damages the protoplasmic structure, and growing ice crystals kill the cell. When ice forms outside of the cell, the freezing is called extracellular. In this type of freezing, water is withdrawn from the protoplast and the protoplasm becomes dehydrated. The capacity of extracellular spaces for growing ice crystals and the ability of the protoplast to withstand dehydration are key determinants of the freezing resistance in certain species. Even in this case, cooling must be slow; otherwise sudden exposure to low temperatures can cause intracellular freezing and cell death (Guy 1990; Taiz and Zeiger 1991; Fowler and Limin 2002). Plant Responses and Adaptation to Low Temperatures Since plants are unable to move or change their environment, they must adapt to changes in their environment. Plants have developed and used several strategies to tolerate temperature stress induced by low and freezing temperatures. Supercooling and cold acclimation are the two most common low-temperature-tolerance mechanisms by which plants protect themselves from freezing temperatures. Supercooling Aqueous solutions may remain in the liquid state when cooled below the freezing point which is known as supercooling. Solutions may supercool to different degrees before they spontaneously freeze. The temperature at which spontaneous nucleation occurs is termed the supercooling point or temperature of crystallization (Zachariassen and Kristiansen 2000). In the absence of any ice nucleation sites, water will remain in the liquid phase down to -38. f C. In the presence of ice nucleation sites, ice crystals are formed and can grow up to 0°C. In some plants, water in the cell is supercooled and

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8 maintained above the supercooling point to avoid freezing. Presence of solutes in the cell will further lower the supercooling point a few degrees, allowing plants to survive temperatures near -40 to -41 C. Using supercooling, some woody plants can withstand temperatures of approximately -40 C by protecting critically important tissues including dormant buds and xylem ray parenchyma. As temperatures drop in the fall, these plant tissues are hardened by exposure to temperatures below 0 C for several days. Although these processes are not fully understood, some modifications were observed in membrane properties. Lack of ice-nucleating sites prevents formation of ice in the protoplast of some tissues. Thus, some cells avoid freezing and show deep supercooling many degrees below the freezing point (at about -38 °C) (Fitter and Hay 2002). Cold Acclimation It has been shown in many plants that exposure to low non-freezing temperatures below 10C enhances tolerance to subsequent freezing temperatures. This process is called cold acclimation; and is used by many plants to cope with stress induced by low temperatures. Plants have different responses to low temperatures and maximum freezing tolerance in cold-acclimated plants is not constitutive, but is induced in response to low non-freezing temperatures (Thomashow 1998). As temperatures drop in early fall, the freezing tolerance of plants increases gradually. This accelerates in late fall resulting in the maximum hardiness of plants. When the temperatures increase, the cold acclimation process can be reversed, which is called deacclimation. While cold acclimation takes two weeks to several months depending on the species, deacclimation takes approximately a week or shorter. Even though cold acclimation is a long process that takes weeks to

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9 months, molecular changes that could lead to freezing tolerance of plants can be detected at very early stages of cold acclimation (Guy 1990). During cold acclimation, a series of physiological and biochemical changes take place in the plants which results from induction of specific gene expression (Guy 1990). These changes include the accumulation of compatible solutes, such as soluble sugars, betaine, and proline; alterations of membrane lipid compositions; increases in abscisic acid (ABA) concentration; and antioxidant activity (Browse and Xin 2001; Smallwood and Bowles 2002). Accumulation of Compatible Solutes Plants accumulate low-molecular-weight organic solutes such as sugars, proline, and glycine betaine in response to low temperature and other stress conditions that cause depletion of cellular water including drought and high salinity. During cellular dehydration, these compounds increase osmotic pressure and prevent loss of water from cells. These organic solutes are called compatible solutes and have a hypothesized function as cryoprotectants by preventing protein denaturation. They maintain membrane integrity by interacting with polar head groups of phospholipids or forming hydrophobic interactions with the membrane. They are also osmolytes that lower the freezing point of cytosol (Guy 1990; Hare et al. 1998; Smallwood and Bowles 2002). Accumulation of sugars, primarily sucrose and the raffinose families of oligosaccharides (RFO) such as rafinose and stachyose were observed in many plants in response to environmental stresses including exposure to low temperatures. (Hinesley et al. 1992; Holaday et al. 1992; Castonguay and Nadeau 1998). These increases are correlated with the increased activity of enzymes involved in the synthesis of these sugars (Guy 1990). Sucrose-phosphate synthase (SPS) and galactinol synthase (GS) are the key

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10 enzymes in the sucrose and RFO biosynthetic pathways, respectively. A correlation between increased activities of SPS and GS and the levels of sucrose and RFO, respectively was reported in alfalfa (Castonguay and Nadeau 1998). In addition, an increase in the endogeneous levels of glucose, fructose, raffinose, and stachyose was observed at the onset of cold acclimation, in August and their levels reached a maximum in December and January in grapes (Hamman et al. 1996). Sugars might have a role in osmoregulation by increasing intracellular osmotic potential (Crow et al. 1993). This process lowers the freezing temperature and prevents drying of the cells by retaining more liquid water inside the cell, which provides a longer period of metabolic activity during extracellular freezing (Levitt 1980). Sugars also have a role in cryoprotection of cell membranes and proteins (Guy 1990). They act as cryoprotectants by protecting cell membranes from high concentrations of electrolytes (Crowe etal. 1990). Glycinebetaine is another compatible solute that accumulates in response to many stresses including low temperature (Allard et al. 1998), salinity (Grieve and Maas 1984), and drought (Ladyman et al. 1983). Based on in vitro studies, glycinebetaine appears to stabilize the structures of enzymes and complex proteins and maintain the integrity of membranes under stress conditions (Sakamoto and Murata 2001 ; Smallwood and Bowles 2002). It is synthesized from choline by a two step oxidation reaction. During the first oxidation reaction of choline, betaine aldehyde is obtained by the activity of choline monooxygenase enzyme. Then, glycinebetaine is synthesized by the activity of betaine aldehyde dehydrogenase enzyme during the second oxidation step of choline (Russell et al. 1998; Iba 2002). The genes encoding both enzymes have been cloned from many

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11 plants including barley (Ishitani etal., 1995), rice (Nakamura et al. 1997), and spinach (Rathinasabapathi et al. 1997). Since exogenous application of glycinebetaine enhances stress tolerance (Makale et al. 1998), choline monooxygenase and betaine aldehyde dehydrogenase have been introduced into plants separately to obtain glycinebetaine. These studies were not successful because sufficient amounts of choline substrate and betaine aldehyde were unavailable in the cell (Nuccio et al. 1998; Iba 2002). When a heterologous betaine aldehyde dehydrogenase gene was introduced into rice along with exogenous betaine aldehyde, transgenic rice plants accumulated glycinebetaine and showed resistance against salt and low temperatures (Kishitani et al. 2000). Another way to obtain glycinebetaine is via oxidation of choline by choline oxidase (COD) in a one-enzyme catalyzed reaction in the soil bacteria, Arthrobacter globiformis and Arthrobacter pascens. The CodA and cox genes, which encode COD of A. globiformis and A. pascens, respectively, have been introduced into many plants including Arabidopsis (Hayashi and Murata 1998), rice (Sakamoto et al. 1998), tobacco (Huang et al. 2000), and Brassica napus (Huang et al. 2000). These transgenic plants showed improved tolerance to several abiotic stresses including low-temperature stress (Sakamoto and Murata 2001). In the transgenic plants, the phase transition from the liquid crystalline to the gel state was shifted (Sakamoto and Murata 2001; Smallwood and Bowles 2002). Plants accumulate proline in response to different environmental stresses (Hare et al. 1999) including low temperature (Barka and Audran 1997). There are several roles of proline in plant stress tolerance including osmoregulation (Yoshiba et al. 1997) and induction of osmotically regulated genes (Iyer and Caplan 1998), stabilization of proteins

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12 and membranes (Rudolph et al. 1986) as well as protection against reactive oxygen species (ROS) (Smirnoff and Cumbes 1989). Proline accumulation was observed after exogenous application of ABA in many plants including barley (Pesci 1987), and maize (Xin and Li 1993). Based on studies with ABA mutants, proline accumulation can be observed through both ABA-dependent and ABA-independent regulation mechanisms (Nambara et al. 1998). A correlation between the accumulation of proline and improved cold tolerance has been observed in many plants including wheat (Dorfling et al. 1997), grapevine (Barka and Audran 1997), and Arabidopsis (Xin and Browse 1998). In addition, some mutants with constitutively high levels of proline showing enhanced freezing tolerance have been isolated, for example the Arabidopsis eskimol mutant (eskl). After cold acclimation, eskl plants maintained their high level of proline, which is 30-fold higher than cold-acclimated wild-type plants (Xin and Browse 1998). Alterations of Lipid Composition and Membrane Structure Studies of freezing and chilling injuries have indicated that membranes are the primary target Exposure to low temperatures causes damage to the plasma membrane due to cellular dehydration. Several types of membrane damage can result from freezing injury in plants, including expansion-induced lysis, lamellar-to-hexagonal-II (Hn) phase transitions, and fracture jump lesions (Thomashow 1999). In expansion-induced lysis, freezing and thawing induce expansion and eventually lysis of protoplasts. During freezing of the medium, water diffuses out of the protoplast, reducing the tension in the cytoplasm, which results in endocytotic vesiculation of the plasma membrane. Formation of vesicles does not cause injury, but it significantly reduces the surface area of the plasma membrane. When the medium thaws, water diffuses into protoplasts, increasing the cytoplasmic vojume and resulting in re-expansion of the plasma membrane. The

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13 vesicles are not readily re-incorporated, leading to lysis of the protoplasts (Dowgert and Steponkus 1984). Under non-freezing conditions, polar head groups of phospholipids are oriented in the aqueous core of the lipid bilayer of the membrane. During freezing, lowtemperature-induced dehydration causes a phase transition from lamellar to the Hn phase in which three-dimensional discontinuous structures with long tubes of lipids are formed. The formation of the Hn phase disrupts the membrane structure by changing continuity and semipermeability of the plasma membrane, leading to solute leakage and introduction of ice nucleation sites into the cells (Gordon-Kamm and Steponkus 1984). In some plants, instead of an Hn phase transition, fracture jump lesions were observed in localized regions in which the fracture plane in the plasma membrane has 'jumped' to lamellae of the endomembranes that are in close apposition to the plasma membrane. (Webb and Steponkus 1993). Chilling also induces structural changes in cell membranes. It has been reported that membrane permeability was increased in response to chilling. This causes ion leakage and altered ion balance in chilling sensitive tissues. During chilling, phase changes (liquid to solid) occur in membrane lipids, which results in deactivation of membrane-bound enzymes, leading to slower respiration, inhibition of photosynthesis and protein synthesis, and increased degradation of proteins (Taiz and Zeiger 1991; Nishida and Murata 1996; Fitter and Hay 2002). It has been reported that cold acclimation prevents the membrane damage caused by freezing and chilling injuries, including expansion-induced lysis and formation of Hn phase transition in rye (Steponkus et al. 1990). Stabilization of membranes during cold acclimation can be attributed to several factors, including changes in lipid composition, accumulation of sugars, and

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14 dehydrins. It was suggested that dehydrins stabilize the membranes through hydrophobic interaction with phospholipids (Close 1996; 1997) and may protect integrity of membranes by preventing lamellar-to-hexagonal phase transitions (Thomashow 1999). It has been shown that in plants and other organisms, the level of unsaturated fatty acids increases at low temperature. Increases in unsaturation of membrane lipids compensates for the reduced membrane fluidity and enzyme activity caused by low temperature. A correlation between chilling sensitivity of plants and unsaturation of membrane lipids was observed and studies have indicated that plants containing higher percentages of unsaturated fatty acids generally showed more chilling resistance (Nishida and Murata 1996). Therefore, enzymes involved in unsaturation of membranes, such as acyltransferases and desaturases from plants and microorganisms, have been used for improving cold tolerance in plants (Nishida and Murata 1996; Iba 2002). Changes in ABA Level An increase in the level of ABA in response to low temperature was first observed in cold-acclimating Solarium commersonii. This increase was transient and was not observed in non-acclimating plants. In addition, exogenous application of ABA increased the freezing tolerance of S. commersonii at warm temperatures (Chen et al. 1983). Later, it was shown that ABA levels increased in response to low temperature in other plants (Lalk and Dorffling 1985; Luo et al. 1992; Lang et al. 1994), and exogeneous application of ABA increased the freezing tolerance of many plants (Orr et al. 1986; Ishikama et al. 1990). Exogeneous application of ABA also increased the expression of COR genes, including COR78, COR47, COR15a and COR6.6 (Hajela et al. 1990; Kurkela and Franck 1990; Gilmour et al. 1992; Nordin et al. 1993; Gilmour et al. 1992; Yamaguchi-Shinozaki and Shinozaki 1993). It was shown that expression of some cold

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15 regulated genes, such as Arabidopsis RAB18 and rd29A, was dependent on ABA; and that these genes carry putative ABA-responsive elements (ABREs) in their promoters (Lang et al. 1992; Nordin et al. 1993; Welin et al. 1994; Yamaguchi-Shinozaki and Shinozaki 1 994). Gene expression through ABREs is regulated by bZIP transcription factors; and several cold-responsive bZIP proteins were identified in plants, including Arabidopsis (Lu et al. 1996; Choi et al. 2000), rice (Aguan et al. 1993), and maize (Kusano et al. 1995). Studies on the abal mutant in Arabidopsis showed that the ABAinduced accumulation of these COR genes was eliminated; but cold induced accumulation of these transcripts was not affected in this mutant. Consequently, it was concluded that cold-regulated expression of these genes was controlled by an ABAindependent pathway (Gilmour et al. 1991; Nordin et al. 1991). Changes in Antioxidant Activity Active oxygen species (AOS) such as superoxide anions (0~2) and hydrogen peroxide (H202 ) are produced by the reduction of molecular oxygen (02) and the hydroxyl radicals (OH») which are produced in cells under stress conditions (Iba, 2002). Levels of AOS increase at low temperature, causing oxidative stress in the cell. Oxidative stress damages cells in different ways, including lipid peroxidation, membrane deterioration, protein and nucleic acids degradation, and cholorophyll quenching (Scandalios 1990; Bestwick et al. 1998; Pastori et al. 2000). Plants and other organisms produce a variety of antioxidants to control AOS levels and to prevent oxidative stress in their cells. Antioxidants can be divided into three groups. The first group contains lipid soluble and membrane-associated tocophenols. The second group includes water soluble reductants, such as ascorbic acid and glutathione. The last group is includes the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase

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16 (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) (Foyer 1993; Iba 2002). It has been observed that the levels of antioxidants in many plants increase at low temperatures during cold acclimation to prevent or reduce the potential damage of increased levels of AOS. It has been shown that the antioxidant enzymes SOD APX, CAT, MDAR, DHAR, and GS are involved in AOS-detoxification systems in plants (Iba 2002). SOD catalyzes the dismutation of two superoxide radicals into oxygen and hydrogen peroxide. The APX and CAT detoxify the hydrogen peroxide by converting it to water. In addition, APX oxidizes ascorbic acid and MDAR, DHAR, and GS are involved in ascorbic acid recovery. Although CAT is localized only in the microbodies in almost all plant species, the other enzymes are found as multiple isozymes at different locations in the cell. According to the metal cofactor involved, SOD can be divided into three groups, Mn-SOD, Fe-SOD, and Cu,Zn-SOD, which are localized in the mitochondria, chloroplast, and chloroplast and cytosol, respectively (Holberg and Bulow 1998; Iba 2002). It was shown that APX and GR levels increase when H202 accumulates in the cell under low-temperature conditions in Arabidopsis (O'Kane et al. 1996) and Fe-SOD and CuZn-SOD levels increase when tobacco plants are chilled (Tsang et al. 1991). Since these enzyme levels increase in stress conditions, they were overexpressed in transgenic plants to study their roles in stress tolerance. When chloroplast CuZn-SOD was overexpressed, resistance to intense light and low temperature was improved in transgenic tobacco plants (Gupta et al. 1993a; Gupta et al. 1993b). Overexpression of Mn-SOD and Fe-SOD in transgenic alfalfa also resulted in improved resistance to low temperature (McKersie et al. 1999; McKersie et al. 2000).

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17 However, transgenic tomato plants overexpressing Escheria coli GR showed no improvement in chilling tolerance (Bruggemann et al. 1999). Changes in Gene Expression Physiological and biochemical changes leading to freezing tolerance in plants have been associated with changes in gene expression during cold acclimation. Although this has been postulated and demonstrated to a point for many years, changes in gene expression during cold acclimation has been only recently studied extensively in many plants. It was first shown that there is a correlation between accumulation of soluble proteins in cortical bark cells of black locust trees and freezing tolerance (Sminowitch and Briggs 1949). Weiser, (1970), proposed that maximum freezing tolerance of temperate woody perennials is associated with changes in gene expression and synthesis of new proteins during cold acclimation. Guy et al. (1985) demonstrated that expression of a number of genes was altered in spinach during cold acclimation. Since then, many cold-regulated genes have been isolated in many plants including Arabidopsis (Gilmour et al. 1998; Liu et al. 1998), Brassica napus (Saez-Vasquez et al. 1995), Hordeum vulgare (Dunn et al. 1996), Medicago sativa (Castonguay et al. 1994), Spinach oleracea (Guy and Li 1998) and Poncirus trifoliata (Cai et al. 1995). Isolation and characterization of these genes are important not only for understanding the low temperature response and the mechanism of cold tolerance in plants, but also for improving cold tolerance in agricultural crops resulting in increased plant productivity and expanded areas of agricultural production. Cold acclimation has an effect on the expression of many genes; these genes are altogether called cold-regulated genes. The cold-regulated genes have been referred to with different terms in the literature based on the specific response of the gene under

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18 certain conditions or by the preference of the groups working with the genes: responsive to dehydration (rd), early responsive to dehydration (erd), cold induced (kin), cold regulated (cor), and low temperature induced (lti). Most low temperature and dehydration inducible genes are in common because low temperature reduces the water potential of the extracellular compartment which causes loss of water from cell by osmosis leading to dehydration. In addition, low temperature decreases turgor pressure as a result of dehydration, which results in the induction of the plant stress hormone ABA. Thus, some genes can be responsive to cold, dehydration, and the exogenous application of ABA, while others are induced in response to cold and dehydration stress, but not by ABA treatment. It was suggested that cold-regulated genes could be induced by different pathways and it was shown that both ABA-dependent and ABA-independent pathways exist and are involved in cold-regulated gene expression in plants (Shinozaki et al. 2000; Viswanathan and Zhu 2002). The genes induced in response to low temperatures during cold acclimation can be divided into two groups: those directly involved in freezing protection and those that regulate gene expression. Genes in the first group are COR genes involved in freezing tolerance, and include COR6.6, C015a, C047, and COR78 isolated from Arabidopsis (Hajela et al. 1 990). Based on DNA sequence analysis, it was shown that COR6.6, COR15a, and COR78 encode novel hydrophilic polypeptides and COR47 encodes a group II late embryogenesis abundant (LEA) protein (Thomashow 1998) that thought to function in dehydration tolerance (Close 1997). In general, COR genes show biochemical similarities with cryoprotective proteins (Artus et al. 1996), which are plant leaf proteins that were reported for the first time in cabbage and spinach leaves and

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19 function in protecting isolated thylokoid membranes from freeze-thaw damage (Volger and Heber 1975). These COR proteins are hydrophilic and synthesized in response to low temperature and remain soluble upon boiling (Artus et al. 1996). Homologs of these genes and many other cold regulated genes have been identified and isolated from different plants. These genes encode for novel hydrophilic proteins including BN1 15 from Brassica napus (Weretilnyk et al. 1993), CR1 from Hordeum vulgare (Cattivelli and Bartels 1990), CAS 15a from Medicago sativa (Monroy et al. 1993), CAP 160 from Spinach oleracea (Kaye et al. 1998) and hydrophobic polypeptides including RC12A from Arabidopsis (Capel et al. 1997), tacr7 from Triticum aestivum (Gana et al. 1997) or homologs of LEA proteins including ERD14 from Arabidopsis (Kiyosue et al. 1994), casl8 from Medicago sativa (Wolfraim et al. 1993), CAP85 from Spinach oleracea (Neven et al. 1993), and COR15 and COR19 from Poncirus trifoliata (Cai et al. 1995). To test if COR15a had a role in freezing tolerance and whether it had the same function as cabbage and spinach proteins, transgenic plants constitutively expressing COR15a were produced. It was shown that chloroplasts of transgenic plants were 1 C to o 2 C more freezing tolerant than the chloroplasts of wild type plants (Artus et al. 1996). Later, experiments established that COR15a helps stabilize membranes during freezing by decreasing freeze induced lamellar-to-hexagonal II phase transition (Steponkus et al. 1 998). Even though constitutive expression of COR1 5a increased the freezing tolerance of transgenic plants, this increase was small, which was expected considering the quantitative nature of cold tolerance. More comprehensive studies involving microarray analyses of thousands of genes revealed that up to several hundred genes were regulated during cold acclimation. These

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20 genes are involved in a variety of cellular functions including metabolism, transcription, protein fate, transport facilitation, biogenesis, communications and signal transduction, cell rescue and defense, cell death and aging (Seki et al. 2001 and 2002; Fowler and Thomashow 2002). Analysis of the regulatory sequences of COR genes revealed that promoters of COR 15a and COR78 contained a TGGCCGAC regulatory sequence element which was activated in response to low temperature and dehydration stress (Horvath et al. 1993). This element was termed the C-repeat (CRT) (Baker et al. 1994). At the same time, Yamaguchi-Shinozaki and Shinozaki (1994) found that the promoter of RD29A (COR78/LTI78) contained a regulatory TACCGACAT cw-element that has a role in both dehydration and cold responsive gene expression and they called it a dehydration responsive element (DRE). Both CRT and DRE elements have the same five base pair (bp) core sequence, CCGAC, and multiple copies are found in the promoters of many coldand dehydration-regulated genes. Characterization of regulatory sequences of the cold and dehydration response genes provided researchers with necessary information for the identification of the regulatory protein binding to these sequences. A regulatory protein binding to the CRT/DRE element was isolated using a yeast one hybrid screen and was designated the CRT/DRE Binding Factor 1 (CBF1) (Stockinger et al. 1997). It has a molecular mass of 24 kDa, a putative bipartite nuclear localization signal, an AP2 DNA-binding domain which contains a 60 amino acids (aa) motif present in a number of plant proteins including Arabidopsis APETAL2, AINTEGUMENTA, and TINY, as well as the tobacco ethylene element response binding proteins (EREBPs), and an acidic region that

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21 functions as an activation domain (Stockinger et al. 1997). Two additional members of CBF proteins were isolated by screening an Arabidopsis cDNA library prepared from cold-acclimated plants and were designated CBF2 and CBF3. Based on the aa sequences of CBF proteins, they were 88% identical and 91% similar to each other (Gilmour et al. 1998). Independently, Liu et al ( 1 998) isolated two proteins binding to DRE elements, also using a yeast one-hybrid screening technique, and called them DRE-binding proteins (DREB1 A and DREB2A), which were induced in response to low temperature and dehydration and high salt stresses, respectively. Like CBF proteins, both DRE binding proteins contain an EREBP/AP2 domain. Except for the similarities found in the DNAbinding domain, there is no significant sequence homology between the two DREB proteins. Using DREB 1 A sequences, two more DRE binding proteins designated DREB IB and DREB1C were isolated (Liu et al. 1998). The DRE binding proteins DREB IB, DREB1C and DREB 1 A are identical to the CRT binding proteins CBF1, CBF2 and CBF3, respectively, and regulate expression of genes containing the CRT/DRE sequence element in their promoters (Stockinger et al. 1997; Liu et al. 1998; Gilmore et al. 1998; Shinwari et al. 1998) in response to both low temperatures and dehydration through an ABA independent pathway (Yamaguchi-Shinozaki and Shinozaki 1994). These three genes form the CBF/DREB protein family. They were mapped in a direct repeat on chromosome 4 in the order CBF 1 /DREB IB, CBF3/DREB1 A, and CBF2/DREB1C (Gilmore et al. 1998; Shinwari et al. 1998). The activities of all CBF/DREB proteins change depending on the COR promoter sequences. This result indicates that CBF binding to the CRT/DRE sequence depends not only on the CCGAC

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22 core CRT sequence, but also on nucleotides outside of this core sequence (Gilmore et al. 1998). Since CBF/DREB proteins are involved in regulation of cold and dehydration responsive genes, these proteins were expressed in transgenic plants for improving cold tolerance. Overexpression of CBF/DREB proteins under the control of the constitutive CaMV35S promoter increased freezing tolerance in transgenic Arabidopsis plants (JagloOttesen et al. 1998; Liu et al. 1998; Kasuga et al 1999; Gilmour et al. 2000). When CBF1 was expressed in transgenic Arabidopsis plants, it induced the expression of COR genes containing the CRT/DRE element, including COR6.6, COR15a, COR47, and COR78, without a low temperature stimulus, and increased the freezing tolerance of nonacclimated Arabidopsis plants. (Jaglo-Ottesen et al. 1998). Freezing and dehydration tolerance were also observed in transgenic Arabidopsis plants overexpressing DREB1A and DREB2A, which was correlated with the level of expression of the stress inducible genes under unstressed conditions. In these plants, the expression level of RD29A was induced more than in the wild type plant under unstressed or stress conditions including, low temperature, dehydration, high salt and ABA treatment. However, transgenic plants expressing DREB1 A and DREB2A showed severe and mild growth retardation, respectively (Liu et al. 1998). Arabidopsis plants overexpressing CBF3 showed that transcript levels of CBF3 were almost equal in nonacclimated and cold-treated transgenic plants, but were higher than in nonacclimated or cold-treated control plants. In addition, expressions of COR genes COR15a and COR6.6 were similar in nonacclimated and cold-treated transgenic plants as well as in cold-acclimated control plants. These transgenic plants also have a

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23 dwarf phenotype with shorter leaves compared to the control plants and require more time for flowering. Increased freezing tolerance was observed in both nonacclimated and cold-acclimated CBF3 -expressing plants compared to non-acclimated or cold acclimated control plants. They also accumulated proline Al-pyrroline-5-carboxylate synthase (P5CS), which is a key enzyme in determining the proline level in plants; and soluble sugars including sucrose, raffinose, glucose and fructose. These results indicated that over-expressing CBF3 leads to multiple biochemical changes that generally occur in plants during cold acclimation. Thus, CBF3 appears to be a key regulatory gene that functions in activating multiple mechanisms resulting in increasing the freezing tolerance of plants (Gilmour et al. 2000). These overexpression studies demonstrated that freezing tolerance, which is a quantitative trait involving many genes, can be manipulated and improved by the expression of single regulatory genes. However, the expression of these regulatory genes under strong constitutive promoters are physiologically costly to the plant, resulting in undesirable growth phenotypes such as those observed in transgenic plants overexpressing CBF3/DREB1 A. To attempt to reduce adverse affects of overexpression of CBF/DREB proteins in transgenic plants, Kasuga et al. (1999) expressed DREB1 A under the stress inducible promoter of the rd29A gene in Arabidopsis. These transgenic plants showed a greater improvement in drought, salt and cold tolerance, and less or a minimal level of growth retardation compared to transgenic plants overexpressing the same gene under a strong constitutive promoter (Kasuga et al. 1999). Once the importance of CBF/DREB transcription factors in regulation of cold responsive genes was established in Arabidopsis, the presence of this pathway and

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24 homologs of CBF/DREB proteins were investigated in other plants. CBF/DREB-like proteins were identified and characterized in Brassica napus, a close relative of Arabidopsis (Jaglo et al. 2001; Gao et al. 2002) and cold-acclimated wheat, rye (Jaglo et al. 2001), and barley (Choi et al. 2002), as well as tomato which does not cold acclimate (Jaglo et al. 2001). As in Arabidopsis, transcripts of CBF/DREB-like genes in B. napus, wheat, rye, and barley accumulated 15-30 min after exposuring plants to low temperature (Jaglo et al. 2001; Choi et al. 2002). After the expression of these genes, expression of Bnl 15, an ortholog of Arabidopsis COR15a, and Wcsl20/COR39 orthologues of Arabidopsis COR47 were induced in B. napus, wheat, and rye, respectively. In addition, transgenic B. napus plants that constitutively overexpressed Arabidopsis CBF genes showed accumulation of Bnl 15 and Bn28 without a low temperature stimulus and the freezing tolerance of both nonacclimated and cold-acclimated plants was increased. Based on these findings, it was concluded that the CBF/DREB cold response pathway is conserved among a number of plants including cold-sensitive and nonacclimating plants (Jaglo et al. 2001; Choi et al. 2002). In Arabidopsis, transcript levels of all three CBF/DREB proteins increased within 15 min and accumulation of COR gene transcripts was observed at 2 h after transferring plants to low temperature. Based on sequence analyses, promoter regions of the three CBF genes are 30% identical in DNA sequence and they have a number of small deletions and insertions. No CRT sequence element was observed in the promoters of any of the CBF genes, indicating that these genes are not auto-regulated. This was confirmed in transgenic plants overexpressing CBF1 in which no accumulation of CBF3 transcripts was observed. Although CBF promoters do not contain a CRT sequence, they

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25 contain some possible cold regulatory elements including multiple copies of Mycrecognition, CANNTG, and ACGT core G-box sequences. In addition to these cold regulatory elements, all three promoters contain an internally repetitive sequence ACAATT ANN ACAATTT approximately at the same position (Gilmore et al. 1998). Since the expression of CBF/DREB genes was induced within 15 min of cold acclimation it was proposed that an unknown activator of CBF/DREB genes which is called inducer of CBF expression (ICE) should be present in the cell. It was further suggested that ICE would be in an inactive state at warm temperature, and under low temperature conditions, it would be activated and induce transcription of CBF/DREB genes by binding to CBF/DREB promoters (Gilmour et al. 1998). Recently, an inducer of CBF expression 1, (ICE1), which is an upstream transcription factor regulating the transcription of CBF genes in the cold, was identified and cloned. ICE1 encodes a MYClike bHLH transcription factor and binds to MYB recognition sequences in the promoter of CBF3. It was found that ICE1 was a positive regulator of CBF3 and a mutation in ICE1 abolished CBF3 expression and decreased the expression of many CBF-target genes, resulting in a reduction in the chilling and freezing tolerance of the mutant. Expression of CBF1 and CBF2 genes are less affected in the ICE1 mutant. Overexpression of ICE1 in wild type plants induced the expression of the CBF regulon in the cold, which resulted in increasing freezing tolerance of transgenic plants. (Chinnusamy et al. 2003). Methods Used for Identification of Cold Regulated Genes The main goal of molecular cold acclimation studies is to identify genes expressed during cold stress. The most common way to identify genes expressed differentially is to compare expression of those genes under two different conditions. For

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26 identification of cold-regulated genes, expression studies were conducted under normal and col-acclimated conditions in many plants including Arabidopsis (Gilmour et al. 1992; Horvath et al. 1993), alfalfa (Monroy et al. 1993), barley (Dunn et al. 1991; Hong et al. 1992), Brassica napus (Weretilnyk et al. 1993), and wheat (Chauvin et al. 1993). The differences in gene expression under different conditions have been studied using a variety of methods. Northern blot hybridization (Cai et al. 1995), dot blot hybridization (Mraz et al. 1997) semi quantitative (Sorrell et al. 2002) and quantitative reverse transcription-polymerase chain reaction (RT-PCR) (Massonneau et al. 2001) were used for expression studies of single or a few differentially expressed genes. In the last decade, subtractive hybridization (Wang et al. 2001), differential display (Horvath and Olson et al. 1998), serial analysis of gene expression (SAGE) (Matsumura et al. 1999), and sequencing of expressed sequence tags (ESTs) (Zhang et al. 2001) have been developed to study expression levels of many genes between and among different experimental conditions at the same time. More recently, macroarray and microarray analyses have been developed and used for the global analysis of the expression of thousands of genes simultaneously under different experimental conditions (Moody 2001). Although gene arrays are presently the most powerful methods for studying gene expression on a wide scale, they require the availability of sequence information and the necessary equipment is expensive for most laboratories. Sequence information is limited for most agricultural crops, including citrus; therefore, sequences must be generated using different methods, such as construction and sequencing of ESTs and subtractive libraries for specific condition before using macroor microarrays for identifying novel genes.

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27 Subtractive Hybridization Subtractive hybridization is a very efficient method for enrichment and isolation of differentially expressed genes. It was first described in the early 1980s for construction of cDNA libraries (Sargent and Dawid 1983) and preparation of probes (Davis et al. 1984) of differentially expressed genes. The main goal of the subtractive hybridization is to identify differentially expressed genes by hybridizing the cDNA from one treatment (tester) with an excess amount of mRNA from another treatment (driver). During hybridization, transcripts present in both tester and driver form an mRNA/cDNA hybrid; however, cDNA sequences present only in the tester do not hybridize with mRNA from the driver and stay single stranded. Then, single stranded cDNAs representing differentially expressed genes are separated from double stranded nucleic acids by hydroxylapatite chromatography and cloned and used for identification of novel genes. Initially, the use of subtractive hybridization was limited by the requirement of large quantities of mRNA and a bias against the identification of rare transcripts (Moody 2001). To improve the recovery of differentially expressed genes, some modifications, such as tagging the cDNA with biotin (Welcher et al. 1986), or oligo(dT)30-latex (Hara et al. 1991) were introduced into the subtractive hybridization method. The initial amount of mRNA required for subtractive hybridization was greatly reduced by adaptation of generic linkers to cDNA (Duguid and Dinauer 1990) and selective PCR amplification of tester cDNA between hybridization cycles (Hara et al. 1991). In addition to reducing the initial amount of mRNA required for subtractive hybridization, these improvements increased the efficiency of differentially expressed genes. The bias against the identification of rare transcripts was overcome by introduction of the Suppression Subtractive Hybridization (SSH) PCR technique in which differentially

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28 expressed sequences are selectively amplified and amplification of abundant transcripts are suppressed (Diatchenko et al. 1996). The SSH PCR eliminated the need for separation of single and double stranded molecules and improved the efficiency of identification of rare transcripts through normalization of differentially expressed genes by suppressing abundant and enriching the rare transcripts (Moody 2001; Ji et al. 2002). The SSH PCR method has been commercialized and developed as PCR Select cDNA Subtraction Kit by Clontech (Clontech, Palo Alto, CA), which has been used widely in the identification of differentially expressed genes in different organisms (Buchaille et al. 2000; Robert et al. 2000; Leypoldt et al. 2001), including plants (Wang et al. 2001; Matvienko et al. 2001; Wang et al. 2002; Takemoto et al. 2003). Subtractive hybridization has been useful for gene expression studies and for the identification of differentially expressed genes on a global scale without any prior sequence information. Compared to other methods, subtractive hybridization produces fewer false positives; however, it requires further studies for verification of differential expression of identified genes by northern blot analysis with gene specific probes or reverse northern blot hybridization using cDNA array analysis. Array Analysis of Gene Expression A microarray is a grid of DNA spots on a chip, small glass slide, or nylon membrane, which is used for hybridization to determine the level of gene expression. There are three general types of microarrays including oligonucleotide chips, oligonucleotide arrays and cDNA arrays. The oligonucleotide chips contain short oligos directly synthesized and fixed on a glass wafer using photoactivated chemistry. The oligonucleotide arrays contain presynthesized oligos spotted on glass slides or to nylon

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29 membranes. The cDNA arrays contain PCR amplified inserts from cDNA or EST clones placed on glass slides or nylon membranes (Ahoroni and Vorst 2001; Moody 2001). Gene expression profiling is the most widely used application for microarrays; this can be used to obtain functional information for genes with unknown function. Statistical techniques such as hierarchical clustering, principal component analysis (PCA) and selforganizing maps (SOM) are used for grouping genes based on their expression profiles from microarray data. Hierarchical clustering of gene expression analysis uses a bottomup approach to join genes with similar expression profiles to form nodes, which are in turn further joined. The joining process continues until all genes are combined in a single hierarchical tree based on their expression profiles. The data obtained from these analyses can give information about cellular regulatory mechanisms, and can group unknown genes with the same putative function (Aharoni and Vorst 2001). The increasing amount of sequence information available makes microarray analysis one of the most important tools for functional genomics to close the gap between sequence information and functional identification of genes in plants. Therefore, using microarray analysis of differentially expressed plant genes in many developmental stages, different conditions, and genotypes can be classified into different groups based on their expression patterns (Kuhn 2001). In plants, microarray technology was first applied to Arabidopsis to study and compare gene expression in leaf tissue and roots using 48 cDNA sequences (Schena et al. 1995). Later, a cDNA microarray containing 1483 Arabidopsis genes was used to find gene expression profiles in different organs and different developmental stages (Ruan et al. 1998). Recently, microarray analysis has been widely used in Arabidopsis and other plants including rice, maize, petunia,

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30 strawberry, and lima beans, (Vicia spp.) to identify genes induced in plant defense, fruit ripening, circadian clock regulation, phytochorome A signaling, seed development, nitrate assimilation, and environmental stress (Aharoni and Vorst 2001). Microarray analysis has recently been used to identify genes involved in cold and drought response in Arabidopsis (Seki et al. 2001; Seki et al. 2002; Fowler and Thomashow 2002), barley (Hazen et al. 2003), and sugarcane (Nogueira et al. 2003). Since availability of sequence information is a limiting factor for microarray analysis, its application can be extended in different plants as more sequences become available. Cold Response in Citrus Citrus is a fruit crop that grows in tropical and subtropical regions of the world. Most commercial citrus types are susceptible to low temperature; however they are able to cold acclimate to some extent. Among the commercial citrus species, there is variation in cold sensitivity; limes, lemons, and pummelos are the most cold-sensitive types followed by grapefruits, oranges, and mandarins. Pummelo is one of the most coldsensitive types and can survive only at -4 or -5°C. On the other hand, mandarins such as 'Satsuma', which is the most cold-tolerant of the commercial citrus, can survive temperatures as low as -10 °C (Yelonosky 1985; Jackson and Fasulo 1994). Since most citrus species are coldsensitive, production of citrus is mostly limited by low temperatures outside tropical and subtropical regions. Significant economical losses from freezing have been reported in subtropical citrus growing regions including Florida in the last century. These freezes have destroyed significant portions of citrus trees and fruits in certain regions and in some cases several times over. Six major impact freezes between 1835 and 1989 has forced relocation of citrus production farther to the south in Florida (Attaway 2000). The extent

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31 of cold damage in citrus during/following a freeze depends on a number of different factors, including the severity of the freeze, tree/orchard location, tree dormancy, tree vigor, scion and rootstock combination, crop load, and soil conditions, as well as, freeze duration. Freezing induces injury symptoms in different parts of the plant. These symptoms include dark water-soaked areas on the leaves, leaf fall, bark spliting, and freeze cankers on tree trunks. In addition, freezes can cause fruit drop, pitting on the surface of the fruits, and extensive internal fruit injury (Jackson 1994). A number of strategies ranging from the simple burning of lighter wood, to tree wraps, to use of relatively advanced microsprinklers have been used for freeze protection in citrus. Although some of these methods provide protection from freezes, their use requires advanced freeze warnings, which require sophisticated freeze forecasts. Even though these strategies provide a certain degree of freeze protection, better protection against freezes requires development of cold tolerant citrus varieties. A need for the development of cold-hardy citrus varieties was recognized after the freezes of 1 894-95 destroyed a significant portion of the citrus in Florida. A breeding program for improving cold tolerance in commercial citrus was initiated using Poncirus trifoliata. This is an interfertile citrus relative that can withstand temperatures down to o 26 C when cold acclimated. Since that time it has been used in breeding programs to develop cold-hardy citrus varieties. Using Poncirus trifoliata as a parent, several citranges including 'Rusk', 'Morton', 'Savage', and 'Troyer' were developed in the cultivar improvement programs and 'Troyer' citrange has been used as a cold tolerant rootstock (Cameron and Frost 1968; Soost and Cameraon 1975; Soost and Roose 1996). More recently, progenies from open pollinated pummelo x Poncirus trifoliata hybrids have

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32 been generated to produce freezing resistant citrus cultivars (Yelonosky et al. 1993). However, production of commercial scion varieties with good fruit quality and cold hardiness have been largely unsuccessful to date because of biological problems associated with citrus breeding including long juvenility periods, polyembryony, heterozygosity, sterility, selfand crossincompatibility, inbreeding depression, and the quantitative inheritance of cold tolerance. Traditional breeding methods have provided limited information about the genetics of cold tolerance and have not been successful for developing cold tolerance in citrus. To overcome the limitations of conventional breeding and improve understanding of important genetic traits in citrus, molecular markers have been integrated into breeding programs. Using molecular markers, a number of genetic linkage maps were developed in citrus and Poncirus hybrid populations (Durham et al. 1992; Jarrell et al. 1992; Cai et al. 1994; Liou et al. 1996; Sahin-Cevik 1999; Sankar and Moore 2000; Weber et al. 2003). The genetics of cold tolerance was studied using quantitative trait loci (QTLs) mapping in a Citrus grandis x Poncirus trifoliata •\ pseudo-testcross population. In this study, a QTL with a major effect and several more QTLs with smaller effects on cold tolerance were identified using QTL mapping (Weber et al. 2003). Identification of these QTLs is important for the understanding of genetics of cold tolerance and can potentially be used for marker-assisted selection; however, application of this information for producing cold-tolerant plants will require a long time. Even though citrus is a cold-sensitive plant, citrus and its relatives can cold acclimate when they are exposed to low non-freezing temperatures. The cold acclimation process increases the freezing tolerance by inducing changes in carbohydrate metabolism,

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33 protein, lipid, and water content of cells in acclimated plants (Yelenosky 1985). An increasing level of carbohydrates and decreasing water content were observed in sweet orange [Citrus sinensis (L.) Osbeck cv. Valencia] grafted on cold-hardy trifoliate orange [Pocirus trifoliata (L.) Raf.)] during cold acclimation and these changes were correlated with an increasing level of cold tolerance (Yelonosky 1992). When the water content and soluble sugar levels in field grown 'Valencia' orange {Citrus sinensis) were analyzed during the coolest weeks of winter, leaf water content and osmotic potential of fieldgrown trees decreased about 20 to 25%. On the other hand, soluble sugar levels were increased by 100% resulting in an increased level of freezing tolerance (Yelonosky and Guy 1 989). Exposure to low temperatures also resulted in modification of protein content in leaves of sweet orange (Citrus sinensis (L.) Osbeck cv. Valencia). A different o polypeptide composition was observed in 'Valencia' leaves that were exposed to 5 C for one week compared to control plants (Guy et al. 1 988). It was shown that freezing tolerance of seedlings of pummelo and trifoliate orange was increased following cold acclimation. Analysis of these seedlings revealed that the polypeptide content of cold acclimated pummelo and trifoliate orange was different compared to those of nonacclimated seedlings. Although many changes were observed in trifoliate seedlings, the differences in pummelo were limited. In addition, a large polypeptide of 160 kDa was detected only in cold-acclimated trifoliate, but not in pummelo or nonacclimated controls (Durham et al. 1991). Recent developments in molecular biology have enabled identification, isolation and characterization of cold-regulated genes and provided insight into the genetics and regulation of cold tolerance in many plants. Since the identification of the first cold

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34 inducible genes in other plants, Poncirus trifoliata has been also explored for identification of cold-regulated genes for improving cold tolerance in citrus. Six cold inducible cDNA sequences were isolated from a cDNA library from the leaves of coldacclimated Poncirus trifoliata. Further characterization of two of these cDNA sequences, COR1 1 and COR19, revealed that these two proteins are similar to cotton D-l 1 and Group 2 LEA proteins. LEA proteins are expressed at high levels during embryo maturation and high stress conditions resulting from loss of intracellular water. Homologs of these genes were isolated from fruits of Citrus unshiu (Hara et al. 1999) and grapefruit (Porat et al. 2002). Application of genomic techniques for studying cold response in Arabidopsis resulted in identification of several hundred cold-regulated genes (Seki et al. 2001; Seki et al. 2002; Fowler and Thomashow 2002). Expression of many cold-regulated genes has also been identified and characterized in other plants. Identification and characterization of these genes has helped in the understanding of mechanisms of cold acclimation and tolerance. These genes have also been used for improving cold tolerance in transgenic plants. Since citrus is cold-sensitive and cold tolerance is an important trait, understanding the cold tolerance in Poncirus, which is a cold-hardy citrus relative, may provide information necessary for improving cold tolerance in citrus. Although citrus and Poncirus have been bred for developing cold tolerance for many years, characterization of genes involved in cold tolerance has lagged behind in Poncirus. To date, only six cold-regulated cDNAs have been identified in Poncirus. Considering the quantitative nature of cold tolerance in citrus and other plants, and the identification of several hundred cold-regulated genes in Arabidopsis, a more comprehensive study is

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35 necessary for identification of more cold-regulated genes in Poncirus. Identification and characterization of more genes involved in cold tolerance may pave the road for developing cold-hardy citrus plants.

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CHAPTER 3 IDENTIFICATION OF COLD REGULATED GENES FROM Poncirus trifoliata (L.) Raf. USING SUBTRACTIVE cDNA LIBRARIES Introduction Low temperature is one of the limiting factors for cultivation of agricultural crops in certain regions. Economically important crops have been bred for temperature tolerance for many years to improve and extend the growing regions. As for many plants, in citrus maximum freezing tolerance is not constitutive, but induced in response to non-freezing temperatures below 10°C, which is called cold acclimation (Thomashow 1998). During cold acclimation, a series of physiological and biochemical changes take place in plants that lead to the induction of specific genes (Guy 1990). Changes in gene expression between cold-acclimated (CA) and nonacclimated (NA) plants have been studied extensively in many plants using differential screening of cDNA libraries (Gilmour et al. 1992; Chauvin et al. 1993; Monroy et al. 1993; Weretilnyk et al. 1993), differential display (Horvath and Olson 1998), and subtractive cDNA libraries. A number of cold-regulated genes have been identified and characterized in many plants, including Arabidopsis (Gilmour et al. 1992; Horvath et al. 1993), Brassica napus (Weretilnyk et al. 1993), Hordeum vulgare (Dunn et al. 1991), Medicago sativa (Monroy et al. 1993), and Spinach oleracea (Neven et al. 1993). In addition, the regulatory sequences containing CRT/DRE elements have been characterized from some of these genes (Baker et al. 1994; Yamaguchi-Shinozaki and Shinozaki 1994). A transcription 36

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37 factor, the CRT/DRE-binding factor 1(CBF1), involved in regulation of the cold response pathway and the expression of these genes by binding to CRT/DRE element has also been isolated in Arabidopsis (Stockinger et al. 1997; Gilmour et al. 1998; Liu et al. 1998), as has its homologs in Arabidopsis and other plants (Jaglo et al. 2001). More recently, microarray analysis has been developed and used for a global examination of expression of thousands of genes simultaneously under different experimental conditions (Moody 2001). Microarray analysis of thousands of genes in CA and NA Arabidosis showed that up to several hundred genes were regulated by low temperature. A majority of these genes (about 75%) was cold-induced and expression of others (about 25%) was repressed by cold (Seki et al. 2001; Seki et al. 2002; Fowler and Thomashow 2002). Fowler and Thomashow (2002) also demonstrated that only a portion of cold-regulated genes are activated by the CBF pathway, indicating that multiple regulatory pathways are involved in the expression of cold-regulated genes. Therefore, the identification and characterization of more genes in different plants will provide better understanding of cold response pathways and improve cold hardiness in plants. Citrus is one of the most economically important fruit crops in the world, grown commercially in almost every country in tropical and subtropical regions. Cultivation of citrus is mainly limited by low temperatures inside of this region. Low temperatures and freezes also result in significant damage and economic losses in subtropical citrus growing regions. Therefore, cold hardiness is a desirable trait for introduction into commercial citrus varieties. Most commercially important varieties of citrus are not coldtolerant and are susceptible to freezes. Yet, there is significant variation among citrus species and relatives for cold tolerance ranging from very cold-sensitive types,

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38 such as Citrus grandis (pummelo) to the cold-hardy interfertile citrus relative Poncirus trifoliata which can withstand temperatures of -26°C when cold-acclimated. Once Poncirus trifoliata was recognized as cold-hardy, in the previous century, it was used in breeding programs mainly in Florida ((Soost and Cameron 1975) and other countries such as Japan and Russia (Gmitter et al. 1992) in efforts to produce cold-tolerant commercial citrus varieties. Although cold-tolerant rootstocks were produced by crossing Poncirus and Citrus, production of scion varieties with good fruit quality and cold hardiness has been unsuccessful to date, mainly because the tree and fruit characteristics of most of the crosses were undesirable as the fruit contained high levels of poncirin which gives a bitter taste. In addition, biological problems associated with citrus breeding including long juvenility periods, polyembryony, heterozygosity, sterility, selfand crossincompatibility, inbreeding depression, and the quantitative inheritance of cold tolerance have also been limiting factors for producing cold-hardy citrus varieties (Soost and Cameron 1975; Soost and Roose 1996). Use of genomics and molecular biology techniques such as gene cloning, gene manipulation, and genetic transformation can overcome problems associated with breeding and provide new approaches for understanding and improving cold tolerance in citrus. This requires availability of genes that can be used in genetic transformation for improving cold tolerance in Citrus. To isolate such genes, cDNA libraries were previously constructed from CA and NA Poncirus trifoliata. Differential screening of these libraries yielded a few cold-induced genes including pBCORcl 15, pBCORcl 19, P BCORc720, P BCORc410, pBCORcl02, and pBCORc510 (Cai et al. 1995). In the present study, more cold-regulated genes were identified by construction and sequencing of subtractive cDNA libraries from cold-

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39 acclimated and nonacclimated Poncirus seedlings. Cold regulation of these genes was demonstrated by expression analysis using reverse northern and northern blot analyses in cold-acclimated and nonacclimated Poncirus. Materials and Methods Plant Materials Seeds were extracted from Poncirus trifoliata cv. Rubideaux fruits grown in the experimental orchard of the Horticultural Sciences Department at the University of Florida, Gainesville, Florida. The seeds were planted in a soilless medium in 2" x 10" 'containers' in racks of twenty and seedlings were grown and maintained in the greenhouse. Cold Acclimation for Library Construction One-year-old seedlings with two-month-old flushes were transferred from the greenhouse to a controlled environment growth chamber for two weeks under a 16-h light/8-h dark photoperiod at 28°C. Control plants were maintained under the same conditions. For cold acclimation, plants were transferred to another growth chamber equipped with low temperature control managed by the GEC134S Precision Temperature Measurement and Control System (Gaffney Engineering, Gainesville, Florida) which measures the chamber temperature with 13 thermocouples at different locations in the chamber. These plants and maintained at 4°C under a 16-h light/8-h dark photoperiod for 2d. Environmental Stress Treatments for Gene Expression Study Two racks containing 40 plants of two-year-old Poncirus and pummelo with twomonth-old flushes were first transferred from the greenhouse to a controlled environment growth chamber and maintained there for two weeks with 16 h light/8 h dark photoperiod

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40 at 28 C. After leaf samples were collected from Poncirus and pummelo plants for nonacclimated and nondehydrated controls, they were subjected to cold acclimation and dehydration treatments. For cold acclimation, 20 plants from each species were transferred to another growth chamber equipped with the same low temperature control system as described above. The plants were maintained at 4°C under 16-h light/8-h dark photoperiod for seven days. Leaf tissue samples were collected from all of these plants following 1 h, 4 h, 8 h, 24 h, 2 d, 4 d, and 7 d of cold acclimation at 4°C. For dehydration, 20 plants from each Poncirus and pummelo were maintained in the controlled environment growth chamber with 16 h light/8 h dark photoperiod at 28C without irrigation for one week. After one week, water continued to be withheld from the plants and the water content of the soil in each pot was measured with a time-domain reflectometry (TDR) probe. Leaf tissue samples were collected and pooled from at least four different plants with the same water content at specific time points of 7 d, 9 d, 1 1 d, 13 d and 15 d following dehydration. All tissue samples were immediately frozen in liquid nitrogen and stored at 80°C until use. Isolation of RNA Poly A+ RNA was isolated from leaf samples collected from at least ten individual nonacclimated and cold-acclimated plants obtained from two independent cold treatment experiments using the Fast Tract 2.0 Kit for isolation of mRNA (Invitrogen, Carlsbad, CA) according to the manufacturers' instructions. Total RNA was isolated from leaf samples collected and pooled from at least ten individual nonacclimated and coldacclimated plants obtained from another independent cold treatment experiment using Trizol (Gibco BRL, Rockville, MD), according to the manufacturers' instructions.

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41 Poncirus trifoliata Non Acclimated Control ^ ^^^^^^ Poly A* RNA Isolation Poncirus trifoliata Cold Acclimated at 4 T for 2 days tttt AAAAAAA I HI! II AAAAAAA cDNA Synthesis by Reverse Transcriptase Double-stranded cDNA Synthesis "AAAAAAA ' ITTTTTTT -TTTTTTTT "AAAAAAA TTTTTTTT Driver Restriction Enzyme Digestion with Rsa I Tester Fill in the ends PCR Amplification using an Adaptor Primer a and d No amplification b ^ b' No amplification c Linear amplification e Exponential amplification Figure 3-1. Construction of cold-regulated cDNA libraries using a subtractive hybridization method (Adopted and modified from Clontech).

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42 Construction of the Subtractive Library Forward and reverse subtracted cDNA libraries were established with 2 ug of Poly A+ RNA from 2 d cold-acclimated and nonacclimated Poncirus seedlings using a PCRSelect cDNA Subtraction Kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions (Figure 3-1). Subtracted cDNAs were amplified and cloned into the pTADV TA cloning vector using the Advantage PCR Cloning Kit (Clontech, Palo Alto, CA). Plasmids were isolated from selected clones using a 96-well plate plasmid purification kit (QIAGEN, Hilden, Germany). Sequence analysis Randomly chosen cDNA clones from forward and reverse subtracted libraries were sequenced using universal primers. Nucleotide sequences and deduced amino acid sequences of these clones were compared against the GenBank database using BLASTN and BLASTX, respectively. Reverse Northern Blot Analysis of Subtracted cDNAs cDNA clones and two negative controls, 1R and 2R sequences from the human genome provided with the PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA), were amplified by PCR using NP1 (5'TCGAGCGGCCGCCCGGGCAGGT-3') and NP2 (5 '-AGCGTGGTCGCGGGCGAGG T-3') primers complementary to regions flanking both sites of the cDNA inserts and Advantage DNA Polymerase Mix. A PCR reaction was performed at 94°C for 30 s, 95°C for 30 s, and 68°C for 3 min for 23 cycles according to manufacturer's instruction (Clontech, Palo Alto, CA). In addition, two previously characterized cold-induced genes, corl 1 (GenBank accession number: L39005) and corl 9 (GenBank accession number: L39004), and a constitutively expressed

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43 atpB gene encoding p subunit of chloroplast ATP synthase from Poncirus trifoliata (GenBank accession number: AJ238409) were amplified using gene specific primers as positive and internal controls, respectively. PCR products of cDNAs and controls were denatured with 0.4 N NaOH and blotted twice onto a Hybond N+ nylon membrane (Amersham Biosciences) using a Bio-Dot Microfiltration Apparatus (Bio-Rad, Richmond, CA) according to the manual. This entire process was repeated to produce two identical copies of each blot. The duplicate blots were pre-hybridized with PerfectHyb buffer (Sigma, St Louis) with 0.1 mg/ml denatured salmon sperm DNA at 65°C for one hour and hybridized with 32 P-labeled single-strand cDNA probes produced by reverse transcription of 250 ng poly A+ RNA isolated from cold and non-acclimated plants with an oligo-dT primer at 65°C for 16 h. Blots were washed twice with 2 X SSC, 1% SDS at 65°C for 20 min, followed by two washes with 0.2 X SSC 0.5% SDS at 65°C for 20 min each. Blots were then exposed to a phosphor screen for 16 h, scanned by the storm phospho-imaging system (Amersham, Uppsala, Sweden), and quantitated using ImageQuant (Amersham, Uppsala, Sweden). Reverse northern blots were repeated three times using cDNA probes prepared from poly A + RNA samples of two independent experiments. Data Analysis Data was collected from two independent reverse northern blot hybridizations containing all cDNA samples and controls in duplicate membranes each containing two spots of the each individual sample. Values of duplicate spots for each individual sample were determined after background subtraction. Since the reverse northern blot hybridizations were done on multiple membranes at different times and the experiment

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44 was repeated three times, values for each sample were normalized using ATP synthase as the constitutive control in order to compare the data sets. Normalization was done by dividing the values of each sample with the value of the constitutive control on the same blot. The ratio of the normalized data for individual genes in cold-acclimated and nonacclimated blots was used to determine fold induction of each clone. Logio values of the normalized data were then used to determine the statistical differences in expression of each clone with cold and non-acclimated probes by a t-test. Clones showing more than a 2-fold change in expression and p < 0.05 were considered to be cold-regulated. Northern Blot Hybridization Total RNA samples from cold-acclimated and nonacclimated Poncirus and pummelo plants were separated on a denaturing agarose gel, transferred to a nylon membrane and prehybridized for 30 min and hybridized with Digoxigenin (DIG)-labeled DNA probes prepared by PCR labeling or DIG-labeled antisense RNA probes prepared by in vitro transcription at 50°C (for DNA probes) or 68°C (for RNA probes) for 16 h according to the DIG Application Manual for Filter Hybridizations (Roche Molecular Biochemicals, Mannheim, Germany). The membrane was washed two times with 2 X SSC and 0.1% SDS at room temperature for 5 min, followed by two washes with 0.1 X SSC and 0.1% SDS at 50°C (for DNA probes) or 68°C (for RNA probes) for 15 min and subjected to DIG-labeled DNA probes or DIG-labeled RNA probes to detect RNA targets on a northern blot using the DIG Chemiluminescent Detection Kit (Roche Molecular Biochemicals, Mannheim, Germany). Membranes were also hybridized with an 18S ribosomal RNA (rRNA) probe for loading and transfer control.

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45 Results To identify cold-regulated genes in Poncirus, reverse and forward subtracted cDNA libraries were prepared using cold-acclimated and nonacclimated Poncirus seedlings. PCR amplification of selected clones demonstrated that the libraries contained fragments ranging from 250 to 1000 bp. A total of 192 randomly picked colonies, 136 from forward (clone number starting with C, cold-induced) and 56 from reverse (clone number starting with N, cold-repressed) subtracted libraries were sequenced. The nucleotide and deduced amino acid sequences were compared using BLASTN and BLASTX, respectively. The sequence analysis revealed that a number of cDNA clones showed homology to previously characterized cold response genes in other plants including Arabidopsis, tobacco, tomato, and potato. Cold response genes identified in this study have homology to different groups of genes, including transcription factors and DNA binding proteins, heat shock proteins, late embryogenesis proteins and some metabolic genes (Table 3-1). To study differential expression in cold-acclimated and nonacclimated plants, these cDNA clones were analyzed by reverse northern blot hybridization. Two previously characterized cold-induced genes from Poncirus, corl 1 and corl9, and two nonhomologous genes from the human genome were included in the blot as positive and negative controls, respectively, to confirm the validity of reverse northern analysis. In addition, a housekeeping gene, choloroplast ATP synthase from Poncirus, whose expression is not affected by cold acclimation, was also included as an internal control to compare and analyze different blots. Reverse northern blot analysis showed that expression of both COR genes was strongly induced by cold acclimation as expected, but their expression was minimal in the non-acclimated control. On the other hand, no

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46 Figure 3-2. Differential screening of forward and reverse subtracted cDNA libraries of cold acclimated Poncirus by reverse northern analysis. PCR amplified cDNAs from forward and reverse subtracted libraries were spotted onto 32 duplicate blots. Blots were hybridized differentially with P label coldacclimated and nonacclimated cDNA probes. The Roman numerals indicate the duplicate blots. Capital letters and numbers are used for rows and columns, respectively to identify the location of individual cDNAs in each blot. The location of the water control, corl 1 cor 19 positive controls, 1R and 2R negative controls, and the ATP synthase constutitive control, are indicated by black, red, blue and green boxes, respectively. Blots I-III contain cDNAs only from the forward subtracted library. The blot V contains cDNAs only from the reverse subtracted library. In blot IV, rows A-C, and E-G contain cDNAs from forward subtracted library, but rows D and H contain cDNAs from the reverse subtracted library.

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47 Cold Acclimated Non Acclimated III IV 1 2 3 4 5 6 ' l 8 9 10 1 1 12 1 2 3 4 5 6 7 8 9 10 11 12 A 1 II* 1 • 1 « 1 1 1 m • • • A B • • • • • B C C D • # • • • • i > • • • • • • • • D E 1 II* 1 •1 * > • • * 1 |L r — I • * • • E F • Ik • • • * > F G • • • • • • 4 • • * • • • • • * U H • • H A 1 II* • 4 • • 1 1 * • • • A B m • B C • • • • • • 4 • • • • • • * • • * • C D • • • • 4 » • • • • • m • • • • • • • D E 1 II* •in 1*1 < I • • • • • 1 II • • • o E F F G G H • • • • • i > • • • • • • • • • • • • 0 i H A 1 II* 1*1 1 • • • I II 1 • • • A B • • • • • • 4 • • • • • • B c • • « • • C D • • • • • • 4 » • • • • • o • • • • D E 1 II* !•! < • • • 1 II 1 • • E F • • • • • • i > • • • • • • • • • 3 F G it G c » • 9 • G H • • • • • • • • • • • 9 m • H A A 1 II* • • • 1 . • • • • B • • • B C D • • • • • • < • * • • • • • • a • • • • c D E 1 II* •II 1*1 i • • • 1 1 1 • • • • F G • • • • • • • G H • • H A 1 1 (—— Jl "ill — .1 •1 4 ) 0 | | • • • • A D D R C c D » • • • > • • • • • • 4» • • D E 1 1 1 1 1 _IH • > * E F • F G G H • • • • • » • • • • • • • o H 1 2 3 4 5 6 7 8 9 10 1 1 12 1 2 3 4 5 6 7 8 9 10 1 1 12

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48 Table 3-1. Summary of sequence and expression analysis identified from cold-acclimated Poncirus cDNA gene expression ratios was determined by a t-test at of cold-regulated genes libraries. Significance of p < 0.05. Clone* number Location on the blot" GenBank Match' E value d Fold induction" t-test p-value r Transcription C6 I-B6, F6 CCHH finger protein 4 Arabidopsis thaliana 0.36 22 0.001 C12 I-B12. F12 Putative bZIP Transcription Factor Arabidopsis thaliana 5e-18 24 0.001 C17 I-C5, G5 AP2 domain protein homolog / potato 8e-33 3 0.05 C32 I-D8, H8 Gene for glycine-rich RNA-binding protein Citrus unshiu (2) e-l 12 10 0.01 C34 I-D10, H10 Putative DNA binding protein (RAV2-like) Arabidopsis thaliana 6e-21 11 0.02 C82 III-B6, F6 DNA-binding protein WRK.Y 1 -parsley 4e-21 5 0.00004 Cell rescue, defense, cell death and aging C5 I-B5, F5 Wound\induced protein (W112) mRNA Mesembryanthemum crystallinum le-11 4 0.02 C7 I-B7, F7 CORcl 1 5 cold stress protein mRNA Poncirus trifoliata (5) 4e-89 49 0.0003 C22 I-C10.G10 Hsc70 mRNA for heat shock protein 70 cognate Salix gilgiana (3) 5e-94 19 0.008 C33 I-D9, H9 Late Embryogenesis protein homolog / tomato (7) 3e-ll 19 0 006 C36 [-D12. H12 DHN 1 , cold regulated LTCOR 1 8 Populus euremericana 5e-08 10 0.0 1 C42 II-B6. F6 CORcl 19 cold stress protein mRNA Poncirus trifoliata le-38 15 0.001 C44 II-B8. F8 Elicitor inducible protein Nicotiana tabacum 9e-17 7 0.01 C58 II-C6, G6 Putative leucine rich protein Oryza sativa 7e-23 4 0.05 C63 II-C1 1, Gl 1 Late embryogenesis abundant protein 5 mRNA Nicotiana tabacum (2) 0.003 8 0.01 C64 II-C12, G12 bdnl mRNA Boea crassifolia (2) 2e-10 16 0.001 C72 II-D8. H8 Seed maturation protein PM39 Glycine max 3e-ll 5 0.03 C119 I11-A7, E7 High molecular weight heat shock protein (Hsp2) Malus domestica 5e-87 8 CI 20 III-A8, E8 Avr-9/Cf-9 rapidly elicited protein 65 Nicotiana tabacum 7e-05 3 0.02 Cellular communication and signal transduction C9 I-B9, F9 Nine-cis-epoxycaratenoid dioxygenase / tomato 6e-19 22 0.0005 C21 I-C9, G9 ACC oxidase Citrus sinensis e-l 29 26 0.0003 C40 II-B4, F4 Arginine decarboxylase Vitis vinifera (2) 7e-08 9 0.001 C78 III-B2, F2 Putative RING zinc finger protein Arabidopsis thaliana 5e-28 9 0.0001 C94 III-C6, G6 Putative ripen ing|related protein Vitis vinifera 8e-37 2 0.04 C96 III-C8, G8 Phosphoribulokinase Pisum sativum 3e-93 4 0.01 CI 17 II-A11, Ell Putative protein kinase Oryza sativa 0.48 7 0.01 Metabolism C3 I-B3, F3 NADP-isocitrate dehydrogenase mRNA Citrus limon e-l 10 10 0.003 C27 I-D3, H3 Nitrate reductase (2) 5e-21 17 0.0002 C65 II-D1, HI relA/spoT|like protein RSH1 Nicotiana tabacum 5e-62 7 0.02 C71 II-D7, H7 Isoflavone reductase related protein Pyrus communis 2e-82 4 0.03 C79 III-B3, F3 Putative protein translation factor le-21 9 0.0003 C87 III-B1 1, Fl 1 Hydroxymethylglutaryl coenzyme A synthase Hevea brasiliensis 9e-49 3 0.01 C100 III-C12,G12 Rieske FeS precursor protein mRNA for Spinach le-63 5 0.0007 C107 III-D7, H7 mRNA for 1 |deoxyxylulose 5|phosphate synthase Catharanthus roseus 2e-14 6 0.03 C113 I1-A7. E7 Glutathione S|transferase Euphorbia esula 2e-18 4 0.04 C122 III-A10, E10 Aminomethyltransferase 4e-66 3 0.0002 C133 IV-B9, F9 Thiamin biosynthesis protein Citrus sinensis 4e-95 5 0.01 Transport facilitation C8 I-B8, F8 ABC1 Protein Nicotiana plumbaginifolia 4e-09 19 0.01 C88 IH-B12, F12 Hexose carier (Hex9) mRNA Ricinus communis 6e-57 4 0.0001 C108 III-D8, H8 Putative ABC transporter Arabidopsis thaliana 3e-10 9 0.01 Energy C61 II-C9, G9 Phosphoenolpyruvate carboxykinase Flaveria pringlei 3e-24 4 0.03 C121 UI-A9, E9 fructose|bisphosphate aldolase | like protein Arabidopsis thaliana 7e-40 9 0.008 Cellular biogenesis C60 II-C8, G8 Peroxisomal membrane protein Arabidopsis thaliana 9e-16 13 0.0009 C75 II-D1 1, HI 1 Similar to glucose|6|phosphate/phosphate|translocator Arabidopsis thaliana 3e-21 5 0.02

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49 Table 3-1. Continued. Clone* number Location on the blot" GeneBank Match' E value d Fold induction' t-test p-value r Protein fate C74 II-D10, H10 ATP-dependent Clp protease (C04B) Lycopersicon esculentum 8e-12 5 0.01 C90 III-C2, G2 Ubiquitin-conjugating enzyme 3e-06 13 0.000003 CI I-B1.F1 Alpha-crystallin-related protein mRNA Trichohyton mentagrophytes (5) 9e-06 9 0.01 Unknown role C19 I-C7, G7 Putative MtN3-like protein mRNA Dianthus caryophyllns (5) 5e-04 18 0.0006 C43 II-B7, F7 Metallopanstimulin mRNA Strongyloides ratti (4) le-08 9 0.01 Proteins with unknown function C4 I-B4, F4 Unknown protein 7 0.02 CIO I-B10, F10 Unknown protein 7 0.07 C16 I-C4, G4 Unknown protein 12 0.01 C28 I-D4, H4 Unknown protein 7 0.01 C35 1-D11,H11 Putative protein 5 0.008 C39 II-B3, F3 Unknown Protein 14 0.001 C49 I-A7, E7 Unknown protein 9 0.01 C53 II-C1.G1 Unknown protein 7 0.01 C62 II-C10.G10 Unknown protein 7 0.007 C66 II-D2, H2 Hypothetical protein 9 0.009 C76 II-D12, H12 Unknown protein 7 0.02 C77 II1-B1,F1 Unknown protein 6 0.0003 C83 III-B7, F7 Unknown Protein 7 0,002 C95 1II-C7. F7 Unknown Protein 6 0.01 C99 III-C1 1, Fl 1 Unknown Protein 2 0.03 C101 III-DJ, HI Unknown Protein 10 0.001 C109 III-D9, H9 Unknown Protein 5 0.002 C123 Ill-All, Ell Unknown Protein 2 0.003 C129 IV-B5, F5 Unknown Protein 2 0.03 C130 IV-B6. F6 Unknown Protein 4 0.008 N8 IV-A8, E8 Unknown Protein 3 0.01 * C indicates cold induced and N indicates cold repressed cDNA. b The location of the individuals cold regulated clones on the reverse northern blot shown in Figure 3-2. c The GenBank sequences showing the highest nucleotide and/or amino acid sequence homology with the individual cDNA clones. The numbers in the parentheses indicate the number of clone showed homology with the specific sequence in the databases. d The Expect (E) value is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. e Fold induction indicates the ratio of the normalized values of individual genes obtained from two independent reverse northern blots with coldand non-acclimated cDNA probes. The statistical differences in expression of each clone was determined by comparison log 10 values of the normalized data from cold and non-acclimated probes by t-test. The clones showing at least 2-fold change in expression and p < 0.05 for t-test are shown.

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50 expression of the human genes, 1R and 2R, was detected. Although no difference was observed in the level of expression of ATP synthase, visible differences were observed in the expression levels of most cDNAs from the forward subtracted library in the coldacclimated and nonacclimated blots (Figure 3-2). Statistical analysis of the reverse northern blots demonstrated that 97 of 1 92 clones showed differential expression in cold-acclimated and nonacclimated conditions.Differential expression of cDNAs was detected by fold changes in their expression in two independent experiments and confirmed with t-tests (p < 0.05). Among the 97 cold-regulated cDNA clones, 96 were induced and only one cDNA was repressed by cold acclimation. Of the 96 cold-induced cDNAs, 76 showed homology with genes with known function(s) in plants or other organisms; however, 20 cDNAs showed homology with genes of unknown function or did not show homology with any previously sequenced genes in GenBank. The expression level of the cold-induced genes ranged from two to 49-fold, indicating that different cDNAs were induced at different levels. Two of the genes identified in this study were identical to previously identified cold response genes in Poncirus, corl 1 and cor 19, indicating that our subtractive library and reverse northern analysis functioned properly to identify coldinducible genes. Newly identified cold-inducible genes in Poncirus showed homology to cold-regulated genes encoding functional proteins in Arabidopsis and other plants including late embryogenesis-abundunt proteins (LEA), heat shock proteins, dehydrins, and those involved in signal transduction, cell defense, metabolism, transport facilitation, and cellular biogenesis. In addition, cDNAs showing homology with previously identified transcription factors involved in environmental stress response in Arabidopsis such as

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51 bZIP, zinc finger, RAV2-like, WRKY1, and AP2 domain containing proteins were identified in Poncirus. The complete list of cold-induced genes and their possible functions are shown in Table 3-1 . The expression level of the cold-repressed gene was reduced 3 fold during cold acclimation. This gene did not show homology with a protein of known function (Table 3-1). To confirm differential expression of cold-regulated genes identified by reverse northern analysis, 17 cold induced cDNAs including C6, C7, C8, C9, CI 2, CI 5, CI 7, C21, C22, C28, C33, C34, C40, C42, C60, C64, and C78 were selected for northern blot analysis. Total RNA from two-day cold-acclimated and nonacclimated plants was analyzed for expression of these genes and ATP synthase using gene specific probes. Northern blot analysis demonstrated that all cold-induced genes were expressed differently in cold-acclimated and nonacclimated plants. The difference in the expression of cold-inducible genes was not due to differences in amount of RNA or experimental variation since the 1 8S RNA was similar and no significant change in expression of ATP synthase was observed in both cold-acclimated and nonacclimated plants. In northern blot analysis C7, which is homologous to the previously characterized cor 19 gene in Poncirus, showed the highest level of expression. This result was consistent with reverse northern analysis where its expression increased 49 fold in coldacclimated plants. A number of other cDNAs including C12, C06, C8, C33, C78, C15, C64, C34, C42, C22, C4, and C40 showed little or no expression in nonacclimated plants; however, their expression levels were highly induced in 2 d cold-acclimated plants (Figure 3-3). High levels of expression of these genes in northern blots were correlated with their fold induction in reverse northern blot analysis. On the other hand, expression

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52 Figure 3-3. Northern blot analysis of selected cDNAs from the cold-induced library. Total RNA from 2 d cold-acclimated (CA) and nonacclimated (NA) Poncirus seedlings were separated on a denaturing agarose gel and blotted onto a nylon membrane. Blots were hybridized with DIG-labeled DNA probes specific to individual genes followed by DIGlabeled 1 8S rRNA probe for loading and transfer control. The cDNAs show homology with gene encoding following proteins. C34 RAV-like protein, C78 RING zinc finger protein, C 12 bZIP protein, C42 Sodium antiporter, CI 5 Nitrate Reductase, C22 Heat shock protein 70, C06 CCHH finger protein, C17 AP2 domain protein, C07 Cor 19 homologue dehydrin, C04 Unknown protein, C60 Peroxisomal membrane protein, C21 ACC oxidase, C3 3 LEA homolog protein, C08 ABC 1 transporter protein, AS ATP synthase, C64 bdnl homologue dehydrin, C40 Arginine Decarboxylase, C28 Unknown protein. \

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53 NA CA C34 • • 18S rRNA C78 18S rRNA < < £ u 1 8S rRNA 1 8S rRNA ^1 i C6 18S rRNA C17 1 8S rRNA C7 18S rRNA C4 1 8S rRNA C60 18S rRNA C21 18S rRNA C33 18S rRNA C8 1 8S rRNA AS 18S rRNA C64 1 8S rRNA C40 18S rRNA C28 1 8S rRNA

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54 of some cDNAs including, C60, C21, and C28 were detected in nonacclimated and coldacclimated plants by northern blot analysis, but expression of these cDNAs was slightly higher under cold acclimation. Although differences in expression can be detected under different conditions, levels of expression were not correlated with their fold induction in reverse northern blot analysis. Only one cDNA, CI 7, which was induced only three-fold on reverse northern blots, showed little expression in nonacclimated plants, but very high expression in cold-acclimated plants on northern blots. Among the cold-induced genes, CI 2, which is homologous to a bZIP transcription factor from Arabidopsis, was selected for further study. This cDNA was induced 24 fold in reverse northern blot analysis and its expression was confirmed by northern blot analysis in 2 d cold-acclimated and nonacclimated Poncirus plants. C12 expression at different time points following cold and drought treatments was studied in cold-hardy Poncirus and cold-sensitive pummelo. Northern blot analysis revealed that in cold-hardy Poncirus, expression of this gene was induced at 4 h, reached peak level at 2 d, and remained at this high level at 7 d of cold acclimation (Figure 3-4). On the other hand, only slight induction of expression was observed in cold-sensitive pummelo in response to cold starting at 8 h of cold acclimation. Based on northern blot analysis, the level of expression and induction of C 12 by cold was insignificant in pummelo compared to expression in Poncirus. When expression of C12 was studied in dehydrated Poncirus and pummelo seedlings, no significant changes in expression of CI 2 were observed, indicating that expression of C12 is only induced in response to cold, but not dehydration.

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55 A Cold Acclimation Poncirus trifoliata i_ >, >> >^ i_ i_ i_ SZ CO (0 (0 -C -C -C ^. T3 T3 T5 O tTt CO CNCM^J-hDrought D T) r N 0) t"O "D co m — C12 18S rRNA B Citrus grandis Cold Acclimation Drought CO !_>,>»>. .C CO CO CO ^ -o -o -o CM CM S > >> nj co co "o T3 ^ CO CO "O "D CO IT) fate. ^->i ! C12 18S rRNA Figure 3-4. Northern blot analysis of C12 expression in response to environmental stresses. (A) Expression of CI 2 in response to cold acclimation and drought in Poncirus detected by antisense DIG-labeled riboprobe. (B) Expression of C12 cDNA in response to cold acclimation and dehydration (drought) in pummelo detected by antisense DIG-labeled riboprobe. The type of environmental stress treatment and the duration of the treatment are indicated above each blot. The expression of 18 S rRNA was used as a loading and transfer control and is shown below the expression of the specific gene.

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56 Discussion It has been shown that cold hardiness is a quantitative trait involving hundreds of genes. Recent developments in genomic techniques and their application for studying cold response in Arabidopsis has confirmed the quantitative nature of cold hardiness by identifying several hundred coldregulated genes (Seki et al. 2001; Seki et al. 2002; Fowler and Thomashow 2002). These studies not only identified genes, but also provided insight into the regulation of these genes in response to low temperature. Although not to the same extent as in Arabidopsis, the expression of many cold-regulated genes has also been studied and characterized in other plants. Only one previous study has been done on cold-regulated genes in Poncirus, which resulted in the identification of seven cDNAs and the characterization of two genes coding for group II LEA proteins (Cai et al. 1995). To identify more cold-regulated genes in Poncirus, our study used the subtractive hybridization method for construction of cold-regulated cDNA libraries. Expression of 192 cDNAs was analyzed in cold-acclimated and nonacclimated Poncirus using reverse northern blot analysis. In these experiments, several control strategies were used to ensure the validity of results. Plant materials from which RNA was isolated were pooled from at least ten similarly treated individual plants to eliminate biological variation. Thus, the changes in gene expression reflect the common response of all plants rather than the response of a single plant. Two previously identified coldinducible genes from Poncirus were used as positive controls, and two human genes with no homology to plant genes along with a water negative control were included in expression studies by reverse northern analysis to confirm the specificity of hybridization. Expression studies were repeated at least twice with two different RNA

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57 preparations from two independent cold acclimation experiments. Each blot contained two replicates of each cDNA sample at different locations in the blot. To eliminate experimental differences in different blots, and to compare results from different experiments, expression data was normalized with an internal control, ATP synthase from Poncirus. In addition, expression of a number of selected genes was studied by northern blot analysis of RNA from another independent cold acclimation experiment. Sequencing and expression analysis of a small fraction (192 clones) of the forward and reverse subtracted libraries resulted in identification of a total of 97 cold regulated cDNAs in Poncirus. These genes included two previously characterized cold response genes, corl 1 and cor 19 in Poncirus (Cai et al. 1995) and showed homology with previously identified environmental stress response genes, especially cold response genes in Arabidopsis (Seki et al. 2001 ; Seki et al. 2002; Fowler and Thomashow 2002) and other plants including tomato, potato, rice, maize, wheat, and barley. Identification of genes homologous to previously characterized genes suggests that the subtractive hybridization and expression analysis functioned properly. Since 97 cold-regulated genes were identified from the screening of only 192 cDNAs, sequencing and expression analysis of more cDNAs from the forward and reverse subtracted libraries could result in identification of many more cold-regulated genes. This study demonstrated that construction of a subtractive library is a useful method for identification of differentially expressed genes and that this method could be used for identification and expression analysis of novel genes under different conditions in Poncirus, Citrus and other plants which have limited sequence information.

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58 Cold-induced genes identified in this study show homology with genes involved in a variety of cellular functions ranging from transcription to transport facilitation. The majority of genes reported here and their functions are similar to cold-induced genes identified in Arabidops is by microarray analysis (Seki et al. 2001; Seki et al. 2002; Fowler and Thomashow 2002). As in Arabidopsis and other plants, cold-induced genes in Poncirus can be divided into two groups, genes encoding regulatory and functional proteins. Regulatory proteins are involved in cellular communication, signal transduction, and regulation of gene expression. A number of genes showing homology to transcription factors such as bZIP, AP2 domain, RAV2-like, WRKY1 DNA-binding and zinc finger proteins regulating expression of cold-regulated genes as well as glycine rich RNA binding protein were identified. Although the number of genes encoding regulatory proteins is limited in Poncirus compared to Arabidopsis, the presence of these coldresponsive transcription factors in Arabidopsis and Poncirus indicates that similar regulatory pathways are activated during cold acclimation. Since we identified genes induced at 2 d of cold acclimation and only a limited number of cDNAs was analyzed, gene expression studies at different time points and sequencing and expression analysis of more cDNAs from the cold-induced library may result in the identification of more regulatory proteins. Genes encoding functional proteins such as late embryogenesis abundant proteins (LEA), heat shock proteins, sugar metabolism and oxidative stress related proteins identified in this study may be involved in cold tolerance. LEA proteins stabilize membranes and proteins through detergent-like or chaperone activities; thus, they protect the integrity of cell (Close 1996 1997; Wisniewski et al. 1999). We identified several

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59 cDNAs in Poncirus showing homology to LEA proteins in different plants indicating that Poncirus is expressing a similar group of genes during cold acclimation to adapt to the changes imposed by low temperature. Heat shock proteins (HSPs) act as chaperones to stabilize the proteins by refolding the denatured proteins and preventing protein aggregation (Sung et al. 2001). In response to stress conditions, HSPs are needed more and thus, their expression increases in stressed plants. Two different HSPs were identified in response to cold stress in Poncirus which were also cold-induced in other plants. Sugars are involved in osmoregulation by increasing intracellular osmotic potential and act as cryoprotectants by protecting cell membranes and proteins (Guy 1990; Crowe et al. 1990, Crow et al. 1993). Expression of sugar transporters, such as hexose carrier and glucose-6-phosphate/phosphate translocator and enzymes involved in sugar metabolism, such as fructose bisphosphate aldolase and phoshoenolpyruvate carboxykinase were increased in cold-treated Poncirus. Cold stress also increases active oxygen species (AOS) which causes oxidative stress in the cell. Glutathione Stransferase (GST) is a detoxification enzyme and may have a role in protecting cells from oxidative stress. Expression of GST was reported to be induced in response to cold treatment to alleviate the effect of AOS in the cell (Seppanen et al. 2000). A cDNA homologous to GST showed increased expression in cold-acclimated Poncirus, suggesting that detoxification of AOS is being used by Poncirus to cope with oxidative stress-induced by cold treatment. Changes in protein expression, stability, and turnover were observed in response to cold treatment in plants. ATP dependent Clp protease and ubiquitin conjugating enzyme are involved in removal of damaged or inactivated proteins as well as turnover of specific

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60 proteins to control the metabolic and developmental processes (Rechsteiner 1987; Vierstra 1993). These two proteins were induced during cold acclimation of Poncirus and Arabidopsis (Seki et al. 2002; Zheng et al. 2002). There is a relationship between cold response and ABA level where ABA levels increased in response to low temperature in plants (Luo et al. 1992; Lang et al. 1994), and exogeneous application of ABA increased the freezing tolerance of many plants (Orr et al. 1986; Ishikama et al. 1990). Therefore, increased expression of genes involving ABA biosynthesis and ABA signaling pathway were reported. Nine-cis-epoxycaretenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis which catalyzes the conversion of 9-cis-epoxycaretenoids to xanthoxin (Iuchi et al. 2001), was induced by cold in Poncirus. In addition, a bZIP transcription factor was identified as a cold-responsive gene in this study. Since the bZIP transcription factor induces gene expression through cw-elements that include the ABA response element (ABRE) (Jakoby et al. 2002), increased expression of these genes indicates that an ABA-dependent pathway is activated during cold acclimation in Poncirus. Ethylene is also increased when plants are exposed to cold. In this study, a cDNA showing homology to 1-aminocyclopropene-l-carboxylate (ACC) oxidase, which is responsible for converting ACC to ethylene, was increased in cold-acclimated Poncirus. In addition, increased expression of a ripening-related protein was observed in Poncirus indicating that cold response and ethylene response pathways may interact. Polyamines (PAs) are commonly found in plants and their levels are increased in response to stress conditions, such as low temperature. Arginine decarboxylase (ADC) is the key enzyme involved in the synthesis of PAs and expression of the gene encoding ADC increases in response to environmental stresses including cold (Mo and Pua 2002).

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61 We found that expression of ADC was increased during cold treatment in Poncirus and a similar result was reported in Arabidopsis (Seki et al. 2002). Northern blot analysis of expression of a number of selected genes identified by reverse northern blot hybridization confirmed that these genes were all induced in response to 2 d cold acclimation in Poncirus. This shows that the reverse northern blot analysis and statistical analysis performed in this study were effective methods for identification of cold-regulated genes. An expression study of one cDNA, CI 2, possibly encoding a bZIP transcription factor, in cold-hardy and cold-sensitive species showed that the expression of this gene is gradually increased and reached peak level at 2 d in response to cold. However, no change in expression was observed in response to drought. This result not only revealed the expression pattern of potentially important regulatory gene, but also suggested that genes selected by reverse northern analysis in this study are likely to be cold-regulated. Identification of a number of cold-regulated genes in this study indicated that although only a fraction of the forward and reverse subtracted libraries were sequenced and analyzed, we identified a number of genes significantly up-regulated during cold acclimation. However, only one down-regulated gene was identified. This may be due to the lack of down-regulated genes in response to low temperature in Poncirus or to screening of a limited number of clones. Since we analyzed expression of only 56 cDNAs from reverse subtracted library, compare to 136 cold-induced, the number of cDNAs was limited. Microarray studies with 7000 and 8000 cDNAs showed that a majority (75%) of cold-regulated genes was induced in response to cold in Arabodopsis. However, only 25% of them were repressed in response to cold indicating that the

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62 number of genes induced by cold is much higher than the repressed ones. Therefore, it is likely that analysis of limited number of clones and down regulation of fewer genes in response to cold in plants contributed to identification of a single down-regulated gene in Poncirus.

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CHAPTER 4 ISOLATION AND CHARACTERIZATION OF COLD INDUCED PUTATIVE TRANSCRIPTION FACTORS IN Poncirus trifoliata (L.) Raf. Introduction During cold acclimation, a series of physiological and biochemical changes take place in plants which results from induction of specific gene expression (Guy 1990). Expression analyses of cold responsive genes have revealed that several hundreds of genes are induced during cold acclimation in Arabidopsis (Seki et al. 2001, 2002; Flower and Thomashow 2002). These studies and others have shown that cold-induced genes are involved in a variety of different cellular functions, including transcription, metabolism, protein fate, transport facilitation, biogenesis, cellular communications and signal transduction, cell rescue and defense, cell death and aging (Seki et al. 2001 and 2002; Fowler and Tomashow 2002; Hazen et al. 2003). Further characterization of cold-induced genes demonstrated that some of these genes encode functional proteins involved in increased biosynthesis of compatible solutes (Sakamoto and Murata 2002), alterations of lipid composition and membrane structure (Nishida and Murata 1996), increased levels of antioxidant activity (Iba 2002), as well as biosynthesis of stress hormones, all of which lead to an increase in cold tolerance. Additionally, some cold-induced genes encode regulatory proteins involved in signal transduction and regulation of gene expression during cold acclimation. Since physiological and biochemical changes during cold acclimation are associated with changes in gene expression, transcriptional regulation of cold responsive 63

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64 genes has been explored for a better understanding of cold response pathways and improving cold tolerance in plants. Expression of a number of transcription factors or DNA-binding proteins increase during cold treatment of Arabidopsis and other plants. Most of these transcription factors contain conserved DNA-binding domains found in eukaryotes, such as a basic-leucine zipper (bZIP), basic-helix-loop-helix (bHLH), RING zinc finger, zinc finger, MYB, homo-box, MADS box, as well as AP2/ERF, AP2/B3, and WPJCY DNA-binding motifs found in plants (Kagaya et al. 1999; Chen et al. 2002; Seki et al. 2001, 2002; Flower and Thomashow 2002; Sakuma et al. 2002). One of the most important research developments in cold acclimation and cold response in plants was identification of the CBF/DREB1 transcription factors that regulate an ABA-independent cold response pathway in Arabidopsis (Stockinger et al. 1997; Gilmour et al. 1998; Liu et al. 1998). The CBF/DREB1 proteins induce expression of coldand dehydration-regulated genes containing a CRT/DRE (C-repeat/dehydration responsive element) CCGAC core sequence in their promoter region (Baker et al. 1994; Yamaguchi-Shinozaki and Shinozaki 1994). CBF/DREB-like proteins were also identified and characterized in Brassica napus, a close relative of Arabidopsis (Jaglo et al. 2001; Gao et al. 2002) and cold-acclimated wheat, rye (Jaglo et al. 2001), and barley (Choi et al. 2002) as well as tomato, which does not cold acclimate, (Jaglo et al. 2001) indicating that the CBF/DREB cold response pathway is conserved among a number of plants including cold-sensitive and nonacclimating plants (Jaglo et al. 2001; Choi et al. 2002). Transcription factors containing an AP2/ERF domain belong to a plant specific transcription factor family with more than a hundred members. Based on the number of

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65 AP2/ERF domains present in the protein, these transcription factors are divided into two subfamilies (Riechmann and Meyerorowitz 1998; Riechmann et al. 2000). The first subfamily contains transcription factors with two AP2/ERF domains, including APETALA2 (AP2) (Jofuku et al. 1994) and AINTEGUMENTA (ANT) (Elliott et al. 1996) from Arabidopsis and Glossy 15 (Moose, et al. 1994) from maize, all of which are involved in flower and seed development. The transcription factors containing only one AP2 domain include ERF, TINY, DREB1/CBF, DREB2, Ptis, ABM, and many more from Arabidopsis and other plants (Sakuma et al. 2002). These transcription factors show sequence similarity only in the AP2/ERF DNA-binding domain and they function mostly in response to biotic and abiotic stresses including cold. The other subfamily of AP2/ERF transcription factors contains one AP2/ERF and one B3 DNA-binding domain have also been identified. Two of these proteins, RAVI and RAV2, were isolated from Arabidopsis. The B3 domain in these proteins shows homology to the DNA-binding domain in VIVIPARAOUS1 (VP1) from maize (Suzuki et al. 1997) and its ortholog ABI3 in Arabidopsis (Giraudat et al. 1992). The AP2 and B3 domains of RAVI bind independently to CAACA and CACCTG m-elements respectively, and the presence of both domains increases the binding activity and specificity of the protein (Kagaya et al. 1999). Recently, it was shown that the RAVI DNA-binding protein was induced in response to cold, suggesting that it is involved in cold induced transcriptional activation in Arabidopsis (Fowler and Thomashow 2002). RING (Really Interesting New Gene) zinc finger domain containing proteins are another group of regulatory proteins. The RING finger domain is involved in zincbinding and it has a cysteine rich sequence defined by the consensus sequence of

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66 CX 2 CX ( 9-39)CX ( i-3)HX ( 2-3)CX2CX ( 4^8)CX2C where X can be any amino acid. The RING finger proteins can be divided into two groups, RING-HC and RING-H2, based on the presence of Cys or His at the fifth coordination site, respectively (Freemont 2000; Joazeiro and Weissman 2000). This domain is found in diverse proteins from a variety of species ranging from viruses to eukaryotes, including animals, yeast, and plants and is involved in a variety of cellular functions, including oncogenesis, viral gene expression, signal transduction, peroxisome biogenesis, DNA repair and recombination, and membrane vesicle sorting (Borden and Freeman 1996; Saurin et al. 1996). The function of RING zinc finger proteins was not clear until recently. It has now been demonstrated that they are involved in specific ubiquitination of proteins through a ubiquitin-dependent proteolysis pathway (Freemont, 2000; Joazeiro and Weissman 2000). Since this pathway regulates the specific degradation of a large number of proteins involved in diverse cellular processes including cell cycle regulation, signal transduction, metabolic regulation, cell differentiation, and stress responses (Hershko and Ciechanover 1998), RING zinc finger proteins may have important roles in these processes. A number of RING finger proteins were identified in the Arabidopsis genome, however, no function has been assigned for the majority of these genes (Jensen et al. 1998). Only a few plant RING zinc finger proteins have been thoroughly characterized, including COP1 and HOS1 which are negative regulators of photomorphogenesis in the dark (von Arnim and Deng 1993; Torii et al. 1999) and cold-responsive gene expression (Lee et al. 2001) in Arabidopsis, respectively. Several other plant RING zinc finger proteins involved in seed development (Zou and Taylor 1993; Molnar et al 2002; Lechner

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67 et al. 2002) and pathogen defense (Takai et al 2002; Guo et al. 2003) have been isolated recently and partially characterized. Since some transcription factors and other regulatory proteins are cold-induced and play important roles in cold-responsive gene expression, we selected two cDNAs showing homology with AP2/ERF family proteins and one with a RING zinc finger protein homology from a cold-induced subtracted cDNA library of 2 d cold-acclimated Poncirus trifoliata. For further characterization, the full-length cDNAs were isolated by random amplification of cDNA ends (RACE) and expression of these genes was studied in response to cold and drought stress in Poncirus and Citrus. Materials and Methods Plant Materials Poncirus trifoliata cv. Rubideaux and Citrus grandis cv. DPI 6-4 (Pummelo) seeds were extracted from trifoliate orange fruits produced in the experimental orchard of the Horticultural Sciences Department at the University of Florida, Gainesville, Florida and pummelo fruits produced in the experimental orchard of University of Florida Citrus Research and Education Center in Lake Alfred, Florida, respectively. The seeds were planted in a soilless medium in 2" x 10" 'containers' in racks of twenty and seedlings were grown and maintained in the greenhouse. Environmental Stress Treatments Two racks containing 40 plants of two-year old Poncirus and pummelo with twomonth old flushes were first transferred from the greenhouse to a controlled environment growth chamber and maintained there for two weeks with 16 h light/8 h dark photoperiod o at 28 C. After leaf samples were collected from Poncirus and pummelo plants for nonacclimated and non-dehydrated controls, they were subjected to cold acclimation and

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68 dehydration treatments. For cold acclimation, 20 plants from each species were transferred to another growth chamber equipped with low temperature control managed by the GEC134S Precision Temperature Measurement and Control System (Gaffhey Engineering, Gainesville, Florida). The plants were maintained at 4 C under 1 6-h light/8h dark photoperiod for seven days. Leaf tissue samples were collected from all of these plants at 1 h, 4 h, 8 h, 24 h, 2 d, 4 d, and 7 d of cold acclimation at 4°C. For dehydration, 20 plants from each Poncirus and pummelo were maintained in the controlled o environment growth chamber with 16 h light/8 h dark photoperiod at 28 C without irrigation for one week. After one week, water continued to be withheld from the plants and the water content of the soil in each pot was measured with a time-domain reflectometry (TDR) probe. Leaf tissue samples were collected from at least four different plants with the same water content at specific time points of 7 d, 9 d, 1 1 d, 13 d and 15 d of dehydration. All tissue samples were immediately frozen in liquid nitrogen and stored at 80 C until use. Isolation of RNA Total RNA was isolated from the various leaf samples using Trizol Reagents (Gibco BRL, Rockville, MD) according to the manufacturer's instructions. Total RNA concentration was determined spectrophotometrically and the samples were stored at 80°C until used. Rapid Amplification of cDNA Ends (RACE) The partial sequences of the selected cold-induced cDNAs were used for designing reverse and forward primers to obtain the 5' and 3' ends of the full-length cDNAs, respectively. Total RNA from 2 d cold-acclimated Poncirus seedlings was used as

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69 template for generating the 3' and 5' RACE ready cDNAs for PCR amplification. The RACE ready cDNAs were used with gene specific reverse and forward primers to obtain 5' and 3' end sequences of the specific genes. All steps of RACE were performed using the Smart RACE cDNA Amplification Kit according to the manufacturer's instructions (Clontech, Palo Alto, CA). Sequence Analysis The partial sequences of cold-induced cDNAs obtained from the subtractive library and the 3' and 5' sequences obtained from RACE were assembled and aligned to determine full-length sequences. The assembled sequences were analyzed for open reading frames using Vector NTI suite (InforMax, Frederick, MD). The full-length cDNA sequences and the deduced amino acid sequences were then compared with previously characterized DNA and protein sequences in GenBank. The conserved domains within the protein sequences of cold-induced cDNAs were detected by a conserved domain search and compared with proteins containing the same or similar domain(s). Northern Blot Hybridizations Total RNA samples from environmental stress-treated and control Poncirus and pummelo plants were separated on denaturing agarose gels and transferred to nylon membranes. The membranes were prehybridized at 68 C for 30 min and hybridized with DIG-labeled antisense RNA probe specific to the 3' half of the individual genes prepared using a DIG RNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) at 68 C for 16 h. The membranes were then washed two times with 2 X SSC and 0.1% SDS at room temperature for 5 min, followed by two washes with 0.1 X SSC and 0.1% SDS at

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70 68°C for 15 min and subjected to detection of DIG-labeled RNA probes to detect RNA targets on northern blots using the DIG Chemiluminescent Detection Kit (Roche Molecular Biochemicals, Mannheim, Germany). After hybridizations with gene specific probes, the membranes were also hybridized with an 1 8S ribosomal RNA (rRNA) probe for loading and transfer control. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) cDNA was synthesized from 1 .5 ug total RNA by Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) using oligo-dT primer according to manufacturer's instructions. PCR amplification was conducted in a 50 ul reaction mixture containing PCR buffer [50 mM KC1, 10 mM Tris-HCl (pH 9.0), 1% Triton X100], 2.5 mM MgCl 2 0.4 mM dNTPs, 50 pmole gene specific primers, 2.5 U Taq DNA polymerase (Promega, Madison, WI) and 1 ul cDNA template. The amplification reaction was carried out in a PTC100 Thermocycler (MJ Research, Waltham, MA). The thermocycler was programmed at 94 C for 5 min initial denaturation for one cycle and 25 or 35 cycles at 94°C for 30 s denaturation, 55°C for 30 s primer annealing, 72°C for 1 min primer extension followed by one cycle of final primer extension at 72°C for 10 min. PCR products were separated in 1 .5% agarose gel by electrophoresis in TAE buffer [0.5 M Tris-Base, 0.5 M EDTA (pH 8.0), 12.6% glacial acetic acid]. Products were stained with ethidium bromide and visualized and photographed under UV light using the IS1000 digital gel imaging system (Alpha Innotech, San Leandro, CA).

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71 Results Cold-Induced PI-B05 cDNA from Poncirus Encodes an AP2 Domain Containing Protein The partial cDNA sequence of PI-B05 (CI 7) was isolated from a subtractive cDNA library of 2 d cold-acclimated Poncirus. Reverse northern analysis revealed that PI-B05 showed three-fold induction in response to cold and partial deduced amino acid sequences of this cDNA showed homology with AP2 domain containing proteins from other plants. Since a number of AP2 domain containing proteins are involved in environmental stress, PI-B05 may be important for understanding cold response in Poncirus. The full-length sequence of PI-B05 cDNA was obtained using 5' and 3' RACE. This cDNA was 1279 bp in length, consisting of a 146 bp 5'untranslated region (UTR), a complete open reading frame (ORF) of 984 bp encoding a polypeptide of 328 amino acids, followed by a 3'UTR of 147 bp (Figure 4-1). Alignment of the protein encoded by this full-length cDNA sequence with other homologous proteins is shown in Figure 4-2. The multiple sequence alignment demonstrated that PI-B05 shares significant sequence homology with previously characterized and putative AP2 domain containing proteins from different plants. The most conserved region of the protein was between amino acids 85-145 which contains the AP2 DNA binding domain. Sequence alignment of the AP2 domain from PI-B05 and the other plant proteins showed that the AP2 domain of PI-B05 was almost identical to the consensus sequence of AP2 domains (Figure 4-3), indicating that PI-B05 possibly contains a functional AP2 DNA binding domain. The expression of PI-B05 in cold-acclimated and nonacclimated Poncirus and pummelo was studied by northern blot analysis to determine the expression pattern of this

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72 PI-B05 ORF (984 bp) 5' UTR. UTR 3' 146 bp PI-B05 cDNA 1279 bp 147 bp B 1 AGCGTGGTCG CGGCCGAGGT ATTAATTCGT AAAACCCCTT GAGTTCTCTC GCATCTTCTT TTCTCTTACT AATCGCCAAA ACCATAGCCT GACCTGATCG TCGCACCAGC GCCGGCTCCA TAATTAAGCA TTTTGGGGAA CTCAAGAGAG CGTAGAAGAA AAGAGAATGA TTAGCGGTTT TGGTATCGGA CTGGACTAGC MC G G A I L G DFFP R H G RCR 101 CTAGTTTAGT TTCCGTGCTT TATTATTACA TTTACTTGCG AAAAGAATGT GTGGCGGTGC TATCTTAGGA GACTTCTTTC CTCGCCACGG CCGCTGCCGC GATCAAATCA AAGGCACGAA ATAATAATGT AAATGAACGC TTTTCTTACA CACCGCCACG ATAGAATCCT CTGAAGAAAG GAGCGGTGCC GGCGACOGCG VTAS DIW P N S P F A A TKQ LPH NFES TPF SDE HQS 201 GTCACCGCTT CCGACATCTG GCCCAACTCA CCCTTCGCTG CTACTAAACA ACTCCCTCAC AACTTTGAAT CTACTCCGTT CTCCGATGAA CACCAGTCAC CAGTGGCGAA GGCTGTAGAC CGGGTTGAGT GGGAAGCGAC GATGATTTGT TGAGGGAGTG TTGAAACTTA GATGAGGCAA GAGGCTACTT GTGGTCAGTG L A K I KRP Q P P S SLN SSS ASGD E R K PKR Q R K N LY 301 TGGCCAAAAT CAAACGCCCT CAACCCCCTT CATCTCTAAA CTCCTCTTCA GCTTCAGGTG ATGAGAGAAA GCCCAAGAGG CAGAGGAAGA ATCTCTACAG ACCGGTTTTA GTTTGCGGGA GTTGGGGGAA GTAGAGATTT GAGGAGAAGT CGAAGTCCAC TACTCTCTTT CGGGTTCTCC GTCTCCTTCT TAGAGATGTC RGIR QRPW GKW A A E IRDP RKG VRV WLGT FNT AEE 401 GGGAATAAGG CAACGCCCCT GGGGCAAATG GGCAGCTGAG ATTCGTGACC CAAGGAAAGG AGTTCGAGTC TGGCTCGGCA CATTCAACAC TGCCGAAGAA CCCTTATTCC GTTGCGGGGA CCCCGTTTAC CCGTCGACTC TAAGCACTGG GTTCCTTTCC TCAAGCTCAG ACCGAGCCGT GTAAGTTGTG ACGGCTTCTT A A R A Y D K EAR KIRG KKA K V N FPNE E D V F T V T P A 501 GCAGCTCGAG CCTATGACAA AGAAGCCCGC AAGATTCGCG GCAAGAAAGC CAAAGTCAAC TTCCCCAACG AAGAGGACGT CTTCACCGTC ACCCCTGCTG CGTCGAGCTC GGATACTGTT TCTTCGGGCG TTCTAAGCGC CGTTCTTTCG GTTTCAGTTG AAGGGGTTGC TTCTCCTGCA GAAGTGGCAG TGGGGACGAC A A A H A Y Y HHQH NPN PTP SFLP SLS QED LFSR N N 601 CTGCCGCCCA TGCTTACTAT CATCATCAGC ATAATCCTAA TCCGACTCCC AGTTTCCTTC CTTCTCTATC CCAAGAAGAT CTnTTAGTC GTAATAACTC GACGGCGGGT ACGAATGATA GTAGTAGTCG TATTAGGATT AGGCTGAGGG TCAAAGGAAG GAAGAGATAG GGTTCTTCTA GAAAAATCAG CATTATTGAG SS1S SVGF DLY GYD LNPQ IVT SGG DENS GTG SGS 701 ATCTATAAGT AGTGTTGGTT TCGACTTGTA TGGTTATGAT CTGAACCCCC AGATTGTTAC TTCTGGTGGT GACGAGAATT CCGGGACTGG TTCTGGTTCT TAGATATTCA TCACAACCAA AGCTGAACAT ACCAATACTA GACTTGGGGG TCTAACAATG AAGACCACCA CTGCTCTTAA GGCCCTGACC AAGACCAAGA V SEG GYD STE L M L N C N Q NVN DGSF A Q M KVK AEE 801 GTTTCAGAAG GTGGGTACGA TTCCACAGAA CTTATGCTGA ATTGCAACCA GAATGTCAAT GATGGTTCGT TTGCTCAAAT GAAGGTGAAA GCCGAAGAAC CAAAGTCTTC CACCCATGCT AAGGTGTCTT GAATACGACT TAACGTTGGT CTTACAGTTA CTACCAAGCA AACGAGTTTA CTTCCACTTT CGGCTTCTTG Q E E E EKR KAEE EEN E V R KLSE ELL AYE NFMK FY 901 AAGAAGAAGA AGAAAAGCGA AAAGCTGAAG AGGAAGAGAA CGAAGTGCGA AAGCTGTCTG AGGAGCTTTT GGCGTACGAG AATTTCATGA AGTTCTATCA TTCTTCTTCT TCTTTTCGCT TTTCGACTTC TCCTTCTCTT GCTTCACGCT TTCGACAGAC TCCTCGAAAA CCGCATGCTC TTAAAGTACT TCAAGATAGT QLPY L D G Q SPV SNN NNNN NNI VQE GVVG NLW SFD 1001 GCTTCCGTAT CTCGATGGGC AATCACCAGT GTCTAATAAT AATAATAATA ATAATAATAT TGTTCAAGAA GGTGTTGTCG GTAATCTTTG GAGCTTTGAT CGAAGGCATA GAGCTACCCG TrAGTGGTCA CAGATTATTA TTATTATTAT TATTATTATA ACAAGTTCTT CCACAACAGC CATTAGAAAC CTCGAAACTA DVTV APA PAN SKAL 1101 GATGTTACTG TTGCCCCTGC CCCTGCGAAC TCCAAGGCTC TGTAACTGTG TCTTTGATTT ATTTGTTATT CCTGTATTTC TTTTTGATTT ATTTGTTAAT CTACAATGAC AACGGGGACG GGGACGCTTG AGGTTCCGAG ACATTGACAC AGAAACTAAA TAAACAATAA GGACATAAAG AAAAACTAAA TAAACAATTA 1201 CCTGTATTTC TTTTTTATTT AAATGTTTAT AAATTCAGTG GCATTGATAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA GGACATAAAG AAAAAATAAA TTTACAAATA TTTAAGTCAC CGTAACTATT TTT T TT TTT T TITITlTm 1 ITITITm Figure 4-1. Full-length sequence of Poncirus cDNA PI-B05. A) The map of the fulllength cDNA sequence showing open reading frame and the 5' and 3' untranslated regions. B) The full-length cDNA sequence and the predicted translation of the PI-B05 open reading frame. The amino acid sequences are shown on the top of the cDNA sequence.

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73 Consensus (DMCGGAILSDIIPP LEKTKKQQQQKKKNKGSiRLPLRQE f 35 iWGTSDLNKKKKNPSNYAPLRS wf SLR KR Fjfip . GDRKKPI SGKR F«PV VD! -a jm 10 to 99 Yl*PLRSKFIDI^DJ|FEADf QHf KDN Sfl -PHP LlSBjl lREtKDDSEIEDVDEf] -fBpV VD JBSlDFQGFKDE EESDI1 wlNSPFtATKQ PHNFESTPFSDiH eeIevdsgewevesbaSakplaaS HP ISTQNVPUif wqtssf— skpistqhv* qhI kdn sSdHovkaf REf KDDSE I EDVDC(dSdEEELKKB FGFSRSSNK KDE BESDICBEVLVQDvCfTFSAPPRRLTA DLWP SK DDDFEADF f D DE PKP AP2 Domain (101) i°i ERF-like-Os (86) ERF2-U (41) AP2 protein-St (49) JERFl-Le (81) ERFGm (86) ASKPLSRl ERF protein Fs {53) SS PI-B05-PI (61) Consensus (101) igirqrpwgkwaaeirdpr omkkrqrkn l|rg i rqr pwgkwaae i rd pr :girorpwgkwaaeirdprkr|rvwlgt YE§B YDREARllRGKK eaar-aydrear irgkk rgirqr pwgkwaae irdpr: rgirqrpwgkwaaeirdprk^rvwlgtfsj rgirqrpwgkwaaeirdprj rg i ror pwgkwaae i rdpri |nfpdgap|asq--;n|dodhyc--nIdddhy I RaKKlKtNF D|AP|SVSRRA IRGKK k|nFPd|pSGAASSKR IRGKK KQN F PMP LPl PKRS K t NlEDtFTVTPA AER AKRKRKNQYRG I RQR PWGKWAAE I RDPRKGVRVWLGTFNTAEEAARAYDAEARR I RGKKAKVNFP E V QOO) 200 | 21f) £20 ,230 ,240 gSO 26(1 ,270 £S0 299 ERF-like-Os (180) -BaJpssBnMPAF|BEK PAVMSAGNKTMYNTNAYAYtA EYTlj|PFVQIQNVSFtPAMNAI BDTFVNl QtNDlj ERF2-Le (126) --YfejppptNiACD Ttvif nH snn CYppBlHv;:p.'r-fcCi|:. AP2 protein-St (132) -y|BpPp|nIVYE|YD TtST M---SNN CYp f|I J T P f t flA KN SGSJERFl-Le (170) Al|T: K KALREETIJ«Q|NMTYISNLrX5GSDDSFSFFEEKIaIkQ GFBNVS FTAVDMGEGS VS PS AGTNVflHBHAflHHCBpB w a! pca! ERFGill (185) RlM: H AQ PfKKN LN1B Kl K I NQMFNFGDN LEG YYS P I DQVEQKl LV NQ V§l APFAGNGVGJSPTCPSADVTaMBs^^By^JwGIOVpI ERF protein Fs 1176) -VM_ L KPLAKANUflSQINLNQNFNFMNSDQDYTMGLMEE-KfftNQ GYM DS I PVNADVGLKSFASNNTApH' fcoHUBwcloGsl PI-B05-Pt (1S1) AAABaYYHHQHNPNP--3sFLPSLS QEDlfS'-fiNSSISSVGFnlYGYDLNPQIVTSG :TBs ; : :S SV e|gy|sTEL Consensus COO) KAHPE lsvp ptyqe l yfssen snsfd sdfg e K ERF-like-Os pijERF2-U 0 71) AP2 protein-St (186) JERFl-Le (266) ERFGm (283) ERF protein Fs (271) PI-B05-PI (229) Consensus (299) KTPEISSVISC 330 ,330 340 ASCAMVP1 ---NRWEE] NRIVEEEEK' THFEECS PEKKLKSCSSTSLTVB KAADClCKll NSgCWAACjcS K' GEjSQFIEDAVPTKKLKSESGNAVFIErJNX'KI IKVK EEC' EEEKRKAEEBIneIRK N E V LSEELAAYES MKFY IP DGCS VAA LN A C G SMLXWSFCCVF L G Y Figure 4-2. Multiple alignment of predicted amino acid sequences PI-B05 with other plant proteins in GenBank showing homology with PI-B05. The alignment was generated by the AlignX module of Vector NTI suite. GenBank accession numbers for ERF-like protein from rice, (Os), ERF2 from tomato (Le), AP2 domain protein from potato (St), JERFlfrom tomato (Le), ERF from soy bean (Gm) and ERF protein Fagus sylvatica (Fs) are AAF05606, AAO34704, T07784, AAK95687, AAQ 10777 and CAD21849, respectively. Conserved AP2 DNAbinding domain is indicated by the line above the sequence alignment.

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74 5fi I 100 flP2-donain 250 I 300 I 332 B 10 20 30 40 50 60 Consensus 1 SKYRGVRQRPWGKWVAEIRDP RKGTRVWLGTFDTAEEAARAYDVAALKLRGPSAVLNFPNEL 62 PI-B05 82 NLYRGIRQRPWGKWAAEIRDP-RKGVRVWLGTFNTAEEAARAYDKEARKIRGKKAKVNFPNEE 143 GI 21264420 4 KHYRGVRQRPWGKFAAEIRDPAKNGARVWLGTFETAEDAALAYDRAAFRMRGSRALLNFPLRV 66 18 GKYRGVRRRPWGKYAAEIRDSRKHGERVWLGTFDTAEDAARAYDRAAYSMRGKAAILNFPHEY 80 90 KSFRGVRRRPWGKFAAEIRDSTRNGVRVWLGTFDSPEAAALAYDQAAFLMRGTSAILNFPVET 152 GI 1903358 GI 1732406 GI 2245108 GI 3617742 GI 2281635 GI 2281633 GI 7531181 59 KKYRGVRQRPWGKWAAEIRDP-HKATRVWLGTFETAEAAARAYDAAALRFRGSKAKLNFPENV 120 23 IRYRGVRKRPWGRYAAEIRDP-GKKTRVWLGTFDTAEEAARAYDTAARDFRGAKAKTNFPTFL 84 4 5 KLYRGVRQRHWGKWVAEIRLP RNRTRLWLGTFDTAEEAALAYDKAAYKLRGDFARLNFPNLR 106 96 KKFRGVRQRPWGRWAAEIRDP-TRGKRVWLGTYDTPEEAAWYDKAAVKLKGPDAVTNFPVST 157 Figure 4-3. Analysis of amino acid sequences of PI-B05 cDNA. A) The location of the conserved AP2 domain in the PI-B05 amino acid sequence detected by Conserve Domain Search in the GenBank. B) Alignment of the amino acid sequences of the conserved AP2 domain from Poncirus PI-B05 with AP2 domain of proteins from other plants. gi2 1264420, gi 1903358, gi 1732406, gi2245108, gi36 17742, gi2281635, gi2281633, gi7531 181 are the GenBank id numbers for ERF1 from Arabidopsis, similar to Nicotiana EREBP-3 from Arabidopsis, S25-XP1 DNA binding protein from tobacco, EREBP-4 like protein from Arabidopsis, RAP2.6 Arabidopsis, AP2 domain containing protein RAP2.5 Arabidopsis, AP2 domain containing protein RAP2.4 Arabidopsis, PTI6 from tomato.

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75 gene in two closely related species with cold-hardy and cold-sensitive phenotypes. Northern blot analysis revealed that in cold-hardy Poncirus, expression of this gene was induced at 1 h, reached peak levels at 2 d and remained at this high level following 4 d of cold acclimation, but decreased after 7 d of cold acclimation (Figure 4-4). However, only a small increase in expression was observed in cold-sensitive pummelo in response to cold starting at 1 d of cold acclimation remained relatively unchanged over the time points tested. Based on northern blot analysis, the level of expression and induction of PI-B05 by cold was insignificant in pummelo compared to expression in Poncirus. When expression of PI-B05 was studied in dehydrated Poncirus and pummelo seedlings, no significant changes in expression of PI-B05 were observed, indicating that PI-B05 is only expressed in response to cold, but not dehydration. Cold-Induced PI-C10 cDNA from Poncirus Encodes a RAV-like Protein with two Different DNA-binding Domains Reverse northern analysis of the partial cDNA sequence of PI-C10 (CIO) showed an 1 1 -fold induction and partial deduced amino acid sequences of this cDNA showed homology with the RAV-like protein from Arabidopsis. Since RAV and RAV-like proteins contain two different DNA-binding domains and it was shown that RAVI was induced in response to cold in Arabidopsis, PI-C10 may be involved in cold-responsive gene regulation in Poncirus. The full-length sequence of PI-C10 cDNA was obtained by 5' and 3' RACE using gene specific primers obtained from partial cDNA sequence. The full-length PI-C10 cDNA was 1546 bp, consisting of a 159 bp 5'UTR, a complete ORF of 1 1 19 bp encoding a polypeptide of 373 amino acids, followed by a 3'UTR of 268 bp (Figure 4-5).

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Figure 4-4. Northern blot analysis of expression of PI-B05 cDNA in response to environmental stresses. A) Expression of PI-B05 cDNA in response to cold acclimation and drought in Poncirus detected by antisense DIG-labeled riboprobe. B) Expression of PI-B05 cDNA in response to cold acclimation and dehydration (drought) in Pummelo detected by antisense DIG-labeled riboprobe. The type environmental stress treatment and the duration of the treatment are indicated on the top. The expression of 18 S rRNA was used as a loading and transfer control and is shown below the expression of the specific gene.

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77 Sequence analysis revealed that the protein sequence encoded by this cDNA showed sequence similarity with RAVI, RAV2, RAV2-like, and RAP2.8 proteins from Arabidopsis. Multiple alignment of PI-C10 with these proteins presented in Figure 4-6 showed that this protein is highly homologous to this group of proteins encoding transcription factors, some of which are involved in cold-responsive gene expression. PICK) showed high sequence identity in four different regions of RAV and RAV-like proteins. Sequence analysis showed that two of these regions were previously characterized AP2 and B3 DNA binding domains. The AP2 and B3 domains are located towards the N terminus and C terminus of the protein, respectively (Figure 4-6). Multiple sequence alignment of AP2 and B3 domains of Poncirus PI-C10 revealed that both domains contain conserved amino acid sequences involved in DNA-binding (Figure 4-7) indicating that PI-C10 may have two functional DNA-binding domains. To determine the expression pattern of this gene in two closely related species with cold-hardy and cold-sensitive phenotypes, the expression of PI-C10 in cold acclimated and nonacclimated Poncirus and pummelo was studied by northern blot analysis. Expression of this gene was induced at 4 h and reached its highest level at 2 d, while it began declining starting at 4 d of cold acclimation in cold-hardy Poncirus (Figure 4-8). However, no expression was detected in response to cold in cold-sensitive, pummelo (Figure 4-8) indicating that this gene is absent or its expression is too low to detect in pummelo by northern blot analysis. When expression of PI-C10 was studied in dehydrated and nondehydrated Poncirus and pummelo plants, no expression of PI-C10 was detected in Poncirus or pummelo (Figure 4-8) indicating that PI-C10 is only expressed in response to cold, but not dehydration.

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78 A PI-C10ORF(1119bp) 5MJTR^ ^^^^^^^^^^ TTTR 3 ' 159 bp PI-ClOcDNA 373 b P 1546 bp B 1 GATTCTAATA CGACTCACTA TAGGGCAAGC AGTGGTATCA ACGCAGAGTA CGCGGGGATT CAAAACAAAT ACACAATTCA GCATCTCTCT CTCTTTCTTT CTAAGATTAT GCTGAGTGAT ATCCCGTTCG TCACCATAGT TGCGTCTCAT GCGCCCCTAA GTTTTGTTTA TGTGTTAAGT CGTAGAGAGA GAGAAAGAAA M DGS C M D ESTT T D 101 CTTTCTTTCT TGTACCAGAA CCTCTTGTAG ACACACATAA CACACATAGA AGAGAAGAAA TGGACGGAAG TTGCATGGAC GAAAGCACGA CAACTGATTC GAAAGAAAGA ACATGGTCTT GGAGAACATC TGTGTGTATT GTGTGTATCT TCTCTTCTTT ACCTGCCTTC AACGTACCTG CTTTCGTGCT GTTGACTAAG SAST SPPI TPP A S I PLTM SPE RLC RVGS GAS SVI 201 AGCATCAACA TCCCCGCCAA TAACTTTCCC TGCGAGCATC CCGCTGACCA TGTCGCCGGA GAGACTTTGC CGAGTCGGCA GCGGAGCCTC CAGCGTGATT TCGTAGTTGT AGGGGCGGTT ATTGAAAGGG ACGCTCGTAG GGCGACTGGT ACAGCGGCCT CTCTGAAACG GCTCAGCCGT CGCCTCGGAG GTCGCACTAA L D S E A G V E A E S R K L PSS KYK GVVP QPN GRW GAQ 301 CTCGACTCGG AAGCCGGCGT GGAGGCCGAG TCGAGGAAGC TGCCGTCTTC CAAATACAAA GGGGTGGTCC CACAGCCGAA TGGGAGGTGG GGCGCCCAGA GAGCTGAGCC TTCGGCCGCA CCTCCGGCTC AGCTCCTTCG ACGGCAGAAG GTTTATGTTT CCCCACCAGG GTGTCGGCTT ACCCTCCACC CCGCGGGTCT IYEK H 0 R VWLG T F N EEE EAAR A Y D I A A QRFR GR 401 TATACGAGAA GCACCAAAGG GTCTGGCTCG GCACCTTCAA CGAGGAAGAG GAAGCTGCCA GGGCCTACGA TATCGCTGCG CAGCGTTTCC GCGGACGCGA ATATGCTCTT CGTGGTTTCC CAGACCGAGC CGTGGAAGTT GCTCCTTCTC CTTCGACGGT CCCGGATGCT ATAGCGACGC GTCGCAAAGG CGCCTGCGCT GAVT NFKQ MSC A G T SSDE GDI E M A FLSS HSK SEI 501 TGCCGTCACA AACTTCAAGC AAATGTCTTG TGCTGGAACG TCCTCCGACG AGGGTGATAT CGAAATGGCG TTCTTGAGCT CCCACTCCAA GTCCGAGATC ACGGCAGTGT TTGAAGTTCG TTTACAGAAC ACGACCTTGC AGGAGGCTGC TCCCACTATA GCTTTACCGC AAGAACTCGA GGGTGAGGTT CAGGCTCTAG VDML RKH TYK DELE QSK R N Y GLDA NGK RVI KHG 601 GTCGATATGC TGAGGAAGCA CACTTACAAA GACGAGCTCG AGCAGAGCAA AAGAAACTAC GGCCTCGATG CCAACGGCAA GCGTGTAATC AAACACGGGG CAGCTATACG ACTCCTTCGT GTGAATGTTT CTGCTCGAGC TCGTCTCGTT TTCTTTGATG CCGGAGCTAC GGTTGCCGTT CGCACATTAG TTTGTGCCCC EGDG A A T GFGS DRV L K A RDQL FEK A V T PSDV GK 701 AAGGCGATGG TGCTGCCACT GGCTTCGGTT CGGACCGGGT CCTCAAAGCG CGTGACCAGC TTTTTGAAAA GGCCGTTACC CCGAGTGACG TGGGGAAGCT TTCCGCTACC ACGACGGTGA CCGAAGCCAA GCCTGGCCCA GGAGTTTCGC GCACTGGTCG AAAAACTTTT CCGGCAATGG GGCTCACTGC ACCCCTTCGA LNRL VIPK QHA E K H FPLQ SGS TSK G L L L NFE DVT 801 GAACCGGCTC GTGATACCCA AGCAGCACGC GGAGAAGCAC TTCCCTTTAC AGAGTGGAAG CACTTCCAAG GGACTGCTCT TGAACTTTGA GGATGTCACC CTTGGCCGAG CACTATGGGT TCGTCGTGCG CCTCTTCGTG AAGGGAAATG TCTCACCTTC GTGAAGGTTC CCTGACGAGA ACTTGAAACT CCTACAGTGG G K V W R F R YSY WNSS QSY VLT KGWS RFV KEK N L K 901 GGCAAAGTTT GGAGGTTTCG GTACTCCTAC TGGAACAGCA GTCAAAGCTA CGTTTTGACC AAAGGGTGGA GCCGGTTCGT CAAGGAGAAG AATTTGAAAG CCGTTTCAAA CCTCCAAAGC CATGAGGATG ACCTTGTCGT CAGTTTCGAT GCAAAACTGG TTTCCCACCT CGGCCAAGCA GTTCCTCTTC TTAAACTTTC A G D I VSF HRST GGD R Q L YIDW KAR TGP VENP VE 1001 CCGGTGACAT TGTTAGCTTT CACAGATCGA CCGGTGGGGA TAGGCAGCTT TACATTGATT GGAAAGCCAG AACAGGACCG GTCGAGAACC CGGTCGAGCC GGCCACTGTA ACAATCGAAA GTGTCTAGCT GGCCACCCCT ATCCGTCGAA ATGTAACTAA CCTTTCGGTC TTGTCCTGGC CAGCTCTTGG GCCAGCTCGG PVQM MRLF GVN I F K IPGN GLV G V D KIVG CNN INN 1101 GGTCCAGATG ATGAGGCTTT TTGGGGTCAA CATTTTCAAA ATTCCTGGGA ATGGTCTTGT TGGTGTCGAT AAGATTGTTG GGTGCAACAA CATTAATAAT CCAGGTCTAC TACTCCGAAA AACCCCAGTT GTAAAAGTTT TAAGGACCCT TACCAGAACA ACCACAGCTA TTCTAACAAC CCACGTTGTT GTAATTATTA NNGK RLR E M E LLSL E C T KKQ RMIGAS 1201 AATAACGGCA AAAGGCTTAG A GAAA TGGAG CTCTTGTCTC TAGAGTGCAC CAAAAAACAA AGAATGATTG GAGCTTCGTA ACATCTTTTT TTTTTCTTTT TTATTGCCGT TTTCCGAATC TCTTTACCTC GAGAACAGAG ATCTCACGTG GTTTTTTGTT TCTTACTAAC CTCGAAGCAT TGTAGAAAAA AAAAAGAAAA 1301 TTTTTTTTAG TAATCTTATT TCTTGTTGTA ATTTTTCTTT TTTTTTTTTG GAGTTTTTGG AATTTTGAGG AGGAGGTTAA AAGAAGGGGA AATAAGAAGA AAAAAAAATC ATTAGAATAA AGAACAACAT TAAAAAGAAA AAAAAAAAAC CTCAAAAACC TTAAAACTCC TCCTCCAATT TTCTTCCCCT TTATTCTTCT "01 GACTGGGGAA AAGTGGTGCT GCTAGTAGTT GCAAGTTGCA AATGAAAAGG AAAAAGGTTG AATTGTAAAT TACAAGTGAC AAAAGCTGAA ATTAGGTTGT CTGACCCCTT TTCACCACGA CGATCATCAA CGTTCAACGT TTACTTTTCC TTTTTCCAAC TTAACATTTA ATGTTCACTG TTTTCGACTT TAATCCAACA 1501 AACAAAATTT TATAATCAAA AAAAAAAAAA AAAAAAAAAA AAAAAA TTGTTTTAAA ATATTAGTTT TTTTTTTTTT 'ITITriTITI' mm ' Figure 4-5. Full-length sequence of Poncirus cDNA PI-C10. A) The map of the fulllength cDNA sequence showing open reading frame and the 5' and 3' untranslated regions. B) The full-length cDNA sequence and the predicted translation of the PI-C10 open reading frame. The amino acid sequences are shown on the top of the cDNA sequence.

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79 AP2 Domain PI-C10-P1 RAVI -At AM-TFl-Al AP2-TF2Al RAV2-like-Al (I) -. AP2TF3-A1 (7) RAV2-AI (I) RAP28-AI (I) Consensus 70 ao H 100 EAESRKLFS -C|E; 'ESBKLf! E/ESPKLPS E'*ESRKLPI UKLF: KLFS1 SRKLFS KKLFS! KGWPCFNGRWGAC IYEKHCRVNLCi: KGVVPCFN3RWGACIYFJ(HCRVWLGT: PC FNjRWGAC I YEKHCRVWLG*! PCENGRWG7«IYEKBCRVWLOT: XGWPCENGRWGAC XGWPCFNGRWGKIYFJOHCRVWLCT! KGVVPCPN3)WG»CIYEKHCRVViLGT! KGWPCFM3JWGAC IYEKHCRVWLCTFNB 120 133 FfltRCMlNFlSs WFRBRCAVINFICllt H 1 H • I iCRFK RLAVUFKM r t "a !FR RLAV.UFkM| FR REAV.NFkH ECSSSVEESSTSI AftTIAKKLSFPFAAALFiLYRWGSGGSSWIXSENG VFIF3?KLFSSKYKGWPCPNGFWjACI^EKHCFA/WL^FNEEEEAARAVEIAA RFRGRLAVTNFKNVL B3 Domain (134) HI ,140 PI-CIO-PI (124) RAVl-Al (120) MAP2-TF1-AI (119)1AP2-F2-AI (119)tRAV2-likc-Al (123)1AP2-TF3-AI (130) |G RAV2-AI (123)1RAP2 8-At (105)1Consensus (134) E 150 .160 : 70 200 210 220 330 .240 rvmUKHTY; IVCMLRK] HSKfcrvEMiRKbm: HSK EIVEMLRKHTYi .hsk e ivcmlrk] hsk eivlmlrk: hsk eivemlrk: ,hsk|eivcmlrk] c(siaflfahskaervemlrl
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80 A 1 SO 100 ISO 200 250 300 350 373 ' ' ' ' ' ' ' 11 flP2-donain 1 B3 B Consensus PI-C10 GI 21264420 GI 1903358 GI 3617742 GI 2281635 GI 7531181 GI 2281647 GI 1171429 GI 2281641 10 20 30 40 50 60 I *-...|....| * |....* J....* | 1 SKYRGVR QRPWGKWVAEIRDP RKGTRVWLGTFDTAEEAARAYDVAALKLRGPSAVLNFPNEL 62 64 SKYKGWPQPNGRWGAQIYE KHQRVWLGTFNEEEEAARAYDIAAQRFRGRDAVTNFKQMS 123 4 KHYRGVR QRPWGKFAAEIRDPAKNGARVWLGTFETAEDAALAYDRAAFRMRGSRALLNFPLRV 66 18 GKYRGVR RRPWGKYAAEIRDSRKHGERVWLGTFDTAEDAARAYDRAAYSMRGKAAILNFPHEY 80 59 KKYRGVR QRPWGKWAAE I RD P HKATRVWLGTFETAEAAARAYDAAALRFRGSKAKLNFPENV 120 23 IRYRGVR KRPWGRYAAEIRDP GKKTRVWLGTFDTAEEAARAYDTAARDFRGAKAKTNFPTFL 84 96 KKFRGVR -QRPWGRWAAEIRDP TRGKRVWLGTYDTPEEAAWYDKAAVKLKGPDAVTNFPVST 157 22 TKFVGVR QRPSGKWVAEIKDT -TQKIRMWLGTFETAEEAARAYDEAACLLRGSNTRTNFANHF 83 384 SIYRGVTRHHQHGRWQARIGRV AGNKDLYLGTFGTQEEAAEAYDVAAIKFRGTNAVTNFDITR 446 4 5 SKYKGW PQPNGRWGAQIYEKHQRVWLGTFNEQEEAARSYDIAACRFRGRDAWNFKNVL 104 Consensus PI -C10 GI 12328S60 GI 12328553 GI 2281641 GI 3522951 GI 4678220 GI 9758405 GI 3695373 GI 6069650 Consensus pi CIO GI 12328560 GI 12328553 GI 2281641 GI 3522951 GI 4678220 GI 9758405 GI 3695373 GI 6069650 50 10 20 30 40 .|....«....|....«....|....*....|.........|.........| 1 LFEKAVTPSDVGKLNRLVIPKQHAEKHFPLPS EPKGILLNFEDGRGK 201 LFEKAVTPSDVGKLNRLVIPKQHAEKHFPLQS GSTSKGLLLNFEDVTGK 181 LFEKAVTPSDVGKLNRLWPKQHAEKHFPLRRAASSD SASAAATGKGVLLNFEDGEGK 177 LFEKAVTPSDVGKLNRLWPKQQAERHFPFPLRRHS SDAAGKGVLLNFEDGDGK 169 LFEKAVTPSDVGKLNRLVIPKQHAEKHFPLPSPS PAVTKGVLINFEDVNGK 34 MFDKVVTPSDVGKLNRLVIPKQHAERFFPLDSS SNEKGLLLNFEDLTGK 22 LFEKPLTPSDVGKLNRLVI PKQHAERYFPLAAAA ADAVEKGLLLCFEDEEGK 4 5 LFEKSLTPSDVGKLNRLVI PKQHAEKYFPLNAVLVSSAAADTSSSEKGMLLSFEDESGK 30 MFDKVLTPSDVGKLNRLVIPKQHAENFFPLED NQNGTVLDFQDKNGK 36 MFEKWTPSDVGKLNRLWPKHYAEKYFPLGPAA RTSPAGTVLCFEDARGGD 60 70 80 VWRFRYSYWNSSQSYV 61 VWRFRYSYWNSSQSYV 265 VWRFRYSYWNSSQSYV 2 54 VWRFRYSYWNSSQSYV 246 VWRFRYSYWNSSQSYV 23 5 SWRFRYSYWNSSQSYV 98 PWRFRYSYWNSSQSYV 89 SWRFRYSYWNSSQSYV 119 MWRFRYSYWNSSQSYV 92 STWRFRYSYWNSSQSYV 104 90 110 120 I 100 — — I — — I — — I... 62 LTKGWSRFVKEKGLDAGDTVSFHRSGRGDSGRL FIDWRRRPAS 266 LTKGWSRFVKEKNLKAGDI VSFHRSTGGDRQLY IDWKARTGPV 255 LTKGWSRFVREKGLRAGDTIVFSRSAYGPDKLL FIDCKKNNAA 247 LTKGWSRFVREKGLRPGDTVAFSRSAAAWGTEKHL LIDCKKMERN 236 LTKGWSRFVKEKNLRAGDWTFERS TGLERQL YIDWKVRSGP 99 MTKGWSRFVKDKKLDAGDIVSFQRC VGDSGRDSRLFIDWRRRPKV 90 LTKGWSRYVKEKHLDAGDWLFHRH RSDGGRFFIGWRRRGDS 120 LTKGWSRFVKDKQLDPGDWFFQRH RSDSRRLFIGWRRRGQG 93 MTKGWSRFVKEKKLFAGDTVSFYRGY I PDDNAQPER 105 ITKGWSRYVRDKRLAAGDTVSFCRAG ARL FIDCRKRAAS 103 308 2 97 291 277 ] i 1 131 161 128 143 Figure 4-7. Conserved DNA-binding domains of PI-C10. A) The location of the conserved AP2 and B3 domains in the PI-C10 amino acid sequence detected by Conserve Domain Search in the GenBank. B) Multiple sequence alignment of AP2 domain of PI-C10 with AP2 domain of other plant proteins in GenBank. The GenBank identification numbers GI 21264420, GI 1903358, GI 3617742, GI 281635, GI 2281647, GI 1171429, GI 2281641 and GI 7531 181, represents ERF1, Similar to Nicotiana EREBP-3, RAP2.6, RAP2.1 1, RAP2.5, CKC, RAP2.8 proteins from Arabidopsis thaliana and PTI6 tomato, respectively. C) Multiple sequence alignment of B3 domain of PI-C10 with AP2 domain of other plant proteins in GenBank. The GenBank identification numbers GI 12328560, GI 12328553, GI 6069650, GI 2281641, GI 3522951, GI 4678220, GI 9758405 and GI 3695373 represent RAV2, putative RAV2-like, RAV2-like and P0514G12.9 proteins from Oryza sativa, RAP2.8 RAV-like B3 domain, putative RAV2, RAV2-like, and RAP2.8-like proteins from Arabidopsis thaliana, respectively.

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81 Since no expression of PI-C10 was detected in pummelo by northern blot analysis, RT-PCR was performed using primer sets specific to 5' and 3' regions of the full-length cDNA to determine if this gene is absent or present in pummelo. RT-PCR amplification from total RNA of nonacclimated Poncirus and pummelo plants showed that the expression of PI-C10 was seen after 25 cycles of amplification in Poncirus and expression was only detected after 35 cycles of amplification in pummelo indicating that basal expression of this gene is very low in both plants (Figure 4-8 lanes 1-2 and 5-6). When the expression of this gene was studied in 2 d cold-acclimated plants, expression of PI-C10 was detected in both 25 and 35 cycles of PCR amplification in pummelo and Poncirus indicating that the PI-C10 isolated from Poncirus is also present in pummelo. However, the level of expression was significantly higher in Poncirus than pummelo at both 25 and 35 cycles of PCR amplification (Figure 4-8 lanes 3-4 and 7-8). These results suggested that although this gene is present in both species, its expression in response to cold acclimation of cold-hardy Poncirus and cold-sensitive pummelo is different. PII-C02 cDNA from Poncirus Encodes a RING Zinc Finger Protein and is Induced in Response to Cold and Drought Reverse northern analysis had revealed that PII-C02 (C78) showed a nine-fold induction upon cold acclimation. The partial deduced amino acid sequences of this cDNA showed homology with RING zinc finger proteins from other plants. At least one RPWG zinc finger protein from Arabidopsis is reported to be induced in response to cold. Thus, to determine whether the putative PII-C02 RING zinc finger protein might be involved in response to cold in Poncirus, the partial sequence was used for isolating fulllength cDNA sequence of this gene. The full-length sequence of PII-C02 cDNA was

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82 Cold Acclimation Drought Poncirus trifoliata t00 C\l >» >> >» co co co "O "O "O CM >-. >» CO CO CO X) "O T3 ^ o a> t>, >» 5* CO CO "D "O co m PI-C10 18S rRNA B Citrus grandis Cold Acclimation Drought i_ XI CO CO CO r ^ -D 13 T3 (O (M (M t N >.>.>> >> >» CO CO CO CO CO "O "D "D "° "° -rco m O NCD rttI PI-C10 18S rRNA Citrus grandis Poncirus trifoliata Figure 4-8. Expression of PI-C10 cDNA in response to environmental stresses. A) Northern blot analysis of expression of PI-C10 cDNA in response to cold acclimation and drought in Poncirus detected by antisense DIG-labeled riboprobe. B) Northern blot analysis of expression of PI-C10 cDNA in response to cold acclimation and drought in Citrus grandis (pummelo) detected by antisense DIG-labeled riboprobe. The type of environmental stress treatment and the duration of the treatment are indicated on the top. The expression of the 1 8S rRNA was used as a loading and transfer control and is shown below the expression of the specific gene. C) RT-PCR analysis of expression of PI-C10 cDNA in non-acclimated (NA) and 2 day coldacclimated (CA) Citrus grandis (pummelo) and Poncirus trifoliata. The number of PCR cycles is indicated on the top and the size of the DNA marker (M) closest to the size of the amplified region of PI-C10 cDNA shown on the left side.

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83 obtained by 5' and 3' RACE using gene specific primers. This cDNA was 1043 bp, consisting of a 153 bp 5'UTR, a complete ORF of 534 bp encoding a polypeptide of 178 amino acids, followed by a 3'UTR of 416 bp (Figure 4-9). Sequence analysis demonstrated that the deduced amino acid sequence of this full length cDNA showed homology with RING zinc finger proteins from Thellungiella halophila and Arabidopsis. Alignment of the protein encoded by this cDNA sequence with other RING zinc finger proteins is shown in Figure 4-10. The multiple sequence alignment demonstrated that PII-C02 shares significant sequence homology with previously characterized and/or putative RING zinc finger proteins from Arabidopsis and Thellungiella halophile. It contains a signature sequence motif for a RING zinc finger at the C terminus of the protein (Figure 4-10). The alignment of the PII-C02 RING zinc finger domain with proteins from plants and other organisms including human and fission yeast containing similar domains showed that the key amino acids in this domain were conserved in PII-C02, indicating that it might be a functional RING zinc finger protein (Figure 4-10). Since a histidine residue was found at the fifth coordination site of PHC02, this protein is likely to belong to the RING-H2 subgroup. The expression of PII-C02 was studied in response to cold and drought stresses to determine the expression pattern of this gene in two closely related species, Poncirus and pummelo. Northern blot analysis showed that expression of PII-C02 increased inconsistently starting at 1 h of cold acclimation in Poncirus. Although the expression level was variable at different time points, expression reached its highest level between 24 h and 2 d of cold acclimation and decreased thereafter (Figure 4-11). Expression of PII-C02 was at a basal level between 0 and 8 h, increased between 8 to 24 h of cold

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84 PII-C02 ORF (534 bp) 5 ' UTR 153 bp UTR 3' PI-B05 cDNA 1043 bp 416 bp B 1 CTAGTGATTC TAATACGACT CACTATAGGG CAAGCAGTGG TATCAACGCA GAGTACGCGG GGCAGTCACA AGATTTTGCT TGATTTCTGA TTCAGTATCT GATCACTAAG ATTATGCTGA QTGATATCCC GTTCGTCACC ATAGTTGCGT CTCATGCGCC CCGTCAGTGT TCTAAAACGA ACTAAAGACT AAGTCATAGA M G L ASM PSA S E G M LCL 101 CAAATTTAGT ACAAAATTGG GTATCTGTAA ATTTAAAATT TTAATTCGAA AACATGGGCC TCGCTAGTAT GCCGTCCGCA TCAGAAGGAA TGCTATGCTT GTTTAAATCA TGTTTTAACC CATAGACATT TAAATTTTAA AATTAAGCTT TTGTACCCGG AGCGATCATA CGGCAGGCGT AGTCTTCCTT ACGATACGAA I L M NTAM PIS IVK G I F R SIL KVV G F Q L AES SST 201 GATTCTAATG AACACTGCTA TGCCAATCTC AATCGTCAAA GGCATATTCA GATCAATCCT CAAGGTTGTC GGTTTCCAGC TTGCTGAATC ATCATCGACA CTAAGATTAC TTGTGACGAT ACGGTTAGAG TTAGCAGTTT CCGTATAAGT CTAGTTAGGA GTTCCAACAG CCAAAGGTCG AACGACTTAG TAGTAGCTGT P Y S Y FAS PQV VSAE PYD VNL SPPL SYV EEF R N Q 301 CCGTATTCAT ATTTCGCTTC ACCTCAAGTT GTCTCCGCAG AGCCATATGA TGTAAATTTA AGTCCTCCCC TTAGCTATGT TGAGGAGTTC CGAAACCAGA GGCATAAGTA TAAAGCGAAG TGGAGTTCAA CAGAGGCGTC TCGGTATACT ACATTTAAAT TCAGGAGGGG AATCGATACA ACTCCTCAAG GCTTTGGTCT N P A I KYE TLLH CED AEH DCSV CLT EFE PQSD INN 401 ACCCTGCAAT CAAGTATGAA ACATTGCTCC ATTGTGAAGA TGCAGAGCAT GACTGTTCTG TGTGTTTGAC CGAGTTTGAG CCTCAATCTG ATATAAATAA TGGGACGTTA GTTCATACTT TGTAACGAGG TAACACTTCT ACGTCTCGTA CTGACAAGAC ACACAAACTG GCTCAAACTC GGAGTTAGAC TATATTTATT LSC OHLF HKV CLE KWLD YLN VTC PLCR TPL IPE 501 CTTGTCTTGT GGACATTTGT TTCATAAAGT GTGCTTGGAG AAGTGGCTGG ACTATTTGAA TGTCACGTGC CCGCTTTGCA GGACACCTCT AATTCCTGAG GAACAGAACA CCTGTAAACA AAGTATTTCA CACGAACCTC TTCACCGACC TGATAAACTT ACAGTGCACG GGCGAAACGT CCTGTGGAGA TTAAGGACTC FEDD PSC FW 601 TTCGAAGATG ATCCCTCTTG TTTCTGGTGA GAGTGTTTTA TGAGTTTGTC TAGTTGTGGA GACTTCCATG TACAGCATGT AGTGTACAGG TATTTACTAA AAGCTTCTAC TAGGGAGAAC AAAGACCACT CTCACAAAAT ACTCAAACAG ATCAACACCT CTGAAGGTAC ATGTCGTACA TCACATGTCC ATAAATGATT 701 TGCATCGGCT GGAGTGTAGT GTTGTTTACA CGCCTTCTGT GTGTGAGTTA AATCTCGAGT CCTTTTGAAG GCTTGTTGAG AAAACCAGAA TTCTGTTGTA ACGTAGCCGA CCTCACATCA CAACAAATGT GCGGAAGACA CACACTCAAT TTAGAGCTCA GGAAAACTTC CGAACAACTC TTTTGGTCTT AAGACAACAT 801 AATATTGTGA GGTTTCTGGT TGTTTTATGG CATATAATCT GACTTTTGAT CTTCAGCTTT CTTTAAAGTT CATATTAGTG ACTTTGGTTT CCATCTTTTC TTATAACACT CCAAAGACCA ACAAAATACC GTATATTAGA CTGAAAACTA GAAGTCGAAA GAAATTTCAA GTATAATCAC TGAAACCAAA GGTAGAAAAG 901 TTTAATGAGT TGTATGTGAC TGAATATGGA GAAGTTGATG AGGCTTTCTA GTTTCCATGC TAGAATTGCT CAAAAAGAAG TTTGAATTTT ACTTGAGTTT AAATTACTCA ACATACACTG ACTTATACCT CTTCAACTAC TCCGAAAGAT CAAAGGTACG ATCTTAACGA GTTTTTCTTC AAACTTAAAA TGAACTCAAA 1001 ACCAAACAAG CATTTGACAA AAAAAAAAAA AAAAAAAAAA AAA TGGTTTGTTC GTAAACTGTT TTTTTTTTTT TTTTTTTTTT TTT Figure 4-9. Full-length sequence of Poncirus cDNA PII-C02. A) The map of the fulllength cDNA sequence showing open reading frame and the 5' and 3' untranslated regions. B) The full-length cDNA sequence and the predicted translation of the PII-C02 open reading frame. The amino acid sequences are shown on the top of the cDNA sequence.

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85 (1)1 10 2_0 1 30 ,40 £0 £0 70 81 RING Zn Finger-Th (1) MGl|s|pBsEGMLC|il|nTaHiSIFKGI|rs|lhBgIrl|qSSS|p|.sMsMpjKBrjMBHi'lrMK ring Zn Finger-At (l) mgl|s|pHsegmlc|i JntaBisiIkgiIrs: :.gS Is: Ip i | BnssB I ! C I M PII-C02-PI (l)MGL§SiPSASEGMLCln^TA^ISl|KGIFRS|LKBGFQLiESSsipYSYF|s[ '.fv-BEBDVNBlPPLsBEEFR Consensus (1) MGLSSLPGPSEGMLCVILVNTALSISIVKGIVRSIL WGI LS SSSSPSSVTASSEI SSEPFDFRVS PESYLEEFR RING Zn Finger-Th RING Zn Finger-At PII-C02-Pt Consensus (83) i (82) j (83) j RING Zinc Finger Domain 90 100 110 12 0 130 .140 .150 IMS CF KKHEONE S |lloj|| 1 (82) QNPAH|EiLLHCED-H H |cSVCL (83) KTPTLRYESLCRCKK ADNECSVCLSKFE DSE I NKLKCGHLFHKTCLEKW I DYWN I TCPLCRTPL VW ED QLSSNVW B 1 20 40 60 80 100 120 140 158 RING c 10 20 30 40 50 60 70 80 .... .....|......... |.........|.... «....|....*....|.... *....|....«. ...|... ...... |... # # ###« ** Consensus 1 ECAVCLEEFE EIRKLRN CGHLFCRSCLDRWLDY GKNTCPLCRTPI 45 PII-C02 100 DCSVCLTEFE POSDINNLS CGHLFHKVCLEKWLDY LNVTCPLCRTPL 146 IFBV A 334 LCKICAENDK DVKIEP CGHLMCTSCLTSWQES EGQGCPFCRCEI 377 GI 7492802 17 ECI ICLSNLPNCPI.DQWDSSSVPASI SSTLDGLRIAKIP CGHYFHNHCLESWCRV ANTCPLCRTEF 82 GI 15238072 104 CCAVCLHEFEN DDEIRRLTN CQHIFHRSCLDRWMMGY NQMTCPLCRTPF 152 GI 16209696 T AVCLGDLED GDEVRELRN CSHMFHRECIDRWLDYECCGGDENNEGEEDNHRTCPLCRTPL 142 GI 15229284 82 MCAVCLGDLED EDEIRELRN CTHVFHRDCIDRWLDYECCG -GDDDNHRTCPLCRTPL 137 GI 15233117 93 NCAVCLYEFEG EQEIRWLRN CRHIFHRSCLDRWMDH DQKTCPLCRTPF 140 GI 15809840 133 DCAVCLCEF1TEDKLRLLPKCSHAFHMDCIDTWLLS HSTCPLCRSSL 179 GI 15228108 '. . E SV LS DSEINKLK CGHLFHKTCLEKWIDY WNITCPLCRTPL 148 Figure 4-10. Analysis of predicted amino acid sequences ofPoncirus PII-C02 cDNA. A) Multiple alignment of amino acid sequences PII-C02 with other plant proteins in GenBank showing homology with PII-C02. The GenBank accession numbers for RING Zn FingerAAM 19707 and AAG41449 represent putative RING zinc finger protein-like protein from Thellungiella halophile and Arabidopsis thaliana. B) The location of the conserved ring zinc finger domain in the PII-C02 amino acid sequence detected by Conserve Domain Search in the GenBank. C) Multiple sequence alignment of the amino acid sequences of the conserved RING zinc finger domain from Poncirus PII-C02 and other proteins containing similar domain. The conserved amino acid residues of the signature motif are indicated by # sign. The GenBank identification numbers GI 10120668, GI 7492802, GI 15238072, GI 16209696, GI 15229284, GI 15233117, GI 15809840 and GI 15228108 represent Cbl-Ubch7 Ubiquitin-Protein Ligases from human, PHD-type RING zinc finger from fission yeast, RING-H2 zinc finger, putative RING-H2 zinc finger F22D1-50, C3HC4-type RING zinc finger, BRH1 RING zinc finger, putative RING-H2 zinc finger Fl 7123-260 and C3HC4-type RING finger proteins from Arabidopsis thaliana

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86 acclimation and dropped to the initial level after 2 d of cold acclimation in pummelo, never reaching the levels attained in Poncirus (Figure 4-11). When the expression of PIIC02 was studied in response to drought, more significant changes in gene expression were observed both in Poncirus and pummelo. In Poncirus, the expression of PII-C02 was induced at 7 d and reached its highest level at 9 d of dehydration; it started to decrease at 1 1 d and returned the initial level at 13 d. On the other hand, in pummelo, the expression of PII-C02 was induced at 7 d and reached its highest level between 9 and 1 1 d and started to decrease after that time. The apparent high level of expression observed at 1 5 d of dehydration may be due to a higher amount of RNA in that sample since the amount of 18S ribosomal RNA is also higher in that sample. These results showed that PII-C02 encoding a putative RING zinc finger protein is induced by both cold and drought; however, the level of induction in response to drought is much higher than the cold-responsive expression. Discussion The gene expression studies in cold acclimated and nonacclimated Arabidopsis and other plants have shown that expression of several hundred genes is changed in response to cold. Genes induced in response to cold are involved in a variety of cellular functions including transcriptional regulation. A family of AP2 domain containing transcription factors, the CBF/DREB family, was isolated from Arabidopsis and shown to regulate cold-responsive gene expression (Stockinger et al. 1997; Liu et al. 1998; Gilmour et al. 1998). Homologs of CBF/DREB genes were identified and isolated from several groups of plants, indicating that the CBF/DREB pathway is conserved at least among some quite disparate plants (Jaglo et al. 2001). However, the presence of the CBF/DREB pathway in other plants including Poncirus is still unknown. In addition to

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87 Cold Acclimation Drought Poncirus trifoliata Figure 4-11. Northern blot analysis of expression of PII-C02 cDNA in response to environmental stresses. A) Expression of PII-C02 cDNA in response to cold acclimation and drought in Poncirus detected by antisense DIG-labeled riboprobe. B) Expression of PII-C02 cDNA in response to cold acclimation and drought in pummelo detected by antisense DIG-labeled riboprobe. The type environmental stress treatment and the duration of the treatment are indicated on the top. The expression of 18 S rRNA was used as a loading and transfer control is shown below the expression of the specific gene.

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88 the CBF/DREBs, expression of a number of other transcription factors with conserved DNA-binding domain(s) such as bZIP bHLH, zinc finger, MYB, home-box, MADS box, as well as AP2/ERF, AP2/B3, and WRKY proteins were induced in response to cold (Kagaya et al. 1999; Chen et al. 2002; Seki et al. 2001, 2002; Fowler and Thomashow 2002; Sakuma et al. 2002). Expression of some of these transcription factors is controlled by CBF/DREB; however, the expression of other transcription factors is independent of CBF/DREB, indicating that pathways other than CBF/DREB are activated in response to cold (Fowler and Thomashow 2002). Three partial cDNAs, PI-B05, PI-C10 and PII-C02, showing homology to transcription factors were selected from a cDNA library prepared from 2 d coldacclimated Poncirus trifoliate The full-length sequences of these cDNAs were obtained by 3' and 5' RACE. Analyses of nucleotide and deduced amino acid sequences of genes encoded by these cDNAs revealed that PI-B05, PI-C10 and PII-C02 encode proteins containing an AP2 DNA binding domain, AP2 and B3 DNA-binding domains, and a RING zinc finger domain, respectively. Sequence analysis of PI-B05 demonstrated that it contains an AP2 DNA-binding domain that is similar to a DNA-binding domain of previously characterized and putative proteins from Arabidopsis and other plants, including potato, tomato, and rice. Since this protein contains only one AP2 domain, it can be classified along with single AP2/ERF containing proteins, including ERF, TINY, DREB1/CBF, DREB2, Ptis, and ABM (Riechmann and Meyerorowitz 1998; Riechmann et al. 2000; Sakuma et al. 2002). Closer analysis of the conserved sequences in the AP2 domain revealed that PI-B05 is most similar to the ERF group of proteins because unlike the DREB1/CBF group, this

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89 group contains alanine and aspartic acid at position 14 and 19, respectively. It was shown that these two amino acids in the AP2 domain determine DNA-binding specificity of proteins (Sakuma et al. 2002). The DREB1/CBF group contains valine and glutamic acid at position 14 and 19, respectively and binds specifically to the CRT/DREB binding element A/GCCGAC. On the other hand, ERF-like proteins bind to the GCC box containing the core AGCCGCC sequence demonstrating that a single base change determines the specificity of these two groups of AP2/ERF proteins (Sakuma et al. 2002). The GCC-box binding specificity of the ERF proteins ERF1 from Arabidopsis and ERF2 from tomato, which contain DNA-binding domains highly similar to PI-B05, have been demonstrated (Allen et al. 1998; Tournier et al. 2003). The 3-dimensional structural analysis of the GCC-box binding domain of ERF 1 from Arabidopsis with the GCC-box sequence revealed that it contains a three-stranded anti-parallel beta-sheet and an alphahelix packed approximately parallel to the beta-sheet. The domain recognizes the target sequence through a P-sheet structure (Allen et al. 1998). Since PI-B05 contains an AP2 domain very similar to this protein, it is likely that it has the same domain structure and binds to a GCC-box. The expression of ERF genes was differentially induced by ethylene and by abiotic stress conditions, including cold, high salinity, or drought, through ETHYLENE-INSENSITIVE2 (EIN2)-dependent or EIN2-independent pathways (Fujimoto et al. 2000). A number of AP2/ERF proteins are also involved in expression of pathogenesis related genes. Thus, a group of AP2/ERF proteins have roles in biotic and abiotic stress-induced gene regulation. Expression analysis of PI-B05 in response to cold and drought in Poncirus and pummelo demonstrated that this gene is only induced by cold in Poncirus, strongly suggesting that it regulates cold-responsive gene expression in

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90 Poncirus under cold stress. Since the AP2 domain of this protein may have binding specificity to a GCC-box sequence, genes regulated by this protein should contain such a GCC-box regulatory sequence. Sequence analysis of PI-C10 showed that this protein contains two different DNA binding domains, AP2 and B3, primarily found in the plant transcription factors RAVI and RAV2 from Arabidopsis (Kagaya et al. 1999). Searches in the sequence database identified a number of other RAV-like proteins from Arabidopsis and other plants. However, these genes have not been characterized. Analysis of the DNA-binding activity of RAVI protein by deletion and electrophoretic mobility shift assays demonstrated that the AP2 and B3 domains independently bind to CAACA and CACTCG sequence motif, respectively. The presence of two DNA-binding domains provides higher binding affinity and specificity to the RAVI protein (Kagaya et al. 1999). Since the amino acid sequence of AP2 and B3 domain of Poncirus PI-C10 is almost identical to the respective domains from RAVI and RAV2 from Arabidopsis, it is likely that they will have the same DNAbinding activity and will bind to CAACA and CACTCG sequence motifs. Currently, the target genes of RAV proteins are not known; however, recent microarray studies show that at least RAVI from Arabidopsis is induced in response to cold acclimation (Fowler and Thomashow 2002), indicating that it is involved in regulation of cold-responsive gene expression. Expression analysis of RAV-like PI-C10 by northern blot hybridization in this study demonstrated that it is induced only in response to cold in cold-hardy Poncirus, but not to drought and no expression was detected in pummelo in response to both cold and drought. This result confirms the suggestion that the RAV proteins are involved in cold-regulated gene expression and PI-C10 most likely regulates gene

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91 expression specifically under cold stress in Poncirus. Further expression analysis of PICK) by RT-PCR showed that this gene is also present in cold-sensitive pummelo; however, the level of expression is significantly higher in cold-hardy Poncirus. The differences observed in expression of this gene in pummelo between northern blot and RT-PCR analyses may be due to the sensitivity of the two detection methods or sequence variations within this gene in pummelo and Poncirus. Since hybridization stringency used in northern blot analysis allows 20% sequence variations and two different regions of PI-C10 were amplified by PCR using two different sets of primer designed from nonconserved regions of this gene, it is unlikely the differences are due to sequence variations. Thus, it is likely that the differences in expression of PI-C10 in pummelo are due to the increased sensitivity provided by RT-PCR. Since the expression signal is amplified exponentionally by PCR, RT-PCR is much more sensitive than the northern blot analysis and it can detect lower amounts of RNA signal. Database searches with the predicted amino acid sequence of PII-C02 from Poncirus identified two Arabidopsis proteins showing high sequence identity. It also showed homology with a number of other proteins from plants and other organisms. The most similar regions of these proteins contained a RING zinc finger motif found near the C terminus suggesting that PII-C02 is a RING zinc finger protein. The alignment of the amino acid sequences of the RING zinc finger domains shows that PII-C02 contains key conserved amino acids in the correct positions. It also revealed that PII-C02 has a histidine residue at the fifth coordination site, suggesting that it is a member of the H2 subclasses of RING zinc finger proteins which is mostly found in plants.

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92 RING zinc finger proteins are thought to function as transcription factors because of the presence of zinc finger motifs in their sequences. Although the exact function of most RING zinc finger proteins is still unknown, recent studies with some mammalian RING zinc finger proteins demonstrated that they are involved in specific ubiquitination of proteins and suggested that these proteins act as ubiquitin protein ligases (Freemont 2000; Joazeiro and Weissman 2000). Since this pathway is conserved in all organisms and regulates the specific degradation of a large number of proteins involved in diverse cellular processes ranging from cell cycle regulation to stress responses (Hershko and Ciechanover 1 998), RING zinc finger proteins may have important roles in these processes. The Arabidopsis genome contains a number of RING finger proteins (Jensen et al. 1998). Although a majority of these proteins have not been characterized and no functions have been assigned to them, more and more studies are being conducted to elucidate the function of some of these proteins. Recent characterization of a small number of plant RING zinc finger proteins revealed that they are involved in key biological processes of plants including photomorphogenesis (Torii et al. 1999), seed development (Molnar et al 2002; Lechner et al. 2002), pathogen defense (Takai et al 2002; Guo et al. 2003) and cold-responsive gene expression (Lee et al. 2001). Although ubiquitin ligase activity has not been shown for any of these proteins, current data suggests that these proteins function through protein-protein interactions and the RING zinc finger domain is essential for their activity (Torii et al. 1999; Molnar et al 2002; Lechner et al. 2002; Takai et al 2002; Guo et al. 2003; Lee et al. 2001). HOS is a RING zinc finger containing protein that functions as a repressor of cold-induced gene expression through CBF/DREB and possibly other pathways in Arabidopsis (Lee et al.

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93 2001). More recently micro-array analysis of a large portion of the Arabidopsis genome revealed that expression of at least one RING zinc finger protein was induced in response to cold (Fowler and Thomashow 2002), indicating that RING finger proteins may also be involved in regulation of cold or other environmental stress-induced gene expression. In this study, expression of a RING zinc finger domain containing PII-C02 was induced in response to both cold and drought in Poncirus and pummelo. Since the expression level of PII-C02 was much higher in dehydrated plants then the cold-acclimated plants, PIIC02 regulation of gene expression may be more important in Poncirus and pummelo under drought stress than cold stress. Three genes identified and characterized in this study showed cold-responsive induction and differential expression in cold-hardy Poncirus and cold-sensitive pummelo. Since the expression of all three genes is significantly higher in cold-acclimated Poncirus than pummelo, they may contribute to cold hardiness in Poncirus. Since two of these genes encode proteins with highly conserved DNA-binding domain(s), they are likely to be involved in transcriptional regulation of other cold-induced genes in Poncirus. Therefore, not only these genes, but downstream genes potentially activated by these proteins could contribute to cold hardiness in Poncirus. Although this study clearly establishes the cold-regulated nature of these genes, further studies are needed for determination of their actual functions and involvement of these genes in cold hardiness in Poncirus and their potential use for improving cold tolerance in Citrus.

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CHAPTER 5 SUB-CELLULAR LOCALIZATION OF COLD REGULATED GENES COR1 1 AND COR19 Introduction Dehydrins are a family of proteins that are commonly induced in response to environmental stress such as dehydration, low temperature, salinity, as well as in response to ABA treatment and during embryo development (Close 1996). They are also referred to as group 2 late embryogenesis abundant (LEA) proteins since they were first identified during embryo development in cotton (Baker et al. 1988). Dehydrins are evolutionary conserved among photosynthetic organisms as well as in yeast. They have been isolated from gymnosperms and angiosperms and the existence of dehydrins has also been detected in algae, cyanobacteria and yeast by immunological analysis using antibodies specific to dehydrins (Close 1997; Mtwisha et al. 1998). The common features of this family of proteins include high hydrophobicity, thermostability and presence of multiple tandem repeats containing highly conserved consensus sequences. Dehydrins are characterized by conserved Y-, S-, Kand O segments found in their amino acid sequences (Close 1996). The numbers of these various segments can be used for determining sub-classes of dehydrins and for comparison between alleles of individual dehydrin genes (Campbell and Close 1997; Choi et al. 1999). The Y-segment is composed of V/TDEYGNP and one to three copies of it is usually found close to the N-terminus of many dehydrin proteins. The S-segment contains five to seven serine residues followed by three acidic amino acids and is located downstream of the Y94

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95 segment of some dehydrins (Close 1996). The S-segment is phosphorylated and may be involved in nuclear localization of maize dehydrin RAB17 (Goday et al. 1994) and tomato dehydrin TAS14 (Godoy et al. 1994). The K-segment consists of the 15-amino acid lysine-rich sequence EKKGIMDKIKEKLPG and is found near the C-terminus of all dehydrins, repeated one to 1 1 times. The K segments are proposed to form class A amphipathic ot-helices (Dure 1993; Close 1996) which may act as an interphase between hydrophobic surfaces of membrane phospholipids and the cytosol to protect membrane function. In addition, they may interact with partially denatured proteins to prevent aggregation of proteins and to protect functions of proteins under environmental stress (Close 1996; 1997). The majority of dehydrins contain a O-segment which is rich in glycine and polar amino acids and is tandemly repeated between K-segments. It has been proposed that the highly polar O-segment of dehydrins interacts with polar groups on macromolecules in the cytoplasm and nucleus to prevent coagulation of macromolecules (Close 1997). Since dehydrins are induced in response to environmental stress, it was suggested that they have a role in dehydration induced stress tolerance in plants. Dehydrins protect integrity of cells by stabilizing membranes and proteins through detergent or chaperone like activities (Close 1996; 1997; Ismail et al. 1999). Increased chilling tolerance in cowpea is not correlated with reduced electrolyte leakage, indicating that the 35 kDa dehydrin might not be protecting the plasma membrane, but that it reduces dehydration induced damage by interacting with internal membranes of the cells instead (Ismail et al. 1999). It was also proposed that COR15am dehydrins from Arabidopsis interact with plant membranes and protect them from freezing induced lamellar-to-hexagonal phase

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96 transitions (Thomashow 1999). Recently it was suggested that the COR19 from Citrus unshiu enhanced cold tolerance in transgenic tobacco by inhibition of lipid peroxidation (Hara et al. 2003). Dehydrins display diverse tissue specific and sub-cellular localization patterns. Immunolocalization and sub-cellular fractionation studies with a number of dehyrins demonstrated that they are mostly localized in the nucleus and cytoplasm (Close 1996). However, some dehydrins have been found to be associated with plasma membranes (Danyluk et al. 1998) chloroplasts (Wishiewski et al. 1999) and mitochondria (Borovskii et al. 2000; 2002; Hara et al. 2003). Many dehydrins localized in the nucleus contain putative bipartite nuclear localization signals (NLS) (Monroy et al 1993; Goday et al. 1994; Godoy et al. 1994; Cai et al 1995; Houde et al. 1995). The nuclear localization of some dehydrins is tissue specific (Asghar et al. 1994), some such as RAB17 from maize and TAS14 from tomato require posttranslational modification (Goday et al. 1994; Godoy et al. 1994) and others can only be localized in the nucleus under certain environmental stress such as cold (Rinne et al. 1999) . Two cold regulated genes COR1 1 and COR19 were isolated from seven-day cold acclimated Poncirus trifoliata which is a cold-hardy citrus relative (Cai et al. 1995). Expression of both genes was induced in response to cold, but repressed in response to drought and flooding. Both genes contained repeats of conserved lysine-rich K-segment within the protein and S-segment similar to other dehydrins near the C-terminus. In addition, three Q-cluster regions containing a conserved H2Q3-5YR sequence motif and a putative bipartite NLS were also found in both COR1 1 and COR1 (Figure 5-1) (Cai et al. 1995). To determine if the NLS found in the cold regulated genes from Poncirus are

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97 functional, sub-cellular localization of the products of these genes was analyzed in onion epidermal cells. Materials and Methods Amplification COR Genes and Cloning of cor::gus Fusion Constructs COR1 1 and COR19 genes were amplified by RT-PCR using specific primers. First, cDNA was synthesized from total RNA isolated from two-day cold-acclimated Poncirus trifoliata using Superscript II reverse transcriptase and oligo-dT primer (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Then, complete open reading frames of COR1 1 and COR19 were amplified from the cDNA by PCR with Pfu DNA polymerase using primers specific to the 3' and 5' ends of the individual genes. Ncol and Bglll sites were incorporated at the 5' and 3' ends of both genes, respectively, and the stop codons of both genes were deleted during PCR amplification. Since the putative NLS signal in the C-terminal half of the COR1 1 and COR19 genes was almost identical, the N-terminal half of COR1 1 was deleted by PCR amplification of the Cterminal half of this gene by PCR with Pfu Polymerase using an internal primer with an Ncol site and the same 3 'terminal primer with a Bglll site and without the stop codon. The PCR products were purified using the Qiquick PCR Purification Kit (QIAGEN, Hilden, Germany), digested with Ncol and Bglll restriction enzymes (Invitrogen, Carlsbad, CA) and cloned into pCAMBIA 1301 binary vector cut with the same restriction enzymes to fuse the COR sequences to a P-glucuronidase (GUS) reporter gene in frame (Figure 5-1). The resulting plasmids were designated as pCAMBIA 1301 CORll,COR19andCORllC.

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98 A C0R19 MAGVIHKIGEALHVGGGQKEEDKSKGEHQSRDHHTTDVHHQQQYHGGEHREGEQKEGLVD C0R11 MAGVIHKIGEALHVGGGQKEEDKRKGEHQSGDHHTTDVHHQQPYHG COR11C -*********************** ****** *********** *** COR1 9 KI KQQI PGAGTADVHHQQQQQYRGGEHREGEHKEGLVDKI KQQI PGAGTTDVHHQQQQYR COR11 COR11C COR19 GGEHREGEQKEGLVDKI KQKIPGVGGGEGATHA-QGEKKKKKEKKK-HEDGHESSSSSDSD COR11 -GEHREGEQKEGLVGKI KQKI PGVGGGEGATHAHGGEKKKKEKKKKKHEDGHESSSSSDSD COR11C -GEHREGEQKEGLVGKI KQKI PGVGGGEGATHAHGGEKKKKEKKKKKHEDGHESSSSSDSD ************* ****************** *********** ************** B COR 19 COR 11 COR11C pBR322own T Bow (right! pVS' ste \ DVS I rep Figure 5-1. Sequence alignment and cloning of COR genes for sub-cellular localization study. A) Amino acid sequence alignment of full length COR1 1, COR19 and C-terminal half of COR1 1 (COR1 1C). The asterisks and dashes indicate identical or absent amino acid residues, respectively. The sequences of bipartite NLS signals are shown in blue. B) Cloning of COR 1 1, COR 19 and COR1 1C into the pCAMBIA 1301 binary vector to generate cor::gus fusion constructs. The red boxes indicate the genes or portions of genes cloned into the binary vector and the small blue boxes show the approximate locations of bipartite NLS in individual genes. The components of binary vector are shown in the vector map.

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99 Particle Bombardment of Onion Epidermal Cells The plasmids containing cor::gus fusion constructs pCAMBIA 1301-COR1 1, COR19 and COR1 1C as well as control plasmid pCAMBIA 1301 were purified using Qigene Miniprep Kit (QIAGEN, Hilden, Germany) and concentrations of DNAs were determined spectrophotometrically. Tungsten particles (30 mg) were incubated in 70% ethanol for 15 min, washed three times with sterile water, and resuspended in 50% glycerol to obtain the concentration of 60 mg/ml. A 50 ul tungsten suspension was mixed with 5 ug DNA from each construct, 16 mM spermidine, and 10 mM CaCl 2 , vortexed for 3 min, and incubated for 1 min at room temperature. After two washes with 70% ethanol, the tungsten particles coated with DNA were resuspended in 48 ul of 100% ethanol. An aliquot (8 ul) of tungsten particles was placed onto a microcarrier for particle bombardment. Inner epidermal layers of onion (Allium cepa) were peeled and placed on petri dishes containing Murashige-Skoog media (Sigma, St. Louis, MO) pH 5.7 [4.3 g MS salts (Invitrogen, Carlsbad, CA) , 1 mg thiamine, 10 mg myo-inositol, 180 mg KH 2 P0 4 , 30 g sucrose, and 6 g agar] with 2.5 mg/1 amphotericin B (Sigma). Epidermal cell layers of onions were bombarded at 1 100 psi using the Biolistic PDS-1000/He System (Bio-Rad, Richmond, CA). Bombarded onion samples were maintained at 28 °C for 24-48 h and analyzed for the expression of GUS or they were first maintained at 28 °C for 24-48 h then transferred to 4 °C for up to five days before analysis for GUS expression. Subcellular Localization of COR Gene Products in Onion Epidermal Cells Bombarded onion epidermal cells were removed from media and fixed in 50 mM phosphate buffer pH 7.0 solution containing 1.5% formaldehyde, 0.05% TritonX-100 for

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100 30 min at room temperature. After washing four times with 50 mM phosphate buffer for 15 min each, fixed epidermal cells were transferred to (5-bromo-4-chloro-3-indolyl-betaD-glucuronic acid (X-gluc) solution containing 0.5 mM X-gluc, 50 mM phosphate buffer pH 7.0, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, and 0.1% TritonX100 and incubated at 37 °C for 6-16 hours. After GUS staining, samples were transferred to water and analyzed for expression and localization of GUS in the epidermal cells by light microscopy (Zeiss). Glass microscope slides were prepared from GUS stained samples and a 4',6-Diamidino-2-phenylindole (DAPI) solution (20 pg/mL DAPI, 0.1X PBS, 10 mM Na azide, 90% glycerol) applied onto the slides to localize the nuclei as previously described by McLean et al. (1990). The slides were examined under light microscopy to observe GUS expression and localization of the nuclei stained with DAPI and photographed using a camera attached to the microscope. Results To determine if the previously reported bipartite nuclear localization signals (NLS) of two cor genes of Poncirus, COR1 1 and COR 19, are functional, these genes were fused to a GUS reporter gene in the pCAMBIA 1301 binary vector (Figure 5-1). Epidermal cells were bombarded with control plasmid 1301 and plasmid containing the fusion constructs, 1301-COR1 1, COR19, and COR1 1C. Histochemical GUS assays showed that all constructs resulted in GUS expression in the onion epidermal cells, indicating that all constructs were functional and the particle bombardment was successful. Analysis of GUS expression and its distribution in the cells with microscopy demonstrated that in onion epidermal cells bombarded with control plasmid pCAMBIA 1301 blue GUS staining was evenly distributed in the cytoplasm, indicating that the GUS localized in the cytoplasm and did not reach the nucleus by diffusion (Figure 5-2). In contrast to 1301,

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101 Figure 5-2. Sub-cellular localization of COR genes in onion epidermal cells. The maps of partial T-DNA regions of the control plasmid and plasmids containing COR gene sequences fused to the GUS reporter gene are shown on the left. Pictures showing the expression and distribution of GUS in onion epidermal cells bombarded with control and nuclear localization constructs are presented on the right side of the individual constructs. Pictures on the left panel present the histochemical GUS staining showing the expression and distribution of GUS in the onion epidermal cells bombarded with specific constructs. Pictures on the right panel present the DAPI staining showing the location of the nucleus in the onion epidermal cells

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102 the expression of GUS was concentrated mainly in the nucleus, with some GUS expression observed in the cytoplasm of the epidermal cells of onion bombarded with COR1 1 and COR19 fusion constructs. The results demonstrated that the COR genes are able to localize GUS in the nucleus of onion epidermal cells indicating that the previously proposed nuclear localization signals of these two genes are functional. When the GUS staining was done for a short period of time (6 h) and/or the expression of GUS in the individuals cells was low, GUS expression was first seen in the nucleus and as the expression increased, the GUS staining started to appear in the cytoplasm (data not shown), indicating that the nucleus is the primary target for these proteins. To determine if temperature has any effect on nuclear localization of these gene products, after initial incubation at 28°C, the bombarded epidermal cells were transferred to 4°C. No difference in GUS staining was observed between the samples kept at 28°C and the ones transferred to 4°C, indicating that temperature has no effect on nuclear localization of these proteins in onion epidermal cells. Since the nuclear localization signals of COR1 land COR19 were found close to the C-terminus of the proteins, the Nterminal half of COR1 1 was deleted and only the Cterminal half of the protein was fused to GUS gene in the binary vector to generate pCAMBIA 1301-COR1 1C (Figure 5-1). When the onion epidermal cells were bombarded with this construct, a high level of GUS expression was observed in nuclei which were identified by DAPI staining and some GUS expression was also found in the cytoplasm, showing that the C-terminal half of the COR1 1 is sufficient for the nuclear localization of the fusion GUS reporter gene (Figure 5-2). As the other constructs, temperature had no effect on nuclear localization of this construct.

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103 Discussion Two cold regulated genes, COR1 1 and COR 19, showing homology to dehydrins were previously isolated from Poncirus trifoliata. These genes not only contained conserved signature domains of Kand S-segments but also contain sequences similar to previously characterized bipartite NLS (Cai et al. 1995). Since it has been proposed that dehydrins are involved in protection of membranes and proteins of cells under environmental stress conditions (Close 1996; 1997; Ismail et al. 1999), the sub-cellular location of these proteins are important for understanding their function in the cell. Therefore, tissue distribution and sub-cellular localization of dehydrins have been explored extensively to assign function(s) for specific dehydrins isolated from different plants. Immunolocalization and sub-cellular fractionation assays revealed that the majority of dehydrins are localized both in the nucleus and cytoplasm of different plant cells (Monroy et al 1993; Goday et al. 1994; Godoy et al. 1994; Houde et al. 1995; Close 1996; 1997). Sub-cellular localization of COR1 1 and COR19 in onion epidermal cells showed that these proteins are localized in the cytoplasm and nucleus indicating that in addition to sequence homology, Poncirus dehydrins may be functionally similar to previously characterized dehydrins from other plants which are localized in the nucleus and cytoplasm (Monroy et al 1993; Goday et al. 1994; Godoy et al. 1994; Houde et al. 1995; Close 1996). Localization of these proteins in the nucleus and cytoplasm suggests that they may be involved in the protection of membranes and macromolecules in the nucleus and cytoplasm as was proposed for other dehydrins (Close 1997). The expression level of the GUS reporter gene was consistently and significantly higher in the nucleus than the cytoplasm of onion epidermal cells bombarded with all three constructs in repeated

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104 experiments. In addition, when GUS expression was analyzed early following bombardment (6-hr) and/or the level of GUS expression was low in individual cells, the proteins were primarily localized in the nucleus. These results suggested that although COR1 1 and COR19 were found both in the nucleus and cytoplasm, the primary target of these proteins is the nucleus. Accumulation of COR1 1 and COR19 primarily in the nucleus implies that they may be involved in protection of macromolecules in the nucleus such as regulatory proteins and/or transcriptional machinery under cold stress. A similar function was also suggested for a wheat dehydrin, WCS120, which is localized in the nucleus and cytoplasm of wheat crown tissue (Houde et al. 1995). Some dehydrins are localized in the nucleus after posttranslational modification of the proteins through phosphorylation of S residues in the S-segment (Goday et al. 1994; Godoy et al. 1994) and others require certain environmental stress such as cold for nuclear localization (Rinne et al. 1999). Since maintaining bombarded cells at 28 and 4 °C did not have any effect on the distribution of the proteins in the cell, unlike the RAB16 dehydrin from birch, (Betula pubescens) (Rinne et al. 1999) neither of the Poncirus dehyrins require change in temperature for their localization in the nucleus. The phosphorylation status of the S-segment of COR1 1 and COR19 and its effect on the nuclear localization was not studied and is still unknown. It has been reported that dehydrins from some plants such as RAB21 from rice and CAP85 from spinach were principally or exclusively localized in the cytoplasm (Mundy and Chua 1988; Neven et al. 1993). More recently it has been shown that dehydrins from some plants localize primarily in chloroplasts (Wisniewski et al. 1999) or plasma membranes (Danyluk et al. 1998). In this study, no specific localization of COR

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105 genes in cell walls or plasma membranes of onion epidermal cells was observed. Other dehydrins, including the COR19 from Citrus unshiu (CuCOR19) which is a homologue of COR 19 from Poncirus used in this study, were mainly localized in the mitochondria (Borovskii et al. 2000; 2002; Hara et al. 2003). Sub-cellular fractionation of transgenic tobacco expressing CuCOR19 showed that although the main location of the CuCOR19 was the mitochondria some of the protein was found in the nucleus (Hara et al. 2003) indicating that even very similar dehydrins from closely related species may show different sub-cellular localization. The variation in the localization patterns of these two proteins may be due to the differences in the method or the type of plant cell used for sub-cellular localization studies. Constitutive expression of CuCOR19 in tobacco decreased electrolyte leakage by reducing lipid peroxidation and the CuCOR19 protein expressed in bacteria prevented peroxidation of soybean liposomes in vitro. Based on these results it was suggested that this dehydrin increased cold tolerance by protecting the integrity of membranes of the cell by decreasing peroxidation in tobacco. This study was focused on sub-cellular localization of two dehydrins from Poncirus and provided experimental evidence for localization of these proteins in the nucleus and cytoplasm. Understanding of the role of Poncirus COR1 1 and COR19 genes in cold tolerance and their possible mechanism of action will require further experiments in Poncirus or other plants.

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CHAPTER 6 SUMMARY AND CONCLUSIONS Citrus is one of the most important fruit crops in the world with an annual production exceeding one hundred million metric tons. Production of citrus is mostly limited by low temperatures outside the tropical and subtropical regions. Most commercial citrus types are cold-sensitive and subjected to low temperatures and freezes in the subtropical regions. Occasional freezes cause significant damage and economic losses in some citrus growing regions, including Florida. Therefore, improving cold tolerance of commercial varieties is an important goal for the citrus industry. Poncirus trifoliata is a cross compatible Citrus relative and unlike Citrus, it can tolerate temperatures as low as -26 °C when it is cold-acclimated. Thus, it serves as good genetic resource for improving cold tolerance in Citrus. Because of this characteristic, it has been used in breeding programs; however, mechanisms of cold response in Poncirus have not been explored at the molecular level. To identify genes induced during cold acclimation, reverse and forward subtracted cDNA libraries were prepared using cold-acclimated and nonacclimated Poncirus seedlings. A total of 192 randomly picked colonies, 136 from forward and 56 from reverse subtracted libraries were sequenced and they showed that a number of cDNA clones had homology to previously characterized cold response genes in other plants. Expression studies with reverse northern blot analysis demonstrated that expression of 97 cDNAs was changed two to 49-fold, indicating that they are coldregulated. Among them, 96 were induced and only one was repressed by cold. Cold106

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107 induced genes identified in this study have homology to different groups of genes, including transcription factors and DNA binding proteins, heat shock proteins, LEAs, and some metabolic genes. Differential expression of 17 selected cDNAs in response to cold was confirmed by northern blot analysis using gene specific probes in cold-acclimated and nonacclimated Poncirus plants. Gene expression studies in other plants revealed that multiple pathways are involved in regulation of gene expression during cold acclimation. Expression of coldregulated genes is controlled by a number of transcription factors, some of which have already been characterized. A number of cDNAs from our cold-induced subtractive cDNA library show homology to previously characterized or putative transcription factors. Three of these cDNAs, PI-B05, PI-C10, and PII-C02 were selected for further characterization. The full-length sequences of these cDNAs were obtained by 3' and 5' RACE using gene specific primers obtained from partial cDNA sequences. Sequence analysis of the full-length cDNA of PI-B05 encodes a protein containing an AP2 DNA-binding domain and showed homology with previously characterized AP2 domain transcription factors from Arabidopsis and other plants. Northern blot analysis revealed that expression of this gene was induced in response to cold acclimation in Poncirus, but not in pummelo. No changes in expression were detected in response to drought either in Poncirus or pummelo, indicating that this gene is induced only by cold in Poncirus. The full-length cDNA of PI-C10 codes for a RAV2-like protein with two different, AP2 and B3, DNA-binding domains. It showed homology to previously characterized RAVI and RAV2-like proteins from other plants. When the expression of

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108 PI-C10 was studied in response to cold and drought in Poncirus and pummelo, expression of PI-C10 was increased only in response to cold in Poncirus. No expression was detected in response to drought in Poncirus or in response to cold and drought in pummelo suggesting that PI-C10 is a cold-regulated gene in Poncirus. Sequence analysis revealed that the PII-C02 cDNA encodes a RING zinc finger protein that showed homology with two RING zinc finger proteins from Arabidopsis, one of which is induced by cold. Northern blot analysis showed that the expression of PIIC02 was induced in response to cold and drought in both Poncirus and pummelo. The increase in gene expression was more significant in response to drought in both plants. Sub-cellular localization of COR1 1 and COR19 in onion epidermal cells with GUS fusion constructs showed that these proteins are localized in the nucleus and cytoplasm, but accumulated predominantly in the nucleus. Deletion of the N-terminal half of COR1 1 demonstrated that C-terminal half conserved in both genes was sufficient for nuclear localization. This study reports the first comprehensive analysis of gene expression during cold acclimation and identification of over 90 cold-regulated genes in cold-hardy Poncirus. The results of expression analysis of cold-regulated genes by various methods lead to following conclusions: (1) Identification of many genes regulated by cold confirms the quantitative nature of the cold hardiness trait in Poncirus; (2) Cold-regulated genes identified in Poncirus are involved in a variety of cellular functions indicating that cold acclimation induces many biochemical and physiological changes in Poncirus as in other plants; (3) Most of the cold regulated genes identified in Poncirus are similar to coldregulated genes in Arabidopsis and other plants suggesting the presence of similar cold

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109 responsive pathways are activated during cold acclimation; (4) Some of cold-regulated genes are induced in cold-hardy Poncirus, but not in cold-sensitive pummelo indicating that differential expression of these genes and others may be responsible for cold hardiness in Poncirus. The purpose of this project was to study changes in gene expression during coldacclimation in cold-hardy citrus relative Poncirus. Using subtractive hybridization, reverse northern, and northern blot analysis, more than 90 genes were identified. Although this may not be the complete list of genes regulated by cold in Poncirus, this study provides the first comprehensive information about changes in gene expression during cold acclimation. The genes identified in this study, especially the ones encoding regulatory proteins, should be further characterized to elucidate their functions and involvement in cold hardiness. Since two of these genes encode proteins with conserved DNA-binding domain(s) and show homology with previously characterized transcription factors, DNA binding activity of these genes can be demonstrated by electrophoretic mobility shift assay or yeast one-hybrid system. Once the binding specificity is confirmed, the target genes containing the specific regulatory sequences can be identified. Constitutive expression and/or antisense inhibition of these genes in Poncirus may reveal the role of these genes in cold hardiness. In addition, since these genes were not induced or induced little in response to cold in pummelo, overexpression of these genes in cold-sensitive Citrus species may improve their cold tolerance.

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BIOGRAPHICAL SKETCH Mehtap Sahin-Cevik was born in Malatya, Turkey in 1972. Ten years later, she moved to Ankara and completed her high school education there. She graduated from the College of Agriculture at Ankara University with B. S. degree in horticulture in June 1994. One year after graduation, in July 1995, she was awarded a scholarship by the Turkish Ministry of Education to pursue M.S. and Ph.D. degrees in the field of Horticulture in the USA. Before coming to the USA, she studied English for 6 months at Middle East Technical University in Ankara, Turkey. In May 1996, she came to Gainesville, Florida, and attended the English Language Institute at the University of Florida for 1 year. She began her master's degree program in the Horticultural Sciences Department in May 1 997 and worked on molecular markers and QTL mapping in Citrus. After finishing her master's degree program, she started her Ph.D. program in the Horticultural Sciences Department in May 1999. She is expected to graduate in December 2003. 129

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Gloria A. Moore Professor of Horticulture Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Wayn£ B. Sherman Professor of Horticulture Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Miuia Gallo-Meagher Associate Professor of Agronomy This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosop hy. December 2003 Dean, College of Agricultui^ Sciences Dean, Graduate School