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Physiological Responses of Flooded Avocado Trees (Persea americana Mill) to Leaf Removal

Permanent Link: http://ufdc.ufl.edu/UFE0044263/00001

Material Information

Title: Physiological Responses of Flooded Avocado Trees (Persea americana Mill) to Leaf Removal
Physical Description: 1 online resource (101 p.)
Language: english
Creator: Sanclemente Galindo, Maria Angélica
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: avocado -- carbohydrates -- exchange -- flooding -- gas -- leaf -- pruning -- removal -- respiration
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
Electronic Thesis or Dissertation
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )

Notes

Abstract: Effects of leaf removal before or after root zone flooding on the physiology and survival of avocado (Persea americana) trees was quantified. Plants had either no (control), one-third (1/3LR), one-half (1/2 LR), or two-thirds (2/3LR) of their leaves removed before or after flooding. Net CO sub two assimilation (A), stomatal conductance (g sub s), transpiration (E), and plant growth measurements were made for flooded and non-flooded plants. Leaf removal or foliar application of Freeway® (a chemical photosynthetic inhibitor) prior to flooding, resulted in significantly lower A, g sub s and dry weights of flooded compared to non-flooded plants. Survival of flooded plants sprayed with Freeway® was 66.6%, whereas survival of non-sprayed plants was 83.3%. Concentration of the C sub seven sugar, D-mannoheptulose, was higher in non-flooded than in flooded plants. In flooded plants, concentration of the C sub seven sugar alcohol, perseitol, was higher in plants with no leaves removed than those in the 2/3 LR treatment. Flooded plants with no leaves removed tended to have higher root respiration than flooded plants with leaves removed. Leaf removal before flooding resulted in lower A and g sub s compared to plants with no leaves removed. When leaves were removed before flooding, only 16.6% of flooded plants in the 2/3LR treatment survived compared to 50% of flooded plants in the 1/3LR treatment. In contrast, removing the same amount of leaves after flooding resulted in 66.6% survival for the 2/3LR and 83.3% for the 1/3LR treatments. In all experiments, flooding reduced A and gs. However, leaf removal after flooding resulted in A and gs returning to levels similar to those of non-flooded trees several days after plants were unflooded. Leaf removal before flooding increased flooding stress, presumably by reducing carbohydrate reserves in the roots, whereas leaf removal after flooding reduced flooding stress presumably by reducing transpirational area.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Schaffer, Bruce A.
Statement of Responsibility: by Maria Angélica Sanclemente Galindo.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044263:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044263/00001

Material Information

Title: Physiological Responses of Flooded Avocado Trees (Persea americana Mill) to Leaf Removal
Physical Description: 1 online resource (101 p.)
Language: english
Creator: Sanclemente Galindo, Maria Angélica
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: avocado -- carbohydrates -- exchange -- flooding -- gas -- leaf -- pruning -- removal -- respiration
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
Electronic Thesis or Dissertation
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )

Notes

Abstract: Effects of leaf removal before or after root zone flooding on the physiology and survival of avocado (Persea americana) trees was quantified. Plants had either no (control), one-third (1/3LR), one-half (1/2 LR), or two-thirds (2/3LR) of their leaves removed before or after flooding. Net CO sub two assimilation (A), stomatal conductance (g sub s), transpiration (E), and plant growth measurements were made for flooded and non-flooded plants. Leaf removal or foliar application of Freeway® (a chemical photosynthetic inhibitor) prior to flooding, resulted in significantly lower A, g sub s and dry weights of flooded compared to non-flooded plants. Survival of flooded plants sprayed with Freeway® was 66.6%, whereas survival of non-sprayed plants was 83.3%. Concentration of the C sub seven sugar, D-mannoheptulose, was higher in non-flooded than in flooded plants. In flooded plants, concentration of the C sub seven sugar alcohol, perseitol, was higher in plants with no leaves removed than those in the 2/3 LR treatment. Flooded plants with no leaves removed tended to have higher root respiration than flooded plants with leaves removed. Leaf removal before flooding resulted in lower A and g sub s compared to plants with no leaves removed. When leaves were removed before flooding, only 16.6% of flooded plants in the 2/3LR treatment survived compared to 50% of flooded plants in the 1/3LR treatment. In contrast, removing the same amount of leaves after flooding resulted in 66.6% survival for the 2/3LR and 83.3% for the 1/3LR treatments. In all experiments, flooding reduced A and gs. However, leaf removal after flooding resulted in A and gs returning to levels similar to those of non-flooded trees several days after plants were unflooded. Leaf removal before flooding increased flooding stress, presumably by reducing carbohydrate reserves in the roots, whereas leaf removal after flooding reduced flooding stress presumably by reducing transpirational area.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Schaffer, Bruce A.
Statement of Responsibility: by Maria Angélica Sanclemente Galindo.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044263:00001


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1 PHYSIOLOGICAL RESPONSES OF FLOODED AVOCADO TREES ( PERSEA AMERICANA MILL.) TO LEAF REMOVAL By MARIA ANGLICA SANCLEMENTE GALINDO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMEN T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Maria Anglica Sanclemente Galindo

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3 To my parents, Ale, and my best friend and favorite architect, Diana

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4 ACKNOWLEDGMENTS I would like to thank al l my big and great family. They all played a special role during the last two years of my degree To my Aunts Ito and Adriana for hosting me in their ho mes during my research. To my A buelo Sanclemente for spoiling me with his warm arepas and pan debonos whenever I went to visit him after fieldwork. S pecial t hanks to my parents for their endless love and constant support. To Diana, f or her friendship and for being there during the good and bad, sponsoring my whims, and supporting my decisions To A le, for his advice, support and encouragement to always achieve my maximum potential. They are all my motivation and my source of joy and strength. Great many thanks to my graduate committee members for their guidance and dedication during my m ree program, particularly during the writing process of this thesis. Specifically, I thank my advisor, Bruce Schaffer who has supported me and offered me invaluable knowledge and motivation to continue my studies after receiving this degree. I also t hank Frederick Davies for his support and guidance throughout my m degree studies and Jonathan C rane for his great tropical fruit production and research class. Finally, Daisy for her warm company and for her assistance with the data collection and instruments operation. I also thank Robert Dowell from Virginia Tech and Letty Almanza and Manuel Sacramento from the Universidad de Cordoba for giving me my first mentoring experience and for their invaluable help during the expe rimental phase of my research.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 1 0 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 1 2 2 LITERATURE REVIEW ................................ ................................ .......................... 1 5 Avocado ................................ ................................ ................................ .................. 1 5 Flooding and Hypoxia stress ................................ ................................ ................... 1 6 Effect of Low Oxygen Concentration on Avocado Physiology, Growth, and Yield .. 1 7 Effect of Low Oxygen Concentration on Leaf Gas Exchange ........................... 2 3 Interactions Between Above Ground Factors and Flooding Stress in Fruit Crops ................................ ................................ ................................ ............ 2 4 Effect of Low Oxygen Concentration on Sap Flow ................................ ........... 29 S u mmary ................................ ................................ ................................ ................ 3 0 3 LEAF REMOVAL AND FLOODING EFFECTS ON LEAF GAS EXCHANGE, ROOT CARBOHYDRATE CONCENTRATION, GROWTH, AND SURVIVAL OF AVOCADO TREES ................................ ................................ ................................ 3 2 Background ................................ ................................ ................................ ............. 3 2 Materials and Methods ................................ ................................ ............................ 3 5 Results ................................ ................................ ................................ .................. 4 0 Discussion ................................ ................................ ................................ .............. 4 4 4 PRE AND POST FLOODING LEAF REMOVAL EFFECTS ON LEAF GAS EXCHANGE, GROWTH, AND SURVIVAL OF AVOCADO TREES EXPOSED TO ROOT ZONE FLOODING ................................ ................................ ................. 59 Background ................................ ................................ ................................ ............. 59 Materials and Methods ................................ ................................ ............................ 63 Results ................................ ................................ ................................ ................... 68 Discussion ................................ ................................ ................................ .............. 7 0 5 CONCLUSIONS ................................ ................................ ................................ ..... 8 8 LIST OF REFERENCES ................................ ................................ ............................... 9 3

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6 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 10 1

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7 LIST OF TABLES Table page 3 1 Percentage of tree survival and mortality ees on Waldin seedling rootstock in each canopy treatment at the end of Expt. 1 (day 49) ................................ ................................ ................................ ............. 5 5 4 1 Number and p ercentage of tree mortality for flooded and non flooded 3 year stock with in each leaf removal treatment (Expt.1).Plants were harvested 52 days after plants were unflooded Leaf removal treatments were: no leaves removed (control), one third (1/ 3 LR ), one half (1/2 LR ) or two thirds (2/3LR of the leaves removed (2/3 LR ) b efore plants were flooded ................................ ................................ ... 8 6 4 2 Number and p ercentage of tree mortality for flooded and non flooded 2 year Simmonds leaf removal treatment (Expt.2). Plants were harv ested 52 days after flooding treatments were initiated (5 days after plants were unflooded ). Leaf removal treatments were: one third of the leaves removed (1/3 LR ), half of the leaves removed (1/2 LR ), and two thirds of the leaves removed (2/3 LR ) after plant s were flooded ................................ ................................ ................................ ...... 8 7

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8 LIST OF FIGURES Figure page 3 1 Soil redox potential of flooded 2 year old (Expt.1) and 1 year old (Expt. 2) do trees on Waldin seedling rootstock ................................ ... 4 9 3 2 Effect of flooding on net CO 2 assimilation (A) for 2 year avocado trees on Waldin seedling rootstock. ................................ ..................... 5 0 3 3 Effect of flooding on s tomatal conductance to water vapor (g s ) for 2 year old avocado trees on Waldin se edling rootstock ................................ ... 5 1 3 4 Effect of flooding on net CO 2 assimilation (A) for 1 year avocado trees on Waldin seedling rootstock ................................ ...................... 5 2 3 5 Effect of flooding on stomatal conductance to water vapor (g s ) of 1 year old (Expt. 2). ................... 5 3 3 6 Effect of flooding on root, leaf, stem, and total plant dry weight of 2 year old ree canopy treatments (Expt. 1). ................................ ................................ .............. 5 4 3 7 Root respiration of non flooded (NF) and flooded (F) tre atments within the leaf removal treatment s and between the control and leaf removal treatment within each flooding treatment. ................................ ................................ .......... 5 6 3 8 Root carbohydrate concentrations for control and leaf removal treatments for 1 year reatments (Expt. 2) ......... 5 7 3 9 R oot carbohydrate concentrations for non flooded and flooded treatments for 1 year af removal treatments (Expt. 2). ................................ ................................ ................................ ..................... 5 8 4 1 S oil redox potential of flooded 3 year old and 2 year old s (Expt. 2) ................................ ................................ ................................ .............. 7 7 4 2 Net CO 2 assimilation ( A) of flooded and non flooded 3 year avocado trees on Waldin seedling rootstock in each of 4 leaf removal treatments (Expt. 1) ................................ ................................ ............................ 7 8 4 3 Stomatal conductance of water vapor (g s ) of flooded and non flooded 3 year removal treatments (Expt. 1) ................................ ................................ .............. 7 9

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9 4 4 Net CO 2 assimilation (A) of flooded and non flooded 2 year avocado trees on Waldin seedling roots tock in each of 4 leaf removal treatments (Expt. 2) ................................ ................................ ............................ 8 0 4 5 Stomatal conductance of water vapor (g s ) of flooded and non flooded 2 year four le af removal trea tments (Expt. 2) ................................ ................................ ........ 8 1 4 6 Transpiration (E) of flooded and non flooded 2 year trees on Waldin seedling rootstock in each of four leaf removal treatments (Expt. 2) ................................ ................................ ................................ .............. 8 2 4 7 Roo t, leaf, stem, and total plant dry weights of flooded and non flooded 3 year four leaf removal tre atments (Expt. 1) ................................ ................................ 8 3 4 8 Root, leaf, stem, and total plan t dry weight of flooded and non flooded 2 year four leaf removal t reatments (Expt. 2) ................................ ............................... 8 4 4 9 Daily sap flow of flooded and nonflooded trees of 2 year avocado trees on Waldin seedling rootstock (Expt. 2) ................................ ........ 8 5

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10 Abstract of Thesis Presented to the Graduate School of the University of F lorida in Partial Fulfillment of the Requirements for the D egree of Master of S cience PHYSIOLOGICAL RESPONSES OF FLOODED AVOCADO TREES ( PERSEA AMERICANA MILL) TO LEAF REMOVAL By Maria Anglica Sanclemente Galindo May 2012 Chair: Bruce Schaffer Major: Horticultural Science Effects of leaf removal before or after root zone f looding on the physiology and survival of avocado ( Persea americana ) trees was quantified. Plants had either no (control), one third (1/3LR), one half (1/2 LR), or two thirds (2/3LR) of their leaves removed before or after flooding. Net CO 2 assimilation ( A), stomatal conductance (g s ), transpiration (E), and plant growth measurements were made for flooded and non flooded plants. Leaf removal or foliar application of Freeway (a chemical photosynthetic inhibitor) prior to flooding, resulted in significantly lower A, g s and dry weights of flooded compared to non flooded plants Survival of flooded plants sprayed with Freeway was 66.6%, whereas survival of non sprayed plants was 83.3%. Concentration of the C 7 sugar, D mannoheptulose, was higher in non flooded than in flooded plants. In flooded plants, concentration of the C 7 sugar alcohol, perseitol, was higher in plants with no leaves removed than those in the 2/3 LR treatment. Flooded plants with no leaves removed tended to have higher root respiration than flooded plants with leaves removed. Leaf removal before flooding resulted in lower A and g s compared to plants with no leaves removed. When leaves were removed before flooding, only 16.6% of flooded plants in the 2/3LR treatment survived compared to 50%

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11 of flooded plants in the 1/3LR treatment. In contrast, removing the same amount of leaves after flooding resulted in 66.6% survival for the 2/3LR and 83.3% for the 1/3LR treatments. In all experiments, flooding reduced A and g s However, leaf removal aft er flooding resulted in A and g s returning to levels similar to those of non flooded trees several days after plants were unflooded. Leaf removal before flooding increased flooding stress, presumably by reducing carbohydrate reserves in the roots, whereas leaf removal after flooding reduced flooding stress presumably by reducing transpirational area.

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12 CHAPTER 1 INTRODUCTION Flooding in agricultural fields may develop due to anthropogenic (e.g., poor site selection, improper irrigation practices) or natural (e.g., flooding, soil compaction) factors leading to altered plant metabolism, growth, and/or yield and plant mortality. Furthermore, an anticipated increase in air temperature and resultant effects on other climatic variables, such as rainfall, as a resu lt of global climate change, may increase the potential for flooding in many areas of the world (IPPC, 2001). Therefore, for agriculture to remain productive in flood prone or potentially flood prone areas, it is important to understand how flooding affec ts crop physiology, growth, yield, and survival. This information should assist with the selection and development flood adapted crops and production systems for areas that experience periodic short term flooding (Schaffer 1998) Avocado ( Persea america na Mill ) is a subtropical to tropical evergreen tree in the Lauracea e (Purseglove, 1968). This crop is grown internationally in Mediterranean, subtropical and tropical regions, and wor ld production area and trade are rapidly increasing (Schaffer et al., 2012). In 2010, w orld production of avocado was estimated to be 3,840,905 tones/year (FAO, 2010). In the United States, the vast majority of avocado production is in California, where there are an estimated 21,108 hectares planted (FAO, 2010). Although there are only 2,995 hectares of avocado planted commercially in Florida, it is second only to citrus in state annual farm gate sales (Evans and Nalampang 2010). Rapidly increasing p roduction of avocado in several regions of the world has resulted in production in marginal sites that are prone to flooding or poor soil drainage

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13 (Schaffer, et al. 2012 ). In South Florida, avocado orchards are often planted in low lying areas with a hi gh water table (1.8 2.3 meters below the soil surface) (Barquin Valle et a l., 2009) and periodic flooding due to heavy rains from tropical storms or hurricanes (Crane et al., 1994). Thus, in Florida and other avocado production areas that are prone to wat erlogging, there is a need to understand the negative impacts of soil flooding on avocado physiology to help devise adequa te crop management strategies to avoid or ameliorate tree and crop losses due to periodic flooding. The root system of avocado is rela tively shallow and does not spread much beyond the tree canopy (Ferreyra et al., 2007). Roots are extensively suberized, with low hydraulic conductivity, few root hairs, poor water uptake and high sensitivity to low soil oxygen concentrations (Ferreyra et al., 2006). These characteristics make avocado trees one of the most susceptible fruit trees to soil flooding (Ferreyra et al., 2006). A few days of flooding avocado trees with a high shoot/root ratio can result in reductions in photosynthesis and yield, a nd a high degree of tree mortality (Schaffer 2008). Part of the suggested pre hurricane practices for avocado (and other subtropical fruit tree species) in southern Florida is to control tree size by removing part of the canopy to reduce wind damage (Cra ne et al., 1994). To mitigate the negative effects of flooding on stress and recovery avocado trees subjected to short term flooding, removing a portion of the tree canopy after floodwaters subside has been recommended. This practice presumably compensates for roots damaged by flooding by bringing the shoot/ root ratio into balance; effectively reducing the transpirational demand of the canopy and helping to avoid plant desiccation (Crane et al., 1994). These pre and post flood pruning recommendations are based solely on observations and not on experimental evidence.

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14 Thus, the effects of the proportion and timing of canopy removal relative to the time of flooding on avocado physiology has not been adequately quantified. The objectives of the research desc ribed in this thesis were: 1) t o determine if reducing the size of the canopy before or after flooding exacerbates stress and hinders recovery of avocado trees exposed to short term flooding; and 2) to quantify fluctuations in carbohydrate concentrations i n avocado trees exposed to short term flooding and pruning. The hypotheses tested were: 1) removing a portion of the canopy prior to the onset of flooding and the subsequent reduction in photosynthesis lowers the carbohydrate availability in the roots. Thu s, there should be less substrate (carbohydrate) for normal root respiration during flooding and floodin g stress is exacerbated; and 2). In contrast to pruning prior to flooding, pruning trees immediately after flooding increases survival of trees by bring ing the shoot/root ratio into balance so that there is sufficient root volume to adequately provide the existing canopy with water and nutrients. The results of this research should provide basic information about the physiological responses of avocado tr ees to root zone flooding and the interaction between leaf removal and flooding. Additionally, the study should enhance the understanding of the role of non structural carbohydrates in responses of avocado trees to low soil oxygen. The practical applicatio n of the results should assist growers by providing quantitative information about how to rehabilitate trees damaged by flooding relative to the amount and timing of canopy removal (pruning).

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15 CHAPTER 2 LITERATURE REVIEW Avocado Avocado ( Persea americana ) is a polymorphic tree species, belonging to the Lauraceae family (Judd, 1999), that evolved in the neotropical rainforest as an overstory canopy tree (Wolstenholme, 2002). It is native to a wide geographical zone including the highlands of central and eas tern Mexico and Guatemala and the Pacific coast of Central America (Knight, 2002). Persea americana consists of several taxa, considered botanical varieties or subspecies that include varieties of commercial interest. The three subspecies, also referred t Persea americana var. americana Mill. (Antillean or West Indian race), Persea americana var. drymifolia Blake (Mexican race), and Persea americana var. guatemalensis Williams (Guatemalan race) (Scora and Bergh, 1990). These t hree races originated wholly or partly within tropical latitudes in Central America and the tree is often considered a tropical species (Wolstenholme, 2002). Although the scientific names for the three races have become imbedded in the literature, it has r ecently been noted that they were not validly published according to standard procedures for taxonomic naming, and they will likely be renamed based on recent genetic analysis (Chanderbali et al., 2012). Avocado trees evolved in andosol soils derived from volcanic ash which are considered the optimum soil type for tree growth due to their physical properties, mainly low bulk density (0.5 0.8 g/cm 3 ), high macro porosity (approx. 46%), high organic matter content, and a low pH between 5 and 6 (Ferreyra et al. 2007). The root system of avocado trees is relatively shallow and does not spread much beyond the tree canopy. Roots are extensively suberized having low hydraulic conductivity, few root hairs, poor

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16 water uptake and sensitivity to low soil oxygen concen trations (Ferreyra et al., 2006; Wolstenholme, 2012). These characteristics may have evolved because of frequent rains that occur in the indigenous rainforest habitat and rapidly drained soils that are conducive to the high root oxygen requirement and sens itivity to poor soil drainage, and the presence of a rich surface organic mulch resulting in a tendency for healthy feeder roots to grow into any decomposing litter layer (Scora et al., 2002). Due to the high root oxygen requirement, avocado trees exposed to root hypoxia, even for short periods as a result of flooding, have decreased leaf expansion, reduced root and shoot growth, root necrosis, and moderate to severe leaf abscission (Ferreyra et al., 2006; Schaffer 2006). Flooding and Hypoxia Stress Abiotic plant stress is defined as any environmental condition that causes harmful biological effects (Salisbury and Ross, 1992). Plants are autotrophic and therefore any change that directly or indirectly reduces the accumulation of biomass should be considered biologically harmful even when it benefits parts of the plant (Salisbury and Ross, 1992). A major environmental factor limiting plant growth and yield worldwide, especially in high rainfall regions, is hypoxia (Bai et al., 2009). Hypoxia refers to the redu ction of oxygen below optimal levels for the normal functioning of the plant, organ, or cell and generally occurs at soil concentrations less than 2 mg O 2 L 1 H 2 O (Gibbs and Greenway, 2003). Whereas, concentrations below 1 mg O 2 L 1 H 2 O or complete lack of oxygen in the soil or medium is referred to as anoxia (Drew, 1997). Low oxygen conditions occur in poorly drained soils or during periods of short term flooding (Drew, 1997). In many areas of the world, including southern Florida, tropical and subtropica l fruit crops, including avocado trees, are grown in areas that often experience flooding from

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17 rising water tables and/or heavy rain from tropical storms or hurricanes (Crane et al., 1994; Schaffer and Whiley, 2002). Although all higher plants require wate r, excess water in the root environment can be injurious or even lethal because it displaces oxygen and other gases in the soil (Drew, 1997; Geigenberger, 2003). Redox potential is an indirect measure of soil oxygen content. Well drained soils are charact erized by redox potentials of +300mV or greater, whereas flooded soils have redox potentials of 200 mV or lower (Kozlowski, 1997). Soil flooding disrupts the metabolism of mesophytic plant species by displacing O 2 from soil pores and promoting O 2 depletion by roots and soil microbes (Nuez Elisea et al., 1999). These processes not only reduce the oxygen content but also increase the concentration of toxic organic and inorganic compounds in the soil (Irfan et al., 2010). This production of toxic metabolites increases during high temperatures and at night, when root respiration increases, together with a simultaneous increase in microbial activity (Irfan et al., 2010). Since respiration increases exponentially with temperature, short term increases or decreas es in soil temperature will decrease or increase the overall requirements of energy associated with root maintenance (Atkin et al., 2000). Effect of Low Oxygen Concentration on Avocado Physiology, Growth, and Yield Reduction of root respiration is one the earliest responses of plants to anoxic soil conditions regardless of whether the plants are tolerant or intolerant to flooding (Liao and Lin, 2001). When the soil oxygen concentration is low, the activity of cytochrome oxidase is limited, thus reducing ade nosine triphosphate (ATP) production during root respiration (Geigenberger, 2003). Reduced availability of oxygen as the final electron acceptor in the mitochondrial electron transport chain mediates a rapid reduction of the ATP / ADP ratio and adenylate ene rgy charge (Serres and Voesenek, 2008). Cells adapt

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18 to this energy reduction by relying on glycolysis and fermentation to generate ATP and regenerate ADP, respectively (Serres and Voesenek, 2008). In plant cells, both aerobic and anaerobic respiration beg ins with glycolysis, where glucose is oxidized to pyruvic acid by a series of reactions. During the conversion of glucose to pyruvic acid a net 2 ATP molecules are produced. After glycolysis, carbon metabolism will proceed to aerobic respiration if suffic ient oxygen is available or otherwise, to anaerobic respiration (also called fermentation) (Geigenberger, 2003; Salisbury and Ross, 1992). There are two types of fermentation defined by the product of the chemical reaction: lactic acid fermentation, where the end product is lactic acid ; and alcoholic fermentation where the end products are CO 2 and ethanol (Taiz and Zieger, 2006). Both types of fermentation occur without producing ATP (Taiz and Zeiger 2006). In the second and third stages of aerobic respira tion, respectively called the tricarbox cylic acid (TCA) cycle and the electron transport chain, up to 36 ATP molecules can be produced (Salisbury and Ross, 1992). Therefore, aerobic respiration produces significantly more energy in the form of ATP than ana erobic respiration (Salisbury and Ross, 1992). Root cell death due to rapid exposure to anoxic conditions has been associated with acidification of the cytoplasm, referred to as cytoplasmic acidosis (Drew, 1997). Sudden exposure to anaerobic conditions re sults in lactic acid fermentation, and protons leaking from the vacuole can significantly lower the cytoplasmic pH (Licausi and Perata, 2009; Schaffer, 2006). In anoxic conditions, cytoplasmic acidosis is believed to be the primary cause of plant cell deat h (Drew, 1997). The production of potentially toxic metabolites in flooded roots as a result of anaerobic respiration has been implicated in

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19 plant cell death (Drew, 1997). Initially, ethanol produced in the roots during alcohol fermentation in anaerobic so ils and transported through the xylem was thought to be toxic to the plant (Drew, 1997). However, the ethanol concentration required to damage plant tissue is extremely high and ethanol readily diffuses out of plant tissues to the surrounding solution wher e it is diluted or metabolized by microorganisms (Drew, 1997; Irfan et al., 2010). Atkinson et al. (2008) found that in Forsythia the ethanol concentration in xylem sap and leaf tissue increased dramatically after four days of flooding ; however, toxicity symptoms were not observed. The immediate biochemical precursor to ethanol, acetaldehyde, is considerably more toxic to plant cells than ethanol and may be a factor in plant cell death during anaerobic root metabolism (Liao and Lin 2001; Schaffer, 2006). P yruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are two enzymes whose activities increase during root submergence, leading to the production of ethanol and regeneration of nicotinamide adenine dinucleotide (NAD+) (Sarkar et al., 2006). The acti vity of PDC is usually many fold lower than that of ADH, suggesting that the rate limiting enzyme for ethanol synthesis in flooded roots is more likely PDC than ADH (Liao and Lin, 2001). However, a high ADH/PDC ratio is required to prevent the accumulation of potentially toxic acetaldehyde (Liao and Lin, 2001; Su and Lin, 1996). In flooded luffa ( Luffa cylindrica ) roots, the acetaldehyde concentration did not rise in proportion to the induction of PDC activity, and it increased only slightly after five days of flooding indicating that a high ADH/PDC ratio is important to avoid accumulation of toxic levels of acetaldehyde (Su and Lin, 1996).

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20 Alcohol dehydrogenase, synthesized under anaerobiosis, is essential for growth and/or survival of plants during hypoxia This enzyme is a key regulator of glycolysis that supplements the depleting demand of NAD+ by catalyzing the conversion of acetaldehyde to ethanol (Keyhani et al., 2006). A significant amount of research with herbaceous plants has shown that increased AD to anoxia. This effect may be due to a role for ADH in avoiding a build up of acetaldehyde by enhancing its conversion to ethanol under anaerobic conditions (Schaffer, 2006). Wignarajah and Greenway (1976) found that ADH activity in the roots of Zea mays was highest with gas mixtures containing 8 13% oxygen in the root zone, intermediate with pure nitrogen, and lowest when the solution was flushed with gas containing 20% oxygen. The products of the fermentative pathwa y (acetaldehyde and ethanol) can be volatized through lenticels (Kozlowski, 1997), or transported to the leaves via the transpiration stream (Atkinson et al., 2008). In flooded mango trees, survival during flooding periods was attributed, at least in part, to the formation of hypertrophic stem lenticels that enhances oxygen diffusion to the roots. These stem openings may serve as excretory sites for the elimination of potentially toxic compounds such as acetaldehyde that results from anaerobic metabolism in flooded roots (Larson et al., 1993). The development of hypertrophic stem lenticels enhances O 2 diffusion to the roots in flooded soils. Hypertrophic stem lenticels have also been observed in Annona species, soybean ( Glycine max ), and forest trees inclu ding Erythrina speciosa (Medina et al., 2009; Nuez Elisea et al., 1999; Shimamura 2010) exposed to flooding of the root zone.

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21 Plant metabolic responses and adaptations to root zone hypoxia and anoxia have been thoroughly reviewed by Irfan (2010), Geigenbe rger (2003), and Kozlowski (1997). In a 1992 review of the effects of low soil oxygen levels on physiology and growth of fruit crops, including avocado (Schaffer et al., 1992), discussion of biochemical and cellular responses was based primarily on resear ch conducted with herbaceous plants. To date, the majority of studies on the effects of low soil oxygen on plant biochemistry and cellular biology still have focused on herbaceous plants and published reports are limited for woody species, including avoca do. The phytohormone, ethylene, has been implicated in a wide range of plant responses and/or adaptations to hypoxia and anoxia in the root zone. These include development of aerenchyma and hypertrophic stem lenticels, development of adventitious roots, a nd leaf epinasty (Drew, 1997; Viser and Voesenek, 2004). Hypoxia generally increases ethylene production, stimulating anatomical or morphological adaptations to low soil oxygen (Yamamoto et al., 1995). In contrast, anoxia decreases ethylene formation due t o the requirement of oxygen for conversion of aminocyclopropane 1 carboxylic acid (ACC) to ethylene, the final step in biosynthesis of ethylene (Yamamoto et al., 1995). In Japanese alder ( Alnus japonica ) seedlings, flooding increased ethylene production i n the submerged parts of the stems, which was related to the formation of adventitious roots and hypertrophied stem lenticels (Yamamoto et al., 1995). Although ethylene has an important role in flooding symptomology for several herbaceous and some woody pl ants, the relationship between ethylene and plant responses to low soil oxygen content in fruit trees remains

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22 unclear (Schaffer et al., 1992) and more work is needed to clarify the role of ethylene in these responses. Root hypoxia inhibits root and shoot growth by affecting many plant physiological processes, including chlorophyll biosynthesis and reactive oxygen species (ROS) (Geigenberger, 2003). Exposure of plants to most adverse conditions causes oxidative stress, which affects plant growth by producti on of ROS, such as superoxide radicals, singlet oxygen, hydroxyl radicals and hydrogen peroxide (Bai et al., 2009). These ROS are all very reactive and can cause severe damage to membranes, deoxyribonucleic acid (DNA), and proteins. Thus, understanding the interaction between enzymatic and non enzymatic ROS scavenging machinery is crucial for identifying key components involved in oxidative stress defense and manipulating hypoxia tolerance in plants. Bai et al. (2009) found that contents of superoxide radic als and hydrogen peroxide significantly increased in Malus hupe h ensis when roots were exposed to hypoxia stress. Elevated activit ies of the antioxidant enzymes superoxide dismutase, peroxidase, and ascorbate peroxidase were also observed. These activities increased after 12 days of hypoxia, in parallel with the accumulation of ascorbic acid and glutathione. These combined responses indicated that M. hupe h ensis may have a protective capacity against oxidative damage by maintaining higher induced activities o f the antioxidant system. Additionally, Keyhani et al. (2006) found that in corms of Crocus sativus the activities of superoxide dismutase and other ROS scavenging enzymes were stimulated under hypoxic or anoxic conditions. Avocado is considered a flood s ensitive species with physiological responses occurring shortly after soil becomes waterlogged (Schaffer et al., 1992). Short periods of

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23 flooding often result in leaf abscission, and leaf size can also be reduced by inhibition of expansion (Ferreyra et al. 2006). In addition to inhibiting shoot growth, flooding of avocado trees generally leads to an inhibition of root growth (Schaffer, 1998), and often root necrosis (Schaffer and Ploetz, 1989). No anatomical, morphological, or physiological adaptations wer e observed in studies of avocado trees in response to low soil oxygen levels (Schaffer, 2006). However, hypertrophic stem lenticels have recently been observed in flooded avocado trees in California (Schaffer, 2012). Ef fects of Low Soil Oxygen Concentratio n on Leaf Gas Exchange In hypoxic or anoxic soils, one of the earliest measurable changes in plants is a decline in leaf gas exchange. Reductions of net CO 2 assimilation in leaves is generally accompanied by decreases of stomatal conductance, transpiration and intercellular partial pressure of CO 2 (Irfan, 2010; Schaffer et al., 1992, 2006). Therefore, leaf gas exchange measurements are useful for determining the degree of flooding stress before any visible symptoms appear (Schaffer, 2006). In fruit crops, the time sequence for reductions of net CO 2 assimilation and stomatal conductance in response to flooding has not been clearly determined, and thus it is unclear which occurs first. Elucidating the temporal separation of these responses as a result of low soil oxygen content would be useful for determining if flood induced reductions of photosynthesis are due to stomatal or non stomatal factors (Schaffer et al., 1992). For avocado trees flooded in calcareous soils, net CO 2 assimilation and transpiration dec lined linearly after trees were flooded, with a mean net CO 2 assimilation of zero by 30 days after trees were flooded. These reductions were attributed to biochemical changes associated with photosynthetic reactions and reduced stomatal conductance rather than a hydraulic

PAGE 24

24 effect, since flooding did not significantly decrease xylem water potential (Schaffer et al., 2006). Similar results have been reported for several other woody and herbaceous plant species when exposed t o different periods of flooding. The se include mamey sapote ( Pouteria sapota ) (Nickum et al., 2010), carambola ( Averrhoa carambola ) (Ismail and Noor, 1996), mango ( Mangifera indica ) (Larson et al., 1996), various A nnona species (Nuez Elisea et al., 1999), Surinam cherry ( Eugenia uniflora ) ( Martin et al., 2009, 2010; Mielke and Schaffer, 2010a,b, 2011), Japanese alder ( Alnus japonica ) (Iwanaga and Yamamoto, 2007), and corn ( Zea mays ) (Yordanova and Popova, 2007). Interactions Between Above Ground Factors and Flooding Stress in Fruit Crop s Du ring the growing season, root growth is assumed to be sustained by current photosynthate (Eissenstat and Duncan, 1992). In container grown plants, a disruption of photosynthesis by defoliating or pruning shoots will decrease or stop root growth and enhance the relative growth rate of the shoots (Eissenstat and Duncan, 1992). Species vary in the response time before root growth is diminished and the length of time required for a plant to recover from defol iation so that its root to shoot ratio is in equilibr ium. Eissenstat and Duncan (1992) found that above ground factors in citrus trees ( Citrus sinensis ) modify carbohydrate partitioning. In normal (adequate oxygen content for normal plant functioning) soil conditions, the total amount of reducing and ketone sugars (including free fructose, sucrose, and fructans) in the fine roots of citrus trees were 24% lower in pruned than in non pruned trees. They also found that 30 days after pruning, 20% of the roots in the pruned trees (at a soil depth of 9 to 35 cm) di ed, but after 63 days, root length of pruned trees was similar to that of the non pruned trees.

PAGE 25

25 However, at that time starch reserves remained 18% l ower in the fine roots of pruned trees than in those of non pruned trees. Since root inundation reduces roo t growth of most woody plants more than it does stem growth, the root/shoot ratio is decreased as a result of flooding (Kozlowski, 1997). One result of a reduced root/shoot ratio is that when flood water drains away and better oxygenated root conditions re turn, previously flooded plants may be less drought tolerant than plants that were never flooded. This may result from less water absorption by diminished root systems that cannot adequately replenish transpirational losses (Kozlowski, 1997). A high level of fermentative metabolism in roots is important for plant survival when soil is flooded because this process can potentially supply a high enough energy charge to sustain root metabolism (Liao and Lin, 2001). A continuous supply of fermentable sugars in roots is critical for long term survival of peas ( Pisum sativum ), pumpkins ( Cucurbita mixta ), and several herbaceous plants exposed to anoxia or flooding (Liao and Lin, 2001). In theory, if the assimilates in leaves are not transported to the roots, carboh ydrate deficiency will occur. Photoassimilate transport to roots can decrease during flooding resulting in accumulation of starch in leaves (Carpenter et al., 2008). Vu and Yelenosky (2006) found that in citrus trees ( Citrus sinensis ), the total nonstructu ral carbohydrate concentrations were high in leaves but drastically reduced in roots as a result of flooding. Root inundation affects not only synthesis of carbohydrates, but also the transport of carbohydrates to meristematic sinks and carbohydrate utiliz ation in metabolism and production of new tissues (Kozlowski, 1997). Carpenter et al. (2008) found that in cuttings of Salix nigra the total nonstructural

PAGE 26

26 carbohydrate pool was reduced in response to complete shoot removal, but increased in response to pe riodic flooding and water stress. These results were attributed to changes in carbon partitioning as indicated by increased soluble carbohydrates in roots and shoots. These results also illustrated the negative effects of drought and to a lesser extent p eriodic flooding on starch mobilization in resprouting of S. nigra. Implications of these findings extend to reduced survival in the field when plants are exposed to the combined stresses of pruning and flooding or drought (Carpenter et al., 2008). The se ven carbon (C 7 ) sugar, mannoheptulose, and a related C 7 sugar alcohol, perseitol, are the major forms of nonstructural carbohydrates in avocado trees (Liu et al., 2002). These C 7 sugars often account for over 10% of the tissue dry weight of avocado, and ca n be found in substantial amounts in all tissues and organs, including the fruit peel and seeds (Liu et al., 2002). In comparison, the nonstructural carbohydrates that occur more commonly in other plant species are based on six carbon hexose units, such as glucose and sucrose. These sugars are found in much lower concentrations in avocado tissues (Liu et al., 2002). In leaves of fruit bearing and non 7 sugars, with mannoheptulose dominating over per seitol and C 6 sugars such as sucrose, glucose and fructose (Bertling and Bower, 2006). Furthermore, leaves of non bearing trees generally have higher concentration s of these sugars than bearing trees (Bertling and Bower, 2006). This could be an indication that C 7 sugars play an important role in fruit growth and development because leaves cannot accumulate sugars when trees carry a heavy fruit load (Bertling and Bower, 2006). Thus, research with avocado that focused only on starch reserves overlooked the im portance of the C 7 sugars in the carbon allocation

PAGE 27

27 of C 7 concentration of sugar alcohols in the mesocarp (Bertling and Bower, 2005). However, such accumulation and/or active transport into the exocarp might be related to a need for stress resistance of these fruit parts, as sugar alcohols like perseitol have been suggested to act as active ox ygen species scavengers (Dennison et al., 1999; Jennings et al., 1998). In addition to their storage role, sugar alcohols are important for protection from oxidative damage as a result of salt and osmotic stress in celery ( Apium graveolens ), olive ( Olea eu ropaea ), and wheat ( Triticum aestivum al., 2007; Abebe et al., 2003). In avocado fruit, Cowan (2004) proposed that C 7 sugars present in the fruit protect certain key enzymes essential for fruit growth and development from damage by reactive oxygen species (ROS). Bertling et al. (2007) found that avocado tissues have different predominant antioxidant systems and similar antioxidant activity except for the mesocarp where the antioxidant activity is lower than in leaves, seeds and the exocarp. The main antioxidant in mesocarp tissue is the C 7 sugar, mannoheptulose (Bertling et al., 2007; Tesfay et al., 2010). Despite the importance of these seven carbon sugars, the exact function of heptose sugars is not completely understood and the number of related research papers published in the past is l imited (Meyer and Terry 2008). Thus, there is a need to study how these sugars respond to environmental stress conditions including root hypoxia or anoxia. Tree mortality can result from a few days of flooding avocado trees with a high shoot/root ratio due to extensive root damage (Schaffer, 1998). Tree mortality can

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28 reportedly be offset by pruning trees after flooding, which is often recommended for avocado trees in Chile and Florida to increase tree survival and recovery from flooding stress (Gil et al., 2008). Removing part or most of the canopy reduces the above ground volume of the tree, making resetting and stabilizing the tree easier. This will also reduce transpirational water lo ss and prevent desiccation (Crane et al., 1994). However, this pruning recommendation for avocado trees exposed to flooding is based on observations and not experimental evidence (Gil et al., 2008). Additionally, as part of pre hurricane practices in Flori da it is suggest ed that growers control tree size by removing part of the canopy to reduce potential wind damage (Crane et al., 1994). The effect of canopy removal on reducing tree damage as a result of low soil oxygen has not been quantified. To the auth quantifies the effects of pruning or the timing of pruning in relation to the flooding period on reducing stress and increasing the growth and survival rate of avocado trees. Gil et al. (2008) found that pruning avocado trees immediately after floodwater drained from the root zone, resulted in a tendency for trees to recover faster than non pruned trees, as indicated by net CO 2 assimilation of pruned trees returning to pre flood levels, whereas those of non pruned trees did not. Pruning the canopy immediately after flooding decreased stress, presumably by lowering the shoot/root ratio, thereby reducing transpirational surface area and transpirational demand, thus reducing potential water stress. In con trast, pruning avocado prior to flooding increased stress compared to flooded, non pruned trees, presumably due to a reduction in leaf area and a subseque n t reduction in photosynthesis and photoassimilate translocation to the roots (Gil et al., 2008). Th e reduced photosynthesis and production and transport of

PAGE 29

29 assimilates likely limited the amount of substrate available for root respiration during flooding (Gil et al., 2008). E ffect of Low Soil Oxygen Concentration on Plant Sap Flow Plant sap flow measurem ents provide direct and continuous measurements of whole plant water use with a high time resolution (Smith and Allen, 1996; Wullschleger et al., 2000). There is little published information on the effects of flooding on continuous measures of plant water status such as stem (xylem) sap flow or fluctuations in trunk diameter in fruit trees (Nicols et al., 2005). Specific plant responses to flooding vary with many factors, including clones and species (Nicoll and Coutts, 1998). Thus, tolerance to floodi ng in fruit crops such as Citrus limon ), apricot ( Prunus armeniaca ) and citrus ( Citrus sinensis ), may depend on the scion/rootstock interactions (Ruiz Sanchez et al., 1996; Domingo et al., 2002, and Garcia avocado, the stem sap flow rate was significantly reduced when trees were grafted onto two different clonal rootstocks, Duke 7 and Toro Canyon, due to differences in the xylem anatomy of each cultivar that causes a discontinuity in the water conduction sy stem that decreases water transport (Fassio et al., 2008). There are no reports of flood tolerant rootstocks in avocado (Schaffer, 1998). However, information on differences in plant sap flow among avocado cultivars and the effects of flooding on stem s ap flow may be useful for future development of flood tolerant avocado rootstocks. Plant age, the properties of the floodwater, and duration of flooding affect the response of trees to soil hypoxia caused by flooding (Kozlowski, 1984). In young apricot tre es, three hours of flooding caused a decrease in sap flow and a significant reduction

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30 in plant hydraulic conductance, decreasing water absorption by the roots (Nicols et al., 2005). Similarly, lemon trees subjected to three days of soil flooding exhibite d a progressive reduction in sap flow and stomatal closure (Ortuo et al., 2007). Flooding also reduced the mean sap flow of Avicennia germinans by 27%, in Rhizophora mangle by 17.3% and in Laguncularia racemosa by 16.7% (Krauss et al., 2007). Internal r egulation of stomatal conductance and stem sap flow have generally been interpreted as a mechanism to restrict water uptake during drought or flooded conditions (Nicols et al., 2005; Ruiz Sanchez et al., 1996 ). Low concentrations of oxygen in the root zo ne reduce the permeability of roots to water (Smith et al., 1990; Zhang and Tyerman, 1991), increasing resistance to water uptake (Domingo et al., 2002). Under these conditions, water loss from the shoots exceeds the supply from the roots, leading to a red uction in leaf water potential and stomatal conductance (Domingo et al., 2002). Most studies o f the effects of waterlogging of fruit trees have concentrated on the detrimental effect that root anoxia has on stomatal activity and growth (Sav and S errano, 1 986; Smith and Ager 1988 ). However, the ability of trees to recover from transient waterlogging of the root system is also important in assessing the effects of short term flooding (Smith et al., 1990). S ummary Flooding results in significant root dama ge to avocado, thereby reducing water uptake (Schaffer and Whiley, 2002). Removal of part of the canopy (pruning) may be a practical method to alleviate or prevent flooding stress to avocado trees. However, the underlying mechanisms that define difference s in responses to leaf removal immediately after flooding compared to leaf removal from avocado trees shortly before

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31 flooding remain unclear. Thus, there is a need to quantify the effect of the timing of leaf removal on whole plant physiology, growth and survival of flooded avocado trees. Measuring the interaction between the time of leaf removal or pruning, with respect to flooding on net CO 2 assimilation, stomatal conductance, and transpiration may provide valuable clues to the mechanisms by which prunin g influences response of avocado trees to flooding because these physiological variables are some of the earliest responses to flooding that can be easily and non destructively measured (Schaffer et al., 2006). This research will provide basic information about the physiological response of avocado trees to root zone flooding and the interaction between pruning and flooding. Additionally, this work should enhance the understanding of the role of non structural carbohydrates in responses of avocado trees to low soil oxygen. The practical application of this study should assist growers by providing quantitative information about how to rehabilitate trees damaged by flooding relative to the amount and timing of canopy removal.

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32 CHAPTER 3 LEAF REM OVAL AND FLOODING EF FECTS ON LEAF GAS EX CHANGE, ROOT CARBOHYDRATE CONCENT RATION, GROWTH AND S URVIVAL OF AVOCADO TREES Background Avocado ( Persea Americana Mill.), a subtropical evergreen tree native to Central American and Mexico, is grown commercially i n Mediterranean, subtropical and tropical climates worldwide (Whiley and Schaffer, 1994). In the United States, the vast majority of avocado production is in California, where there are an estimated 21,108 hectares planted (FAO, 2010). Although there are only 2,995 ha of avocado trees planted commercially in Florida, it is second only to citrus among tree fruit crops in annual farm gate sales (Evans and Nalampang, 2010). Rapidly increasing avocado production in several regions of the world has resulted in orchard establishment on marginal sites that are prone to flooding or poor soil drainage (Schaffer et al., 2012 ). In southern Florida, avocado orchards can become saturated due to capillary water movement from a water table which is not far ( 1.8 2.3 m; Barquin Valle, 2011 ) from the soil surface, and heavy rains from tropical storms and hurricanes (Schaffer and Whiley, 2002; Crane et al., 1994). Flooding displaces oxygen and other gases and increases the concentrations of toxic organic and inorganic com pounds in agricultural soils (Drew, 1997; Geigenberger, 2003; Irfan et al., 2010). Thus, flooding can negatively impact crop physiology, growth, production and even survival of fruit trees, including avocado (Schaffer et al., 1992). Avocado is a flood sen sitive species with physiological responses occurring shortly after soil becomes waterlogged (Schaffer et al., 1992). Responses of avocado trees to even short periods of root zone hypoxia include leaf abscission, root necrosis, and reductions

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33 in net photo synthesis, shoot and root growth, and fruit production (Schaffer and Whiley, 2002). A few days of flooding can result in avocado tree death (Schaffer, 1998) which has bee n attributed to the high canopy to root ratio as a result of damage to roots which o ccurs prior to canopy damage when trees are flooded (Schaffer, 1998). Root water absorption and nutrient uptake are reduced during flooding. As a result, the resistance to water movement across the root cortex is increased. This eventually leads to leaf de hydration, stomatal closure, and tree death (Schaffer et al., 1992, 2002, 2006, 2007). The observation of a high shoot/root ratio as a result of flooded or poorly drained soils has led to the recommendation in southern Florida and Chile to prune or remov e part of the canopy after flooding to mitigate stress from root hypoxia (Gil et al., 2008). Due to extensive root damage of partially uprooted trees after a tropical storm or hurricane, removing part or most of the canopy reduces the weight of the tree, making resetting and stabilizing the tree easier, and also reduces transpiration and prevents desiccation (Crane et al., 1994). Nevertheless, canopy pruning after flooding to mitigate flooding stress is based on observations rather than experimental evide nce. Preliminary studies with avocado trees in containers showed that pruning the canop y immediately after the floodwater subside s reduced plant stress and increased tree survival (Gil et al., 2008). In contrast to leaf removal after flooding leaf remova l before flooding was shown to increase flooding stress of avocado trees in containers (Gil et al., 2008). It was suggested that increased stress of flooded avocado trees due to canopy removal was the result of less carbohydrate produced and translocated to the roots as a result of reduced photosynthesis from the combined effects of less leaf area and root hypoxia.

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34 Thus, it was hypothesized that reducing the leaf area resulted in less carbohydrate transported to the roots to act as a substrate for root re spiration, and thus less ATP was produced to help maintain normal plant metabolism under hypoxic root conditions (Gil et al., 2008). If this hypothesis is correct, reducing carbohydrate production and transport to the roots by means other than leaf removal i.e. the use of a chemical photosynthetic inhibitor, should also increase flooding stress and delay tree recovery after flooding. The seven carbon (C 7 ) sugar, mannoheptulose, and a related C 7 sugar alcohol, perseitol, represent major forms of nonstructu ral carbohydrates in avocado trees (Liu et al., 1999). These C 7 sugars often account for more than 10% of the tissue dry weight and can be found in substantial quantities in all tissues and organs. The concentrations of primary nonstructural carbohydrates based on a six carbon hexose skeleton (i.e., glucose and sucrose) common in most other fruit trees, are in much lower concentrations than the C 7 sugars in avocado tissues (Liu et al., 1999). Research with avocado has often focused only on starch reserve s and thus overlooked the importance of the C 7 sugars in the carbon allocation process. Little is known about how these sugars mediate root / shoot relations or respond to environmental stress conditions, particularly root hypoxia or anoxia. The main objecti ve of this study was to determine if limiting net CO 2 assimilation by leaf removal or foliar application of a chemical photosynthetic inhibitor prior to flooding exacerbates stress and delays or prevent s recovery of avocado trees exposed to short term flo oding. Additionally, the combined effects of leaf removal prior to flooding and root zone hypoxia on root carbohydrate concentrations, particularly C 7 sugars, were determined.

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35 Materials and Methods Study site description This study was conducted in an open air ( o nly the roof was covered ) greenhouse at the University of Florida, Tropical Research and Education Center in Homestead, Florida (25.5 o N and 80.5 o W). Temperature in the greenhouse was recorded with a StowAway, TidbiT sensor/data logger (Onset Comput er Corp., Bourne, Massachusetts, USA) located 15 cm above the canopy. Photosynthetic photon flux (PPF) was measured with a quantum sensor (model Li 190) connected to a LI 1000 datalogger (Li Cor Inc., Lincoln, Nebraska, USA). There were two experiments T he first experiment (Expt. 1) conducted from June August 2010, determined the effects of reduced photosynthesis by either leaf removal or the use of a foliar applied chemical photosynthetic inhibitor prior to flooding on plant stress and recovery from f looding. The second experiment (Expt. 2), conducted during September 2011, focused on determining the effects of reduced photosynthesis by leaf removal prior to flooding on root carbohydrate content, root respiration and plant stress from flooding. Plant m aterial Two year Persea americana Mill) on Walden seedling rootstock growing in potting medium (40% Canadian peat, 10% coir, 40% pine bark and 10% perlite) in 3 L containers were obtained from a commercial nursery (Expt. 1). One year L containers were obtained from a commercial nursery growing in the same type of

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36 potting medium as trees in Expt. 1. Prior to initiating treatments, trees in each experime nt were treated with the fungicide, Alliete (Bayer Crop Science, Morganville, NC, USA), as a soil drench to help prevent Phytophthora root rot. Experimental design E xpt. 1 consisted of a randomized design with a 3 x 2 factorial arrangement of treatments. There were three canopy treatments: two thirds of the leaves removed before flooding (leaf removal); use of Freeway ( alcohol ethoxylates, silicone polyether copolymer, propylene glycol and dimethylpolysiloxane ; Loveland Products Inc., Loveland, CO, USA), a chemical photosynthetic inhibitor sprayed on the canopy (C.P.); or no leaf removal or chemical spray (control); and two flooding treatments: flooded or non flooded. Freeway is a chemical surfactant that was previously found to inhibit photosynthesis o f tropical fruit trees (Schaffer et al., 2010) including avocado (B. Schaffer, University of Florida, 2009, unpublished data There were six single tree replications for each treatment combination. E xpt. 2 consisted of a completely randomized design with a 2 x 2 factorial arrangement of treatments. There were two leaf removal treatments: no leaf removal (control) or two thirds of the leaves removed (leaf removal); and two flooding treatments: flooded and non flooded. There were 6 single tree replications fo r each treatment combination. Flooding treatments Plants were flooded by submerging each entire plant container in a 19 L plastic bucket filled with tap water to 5 cm above the soil surface. Buckets were refilled each day with stagnant water collected on the same day that the flooding treatment was

PAGE 37

37 initiated to avoid re oxygenation of the medium and maintain a constant water level. Trees in the control treatment were not flooded. Plants were unflooded after a 2 assimilation or stomatal conductance to water vapor was observed between flooded and non flooded trees in any of the canopy treatments (Expt. 1) or leaf removal treatments (E xpt. 2). The flooding period was 4 days in each experiment. Trees were irrigated by overhead irrigation for 15 minutes twice each day, except during the flooding period, when non flooded trees were manually irrigated twice each day. Tensiometers (Irrometer Company, Riverside, CA, USA) were installed in five randomly selected containers for plants in the non flooded treatments and soil suction was maintained at 10 15 KPa to ensure that trees were not drought stressed (Kiggundu et al., 2012). Leaf removal tr eatment Two thirds of the leaves were removed the day before the flooding treatments were initiated for plants in the leaf removal treatment in Expts. 1 and 2. The total leaf area was reduced to the desired level by counting the number of leaves per tree a nd then removing two thirds of the total number of leaves in each tree starting from the base of the trunk upwards Trees in the control treatment had no leaves removed. Chemical photosynthetic inhibitor treatment The foliage of p lants in the C.P. treatme nt in Expt. 1 were sprayed with with 2 ml L 1 of Freeway in distilled water to run off with a hand sprayer, covering both the adaxial and abaxial surfaces. Applications started the day before initiating the flooding treatment. During the flooding period, flooded and non flooded plants were sprayed

PAGE 38

38 every other day until plants were unflooded. Plants in the control treatment (flooded or non flooded) were not sprayed with Freeway Leaf gas exchange measurements Net CO 2 assimilation and stomatal conductan ce to water vapor were measured with a Ciras 2 portable gas analyzer (PP Systems, Amesbury, MA, USA) at a light saturated PPF (1000 m m 2 s 1 ), a reference CO 2 concentration of 375 m mol 1 and an air flow rate into the leaf cuvette of 200 ml min 1 Measu rements were made between 900 HR and 1100 HR, starting 2 days prior of the initiation of flooding treatments. Leaf gas exchange was measured daily on the same two fully expanded, mature leaves of each plant. After the flooding period in Expt. 1, measuremen ts were made at 1 2 day intervals to monitor tree recovery and were stopped 45 days after flooding when there were no significant differences in net CO 2 assimilation or stomatal conductance between flooding treatments. Measurements were stopped and plan ts were harvested in Expt. 2 immediately after the flooding period so that root respiration and carbohydrate concentrations could be measured. Plant dry weights Plants were harvested at the end of both experiments. Plant organs (leaves, stems, and roots) w ere collected for dry weight determinations. Roots were separated from the rooting medium by carefully washing them in tap water. Tissue samples were oven dried at 70C to a constant weight and leaf, stem root and whole plant dry weights were determined.

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39 Root respiration Root respiration was measured in Expt. 2 in excised roots (~2 mm in diameter) using a method similar to that described by Burton and Pregitzer (2003). From each plant, 2 g fresh weight of roots were harvested and cleaned with a brush to remove soil particles. Root samples were placed in a polyvinyl chloride (PVC) cylinder sealed to a Plexiglas bottom. The cylinder was covered with a soil respiration chamber attached to a Ciras 2 portable gas analyzer (PP Systems, Amesbury, MA, USA). R oot respiration was determined using an airflow rate into the chamber of 100 m l min 1 After measurements were made, root samples were oven dried at 70C to a constant weight for dry weight determinations. Root carbohydrate concentration s R oot samples from each plant in each treatment combination of Expt. 2 were collected immediately after plants in the flooded treatment were unflooded. Samples were freeze dried at 50C for 50 h in a Freezone 4.5 freeze dryer (Labconco, Kansas City, MO, USA). Freeze dried samples were ground using a Proctor Silex coffee grinder (Hamilton Beach, Southern Pines, NC, USA) to obtain 0.1 g per sample. The samples were sent to the Department of Botany at the University of California, Riverside for extraction and analysis of C 7 s ugars, sucrose, fructose and glucose. Carbohydrates were extracted using the ethanol based method described by Liu et al. (2002). This method is sufficiently sensitive to detect sugar or starch concentrations as low as 0.04% (Chow and Landhusser, 2004).

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40 S oil redox potential and soil temperature Soil redox potential and pH were measured in each container with a metallic ORP indicating electrode for plants in the flooded treatment in both experiments (Accument Model 13 620 115, Fisher Scientific, Pittsburg, PA, USA) connected to a voltmeter. Measurements were made daily in each container during the flooding period by placing the electrode into a polyvinyl chloride (PVC) pipe inserted 10 cm deep into the media of each container. Soil temperature was recorded using a Hobo Tidbit v2 sensor/datalogger (Onset Computer Corp., Pocasset, M A USA) placed 5 cm deep into the container of one randomly selected plant in each treatment. Statistical analyses Data were analyzed by a two way analysis of variance (ANOVA) to a ssess interactions between flooding and canopy (leaf removal, Freeway application, or control) treatments (Expt. 1), or between flooding and leaf removal treatments (Expt. 2). Differences between flooding treatments were compared by repeated measures ANO VA for leaf gas exchange variables, or a T test for root respiration, carbohydrate concentration, and plant dry weights (SAS 9.1, SAS Institute, Cary, NC, USA). Results Soil and air temperature, soil redox potential and light intensity Expt. 1. Mean dail y air temperature in the greenhouse ranged from 23 to 41C with a mean of 29C. Soil temperature in the non flooded treatment ranged from 24 to 39C with a mean of 30C. Soil redox potential for the flooded treatment was slightly

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41 below 200 mV beginning one day after flooding and values continued to decrease to a mean of 18 mV by day 4 (Fig ure 3 1). Expt. 2. Mean daily air temperature in the greenhouse ranged from 20 to 39C with a mean of 28C. Soil temperature in non flooded soil ranged from 21 to 37C wi th a mean of 30C. Soil temperature in flooded soil ranged from 21 to 38C with a mean of 28C. Soil redox potential for the flooded treatment was slightly below 200 mV beginning the after 1 day of flooding and decreased to a mean of 18 mV by day 4 (Fig ur e 3 1). Leaf gas exchange Expt. 1. canopy and flooding treatments for net CO 2 assimilation and stomatal conductance on one or more measurement date(s). Therefore flooding treatments were compared separately within each c anopy treatment. There was no significant effect of flooding on net CO 2 assimilation in the untreated controls on any of the measured dates and stomatal conductance was significantly lower in flooded than in non flooded plants (P < 0.05) on only one date during the recovery period (Fig ure s 3 2, 3 3). Net CO 2 assimilation and stomatal flooded plants in the leaf removal treatment after 4 days of flooding until about 28 days into the recovery period. In plants treated with Freeway net CO 2 a ssimilation and stomatal conductance flooded trees on several dates after the flooding period, beginning 1 and 3 days after plants were unflooded for net CO 2 assimilation and stomatal conductance, respectively (Fig ure s. 3 2, 3 3).

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42 Expt. 2. For plants in the control treatment after 3 and 4 days of flooding net CO 2 assimilation and stomatal conductance, respectively, were significantly lower (P < 0.05) in flooded than in non flooded plants (Fig ure s 3 4, 3 5). Net CO 2 assimilation and stomatal conductance were significantly lower in flooded than in non flooded plants for plants in the leaf removal treatment, (P < 0.05) after 3 and 4 days of flooding, respectively (Fig ure s 3 4, 3 5). Tissue dry weight s and plant survival Expt. 1 Although there was no significant interaction (P > 0.05) between flooding and Freeway treatments for root, leaf or stem dry weights, there was ore, for each plant tissue, differences between flooding treatments were compared separately within each Freeway treatment. Stem dry weight of co ntrol plants was significantly higher the flooded than those in the non flooded trea tment (Figure 3 6). For plants treated with Freeway flooded plants (Figures 3 6c). Total plant dry weight in the leaf removal treatment was ooded than for non fl ooded plants (Figure 3 6b). At the end of the experiment (day 49), 33% of the flooded plants treated with Freeway died. In both the control and leaf removal treatments, 17% of the flooded plants died (Table 3 1). Tissue dry weights an d plant survival were not assessed in Expt. 2 because those plants were harvested immediately after the 4 day flooding period to determine root respiration and carbohydrate concentrations. Dry weight and survival differences

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43 between flooded and non floode d avocado trees can generally not be detected until several days after a short (3 5 day) flooding period (B. Schaffer, University of Florida, 2010, unpublished data). Root respiration There was no significant interaction (P > 0.05) between the flooding an d the leaf removal treatments for root respiration in Expt. 2. Therefore, leaf removal treatments were pooled to compare flooding treatments and flooding treatments were pooled to compare leaf removal treatments. There was no significant effect of floodin g or leaf removal (P > 0.05) on root respiration. However, in the leaf removal treatment flooded plants had a higher mean root respiration than non flooded plants. In addition, control plants in the flooded treatment tended to have higher root respiration than plants with leaves removed (Figure 3 7). Root carbohydrate concentration s There was a significant interaction in Expt. 2 between the flooding treatments and the leaf removal treatments only for sucrose and glucose concentrations (P < 0.05). Therefor e, the effects of flooding treatment on root concentrations of each carbohydrate were analyzed separately within each leaf removal treatment and the effects of leaf removal were analyzed separately within each flooding treatment. The carbohydrate found in the greatest concentration in the root s was D mannoheptulose, followed by perseitol. Sucrose and glucose were detected in the roots, but in much lower concentrations than the C 7 sugars (Figure 3 8). Fructose was

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44 not detected, either because it was not pr esent or its levels were below the detection limit of 0.04% of the tissue dry weight (Liu et al., 2001). D mannoheptulose and sucrose concentrations were significantly higher in non flooded than in flooded plants in the control treatment (Figure 3 8). Fo r plants in the leaf removal treatment, only the D mannoheptulose concentration was significantly higher in non flooded than flooded plants (P < 0.5). In both the leaf removal and control treatments, the D mannoheptulose concentration was nearly twice as high in the non floode d as in the flooded plants (Figure 3 9). For plants in the non flooded treatment, perseitol was significantly lower in the control treatment than in the leaf removal treatment, whereas glucose was significantly higher in the control treatment (P < 0.05; Figure 3.8 ). Flooded control plants had higher perseitol concentrations than plants in the leaf removal treatment (P < 0.05; Figure 3.9). Discussion A decline in net CO 2 assimilation of flooded plants 2 days (Expt. 1) or 4 days (Expt. 2), respectively after flooding, coincided with reductions in stomatal conductance and soil redox potential. This is consistent with previous research that showed that a reduction in leaf gas exchange is the earliest symptom of flooding stress of avocado ( Schaffer et al., 1992), and that this response tends to occur within hours of exposure to root zone hypoxia (Schaffer and Ploetz 1989; Schaffer et al., 1992). Leaf removal for flooded trees, or the use of Freeway prior to flooding resulted in a greater r eduction in net CO 2 assimilation, slower recovery from flooding stress, and more tree death compared to the control treatment. Thus, an inhibition of the photosynthesis before flooding negatively affect ed avocado tree recovery. Reduced

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45 photosynthesis res ults in less carbohydrate available for glycolysis in the roots and thus less energy (ATP) production (Taiz and Zieger, 2010). Based on similar findings to those of the present study, Gil et al. (2008) postulated that pruning avocado trees prior to floodin g increases stress and delays recovery of flooded trees due to reduced ATP production as a result of less carbohydrate being produced and transported to the root. Thus, there is presumably less substrate (carbohydrate) available for root respiration as a result of less photosynthetic surface area. In Expt. 2, D mannoheptolose was found in considerably greater concentrations than any of the other carbohydrates. The reduction in stomatal conductance and net CO 2 assimilation of flooded plants coincided with lower total carbohydrate concentration, primarily D mannoheptolose, in the roots. The C 7 sugar, D mannoheptulose, is the primary photosynthetic product in avocado. This is catalyzed by aldoses in the Calvin cycle to form the storage product, perseitol (L iu et al., 1999). In experiments with tomato ( Solanum lycopersicum) root zone hypoxia resulted in reductions in fructose and glucose concentrations, the primary non structural carbohydrates metabolized in tomato (Horchani et al., 2009). Similar l y, root hypoxia resulted in reductions in sucrose and the sugar alcohol, mannitol, in pigeon pea ( Cajanus cajan ; Kumuta et al., 2008). In the present study with avocado, the concentration of D mannoheptulose was not only reduced by flooding, but also tended to be lower in plants with leaves removed prior to flooding. Additionally, the concentration of perseitol was significantly lower for plants with leaves removed before flooding than in the control plants that were flooded. This suggests that removing

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46 leaves be fore flooding reduces carbohydrate production making plants more susceptible to flooding damage. Root inundation affects not only carbohydrate synthesis, but also photoassimilate transport to meristematic sinks and their utilization in metabolism and pro duction of new tissues (Kozlowski, 1997). Carpenter et al. (2008) found that in cuttings of Salix nigra the total nonstructural carbohydrate pool was reduced in response to complete shoot removal, but increased in response to periodic flooding and water s tress. These results were attributed to changes in carbon partitioning as indicated by increased soluble carbohydrates in roots and shoots. These results also illustrated the negative effects of periodic flooding on starch mobilization and resprouting of S nigra. Implications of these findings extend to reduced survival in the field when plants are exposed to the combined stresses of reduced canopy size and flooding (Carpenter et al., 2008). In theory, if the translocation pathway is blocked, which typic ally occurs with flooding (Carpenter et al., 2008), assimilates in leaves will not be translocated to the roots, thus resulting in carbohydrate deficiency in the roots. A continuous supply of fermentable sugars in roots was found to be critical for long t erm survival of pea ( Pisum sativum) pumpkin ( Cucurbita maxima ), and several herbaceous plants exposed to anoxia or flooding (Liao and Lin, 2001). Fermentable sugars are important for plant survival when soil is flooded because this process can potentially supply a high enough energy charge to sustain root metabolism (Liao and Lin, 2001). However, in the present study with avocado, there was no significant statistical interaction between the flooding and the leaf removal treatment for root respiration. Pres umably, the length of flooding was not long enough to observe a

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47 decrease in root respiration as overall plant decay was observed days after flooding, even in plants in which the root carbohydrate concentration was reduced by flooding. Although, antioxidan t activity was not measured in this study, i t has been suggested that D mannoheptulose acts as a major antioxidant in mesocarp tissue of avocado fruits (Tesfay et al., 2010). This may also explain why the D mannoheptulose concentration of flooded plants wa s higher for those with leaves removed prior to flooding than in the control treatment. Plants in which photosynthesis was inhibited before flooding had less carbohydrate, thus less antioxidant concentration during and after the flooding stress. Sugar alco hols that serve as antioxidants have also been observed in celery ( Apium graveolens ) olive ( Olea europaea ) and wheat ( Triticum aestivum environmental stresses. While in the present study with avocado, flooding reduced net CO 2 assimilation in the leaves and thus presumably carbohydrate mobilization to the roots, plants in the control treatment had more photoassimilate production than trees with leaves removed, as a result of r educed photosynthetic surface area, and thus a higher concentration of the D mannoheptulose in the roots. Based on leaf gas exchange, root respiration, carbohydrate concentration, growth, and plant survival, inhibition of photosynthesis by leaf removal or the application of Freeway prior to flooding exacerbates flooding stress. Reduction of the main photosynthetic products, D mannoheptulose and perseitol, in the roots and possibly the role of the former as an antioxidant appeared to result in flooded plan ts with leaves removed prior to flooding, being more susceptible to flooding stress than plants with their canopies left intact. Therefore, this study provides evidence that reducing

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48 photosynthesis by leaf removal before flooding exacerbates the flooding s tress and decreases plant survival. Further studies are necessary to quantify the effect of prolonged flooding on root respiration and plant biomass of plants with photosynthesis inhibition before flooding.

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49 Figure 3 1 Soil redox potential of flooded 2 year old (Expt. 1) and 1 year old (Expt. 2) on Waldin seedling rootstock. Plants were flooded from 11 June to 14 June 2010(Expt. 1) and from 3 Sept. to 6 Sept. 2011(Expt. 2). Redox potentials below +200 mV indicate that soil conditions are anaerobic (Ponnamperuma, 1984) n=6.

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50 Figure 3 2 Effect of flooding on net CO 2 assimilation (A) for 2 year avocado trees on Waldin seedling rootstock. Treatments included a control, leaf removal, and spraying with Freeway from 10 Sept. to 29 July 2010 (Exp.1). An a sterisk indicate s a significant difference between treatments according to a repeated measures ANOVA

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51 Figure 3 3 Effect of flooding on stomatal conductance to water vapor (g s ) for 2 year old a control, leaf removal, a nd spraying with Freeway from 10 Sept. to 29 July 2010 (Expt.1). An a sterisk indicate s a significant difference between treatments according to a repeated measures ANOVA

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52 Figure 3 4 Effect of f looding on net CO 2 assimilation (A) for 1 year avocado trees on Waldin seedling rootstock. Plants were flooded from 1 Sept. to 6 Sept. 2011. Treatments included a control and leaf removal. An a sterisk indicate s a significant difference bet ween treatments according to a repeated measures ANOVA

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53 Figure 3 5 Effect of flooding on stomatal conductance to water vapor (g s ) of 1 year old trees on Waldin seedling rootstock (Expt. 2). Plants were flooded from 1 Sept. to 6 Se pt. 2011. Treatments included a control and leaf removal the day before flooding. An a sterisk indicate s a significant difference between treatments according to a repeated measures ANOVA (P 6.

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54 Figure 3 6 Effect of flooding on root, lea f stem, and total plant dry weight of 2 year old canopy treatments ( Expt. 1 ). An a sterisk indicate s a significant difference between treatments accordin g to a T

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55 Table 3 1. Percentage of tree survival and mortality Waldin seedling rootstock in each canopy treatment at the end of Expt. 1 (day 49) Treatment Control Leaf removal Freeway Flood ed No. of plants alive 5 5 4 No. of dead plants 1 1 2 Survival (%) 83.3 83.3 66.6 Mortality (%) 16.6 16.6 33.3 Non flooded No. of plants alive 6 6 6 No. of dead plants 0 0 0 Survival (%) 100 100 100 Mortality (%) 0 0 0

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56 Figure 3 7 R oot respiratio n of non flooded (NF) and flooded (F) treatments within the leaf removal treatment and between the control and leaf removal treatment within each flooding treatment. 0.05) between flooding treatments according to a T test (P > 0.05), n = 6

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57 Figure 3 8 Root carbohydrate con centrations for control and leaf removal treatments for 1 year ol within flooding treatments ( Expt. 2 ). An a sterisk indicate s a significant difference between treatments according to a T

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58 Figure 3 9 Root carbohydrate con centratio ns for control and leaf removal treatments for 1 year within flooding treatments ( Expt. 2 ). An asterisk indicat e s a significant difference between treatments according to a T

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59 CHAPTER 4 PRE AND POS T FLOODING LEAF REMOVA L EFFECTS ON LEAF GA S EXCHANGE, GROWTH, AND SURVIVAL OF AVOC ADO TREES EXPOSED TO ROOT ZONE FLOODING Background In agricultural crop production areas, e xcess water produces hypoxic soil conditions (Gambrell and Patrick, 1978) by disp lacing oxygen and other gases in the soil which are necessary for normal root metabolism (Drew, 1997; Geigenberger, 2003). Although all plants require water, excess water in the root environment can be injurious or even lethal. L ow oxygen in the soil due t o flooding is one of the main environmental factors limiting plant growth and yield worldwide, especially in high rainfall regions (Bai et al., 2009). Therefore, for agriculture to remain viable in areas with poor soil drainage or that are prone to floodin g from tropical storms or hurricanes it is important to understand how flooding affects crop physiology, growth, and yield to help identify flood adapted crops and production systems (Schaffer 1998). A vocado production is rapidly increasing worldwide. For example, from 2009 to 2010, avocado production increased by 13,610 ha in Colombia, by 3,380 ha in Peru, and by 2,000 ha in Mexico (FAO 2009, 2010). This rapid expansion of avocado production has resulted in orchard establishment on marginal sites that are prone to flooding or poor soil drainage (Schaffer et al., 2012). In addition, in many places avocado orchards are over irrigated (du Plessis, 1991) which can result hypoxic conditions in the root zone. Flooding of avocado orchards occurs periodically in areas, such as southern Florida, where there are high water tables and heavy rains or tropical

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60 storms and hurricanes (Schaffer and Whiley, 2002; Crane et al., 1994). A vocado trees have a relatively shallow root system that doe s not spread much beyond the tree canopy ( Wolstenholme, 2002 ). Roots are extensively suberized, with low hydraulic conductivity, few root hairs, poor water uptake and sensitivity to low soil oxygen concentrations (Ferreyra et al., 2006). These characterist ics make avocado one of the most susceptible fruit trees to soil flooding (Ferreyra et al., 2006 ; Schaffer et al., 1992 ). Short periods (a few days ) of standing water in the root zone of avocado trees has been shown to negatively impact physiological proc esses such as net carbon assimilation and transpiration, and cause tree mortality ( Schaffer, 1998; Schaffer et al., 1992) Tree mortality in flooded avocado orchards has been attributed to a reduction in root volume as a result of oxygen starvation of t he roots (Schaffer and Ploetz, 1989; Kozlowski 1997). This reduction of root volume caused by flooding can be exacerbated if Phytophthora cinnamomi (the cause of Phytophthora root rot; the major disease of avocado worldwide) is present in the soil (Ploetz and Schaffer, 1989; Schaffer and Ploetz, 1989). When avocado trees are flooded, the reduction in root volume occurs before the canopy is affected (Schaffer et al., 1992). This results in a high canopy volume relative to root volume (Schaffer, 1998). The high shoot to root ratio of avocado trees as a result of orchard flooding has led to the recommendation in some areas, such as South Florida, of reducing the size of the canopy of trees exposed to flooding to bring the shoot to root ratio back into equili brium (Crane et al., 1994). Removing a portion of the canopy decreases transpiration, which compensates for decreased water absorption

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61 due to reduced total root volume as a result of death of some roots from flooding. Therefor e, sufficient water and nutri ents can be supplied by the roots to the canopy if the canopy volume is adequately reduced (Crane et al., 1994). Reducing canopy size in flooded orchards also decreases tree weight, thereby facilitat ing re establishment of trees that may topple as a resu lt of strong winds and flooded soils from tropical storms or hurricanes (Crane et al., 1994). In a preliminary study with avocado trees in containers trees with two thirds of the canopy removed (pruned) after flooding tended to recover faster from floo ding stress than non pruned trees (Gil et al., 2008) Pruned trees exhibited a more rapid recovery of net CO 2 assimilation and transpiration to pre flood levels and had greater root dry weights than non pr uned trees (Gil et al., 2008). It was suggested th at the reduction in leaf area and subsequent decreased transpiration due to pruning compensated for the reduction in viable root volume and the decreased capacity for water absorption by flood damaged roots (Gil et al., 2008). An alternative to removing a portion of the canopy after flooding to mitigate flooding effects on tree vigor and vitality is to remove a portion of the canopy prior to a predicted flooding event if there is sufficient time (Crane et al., 1994). In a study with young avocado trees in containers, Gil et al. (2008) removed a portion of the canopy prior to flooding. In contrast to results observed for post flooding canopy reduction, remov ing a portion of the canopy immediately prior to flooding increased tree stress and mortality (Gil e t al., 2008). It was hypothesized that pre flooding leaf removal as a result of less carbohydrate produc tion and translocat ion to the roots as a result of reduced photosynthesis from the combined effects of decreased leaf area and stress caused by root h ypoxia (Gil et al., 2008). Thus, there was less carbohydrate in the roots to serve

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62 as a substrate for respiration during flooding, and consequently less ATP produc tion for n ormal plant metabolism resulting in increased tree stress ( Chapter 3 of this thes is ). R ecent experiments have shown that the root concentrations of D mannoheptulose and perseitol, the main photosynthetic products in avocado trees, were reduced under flood ed conditions and that concentrations of these carbohydrates tended to be lower i n trees with leaves removed compared to those with no leaves removed before flooding ( Chapter 3 of this thesis ). Therefore, it appears that reducing leaf area before flooding exacerbates flooding stress, whereas leaf area removal after flooding can increas e tree survival. Recommendations related to pruning of avocado trees to reduce flooding stress are based mainly on observations (Gil et al., 2008). Thus, there is a need to quantify the effect s of the amount and timing of leaf removal or pruning on physi ology growth and survival of flooded avocado trees. Additionally, t he underlying basis for the differen t flooding responses between trees with leaves removed before or immediately after flooding remains unclear. The objective of this study was to determi ne the relationship between the timing (pre or post flooding) and quantity of leaf removal on stress and survival of avocado trees exposed to flooding The hypotheses tested were: 1) Leaf removal immediately prior to flooding exacerbates flooding stress d ue to decreased net CO 2 assimilation during flooding; and 2) Leaf removal immediately after flooding decreases water demand by reducing transpirational surface area.

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63 Materials and Methods Study site description This study was conducted in an open air ( O nl y the roof was covered ) greenhouse at the University of Florida, Tropical Research and Education Center in Homestead, Florida (25.5 o N and 80.5 o W). Temperature in the greenhouse was recorded with a StowAway TidbiT sensor/data logger (Onset Computer Corp., Bourne, M A, USA) located 10 cm above the canopy. Photosynthetic photon flux (PPF) was measured with a quantum sensor (model LI? ? 190) connected to a LI 1000 datalogger (Li Cor Inc., Lincoln, Nebraska, USA). There were two experiments: In the first experim ent (Expt. 1), conducted from June August 2011, the effect the percentage of leaves removed prior to flooding on leaf gas exchange and plant survival during and after flooding was determined. In the second experiment (Expt. 2), conducted during October Dec ember 2011, the effect of the percentage of leaves removed after flooding on leaf gas exchange and plant survival during and after flooding was determined. Plant material Expt. 1. Three year in pot ting medium composed of 40% Canadian peat, 10% coir, 40% pine bark and 10% Perlite in 3 L containers were obtained from a commercial nursery. Expt. 2 Two year the same potting medium and the sa me size containers as described for plants in Expt. 1 were obtained from a commercial nursery

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64 Experimental design Expt. 1. The experiment consisted of a completely randomized design with a 4 x 2 factorial arrangement of treatments. There were four leaf removal treatments: no leaf removal (control), one third of the leaves removed (1/3LR), half of the leaves removed (1/2LR), and two thirds of the leaves removed (2/3LR); and two flooding treatments: flooded (F) and non flooded (NF). Leaves were removed 1 day before flooding. There were 6 single plant replications for each treatment combination. Expt. 2. The experiment consisted of a completely randomized design with a 4 x 2 factorial arrangement of treatments. Leaf removal treatments were similar to those in Expt. 1, except that instead of removing leaves before flooding, leaves were removed immediately after trees were unflooded. There were 6 single plant replications for each treatment combination. For each experiment, prior to initiating treatments, tre es treated with the fungicide, Alliete (Bayer Crop Science, Morganville, NC, USA), as a soil drench to help prevent Phytophthora root rot. Leaf removal treatments The desired percentage of leaves was removed by counting the number of leaves per tree an d then removing a percentage of the total number of leaves from each tree starting from the base of the tree upward. N o leaves were removed from trees in the control treatment. Flooding treatments Plants were flooded by submerging entire plant containers in 19 L plastic buckets filled with tap water to 5 cm above the soil surface. Buckets were refilled each day with

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65 stagnant water collected on the same day that the flooding treatment was initiated to avoid re oxygenation and to maintain a constant water l evel. Trees in the control (NF) treatment were not flooded. Flooded plants were unflooded after a statistically 2 assimilation or stomatal conductance of water vapor was observed between flooded and non flooded t rees in any of the leaf removal treatments. In Expt. 1, the flooding period was 3 days and in Expt. 2 the flooding period was 5 days. Trees were irrigated by overhead irrigation for 15 minutes twice each day except during the flooding period when non floo ded trees were manually irrigated twice daily. Tensiometers (Irrometer Company, Riverside, C A USA) were installed in 5 randomly selected containers for plants in the non flooded treatments and soil suction was maintained at 10 15 KPa to ensure that non fl ooded trees were not drought stressed (Kiggundu et al., 2012). Leaf gas exchange measurements Expt. 1. Net CO 2 assimilation, stomatal conductance of water vapor, and transpiration were measured with a ADC3 portable gas analyzer and a PLC3 leaf cuvette wit h a halogen light source (Analytical Development Company, Bioscientific Ltd., Great Amwell, England) at a light saturated PPF of 1000 m m 2 s 1 a reference CO 2 concentration of 375 m mol 1 and an air flow rate into the leaf cuvette of 200 ml min 1 Measu rements were made between 900 HR and 1100 HR, starting 2 days prior to flooding. Leaf gas exchange was measured daily on the same two fully expanded, mature leaves of each plant. After the flooding period, measurements were made at 1 day intervals for 10 d ays after flooding and at 2 day intervals thereafter until 52 days after plants were unflooded when there were no significant differences between flooded

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66 and non flooded plants in net CO 2 assimilation or stomatal conductance within any of the leaf removal treatments. Expt. 2. Net CO 2 assimilation, stomatal conductance of water vapor, and transpiration were measured with a Ciras 2 portable gas analyzer with a broad leaf cuvette and a halogen light source (PP Systems, Amesbury, MA, USA) at a light saturate d PPF of 1000 m m 2 s 1 a reference CO 2 concentration of 375 m mol 1 and an air flow rate into the leaf cuvette of 200 ml min 1 Measurements were made between 900 HR and 1100 HR, starting 2 days prior to flooding. During the flooding period, leaf gas exchange was measured daily on the same two fully expanded, mature leaves of each plant throughout the experiment. After the flooding period, measurements were made at 1 day intervals for 10 days after flooding and at 2 day intervals thereafter until 53 da ys after plants were unflooded when there were no significant differences between flooded and non flooded plants for net CO 2 assimilation or stomatal conductance within any of the leaf removal treatments. Plant biomass measurements Plants were harvested and dry weights were determined at the end of each experiment. Roots were separated from the rooting medium and carefully washed with tap water. Tissue samples were then oven dried at 70C to a constant weight, and leaf, stem, root and total plant dry weig hts were determined. Soil redox potential and soil temperature Soil redox potential was measured in each container with a metallic ORP indicating electrode for plants in the flooded treatment in both experiments (Accument Model 13 620 115, Fisher Scientif ic, Pittsburg, PA, USA) connected to a voltmeter.

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67 Measurements were made daily in each container during the flooding period by placing the electrode into a polyvinyl chloride (PVC) pipe inserted 10 cm deep into the media of each container. Soil temperatur e was recorded with a Hobo Tidbit v2 sensor/datalogger (Onset Computer Corp., Pocasset, M A USA) placed 5 cm deep into the container of one randomly selected plant in each treatment combination. Sap Flow Expt. 2 X ylem sap flow was monitored daily in a su bsample of 3 plants in the following treatment combinations: Control NF, Control F, 2/3P NF, and 2/3P F to determine the effects of treatments on transpirational demand. Xylem sap flow was monitored with a Dynagauge Flow 32 1KTM Sap Flow System (Dynamax In c., Houston, TX, USA). This system is based on the heat balance technique which is a non intrusive, non destructive method to measure sap flow (Dynamax Inc. 2010). Measurements started 2 days before the initiation of the flooding treatment s and stopped the day before plants were unflooded ( day 4 ) Statistical analysis Data were first analyzed by a two way analysis of variance (ANOVA) to test if there were significant interactions between flooding and leaf removal treatment s Differences between flooding t reatments were determined by repeated measures ANOVA for leaf gas exchange variables and with a T test for biomass variables (SAS Institute, Cary, N C USA).

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68 Results Soil and air temperature, soil redox potential and light intensity Expt. 1. Daily air te mperature in the greenhouse ranged from 22 to 40C with a mean of 28C. Soil temperature ranged from 23 to 34C with a mean of 28C. Mean soil redox potential for the flooded treatment was slightly below 200 mV beginning on day 1 and continued to decrease to 94 mV by day 3 (Figure 4 1). Expt. 2. Daily air temperature in the greenhouse ranged from 14 to 35C with a mean of 23C. Soil temperature ranged from 18 to 29C with a mean of 23C. Mean soil redox potential for the flooded treatment was 220 mV beginn ing on day 1 and continued to decrease to 48 mV by day 4 (Figure 4 1). Leaf gas exchange Expt. 1. removal and flooding treatments for net CO 2 assimilation and stomatal conductance on a few of the measurement dates. Therefore, flooding treatments were compared separately within each leaf removal treatment. In each leaf removal treatment, net CO 2 assimilation, stomatal conductance and in flooded than in non flooded trees af ter 4 days of flooding (Figures 4 2, 4 3, 4 4). Net CO 2 assimilation of flooded plants increased to values close to those of non flooded plants 34 days after plants were unflooded only in the 1/3LR treatment. Net CO 2 assimilation of flooded plants did not recover to pre flooding levels in the other leaf removal treatments (Figure 4 2). Expt. 2. removal and flooding treatments for stomatal condu ctance and transpiration on a few of

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69 the measurement dates. Therefore, for all leaf gas exchange variables, flooding treatments were compared separately within each leaf removal treatment. Net CO 2 than in non flooded plants (Figure 4 4). Reductions in net CO 2 assimilation were first observed on the third and fourth day of flooding in the 1/3LR and 1/2LR treatments respectively In the control and 2/3LR treatments, reductions in net CO 2 assimilati on were observed 1 and 8 days after plants were unflooded, respectively. The same pattern was observed for stomatal conductance (Figure 4 5) and transpiration (Figure 4 6) within each of the leaf removal treatment. In control plants, net CO 2 assimilation and stomatal conductance values of flooded plants recovered to those of the non flooded trees 35 days after plants were unflooded. In the 1/3LR and 1/2LR treatments, net CO 2 assimilation returned to levels similar to non flooded plants 45 days after plant s were unflooded. Net CO 2 assimilation of plants in the 2/3LR treatment returned to values similar to those of non flooded plants 30 days after plants were unflooded (Figures 4 4, 4 6). Plant growth and survival Expt. 1 There was a significant statistic flooding and the leaf removal treatments for root dry weight. Therefore, the effect of flooding on leaf, stem, root, and plant dry weights were determined separately within each leaf removal treatment. In the control a nd 1/2LR treatment, flooded plants had leaf and total plant dry weights than non flooded plants. In the 1/3LR treatment, there

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70 were no significant differences (P > 0.05) in dry weights between flooded and non flooded plants (Figure 4 7). At the end of the experiment (day 55), 83% of the flooded plants in the 2/3LR treatment died. In the control and 1/2LR treatments, 67% of the flooded plants died, whereas in the 1/3LR treatment, only 50% of the flooded plants died (Table 4 1). No non flooded plants died. Expt. 2. stem, root, and total plant dry weights than non flooded plants. In the 1/3LR treatment, flooded plant s. In the 1/2LR and 2/3LR treatments, there were no significant differences (P > 0.05) between flooded and non flooded plants for leaf, stem, root or plant dry weight (Figure 4 8). At the end of the experiment (day 57), 33% of flooded plants in the contro l and 2/3LR treatments died. In the 1/3LR and 1/2LR treatments, 17% of the flooded plants died (Table 4 2). Sap Flow There was no significant interaction (P > 0.05) between leaf removal and flooding treatments for daily sap flow. Therefore leaf removal tr eatments were pooled to compare flooding treatments. There were no significant (P > 0.05) differences between flooded and non flooded trees for daily xylem sap flow rate (Figure 4 9). Discussion Removal of avocado leaves prior to flooding (Expt. 1) result ed in significantly lower net CO 2 assimilation and stomatal conductance in flooded than in non flooded plants.

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71 Leaf removal prior to flooding also prevented plant recovery after plants were unflooded as indicated by significantly lower net CO 2 assimilatio n and stomatal conductance in flooded than in non flooded plants with leaves removed until the end of the experiment (day 55). Tree mortality was higher and organ dry weights were lower in flooded plants with leaves removed prior to flooding than in plant s with no leaves removed. In the present study, in order to leave the most photosy n thetically active leaves intact young leaves (Lambers et al., 2008) leaves were removed starting from the base of the trunk upwards until the desired percentage of leaves were removed. In a similar study, when avocado leaves were removed by pruning (from the top of the branches downward) the results were similar to this study. When a portion of the leaves was removed from the base of avocado canop y s immediately after floo ding (Expt. 2), net CO 2 assimilation and stomatal conductance were lower in flooded plants than in non flooded plants during the flooding period. However, in contrast to leaf removal before flooding, removing leaves after flooding facilitated recovery of f looded plants weeks after plants were unflooded as indicated by leaf gas exchange values that were similar to those of non flooded plants. Leaf removal after flooding also resulted in higher organ dry weights and greater tree survival after flooding comp ared to plants with no leaves removed. Similarly, Gil et al. (2008) found that removal of two thirds of the avocado canopy by pruning before flooding exacerbated stress, whereas removal of the same proportion of the canopy after flooding enhanced plant su rvival. Increased stress of avocado trees as a result of leaf removal prior to flooding observed in the present study is consistent with previous research showing that

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72 harvesting (shoot removal) forage legumes causes significant plant stress, especially when roots are in hypoxi c conditions (Hatwig et al., 1987). Similarly, i n alfalfa ( Medicago sativa ) root waterlogging following clipping significantly increased injury of plants grown in greenhouse or the field (Barta and Schmitthener 1986) Increased i njury to plants with leaves removed prior to flooding may have been the result of a reduction in carbohydrate production due to a reduced amount of photosynthesizing tissue Because a continuous supply of fermentable carbohydr ates in root tissues is criti cal for long term survival during flooding, the presence of leaves may be important as an assimilate source for root respiration (Barta, 1987; Liao and Lin, 2001) Root zone flooding also affects transport of carbohydrates to meristematic sinks (Kozlowski, 1997). Therefore if a sufficient amount of assimilates in leaves is not produced and transported to the roots, carbohydrate deficiency will occur (Liao and Lin, 2001). In previous experiments with avocado trees, flooding resulted in a decrease in photo synthesis that coincided with a decrease in the primary photosynthetic products, D mannoheptulose and perseitol in the roots ( Chapter 3 of this thesis ). Additionally, removal of two thirds of avocado leaves prior to flooding resulted in an even greater red uction of these carbohydrates in roots compared to plants with no leaves removed prior to flooding This may explain why in Expt.1 tree mortality was higher in plants with two thirds of the leaves removed before flooding than in any other leaf removal tre atment, including the control plants Reduction in root respiration is one the e arliest responses of plants to root hypoxia (Liao and Lin, 2001) When oxygen is not available for energy production in the roots, plants undergo anaerobic respiration, which produces less energy (ATP) than aerobic

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73 respiration for plant metabolism (Taiz and Zieger, 2010). Thus, the combined effect of reduced transport of photoassimilates and anaerobic respiration is exacerbated by the reduction of carbohydrate reserves when lea ves are removed before flooding, resulting in severe stress and tree mortality Flooding avocado trees reduced stomatal conductance, net CO 2 assimilation root and shoot growth and caused root necrosis These responses appear ed to be greater in plants with two thirds of the leaves removed than in plants with fewer leaves removed ( i.e., one third of the leaves removed), demonstrating that leaf removal before flooding increases flooding stress and reduces tree recovery. Removing leaves immediately after flo oding (Expt. 2) increased post flooding survival of avocado trees. A reduction in net CO 2 assimilation and stomatal conductance were observed in flooded plants as seen in Expt. 1 T ranspiration was also lower in flooded plants, presumably as a result of d ecreased stomatal conductance This is consistent with previous research with young apricot trees ( Prunus armeniaca ) that showed a reduction of transpiration in f looded plants after 50 hours of flooding (Nicols et al., 2005). In avocado trees, r eductions in transpiration have been attributed to reductions in stomatal cond uctance rather than a hydraulic effect since flooding d oes not reduce xylem water potential (Schaffer et al., 1992; Schaffer et al., 2006). Similar results have been observed in other tro pical fruit trees such as mamey sapote ( Pouteria sapota ) (Nickum et al., 2010) and carambola ( Averrhoa carambola ) (Ism ail and Noor, 1996). T he mechanisms for reductions in stomatal conductance in avocado trees as a result of flooding have not been elucidat ed (Gil et al., 2009). It has been hypothesized for other woody plants that an increase in

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74 abs c i s ic acid (ABA) concentration in leaves is the root to sho ot signaling mechanism inducing stomatal closure in flooded plants (Kozlowski, 1997). In the present st udy, ABA concentration was not measured; however, Gil et al. (2009) could not relate stomatal closure to differences in ABA concentrations between flooded and non flooded avocado trees. Therefore, the mechanism for increased stomatal closure of flooded av ocado trees still needs to be elucidated. Internal regulation of stomatal conductance and stem sap flow have generally been interpreted as mechanism s to restrict water movement during drought or flood ing (Nicols et al. 2005 and Ruiz Sanchez et al. 1996 ) Low oxygen concentrations in the root zone have been shown to reduce the permeability of roots to water (Smith et al. 1990 ; Zhang and Tyerman 1991), increasing the resistance to water uptake (Domingo et al. 2002). Under these conditions, water loss fr om the shoots exceeds the supply from the roots, leading to a reduction in leaf water potential and stomatal conductance (Domingo et al. 2002). In woody plants, the differences between water loss via transpiration and water uptake and short term dynamics associated with changes in water status can be determined from measure Citrus limon ) trees subjected to 3 days of flooding exhibited a progressive reduction in sap flow and stomatal closure (Ortuo et al., 2007). Similarly, in young apricot trees ( Prunus armeniaca ), 3 hours of flooding caused a decrease in sap flow and a strong reduction in plant hydraulic conductance, decreasing water absorption by the roots (Nicols et al., 2005). In the same experiment, a close relationship was observed between leaf water potential, leaf conductance and pl ant hydraulic conductance indicating that hydraulic signals are likely to play a dominant role in coordinating the observed responses of the

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75 shoot. In the present study, there was a ten dency for sap flow to decrease as the flooding days progressed Howeve r, a relationship between plant water loss and sap flow was not observed because sap flow measurements were discontinued before the end of the flooding period Sap flow sensors caused a slight browning of the stem a few days after flooding commenced This may have been the result of removing part of the vascular tissue during stem preparation for the sap flow system. M easurements were halted after the symptoms were observed to allow stems to heal and avoid further tree damage. However, this may have affect ed tree survival in control and 2/3LR treatments since tree mortality in both treatments was 66.67%. Tree survival of flooded plants was 83.3% in both the 1/3LR and 1/2LRhalf leaf removal treatments when leaves were removed after the flooding period Des pite high tree mortality, possibly due to stem damage from the sap flow sensors, plants in the 2/3LRl treatment that survived flooding recovered faster than plants with any other leaf removal level. This suggests that removing two thirds of the leaves afte r flooding may be the optimum leaf removal level to ensure tree survival after flooding. Based on leaf gas exchange, tree recovery, and plant biomass, leaf removal before flooding increased flooding stress whereas leaf removal immediately after flooding increased plant survival The results of this study support the observation that leaf removal from avocado trees immediately after flooding can reduce flooding stress of avocado trees (Crane et al., 1994; Gil et al., 2008) Removing two thirds of the canop y immediately after flooding may be the optimum amount of leaf removal to ensure tree survival. On the other hand, leaf removal shortly before flooding can exacerbate flooding stress. F urther studies are necessary to quantify the relationship

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76 between the q uantity of canopy removed and tree survival and recovery of trees pruned before flooding.

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77 Figure 4 1 Mean s o il redox potential of flooded 3 year (Expt. 1) and 2 year avocado trees (Expt. 2) on Waldin seedling rootstock Plants were flooded from 11 June to 13 June 2011 (Expt. 1) and from 13 Oct. to 17 Oct. 2011 (Expt. 2). Redox potentials below +200 mV indicate that soil conditions are anaerobic (Ponnamperuma, 1984), n = 6.

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78 Figure 4 2 N et CO 2 assimilation (A) of flooded and non flooded 3 year avocado trees on Waldin seedling rootstock in each of 4 leaf removal treatments (Expt. 1). Leaf removal t reatments were: no leaves removed ( control ) one third (1/3LR), one half (1/2LR) or two thirds (2/3LR) of the leaves removed before flooding An asterisk indicates a significant difference between flooding 0.05) n=6.

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79 Figure 4 3 Stomatal conductance of water vapor (g s ) of flooded and non flooded 3 year avocado trees on Waldin s eedling rootstock in each of 4 leaf removal treatments (Expt. 1). Leaf removal t reatments were: no leaves removed ( control ) one third (1/3LR), one half (1/2LR) or two thirds (2/3LR) of the leaves removed before flooding An asterisk indicates a significant difference between flooding treatments according to a repeated measures

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80 Figure 4 4 Net CO 2 assimilation (A) of flooded and non flooded 2 year avocado trees on Waldin seedling rootstock in each of 4 leaf re moval treatments (Expt. 2). Leaf removal t reatments were: no leaves removed ( control ) one third (1/3LR), one half (1/2LR) or two thirds (2/3LR) of the leaves removed after flooding An asterisk indicates a significant difference between flooding treatmen 0.05), n = 6.

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81 Figure 4 5 S tomatal conductance of water vapor (g s ) of flooded and non flooded 2 year seedling rootstock in each of 4 leaf removal treatments (Expt. 2). Leaf removal t reatments were: no leaves removed ( control ) one third (1/3LR), one half (1/2LR) or two thirds (2/3LR) of the leaves removed after flooding An asterisk indicates a significant difference between flooding treatments according to a repeated measures

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82 Figure 4 6 Transpiration (E ) of flooded and non flooded 2 year avocado trees on Waldin seedling rootstock in each of 4 leaf removal t reatments (Expt. 2). Leaf removal t reatments were: no leaves removed ( control ) one third (1/3LR), one half (1/2LR) or two thirds (2/3LR) of the leaves removed aft e r flooding An asterisk indicates a significant difference between flooding treatments acco 0.05), n = 6

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83 Figure 4 7 R oot, leaf, stem, and total plant dry weight s of flooded and non flooded 3 year f our leaf removal treatments (Expt. 1). Leaf removal treatments were: no leaves removed (control), one third (1/3LR), one half (1/2LR) or two thirds (2/3 LR) of the leaves removed prior to flooding. There were no significant differences between flooding tr eatments for any dry weight according to a T test (P > 0.05), n = 6.

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84 Figure 4 8 R oot, leaf, stem, and total plant dry weight of flooded and non flooded 2 year k in each of four leaf removal treatments (Expt. 2). Leaf removal treatments were: no leaves removed (control), one third (1/3LR), one half (1/2LR) or two thirds (2/3LR) of the leaves removed after flooding. An asterisk indicates a significant difference between flooding treatments according to a T 0.05), n = 6.

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85 Figure 4 9 Daily sap flow of flooded and non flooded trees of 2 year avocado trees on Waldin seedling rootstock (Expt. 2) Leaf removal t reatments were: no leaves removed ( control ) and two thirds of the leaves removed (2/3LR) after flooding There were no significant differences between flooding treatments on any measurement date (P > 0.05), n= 3.

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86 Table 4 1 Number and p ercentage of tree mortali ty for flooded and non flooded 3 year with in each leaf removal treatment (Expt.1). Plants were harvested 52 days after plants were unflooded Leaf removal treatments were: no leaves removed (cont rol), one third (1/ 3 LR ), one half (1/2 LR ) or two thirds (2/3LR of the leaves removed (2/3 LR ) before plants were flooded Treatment Control 1/3LR 1/2LR 2/3LR Flooded No. of plants alive 2 3 2 1 No. of dead plants 4 3 4 5 Mortality (%) 66.6 50 66.6 83. 3 Non f looded No. of plants alive 6 6 6 6 No. of dead plants 0 0 0 0 Mortality (%) 0 0 0 0

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87 Table 4 2. Number and p ercentage of tree mortality for flooded and non flooded 2 year Simmonds in each leaf removal treatment (Expt.2). Plants were harvested 52 days after flooding treatments were initiated (5 days after plants were unflooded ). Leaf removal treatments were: one third of the leaves removed (1/3 LR ), half of the leaves removed (1/2 L R ), and two thirds of the leaves removed (2/3 LR ) after plants were flooded. Treatment Control 1/3LR 1/2LR 2/3LR Flooded No. of plants alive 4 5 5 4 No. of dead plants 2 1 1 2 Mortality (%) 33.3 16.6 16.6 33.3 Non f looded No. of plants alive 6 6 6 6 No. of dead plants 0 0 0 0 Mortality (%) 0 0 0 0

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88 CHAPTER 5 CONCLUSIONS In several areas of the world where avocados are commercially produced, including southern Florida, trees can experience periodic flooding of the root zone due to poor soil drainage, or heavy rains from tropical storms or hurricanes. The rapid depleti on of oxygen in the root zone as a result of flooding can be injurious or even lethal to plants. Pruning the canopy has been recommended as a method to rehabilitate injured avocado trees after flooding. Since roots are damaged by flooding, reducing transpiration by canopy pruning presumably offsets the reduced water uptake by damaged roots and thus alleviates water stress. Pruning trees prior to flooding (in anticipation of a predicted storm or hurricane or after harvest during hurricane season ) may actually exacerbate flooding stress by reducing carbohydrate concentration from reduced photosynthesis (as a result of less leaf area), thereby reducing the s ubstrate for root respiration. Consequently, reduced respiration in the roots of pruned trees during flooding may result in increased flooding stress compared to non pruned, flooded trees. The objectives of this research were: 1. t o determine if limiting n et CO 2 assimilation by leaf removal or foliar application of a chemical photosynthetic inhibitor prior to flooding exacerbates plant stress and delays or prevents recovery of avocado trees from short term (a few days) flooding; 2. t o determine the combined effects of leaf removal prior to flooding and root zone hypoxia on root carbohydrate concentrations, particularly C 7 sugars; and 3. t o determine the relationship between the timing (pre or post flooding) and quantity of leaf removal on stress and surviva l of avocado trees exposed to short term flooding.

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89 After a few days of flooding, in all experiments soil redox potential measurements indicated that the rhizophere of flooded plants was hypoxic. Decreased net CO 2 assimilation and stomatal conductance were observed for flooded plants in all experiments. This is consistent with previous studies showing that avocado is a flood sensitive species which exhibits reductions in leaf gas exchange and shoot and root growth shortly after plants are flooded (Schaffer et al., 1992). Leaf removal immediately (1 day) or foliar application of the chemical photosynthetic inhibitor, Freeway immediately (1 day) before and when roots were flooded, increased the negative impact of flooding on net CO 2 assimilation, stomatal c onductance, organ and whole plant dry weights and tree survival. This was p resumably due to a reduction of carbohydrate reserves for root respiration during the flooding period. Unlike other fruit crops, D mannoheptulose and a related C 7 sugar alcohol, perseitol represent major forms of nonstructural carbohydrates in avocado (Liu et al., 1999). The nonstructural carbohydrates found in other tree fruit species are based on a six carbon hexose skeleton, such as glucose, sucrose and even starch. While the se six carbon sugars are found also found in avocado trees, they are present in much lower concentrations than the C 7 sugars in avocado tissues (Liu et al., 1999). Flooded plants with no leaves removed prior to flooding had higher D mannoheptulose and per seitol concentrations than plants with leaves removed prior to flooding. Reduction of carbohydrate reserves by leaf removal resulted in carbohydrate deficiency in root tissues and thus less carbohydrate in the roots for use as a substrate for respiration These C7 sugars have also been shown to have antioxidant properties in avocado fruit (Bertling et al., 2007). Although studies have shown that root respiration is reduced in

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90 flooded plants (Liao and Lin 2001; Geigenberger, 2003; Serres and Voesenek, 200 8), significant differences in root respiration of avocado trees between flooding or leaf removal treatments could not be detected in the present study. However, flooded plants with no leaves removed prior to flooding tended to have higher root respiratio n than flooded plants with leaves removed. Based on leaf gas exchange, root respiration, carbohydrate concentration, plant growth, and plant survival, inhibition of photosynthesis by leaf removal or the application of Freeway prior to flooding exacerbate s flooding stress. Reduction of the main photosynthetic products, D mannoheptulose and perseitol, in the roots and possibly the role of the former as an antioxidant (although antioxidant activity was not measured in this study) apparently resulted in plan ts with leaves removed prior to flooding, being more susceptible to flooding stress than plants with their canopies left intact. These observations provide evidence that reducing photosynthesis by leaf removal before flooding can increase flooding stress a nd decrease plant survival. However, additional studies are necessary to quantify the effect of prolonged flooding on root respiration and plant biomass of plants with photosynthesis inhibited, either by leaf removal or with a chemical photosynthetic inhib itor, before flooding. In separate experiment s plants were subjected to varying amounts of leaf removal [0 (control), one third (1/3LR), half (1/2LR), and two thirds (2/3LR)] immediately before or after flooding. Similarl to previous experiments, removal of leaves immediately before flooding resulted in severe stress and significantly lower net CO 2 assimilation and stomatal conductance in flooded than in non flooded trees. Tree mortality of plants in the 2/3LR treatment was 83.3%, whereas tree mortality in 1/3LR treatment was 50%

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91 and in the control and 1/2LR treatments was 66.6%. Plants in the 1/3LR treatment were the only ones that recovered well after flooding as indicated by higher survival and higher organ dry weights than in any other leaf removal tre atment The reasons for high tree mortality in the control treatment remain unclear. Although no leaves were removed from control plants and sufficient quantities of carbohydrate s may have been available for respiration, these plants had higher transpirati onal surface area than plants with leaves removed. Roots damaged by flooding may have been unable to supply enough water to a large canopy, thus preventing recovery and increasing tree mortality. In contrast to leaf removal before flooding, leaf removal after flooding significantly reduced net CO 2 assimilation and transpiration of flooded plants. However, plants with leaves removed after flooding recovered faster than control plants as indicated by greater organ dry weights and tree survival than for plan ts with leaves removed prior to flooding. Tree survival in both the 1/3LR and 1/2LR treatments was 83% and for both the control and 2/3LR treatments was 66.6%. Plants in the 2/3LR treatment recovered faster than any other leaf removal level presumably due to equilibrium between the shoot and root volume Similar results were observed by Gil et al. (2008), who observed that 1 year pruned after flooding compared to non pruned trees This was presumably due to reduced transpirational demand as a result of a reduction in canopy volume of pruned trees that had the root system partially damage d from flooding. This research provides evidence that reducing photosynthesis and carbohydrate re serves by removing leaves or the use of a chemical photosynthetic inhibitor before flooding increases the flooding stress and decreases survival of avocado trees In

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92 contrast to removing leaves prior to flooding, removing leaves from avocado trees immedi ately after a flooding event appears to reduce flooding stress and increases tree survival, presumably by reducing transpiration demand on a root system that has been damaged by flooding. Of the percentages of leaf removal tested, removing two thirds of t he canopy immediately after flooding was the was the most effective amount of leaf removal to reduce tree stress and help trees recover from and survive flooding. Further studies are necessary to quantify the effect of prolonged flooding on root respirati on and biomass of plants with lea ves remov ed prior to flooding.

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101 B IOGRAPHICAL SKETCH Maria Anglica Sanclemente Galindo was born in Cali Colomb ia in 1983 After graduating from high school in 1999, she attended the Universidad del Valle to study biology. After taking her first botany class during the second year of her undergraduate program, Ang lica became interested in plants. In her second yea r as an undergraduate she worked as a student assistant for the basic botany and plant physiology classes offered in her department Additionally, she worked in the herbarium of the university for one year. Upon graduation in 2005, she moved to St. Peter sburg, Florida with her parents. After taking English classes for six months, she started working at the University of South Florida in St. Petersburg. There, s he worked as a research assistant in the D epartment of Internal Medicine, doing research in dia betes, aging, and obesity. During her time there, she enjoyed working with non human primates. In January 2010, she decided to go back to plants and started her M aster of Science degree in h orticultural science at the University of Florida under the mento rship of Dr. Bruce Schaffer. Her m degree thesis focused on the physiological responses of flooded avocado trees to leaf removal. Upo n completing her MS degree, Ang lica will pursue a PhD degree focusing on plant physiology. After her graduate studies, she plans to pursue a career in academia.