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Role of Temperature in Water Uptake of Cold Acclimated 'Hamlin' Sweet Orange

Permanent Link: http://ufdc.ufl.edu/UFE0024941/00001

Material Information

Title: Role of Temperature in Water Uptake of Cold Acclimated 'Hamlin' Sweet Orange
Physical Description: 1 online resource (77 p.)
Language: english
Creator: Barkataky, Smita
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cold, deacclimation, irrigation, relative, water
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: With a crop value of approximately $1.2 billion in 2007-08, citrus is one of the most important horticultural crops grown commercially in Florida. Florida?s climate is characterized by a rainy season where daily irrigation is not needed and a dry season where daily irrigation is required. The dry season occurs during winter where temperatures are often below that which alters citrus physiology in a manner that may affect its irrigation requirements. The following study was conducted to evaluate the effect of cold, non-freezing temperatures on water use of citrus and physiological aspects that may alter water use independent of soil moisture. Two experiments were conducted on potted citrus trees grown in environmental growth chambers at the University of Florida, Southwest Florida Research and Education Center. In the first experiment, the trees were exposed to temperatures that promoted a fully cold hardened state. In the second experiment, the trees were exposed to alternating warm and cold temperatures that simulated temperature fluctuations during winter in Florida. Results of cold-treated trees were compared to trees maintained in growth promoting temperatures. Water use decreased to a third for fully cold hardened trees compared to the controls. Water use was less in the alternating temperature experiment than the controls, but more than for fully cold hardened trees. Although soil moisture was not limiting throughout the two experiments, stomatal conductance was less and root resistance higher for cold treated trees than the controls. These results demonstrate that during periods of cold acclimating temperatures, citrus will decrease water use independent of soil moisture. Current irrigation scheduling of commercial citrus is based on a reference evapotranspiration (ETo) multiplied by a crop coefficient (Kc) that varies from 0.7 in winter to 1.1 during the height of the growing season. The results from this study indicate that the Kc during winter may need to be adjusted to account for differences in water requirements of citrus due to temperatures that promote cold acclimation. By effectively modeling the effect of temperature on water requirements of citrus during winter, irrigation can be optimized, thus saving water and reduce leaching below the root zone.
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.
Statement of Responsibility: by Smita Barkataky.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Morgan, Kelly Tindel.
Local: Co-adviser: Ebel, Robert C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024941:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024941/00001

Material Information

Title: Role of Temperature in Water Uptake of Cold Acclimated 'Hamlin' Sweet Orange
Physical Description: 1 online resource (77 p.)
Language: english
Creator: Barkataky, Smita
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cold, deacclimation, irrigation, relative, water
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: With a crop value of approximately $1.2 billion in 2007-08, citrus is one of the most important horticultural crops grown commercially in Florida. Florida?s climate is characterized by a rainy season where daily irrigation is not needed and a dry season where daily irrigation is required. The dry season occurs during winter where temperatures are often below that which alters citrus physiology in a manner that may affect its irrigation requirements. The following study was conducted to evaluate the effect of cold, non-freezing temperatures on water use of citrus and physiological aspects that may alter water use independent of soil moisture. Two experiments were conducted on potted citrus trees grown in environmental growth chambers at the University of Florida, Southwest Florida Research and Education Center. In the first experiment, the trees were exposed to temperatures that promoted a fully cold hardened state. In the second experiment, the trees were exposed to alternating warm and cold temperatures that simulated temperature fluctuations during winter in Florida. Results of cold-treated trees were compared to trees maintained in growth promoting temperatures. Water use decreased to a third for fully cold hardened trees compared to the controls. Water use was less in the alternating temperature experiment than the controls, but more than for fully cold hardened trees. Although soil moisture was not limiting throughout the two experiments, stomatal conductance was less and root resistance higher for cold treated trees than the controls. These results demonstrate that during periods of cold acclimating temperatures, citrus will decrease water use independent of soil moisture. Current irrigation scheduling of commercial citrus is based on a reference evapotranspiration (ETo) multiplied by a crop coefficient (Kc) that varies from 0.7 in winter to 1.1 during the height of the growing season. The results from this study indicate that the Kc during winter may need to be adjusted to account for differences in water requirements of citrus due to temperatures that promote cold acclimation. By effectively modeling the effect of temperature on water requirements of citrus during winter, irrigation can be optimized, thus saving water and reduce leaching below the root zone.
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.
Statement of Responsibility: by Smita Barkataky.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Morgan, Kelly Tindel.
Local: Co-adviser: Ebel, Robert C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024941:00001


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1 ROLE OF TEMPERATURE IN WATER UPTAKE OF COLD ACCLIMATED HAMLIN SWEET ORANGE By SMITA BARKATAKY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Smita Barkataky

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3 To my mother late Ranjita Barkataky

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4 ACKNOWLEDGMENTS I would like to thank God almighty for guiding, protecting and helping me and my family in every perspective of my life. I take the privilege to express my profound gratitude appreciation and indebtedness to my chairperson, Dr. Kelly T. Morgan, not only for financial and moral support, but also for his sincere guidance, sug gestions and for offering his precious time and knowledge throughout the academic program. I would like to expresses my heartiest thanks and gratefulness to Dr. Robert C. Ebel, for allowing me to use his laboratory, providing me help during data collection, helping with his valuable suggestions and kind inspiration pertaining to this work. I am also thankful to Dr. Edward A. Hanlon, Jr, for his benign help, suggestions and active cooperation throughout the program. I would like to thank Dr. John Dunckelman, Associate Director, and all the employees of South West Florida research and education center, Immokalee, FL, for their help and cooperation throughout my entire program. Special thanks go to Mr. Peter Newman, for making me familiar to the growth chambers and equipments, helping me with data collection and being extremely supportive, helpful and cooperative. I am thankful to Lisa for helping me with data collection throughout the experimental period. Sincere thanks go to Julie for helping me with getting b ooks from library; Naveen, Jawwad and Augustine for their help, particularly for monitoring the growth chambers after a rain or storm events; Rosa for allowing me to use the deep freezer to keep the leaf samples; and Sadie, Jan, Ann and Ruddy for their hel p in collecting weight data. I am also thankful to Shinjiro for demonstrating how to use the Sigma plot software and for being helpful and supportive.

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5 I am grateful to Mrs. Suchi S. Shukla and Dr. Sanjay Shukla for their continuous help, support and encouragement towards me and my family. I am and will always be thankful to my father, Mr. Bipul Barkataky, my mother in -law, Mrs. Surama Goswami, my brother and sister and all other members of my and my in laws family for their love, blessings, moral support and good wishes towards me during every step of my life. My deepest gratitude goes to my husband, Debashish Goswami for his love, active support, and inspiration during the program and throughout my life. Lastly, I acknowledge our three year old son, Samp rit for making each moment of my life full of fun and joy.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 ABSTRACT ........................................................................................................................................ 11 CHAPTER 1 INTRODUCTION ....................................................................................................................... 13 2 LITERATURE REVIEW ........................................................................................................... 18 Citrus Irrigation In Florida .......................................................................................................... 18 Climate And Irrigation Requirements Of Citrus In Florida ..................................................... 19 Water Relations Of Citrus .......................................................................................................... 20 Water Flow Through The Soil Plant Atmosphere Continuum ................................................. 21 Soil Water Potential .................................................................................................................... 22 Root Resistance ........................................................................................................................... 22 Cold Acclimation Of Citrus ........................................................................................................ 23 Citrus Response to Drought Stress ............................................................................................. 25 Effects On Vegetative Growth ............................................................................................ 25 Effects On Reproductive Growth ....................................................................................... 26 Effect Of Stress On Growth And Production ..................................................................... 27 Objectives .................................................................................................................................... 29 Experiment 1: Cold Acclimation Experiment .................................................................... 29 Experiment 2: Alternate Cold Acclimation And Deacclimation Experiment.................. 30 3 MA TERIALS AND METHODS ............................................................................................... 31 Experimental Design ................................................................................................................... 31 Soil And Plant Characteristics .................................................................................................... 31 Insulation Of The Pots ................................................................................................................ 31 Treatments ................................................................................................................................... 32 Acclimation Treatments For The First Experiment ........................................................... 32 Acclimation And Deacclimation Treatments For The Second Experiment ..................... 32 Growth Chambers ....................................................................................................................... 32 Management Practices ................................................................................................................ 33 Watering ............................................................................................................................... 33 Fertilizer Application ........................................................................................................... 33 Pesticide Application ........................................................................................................... 33 Data Collection ............................................................................................................................ 3 4 Calculations And Estimations .................................................................................................... 37

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7 Leaf Area .............................................................................................................................. 37 Root Resistance .................................................................................................................... 37 Hourly Water Use Or Hourly Crop Evapotranspiration .................................................... 38 Relative Water Use .............................................................................................................. 38 Statistical Analysis ...................................................................................................................... 39 4 RESULTS AND DISCUSSION ................................................................................................ 42 Growth Chamber Environmental Measurements ...................................................................... 42 Relative Humidity, Absolute Humidity, Dew Point, And Light Intensity ....................... 42 Mean Temperature ............................................................................................................... 42 Physiological Responses Of Plants During Cold Acclimation And Alternate Warm And Cold Periods ............................................................................................................................. 43 Overall Crop Growth ........................................................................................................... 43 Stem Water Potential ........................................................................................................... 44 Transpiration ........................................................................................................................ 45 Stomatal Conductance ......................................................................................................... 46 Osmotic Potential................................................................................................................. 47 Root Resistance .................................................................................................................... 47 Relative Water Content ....................................................................................................... 49 Citrus Water Use And Water Requirement ............................................................................... 50 Water Use ............................................................................................................................. 50 Relative Water Use .............................................................................................................. 51 5 CONCLUSION ........................................................................................................................... 67 LIST OF REFERENCES ................................................................................................................... 69 BIOGRAPHICAL SKETCH ............................................................................................................. 77

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8 LIST OF TABLES Table page 4 1 Leaf area (cm2) at the beginning and at the end of the cold acclimation experiment. ....... 57 4 2 Leaf area (cm2) at the beginning and at the end of the alternate cold acclimation and deacclimation experiment. ..................................................................................................... 57 4 3 Water use and relative water use of the acclimated plants at different air temperatures during the cold acclimation experiment. ............................................................................... 66 4 4 Water use and relative water use of the acclimated plants at different air temperatures during alternating cold acclimation and deacclimation experiment. .................................. 66

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9 LIST OF FIGURES Figure page 1 1 Commercial Citrus Production Areas in Florida .................................................................. 17 1 2 Distribution of Soil orders in Commercial Citrus Production Areas in Florida. ................ 17 3 1 Plants inside growth chamber with insulation cover. .......................................................... 40 3 2 Plants inside growth chambers showing the irrigation emitters. ......................................... 40 3 3 Leaves wrapped with flexible plastic and aluminum foil before measuring stem water potential. ................................................................................................................................. 41 3 4 Leaf area measurement at the end of the first experiment. .................................................. 41 4 1 Absolute humidity, dew point, and relative humidity in the control and acclimated chambers in cold acclimation experiment. ........................................................................... 52 4 2 Absolute humidity, dew point and relative humidity in control and acclimated chambers during the 1st cycle of the alternate acclimation and deacclimation experiment. ............................................................................................................................. 53 4 3 Average daily light intensity in the control and acclimated chambers in the cold acclimation experiment .......................................................................................................... 54 4 4 Average daily light intensity in the control and acclimated chambers in the alternate cold acclimation and d eacclimation experiment. ................................................................. 54 4 5 Mean daily temperatures in control and acclimated chambers during the cold acclimation experiment. ......................................................................................................... 55 4 6 Mean daily temperatures in control and acclimated chambers during the 1st cycle of alternate cold acclimation and deacclimation experiment. .................................................. 55 4 7 Comparison of overall growth of the control and the acclimated plants at the end of the cold acclimation experiment. .......................................................................................... 56 4 8 Stem water potential during the cold acclimation experiment. ........................................... 58 4 9 Stem water potential during alternate cold acclimation and deacclimation experiment ... 58 4 10 Transpiration during cold acclimation experiment .............................................................. 59 4 11 Transpiration during alternate cold acclimation and deacclimation experiment ............... 59 4 12 Stomatal conductance during cold acclimation experiment. ............................................... 60

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10 4 13 Stomatal conductance during alternate cold acclimation and deacclimation experiment. ............................................................................................................................. 60 4 14 Osmotic potential during cold acclimation experiment. ...................................................... 61 4 15 Osmotic potential during alternate cold acclimation and deacclimation experiment. ....... 61 4 16 Roo t resistance during cold acclimation experiment. .......................................................... 62 4 17 Root resistance during alternate cold acclimation and deacclimation experiment. ........... 62 4 18 Relative water content during the cold acclimation experiment. ........................................ 63 4 19 Relative water content during the alternate cold acclimation and deacclimation experiment. ............................................................................................................................. 63 4 20 Hourly water use during the cold acclimation experiment .................................................. 64 4 21 Hourly water use during the alternate cold acclimation and deacclimat ion experiment. ............................................................................................................................. 64 4 22 Relative water use between acclimated and non acclimated (control) plants during cold acclimation experiment. ................................................................................................. 65 4 23 Relative water use between acclimated and non acclimated (control) plants during alternate cold acclimation and deacclimation experiment. .................................................. 65

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11 Abstract of Thesis Presented to the Graduate School of the Universit y of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ROLE OF TEMPERATURE IN WATER UPTAKE OF COLD ACCLIMATED HAMLIN SWEET ORANGE By Smita Barkataky August 2009 Chair: Kelly T. Morgan Cochair: Rober t C. Ebel Major: Soil and Water Science With a crop value of appro ximately $1.2 billion in 200708, citrus is one of the most important horticultural crops grown commercially in Florida. Floridas climate is characterized by a rainy season where daily irrigation is not needed and a dry season where daily irrigation is re quired. The dry season occurs during winter where temperatures are often below that which alters citrus physiology in a manner that may affect its irrigation requirements. The following study was conducted to evaluate the effect of cold, non -freezing tem peratures on water use of citrus and physiological aspects that may alter water use independent of soil moisture. Two experiments were conducted on potted citrus trees grown in environmental growth chambers at the University of Florida, Southwest Florida Research and Education Center. In the first experiment, the trees were exposed to temperatures that promoted a fully cold hardened state. In the second experiment, the trees were exposed to alternating warm and cold temperatures that simulated temperature fluctuations during winter in Florida. Results of cold-treated trees were compared to trees maintained in growth promoting temperatures. Water use decreased to a third for fully cold hardened trees compared to the controls. Water use was less in the alte rnating temperature experiment than the controls, but more than for fully cold hardened trees. Although

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12 soil moisture was not limiting throughout the two experiments, stomatal conductance was less and root resistance higher for cold treated trees than the controls. These results demonstrate that during periods of cold acclimating temperatures, citrus will decrease water use independent of soil moisture. Current irrigation scheduling of commercial citrus is based on a reference evapotranspiration (ETo) mu ltiplied by a crop coefficient (Kc) that varies from 0.7 in winter to 1.1 during the height of the growing season. The results from this study indicate that the Kc during winter may need to be adjusted to account for differences in water requirements of c itrus due to temperatures that promote cold acclimation. By effectively modeling the effect of temperature on water requirements of citrus during winter, irrigation can be optimized, thus saving water and reduce leaching below the root zone.

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13 CHAPTER 1 INTRODUCTION Citrus trees are perennial evergreen plants that evolved in humid tropic conditions but have become widely adapted to semiarid regions of the world (Kriedemann and Barrs, 1981). Currently, 576,577 acres are in commercial citrus production, with a total production of 203.8 million boxes of citrus which accounts 70% of the 13 million tons of total citrus production of the United States for the 200708 season. With a crop value of $1.2 billion in 200708, citrus is one of the most important horticu ltural crops in Florida. The citrus in Florida is grown in a range of soil types and the climate varies throughout the region, all of which affect production practices. There are four major commercial citrus production regions in Florida (Figure 1 1) and three major soil orders where citrus are grown: Entisols, Spodosols, or Alfisols (Obreza and Collins, 2008). Entisols are found mostly on the Central Florida ridge, while the majority of soils on the Flatwoods of southwest Florida are Spodosols. Florida's Indian River citrus -growing area near the east coast contains a mixture of Alfisols and Spodosols (Figure 1 2, Obreza and Collins, 2008). Climate is one of the most important factors that affect citrus growth and production. Citrus can be grown in a varie ty of arid and humid climates and can withstand temperatures ranging from 2.2 C (28 F) to 40.6 C (105 F). However, citrus performs best in the range between 15.6 C (60 F) and 30 C (86 F) (Parsons and Beck, 2004). Florida has favorable climate for the production of citrus. The northern and the central part of Florida have a humid subtropical climate, whereas the southern part has a tropical climate. The temperature in the state varies from nearly 32 C to 38 C (90 F to 100 F) during the summer mo nths. During late autumn and winter months, Florida has experienced occasional cold fronts that can bring high winds and relatively cooler temperatures for the entire state, with high temperatures between 4

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14 15 C ( 40s and 50s F ) and lows between 0 10 C (30s and 40s F) for few days. Florida has the highest average precipitation of any state, in large part because afternoon thunderstorms are common in most of the state from late spring until early autumn. Florida has a defined rainy season from June through September and dry season from October through May. The soil types and climates lend themselves to climatic stresses throughout the growing season. Records indicate that freezes have always been a risk factor in growing citrus in Florida during the dr y season of winter. Freezing temperatures can cause extracellular ice formation, which lowers apoplastic water potential, dehydrates the symplast, and destabilizes cellular membranes (Steponkus, 1984). In Florida, the freezes in January 1981 and 1982 cause d orange production loss to be a combined total of 3,640,000 tons (39.1%) compared with the 197980 seasons. The heaviest losses were incurred in the northern and northwestern portions of the citrus belt, where several counties experienced a nearly complet e loss (88%) of their commercial citrus trees (Miller, 1991). Drought stress is a nother climatic stress that impacts citrus grown in Florida where rainfall is insufficient to meet evaporative demand during winter, and thus its a standard industry practice to irrigate. Cell growth is very sensitive to drought stress and only a few bars of drought stress can slow down or stop the process of cell growth (Stover et al., 2002). As stress continues to develop, synthesis of a hormone called abscisic acid (ABA) increases, leading to earlier stomatal closure and a decrease in photosynthesis (Stover et al., 2002). Drought stress has a profound effect on the vegetative and reproductive growth of citrus (Kriedmann and Barrs, 1981). The most dramatic and obvious respons e of citrus to drought stress is leaf abscission, reducing the tree canopy (Spurling, 1951; Mathews, 1972; Marsh, 1973; Kaufmann, 1977). Drought stress also reduces top growth to re -establish a more favorable shoot : root ratio when undergoing water deficits (Kaufmann, 1977).

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15 Many cold protection methods have been used over the years. These methods include heaters, wind machines, fog generators, high volume over tree irrigation, and low volume undertree microsprinkler irrigation (Parsons and Boman, 2009). However, irrigation has been reported as the best practice that protects citrus plants against freezing by regulating factors such as heat of fusion and heat of vaporization (Parsons and Boman, 2009). Supplemental irrigation of citrus in Florida came into u se in the 1940s. Various irrigation methods were used, including water wagons, surface flood, subirrigation, sprinkler, and micro irrigation systems. The transition from the older irrigation methods to micro irrigation between 1991 and 2001 resulted in considerable reduction in water use requirements per acre for citrus ((Floridas agricultural water policy, 2003). Microsprinkler irrigation systems have become the standard for Florida citrus. Microsprinkler and drip systems have been collectively referred to as trickle, low volume irrigation, or microirrigation systems. Compared to overhead sprinklers, low volume systems can save water if they are properly managed. Because these systems usually operate at lower pressures than conventional overhead systems, there can also be appreciable savings from reduced energy costs. In addition, microsprinkler irrigation can provide partial frost protection for both young and mature trees. Irrigation recommendations have been based on water use during the growing season, however, the cold acclimation process of citrus alters water use, and the recommendations for winter may not be appropriate. Citrus tree physiology changes dramatically as they acclimate to low temperatures (Dansereau, 2007). Cold acclimation or cold hard ening is the ability of a plant or plant organ to resist freezing or subfreezing conditions (Fuchigami, 1996; Soule, 1985). Citrus trees acclimate slowly and deacclimate rapidly in response to temperature (Yelenosky, 1985). Citrus growth tends to decline w hen ambient temperatures are less than 12 oC, cold acclimates

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16 below 10 oC, and are fully cold hardened when exposed to 10 oC or less for approximately 3 weeks (Yelenosky et al., 1984; Yelenosky, 1985; Young, 1969; Young and Peynado, 1962, 1965).

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17 Figure 1 1. Commercial Citrus Production Areas in Florida (Available at http://edis.ifas.ufl.edu/SS403 ). Figure 1 2. Distribution of Soil orders in Commercial Citrus Production Areas in Florida. (Available at http://edis.ifas.ufl.edu/SS403)

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18 CHAPTER 2 LITERATURE REVIEW Citrus Irrigation In Florida Accurate determination of soil moisture status is the prime factor of a good irrigation management practice. There are different direct or indirect methods to measure soil moisture content. However, there is no universally recognized standard method and no uniform way to compute and pr esent the results (Boman and Parsons, 2002). A major problem still exists in determining the proper amount of water to apply to irrigated fields (Thomson and Ross, 1995). Over the years, some sensor -based scheduling methods have used to indicate how much w ater to apply. These methods observed sensor responses to wetting to adjust subsequent water amounts (Stolzy et al., 1959; Fischbach and Schleusener, 1961; Skinner, 1976). Limitations of and the need to calibrate scheduling models have prompted endorsement by many researchers, particularly with extension interests, of these and similar sensor based approaches to irrigation scheduling. These approaches, however, have been facing some limitations in many sectors of the farm community because sensor readings r equire interpretation and some sensor types require maintenance. Many field calibrated ET models meet the objective of forecasting irrigation dates with continuous weather updating. Evapotranspiration is a combined process of both evaporation from soil an d plant surfaces and transpiration through plant canopies (Irmak and Haman, 2003). In practice, the estimation of the evapotranspiration rate for a specific crop requires first calculating potential evapotranspiration (ETc) or reference evapotranspiration (ETo) and then applying the proper crop coefficients (Kc) to estimate actual crop evapotranspiration (ETa). ETc can be defined as the amount of water transpired in a given time by a short green crop, completely shading the ground, of uniform height and wit h adequate water status in the soil profile. ETo can

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19 be defined as the rate of evapotranspiration from a hypothetical reference crop with an assumed crop height of 0.12 m (4.72 in), a fixed surface resistance of 70 sec m1 (70 sec 3.2ft1) and an albedo of 0.23, closely resembling the evapotranspiration from an extensive surface of green grass of uniform height, actively growing, well -watered, and completely shading the ground (Irmak and Haman, 2003). Crop coefficient ( Kc ) could be estimated using relative water use of plants during the cold temperatures in winter over the warm temperatures in summer. Reported Kc values for central Florida citrus ranged from about 0.6 in winter to 1.1 in summer (Boman, 1994; Fares and Alva, 1999; Rogers et. al., 1983). In F lorida, using the average of these two numbers for Kc (0.85) and the relative water use, the Kc values at different temperature conditions can be estimated. Many models have been developed for use as ETo and Kc to determine water use periods. These models allow growers to accurately schedule irrigation according to Kc and recognized soil depletion values ( Morgan et. a l. 2006). Climate And Irrigation Requirements Of Citrus In Florida The Florida citrus belt is located in a subtropical climate zone that normally receives 48 59 inches of rainfall annually (Tucker et al., 2002). This rainfall amount is adequate to meet citrus water requirements for the states predominantly sandy, low water -holding capacity Ridge soils, and poorly drained, high water table flat woods soils of coastal and southern Florida (Boman, 2002). However, its uneven annual distribution pattern does not actually satisfy peak seasonal demands during the dry season of March through early June. During this period, good irrigation management is particularly critical to reduce stress and associated young fruit drop and yield loss (Tucker et al., 2002). Supplemental water from irrigation has a major impact on yield when rainfall is deficient, particularly in the spring. In arid regions, most of the water needs are met by irrigation, and rainfall is a secondary provider of water (Parsons and Beck, 2004). Moderate

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20 water stress in citrus groves reduces shoot growth and leaf expansion, slows canopy development in young trees, and reduces vegetative grow th needed to support fruit production in mature trees (Stover et al., 2002). Most commercial citrus groves in Florida use irrigation, however scheduling the amount and time for irrigation is important for water use efficiency. Various methods of irrigation have been used in Florida. However, in recent years, nearly all new groves use microirrigation (drip and microsprinklers) systems (Boman et al., 2002). With chemigation, microirrigation systems can also provide an economical method of applying fertilizer and other agricultural chemicals on a timely basis (Boman et al., 2008). Water Relations Of Citrus Citrus is one of the most important irrigated crops in arid and semi arid regions. Citrus water requirements vary with climatic conditions and variety (Roge rs and Bartholic, 1976; Boman, 1994; Fares and Alva, 1999). Hydraulic conductivity of citrus roots is greatly controlled by soil temperature. Bialoglowski (1936) found for rooted leafy lemon cuttings that when root temperature is the only variable, transpi ration during the day was greatest at 25 oC, and declined when the ambient temperatures were either less than 25 oC or greater than 40 oC. However at night when transpiration was very low, the rate was independent of root temperature throughout the range o f 0 to 40 oC. Water relations of citrus are influenced to a large extent by high resistances to water transport within the plant mainly because of low root: shoot ratios and its poorly developed root hairs (Kriedemann and Barrs, 1981). Another feature of c itrus is the sensitivity of its stomata to air humidity, which is associated with high internal resistances at leaves and stems. The evapotranspiration, ET, of Florida citrus typically ranges between 820 and 1280 mm yr 1 (Rogers et al., 1983). Soils lose their ability to conduct water to the surface as they dry (Hillel, 1998). Likewise, citrus ET decreases as the fraction of the soil surface receiving full sunlight decreases and the canopy shades an increasingly larger ground area (Castel and Buj,

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21 1992). T herefore, it is important to assess the ability of citrus to make use of the residual soil water available from the winter season. Hilgeman et al. (1969) found that internal water deficit of Valencia oranges was at a minimum in March and April, however, i n early May there quickly followed a period of high transpiration and large internal water deficits. The authors also found cooler cloudy conditions lowered transpiration in August and transpiration increased again on warmer days in September and early Oct ober, only to decrease once more with the onset of winter due to low temperatures and low vapor deficits. Resistance to water uptake in an irrigated and a water -stressed 22year old citrus tree was examined during 7 weeks by Cohen et al. (1983) who found t hat sap flow in stressed trees decreased with time at a higher rate than the water potential difference between soil and leaf, which implies an increase in the resistance of the transport system. Water Flow Through The Soil Plant Atmosphere Continuum To understand how plants behave in cold temperature, it is necessary to know water movement within the plant. Water moves from the soil, through the plant, and out into the surrounding atmosphere (Nobel, 1991). The most generally accepted mechanism for water mo vement through a plant is the cohesion theory proposed by Henry Dixon and Charles Joly in the 19th century (Nobel, 1991). Water evaporating from the leaves creates a tension in the xylem where hydrogen bonds provide an intermolecular attraction and conti nuity between water molecules (Nobel, 1991). Thus the column of water in the lumen of the xylem is drawn upward to regions with lower hydrostatic pressure (Nobel, 1991) Water movement in the soil -plant atmosphere continuum can be described using an analo gy of Ohms law (Landsberg and Jones, 1981; Van den Honert, 1948), which describes flow as being proportional to driving force (the water potential gradient) and inversely proportional to the resistance in the flow path

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22 ) ( ) ( ) ( ) (soil root stem soil va vs va vsR R r r p p T (2 1) Where, T = transpiration pvs = vapor pressure at the leaf surface pva = the vapor pressure of the atmosphere rvs = stomatal resistance and the inverse is stomatal conductance rva = boundary layer resistance soil = soil water potential stem = stem water potential Rroot = root resistance Rsoil = resistance exerted by soil against water movement from soil to the root surface. Soil Water Potential Total soil water potential is defined as the amount of work pe r unit quantity of pure water that must be done by external forces to transfer reversibly and isothermally an infinitesimal amount of water from the standard state to the soil at the point being considered. Transpiration is driven by evaporative demand (pvs pva), which is controlled by stomata at the leaf air interface and accelerated by wind which reduces rva. As the VPD (vapor pressure deficit) increases, stomatal conductance of citrus tends to decrease. Hall et al. (1975) found higher stomatal conduct ance in a controlled environment when plant transpiration, and soil and root/soil hydraulic conductivity (Chone et al, 2001). Evaporation of water from the stomata l cavities results in development of a plant water deficit due to the stem stem soil, water is drawn from soil into the plant. Root Resistance Root resistance, Rroot represents about 2/3 of total plant hydraulic re sistance (Landsberg and Jones, 1981; Passioura, 1988) and hence is one of the most important factors at the soil plant atmosphere continuum. The water potential gradient from soil to atmosphere is termed as the driving force (Rieger and Litvin, 1999). Sl atyer (1967) suggested that resistance to flow

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23 through roots probably was the major source of plant hydraulic resistance because water in its passage through the plant has to flow through cell membranes in the root endodermis. Castel (1978) reported a tota l hydraulic resistance of 116.7 x 10 9 J kg1 m1 sec1 for rooted Valencia cuttings growing in nutrient solution. Castel also found that citrus root resistance is four times as high as leaf resistance, and three times as high as stem resistance. In a shor t -term experiment (1 hour) conducted by Ramos and Kaufmann (1979), it was found that temperature strongly affected water flow through rough lemon roots from 5 to 35 C. Temperature influenced root membrane permeability, since reduced flow could not be expl ained by changes in water viscosity. Soil temperature has pronounced effects on many physiological processes in plants such as root growth, ion absorption, hydraulic conductivity, and associations with symbiotic organisms (Kramer, 1969; Nielsen, 1974; Syve rtsen et al., 1983). If root temperatures are greater or less than optimum temperatures, the hydraulic conductivity of roots may be reduced and thus may indirectly affect stomatal conductance, CO2 assimilation, and subsequent translocation of carbohydrates (Kadoya et al., 1981). Cold Acclimation Of Citrus A significant component of winter hardiness in cold hardy plants is the capacity to undergo cold acclimation. Almost all temperate perennial and many annual and biennial plants, upon exposure to low nonfre ezing temperatures can alter their tissue and cellular freezing tolerance capacity (Guy, 1990). Levitt (1980) described cold acclimation as a physiological modification that is induced by gradual exposure to chilling temperatures that enable the plant to m aintain homeostasis and ultimately to survive and reproduce in a stressful environment. Gene expression is altered in plants during cold acclimation. Several plant genes induced by low temperatures have been identified in alfalfa (Wolfraim et al., 1993), A. thaliana (Gilmour et al., 1992), and strawberry (Yubero-Serrano et al., 2003). During cold acclimation, several

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24 changes take place within a plant including mRNA transcription, increase in protein synthesis, and qualitative changes in the pattern of synt hesized proteins (Gilmour et al., 1992). Changes in gene expression alter downstream products, especially carbohydrate concentration (Yelenosky and Guy, 1977). They found that carbohydrate increases the fastest and at a constant rate between 15 and 5 oC, b ut decrease at 0oC. Besides the increase in carbohydrates (sucrose, glucose and fructose), cold hardened plants exhibit an increase in proline, ATP, ABA, glutamic acid, ascorbic acid, and unsaturated fatty acids (Kushad and Yelenosky, 1987). Low temperatu re (LT) is one of the most important abiotic factors limiting growth, productivity, and distribution of plants (Boyer, 1982; Sakai and Larcher, 1987). Low temperature decreases biosynthetic activity of plants inhibits the normal function of physiological p rocesses and may cause permanent injuries, leading to death of the plant. Citrus remains uninjured if ice does not form in the tissues during cold acclimation, excluding fruit peel, which is injured at temperature above 0 oC (Eaks, 1960; Purvis and Yelenow sky, 1982). Freezes cause ice to form in the apoplast (Meryman, 1956, 1966; Yelenosky, 1985, 1996). During extracellular freezing, ice first forms in the dilute apoplastic solution and a water potential gradient are established between the extracellular ic e crystal and the intracellular liquid water. The lower water potential of ice as compared to that of liquid cellular water at the same temperature will cause liquid water to move from the cell to the extracellular ice (Levitt, 1980). This process, dependi ng on the temperature and concentration of the cell sap imposes a dehydration stress on the cell. When the cellular water potential of the partially dehydrated cell equals that of the extracellular ice, an equilibrium is established and further dehydration will not occur provided the temperature remains constant (Hansen and Beck, 1988; Rajashekar and Burke, 1982). If the temperature further declines or increases, water will flow out of or back into the cell, respectively. During such equilibrium

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25 freezing a tissue can behave either as an ideal solution or as a non -ideal solution (Hansen and Beck, 1988; Rajashekar and Burke, 1982). In nonideal equilibrium freezing, negative wall pressure is believed to reduce the degree of cell dehydration and solute concentr ation (Rajashekar and Burke, 1982). Cold tolerance of citrus is promoted by cold temperature (Ebel et al., 2005; Yelenosky et al., 1984; Yelenosky, 1978, 1985, 1996), which is accompanied by plant dehydration (Yamada et al., 1985; Yelonosky, 1982; Yelenosk y, 1985), an important mechanism that enhances survival during freeze events. Citrus roots acclimate to cool soil (15 oC) through an increase in conductivity of individual roots (Syvertsen et al., 1983). During cold acclimation, dehydration of citrus is pa rtly caused by the increase in root resistance, which inhibits water uptake (Kadoya et al., 1981; Kramer, 1969; Nielson, 1974; Syvertsen et al., 1983). With the exception from the usual case of stomatal aperture, where maximum dehydration is observed when all the stomata are open; cold acclimated citrus may experience dehydration even though the stomata are closed. This effect may be because of higher root resistance (Kadoya et al., 1981) that inhibits water uptake by the roots resulting in dehydration. It has been recognized that citrus develop plant water deficits during cold acclimation, and development of these plant water deficits may play a role in cold tolerance (Yelenosky, 1985). Citrus Response to Drought Stress Effects On V egetative G rowth Drought stress has a profound effect on the vegetative and reproductive growth of citrus (Kriedmann and Barrs, 1981).The most dramatic and obvious response of citrus to drought stress is leaf abscission (Spurling, 1951; Mathews, 1972; Marsh, 1973; Kaufmann, 1977) However, before widespread abscission occurs, some form of adjustment apparently occurs because of which we see the plants carrying leaves even though the orchard has been abandoned 5 years

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26 ago. Another response due to drought stress includes reduced top growth, by which citrus receives a more favorable shoot: root ratio when undergoing drought stress (Kaufmann, 1977). Levy et al. (1978) suggested that drought stress reduces final leaf size in grapefruit. Maotani et al. (1977) found that growth of the fruit and trunk of Satsuma mandarin was more sensitive to leaf water deficit compared to leaf growth. He found that fruit growth ceases at a leaf water potential below 0.8 MPa, while leaf growth can persist between 1.5 to 2.0 MPa. As a consequence of drought stress, canopy growth is reduced. Tudela and Primo-Millo (1992) reported that the leaf abscission occurred when drought -stressed citrus plants were allowed to rehydrate whereas no abscission was observed in plants in drought stress. Like canopy growth, citrus stem growth is also reported to be adversely affected by water shortage. Continued drought stress can lead to twig dieback (Marsh, 1973). In severe and prolonged drought stressed condition, death of branches or even death of the whole tree can occu r. Fruits and leaves are normally affected first in this process (Mathews, 1972). Drought stress may lead to increased depth of rooting (Marsh, 1973). Even when rooting depth is not increased by water stress, the proportion of feeder roots of orange may be increased (Hilgeman and Sharp, 1970), which is apparently due to the increase in the total weight of the feeder roots. Effects On R eproductive G rowth When water becomes deficient, fruit bud initiation in citrus is generally increased (Magness, 1953). A pe riod of water stress was reported to be favorable for the subsequent flowering in Philippines and similar periods of stress are deliberately imposed to induce flowering of lemons at commercially desirable times in Sicily and Israel (Monselise and Halevy, 1 964). Flower bud initiation is mediated by changes in hormone balance and hormones may result from accumulation of sugars in water -stressed trees (Magness, 1953).

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27 According to Jones and Cree (1965), unlike most deciduous fruit crops, citrus is self thinnin g. Many bud and flowers drop even before fruit is set. The reason for this observed drop is weakening of tissue in a preformed abscission zone at the point of attachment of the base of the ovary to the disc or of the pedicel to the twig (Erickson, 1968). D rought stress leads to increased flowering and dropping of young fruits (Magness, 1953; Shmueli et al., 1973; Van der Weert et al., 1973; Shalhevet et al., 1976); which if sufficiently severe can lead to complete crop loss. Effect Of S tress On G rowth And P roduction Plants develop various mechanisms to withstand drought such as higher shoot to root ratios, fewer and smaller leaves, concentrated solutes or increased activity of oxidative stress enzymes in leaf cells (Lei et al., 2006). As soil dries, accumu lation of solutes to decrease leaf osmotic potential allows plants to maintain a favorable water potential gradient. Thus, lower leaf osmotic potentials can maintain the positive leaf turgor, which is required to keep stomata open and sustain gas exchange Sanchez and Syvertsen, 2006). The accumulation of proline in many plants represents a general response to stresses including water deficit, flooding (Aloni and Rosenshtein, 1982), high salinity, high temperature, freezing, UV radiation and heavy metals (Siripornadulsil et al., 2002; Yin et al., 2005). Drought stress arises from an imbalance between water supply and demand. Demand depends on the leaf -to air vapor pressure gradient (VPG) and is a function of leaf temperature and atmospher ic vapor pressure deficit (Kriedemann and Barrs, 1981). As VPD increases, stomatal conductance of citrus decreases and the stomatal sensitivity to VPD plays a key role in maintenance of favorable leaf water relations. The sensitivity of citrus stomata to V PD ensures that stomatal resistance is usually high compared with other crops (Mantell, 1977); which in turn leads to a tendency for water use efficiency to be higher in citrus than in other C3 crop species

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28 Oranges are usually damaged when the fruit are e xposed to temperatures of 2.22 C (28 F) or lower for 4 hours or more. As the temperature gets colder or durations less than 2.22 C get longer, damage to fruit, leaves, twigs, and eventually large branches increases. More than any other factor, freezes h ave caused some of the most dramatic changes in fruit supply, availability, and price (Parsons and Boman, 2003). Southwick and Evenport (1985) conducted experiments with containerized 'Tahiti' lime (Citrus latifolia Tan.) trees to define conditions needed to induce flowering and reported that cyclical or continuous water stress for 4 to 5 weeks induced flowering. Flowering was also induced in moderate ( 2.25 MPa, midday) or severe ( 3.5 MPa, midday) water stress conditions, as measured by leaf xylem pressu re potential for only 2 weeks. However, the response was more significant in severely stressed trees. Low temperature (18 C day/10 C night) induced a time dependent flowering response much like that of moderate water stress. Significantly negative leaf x ylem pressure potentials as compared to controls were found only with the drought stress treatment, suggesting that a common stress linked event, separate from low plant water potential is involved in floral induction. Leafless, immature cuttings from mature, field grown trees were induced to flower by water stress treatment, suggesting that leaves are not essential for a flower inductive response. Bryla et al. (2001) reported that temperature governs citrus root respiration with moist soil conditions. For example, when soil temperatures fluctuated diurnally, the relationship between root respiration and soil temperature was exponential. Respiration increased by a factor of 1.8 2.0 for every 10 oC increase in soil temperature. Water moves from the soil t hrough the p lant to atmosphere in response to a vapor pressure gradient. Cold temperatures have a profound effect on the water uptake and on the physiological

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29 responses of citrus. Cold temperatures cause stomatal closure and increase root resistance which ultimately reduce water uptake. Reduced water uptake in cold temperatures reflects the fact that plants require less water when the ambient air temperatures are low. Thus to investigate the different responses of citrus trees with respect to water use and the physiological responses during cold acclimation temperatures, two studies were conducted in growth chambers with the following objectives. Objectives Because of the influence of cold acclimating temperatures on citrus physiology with respect to water upta ke, requirements for water during different temperature conditions may be different. Considering the size of the industry and the occasional water shortages that occur in parts of Florida, determining the water requirements would optimize irrigation schedu ling and water use efficiency. The overall objective of these studies was to increase our understanding of the water relations of citrus during cold acclimation, which will allow us to increase our understanding of irrigation requirements of citrus during winter in Florida. Experiment 1: Cold A cclimation E xperiment An experiment was conducted at the University of Florida, Southwest Florida Research and Education Center to evaluate the physiological responses and water relations of Hamlin orange trees to m aximum cold acclimation using growth chambers with the following specific objectives 1. Compare the water relations of coldacclimated Hamlin sweet orange plants with non acclimated control trees, with special reference to stem water potential, osmotic potential, relative water content, transpiration, and stomatal conductance. 2. Using water relations data and leaf area, estimate root resistance (Rroot) of citrus exposed to cold acclimating temperatures when soil water is not limiting.

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30 3. Evaluate hourly water use based on cold weather acclimation and estimate relative water use for trees exposed to cold acclimated temperatures. Experiment 2: Alternate Cold Acclimation And D eacclimation E xperiment Floridas climate is known for its mild temperatures even during the winter months. The state usually does not have continuous cold temperatures. Generally, air temperatures are reduced for only few days during a cold period. The goal of the second experiment was to determine citrus behavior in terms of water use during fluctuating temperatures. The specific objectives of this study were 1. Compare the water relations of Hamlin sweet orange plants exposed to alternating high and low temperature to control trees held at temperatures that promote growth, with spec ial reference to stem water potential, osmotic potential, relative water content, transpiration, and stomatal conductance. 2. Using water relations data and leaf area, estimate root resistance (Rroot) of citrus exposed to alternating temperatures when soil water is not limiting. 3. Evaluate hourly water use based on cold weather acclimation and estimate relative water use for trees exposed to alternating temperatures.

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31 CHAPTER 3 MATERIALS AND METHODS Experimental Design Two experiments were designed and carried out at South West Florida Research and Education Center, Immokalee in a similar manner using the same materials and equipments. In the first experiment, temperature in the acclimated chamber was programmed highest in the first week, and then it was r educed gradually and then held at constant temperatures for several weeks. Immediately after the completion of the cold acclimation experiment, the second experiment with alternate acclimation and deacclimation temperatures was conducted. Soil And Plant C haracteristics The soil for potting the citrus trees was collected from a commercial sugarcane field near Clewiston, Florida, which is classified as Margate sand. The Margate soil series is usually characterized by poorly drained, rapidly permeable soils t hat formed in sandy marine sediments of variable thickness over fractured limestone. Sixteen numbers of Hamlin sweet orange ( Citrus sinensis L. ) budded on Carrizo citrange (C. sinensis L. Osbeck X Poncirus trifoliata L. Raf.) rootstocks were used in these experiments The plants were potted into sixteen 20 x 40 cm (10.60 liter) tree pots (manufactured by Steuwe and Sons, Inc.) filled with 11.79 kg of screened air dried soil. These plants were approximately one year old. Six months before starting the experi ments, plants were maintained in a green house. In the greenhouse, the plants were pruned at a height of 60 cm from the juncture of stalk and scion. Insulation Of The Pots Each pot was enclosed in fiberglass insulation using duct tape to join the edges (F igure 3 1). The pots were then placed inside the growth chambers. The purpose of the insulation was to slow temperature declines of roots.

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32 Treatments Acclimation Treatments For The F irst Experiment Two identical environmental growth chambers (Model: PGR 15; Conviron products of America, Pembina, ND, USA ) were used in the experiments having exterior dimensions, growth area and growth height of 265 cm x 86 cm x 199 cm, 1.4 m2 and 147 cm, respectively. Plants were equally divided between the two environment al growth chambers with desired temperature, lighting, and relative humidity. In the control growth chamber, air temperature was maintained at 25 oC day/20 oC night throughout the experiment. Plants to be cold acclimated were placed in the second chamber a nd were exposed to air temperatures of 25 oC day/20 oC night for 7 days; 20 oC day/15 oC night for 7 days; 15 oC day/10 oC night for 7 days; and 10 oC day/5 oC night for 35 days. Both control and acclimation treatments were subjected to a 12/12 hour light/ dark photoperiod throughout the two experiments. Acclimation And D eacclimation Treatments For The Second E xperiment The control growth chamber was programmed at air temperature of 25 oC day/20 oC night throughout the experiment. In the treated chamber, plants were exposed alternately to 10 days cold and 3 days warm temperature. After the end of the previous experiment, the temperature of that chamber was raised to 10 oC day/5 oC night for 3 days; then the temperature was reduced to 10 oC day/5 oC night for the next 10 days, this pattern was repeated to 3 times. Both acclimation treatments were subjected to a 12/12 hour light/dark photoperiod. Growth C hambers Taking the measurements in one chamber usually took about 2 hours. Therefore, the control chamber was programmed with a 2 hour delay from actual time so that measurements were taken at similar relative time during the light period.

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33 Management Practices Watering The pots were watered using a single 35 ml/min drip irrigation emitter (Figure 3 2) per po t on daily basis. The irrigation emitters were programmed for watering 2 times daily for 10 minutes each time in order to minimize drought stress except on the two consecutive days of data collection. Thus each plant was programmed to receive approximately 700 ml water daily. On the 6th day after temperatures were changed in the cold acclimation treatment, 20 minutes before measuring stem water potential, plants were watered for additional 15 minutes (nearly 535 ml water to each plant). Irrigation emitters were turned off on the second day of data collection because 500 ml of fertilizer solution was applied manually to each plant. Emitters were programmed to irrigate only in the evening on the 1st day of data collections. Fertilizer A pplication A fertilize r solution was prepared by mixing 33.6 grams of a water soluble fertilizer, (Peters professionals, 18 8 17) in two gallons of water at the recommended dose: 16.8 grams in 7.57 liters of water (providing 400 ppm N) and applied 500 ml to each plant daily for the initial two weeks of the 1st experiment. In the following week, concentration of the fertilizer was reduced to the recommended rate and 500 ml/ plant was applied every other day. With the same concentration of the fertilizer solution, plants were fert ilized once a week for the rest of the experiments. Foliar spray of Keyplex (a combination of essential micronutrients) was applied once each month. Pesticide A pplication The plants were observed to have spider mite infestation two months before starting the experiments. Plants were treated with Agri -Mek 0.15 EC (Abamectin) as well as Admire Pro (Imidacloprid) solution. The first pesticide solution was made by mixing 1.0 ml pesticide in

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34 3.784 liters of water and 2% spray oil. This mixture was sprayed on th e foliage using a hand sprayer. Admire Pro solution was prepared by mixing Admire Pro in water using 1.8 milliliters in 3.784 liters water and the soil of each pot was drenched with 820 ml of the solution. The same treatment was given to the plants just be fore placement in the growth chambers. Data Collection Most of the data were collected on the last two day s of each treatment. Data collection included: 1 Stomatal conductance, leaf transpiration, and quantum of 5 leaves per plant using a Steady state poro meter (Model: LI 1600, LI COR.inc., Nebraska). 2 stem, of one leaf per plant were determined using pressure bomb (Model 3005 Plant water status console, Soil moisture equipment corporation, Santa Barbara, CA). 3 Osmotic potential ) stem by using a Dew point Microvoltmeter (Model: HR 33 T, Wescor, INC, 459 South Main Street, Logan, Utah) 4 Fresh weight, turgid weight, and dry weight stem in order to calculate relative water co ntent. 5 Leaf area (cm2stem by using leaf area meter (Model LI 3000A, LI COR inc., 4421 Superior St., Lincoln, Nebraska) 6 Leaf area was measured at the beginning of the experiment. At the end of the experiment, leaf areas of all the remaining leaves were measured. On the day of data collection, plants of the control chamber were watered 2 hours after the start of the light period. Immediately after irrigation, one average matured healthy leaves were selected from each pla nt and wrapped by using a transparent plastic sheet and a piece of aluminum foil (Figure 33). Temperature, relative humidity, light intensity, and soil moisture content were recorded at 10minute intervals by data loggers; and the data were downloaded eve ry week. To verify whether the control and the acclimated chambers experienced nearly equivalent meteorological

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35 conditions during the experiments, data from the logger were plotted to compare the daily relative humidity, absolute humidity, and dew point fo r both experiments. However, due to failure of the loggers after 14 days of the second experiment, no data could be accessed. The steady state porometer was placed inside the growth chamber 40 minutes before the start of data collection to equilibrate the instrument to conditions within the chamber. Two hours after watering, five newly matured leaves were selected randomly from each plant and stomatal conductance, transpiration, quantum, leaf temperature, and relative humidity of the canopy were measured. The average quantum measured for 5 leaves in a plant was considered as representative of light intensity for that particular plant canopy. The average of all the 8 plants in the chamber was considered as the light intensity of the chamber. These light inte nsity values were plotted against time (week) and were used to compare the two chambers. All plants were watered again and left for 15 20 minutes to minimize drought stress stem was measured. Wrapped leaves were cut at the base of petiole with a razor blade and stem was measured using the pressure bomb that was calibrated to be pressurized at 100 bars (10 MPa) per 30 seconds. The growth chamber containing the controls was sampled and the growth chamber containing the treatment was subsequently s ampled. This process was followed each time measurements were completed. stem stem, were unwrapped and weighed to determine fresh weight (FW). Leaf area of each leaf was then measured using a leaf are a meter. One circular disk of 7mm diameter was cut out from each leaf blade by using a hole punch and the remainder of the leaf disk was then soaked in pure water, and placed in glass petri plates. The leaves were kept in the refrigerator for 24 hours. The soaked leaves were then dried by using paper towels and weighed to get the turgid weight (TW). These leaves were then put

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36 into sixteen small paper bags holding one leaf per bag, labeled, put into a bigger paper bag and then dried at 129 oC for 72 hours. T he dried leaves were then weighed to determine dry weight (DW). FW, TW AND DW were used to determine the relative water content (RWC) according to Equation 3 1. 100 ) ( ) ( (%) DW TW DW FW RWC (3 1) Where, FW =fresh weight (g) DW =dry weight (g; after 72 hours at 129 oC) TW = turgid weight (g; after holding samples for 24 hours in distilled water at 4 C) The remaining cut disks were dipped in liquid nitrogen by holding each disk with forceps then put inside a Ziploc bag and kept at 80 oC for at least one week to measure the osmotic ). Osmotic potentials of all leaf samples were obtained at the end of the experiment using a dew point microvoltmeter. The dew point microvoltmeter was calibrated using NaCl solutions with Molality 0.550, 0.157, a nd 0.053 of known osmotic potentials of 2.5, 0.75, and 0.25 MPa. On the following day of data collection, the plants were watered normally. Two hours after watering, the pots were weighed. Transpiration was allowed to occur for 4 hours and pots were re weighed to determine water loss (T) using Equation 34. During the 1st week of the 2nd experiments, few shoots from the control plants were trimmed and leaf area of all trimmed leaves were measured. At the end of the 9 -week experiment, leaf areas of all t he leaves grown during the experiment were measured using a portable area meter (Figure 3 4).

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37 Calculations And Estimations Leaf Area At the beginning of the experiment, leaf area of each plant was measured (L0). The leaves that were developed during the e xperiment were tagged with colored ribbons for both experiments. At the end of the 1st experiment, areas of those leaves developed during the experimental period were measured (L1). After the end of the second experiment, leaf area for the leaves that had grown during the experiment was measured (L2). For the first experiment, initial leaf area is L0 and final leaf area is (L0 +L1). Similarly for the later one, leaf area at start and at end of the experiment were considered to be (L0+L1) and (L0+L1+L2) resp ectively. Based on these data, leaf area per plant per week was calculated using a linear regression equation for each plant. Root R esistance Root resistance of each plant was calculated using Equation 2 1, which when solved for Rroot, gives Equation 3 2. soil stem soil rootR T R ) ( (3 2) soil for soil at water holding capacity is about 0.03 MPa. This value in our study was set to zero introducing a relatively small error. Also, it is assumed that Rsoil was zero, which is a reasona ble assumption for well watered soils. These assumptions simplify Equation 3 2 to Equation 3 3. T Rstem root (3 3) stem is measured and T is determined by Equation 3 4

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38 t A PW PW Tleaf t o) ( (3 4) Where, PWo = pot weight in gram measured 2 hours after watering (g) PWt = pot weight measured about 4 hours later (g) Aleaf = total leaf area of the plant (m2) t = the time elapsed between weight measurement (s) To determine Rroot in units of MPa.s. /m, units that are the most commonly reported in the literature for liquid resistance, T was multiplied by 1 x 106 m3.g1 to convert transpiration units from weight (g.m2.s1) to volume (m3.m2.s1). Hourly Water U se Or Hourly C rop Evapotranspiration Water use of each plant was b ased on loss of pot weight during 4 hours of exposure time to treatment light and temperature. Assuming the pots were at FC 2 hours after watering, water used by each plant per hour was calculated using Equation 3 5. 4 2 1 ) (3weight weight cm Waterusend st (3 5) Wher e, 1st weight = weight of pots measured 2 hours after watering 2nd weight = weight of the pots measured 4 hours after taking the 1st weight Relative Water U se Relative water use (RWU) of the acclimated plants was calculated from the hourly water use data by using Equation 3 6. For both the experiments, the average of all the values for water use for the control plants was considered to be 100 % water use. Percentage of the fraction of the water use by acclimated plants (WUacclimated) to the average water u se of the control plants (WUcontrol) gave the relative water use for the acclimated plants at different temperatures. Using the relative water use data during cold acclimation and alternate acclimation and deacclimation experiments, water requirement by th e Hamlin orange plants in a certain temperature

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39 conditions was predicted. Because the relative water use was calculated from the average water use by the 8 plants per week, no standard error bars were used to verify the differences between control and ac climated plants. 100lim Control ated accWU WU RWU (3 6) Statistical Analysis The design of the experiment could be expected to vary depending upon the similarities or variability between the two chambers in relation to environmental conditions. If all the environmental parameters, except temperature, remained constant for both the chambers, then data could be analyzed as a completely randomized design with two treatments and 8 replications. A ll data are discussed based on graphical r epresentation with differences indicated as error bars. Means were separated using 2x standard error about the mean.

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40 Figure 3 1. Plants inside growth chamber with insulation cover. Figure 3 2. Plants inside growth chambers showing the irrigation emitters. Emitter

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41 Figure 3 3. Leaves wrapped with flexible plastic and aluminum foil before measuring stem water potential. Figure 3 4. Leaf area measurement at the end of the first experiment. Leaves wrapped with foil for measuring stem water potential

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42 CHAPTER 4 RESULTS AND DISCUSSION Growth Chamber Environm ental Measurements Relative H umidity, A bsolute H umidity, D ew P oint, And Light I ntensity Figure 4 1 indicated that the absolute humidity and dew point remained approximately the same throughout the cold acclimation experiment. At the beginning of the exper iment, the absolute humidity and dew point in the acclimated chamber were similar to the control chamber, but with the reduction in temperatures in the acclimated chamber, absolute humidity and dew point decreased in the acclimated chamber compared to the control one. The dew point is associated with relative humidity. A high relative humidity indicates that the dew point was closer to the current air temperature. When the dew point stays constant and temperature increases, relative humidity will decrease. Because the dew point in our experiments were nearly similar to air temperatures in the control and acclimated chambers, the relative humidity for both the chambers were nearly the same (between 80 90%) throughout the experiment. The light intensity (quant um) data for the acclimated chamber was not significantly different from the control chamber (Figure 4 3). The average light intensity in control and acclimated chambers durin g the 22/s respectively. Analogous trends of absolute humidity, dew point, relative humidity and light intensity were observed in the alternating acclimation and deacclimation experiment (Figure 4 4) indicating that the environmental conditions in both chambers were nearly identical during the experiment. Mean Temperature The measured daily mean temperatures confirmed that the growth chambers maintained the target temperatures. Results showed that dai ly temperature remained nearly constant in the

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43 control chamber throughout the experiment, whereas, mean daily temperature gradually declined to 10 0C in the acclimated chamber and then held constant (Figure 4 5). Because of unavailability of data after th e 2nd week of experiment 2, discrete interpretation of temperature during the second experiment after the 1st cycle was not possible. However, looking at the trend for the first 14 days data (Figure 4 6), it has been assumed that the temperature in the co ntrol chamber was constant and temperature in the acclimated chamber changed to an alternate high and low: high for 3 days and low for the following 10 days. Physiological Responses Of Plants During Cold Acclimation And Alternate Warm And Cold Periods Ove rall Crop G rowth The overall growth of a plant can be represented by the per cent increase in leaf area measured at the beginning and at the end of an experiment (Figure 4 7). Leaf area increase for the control plants was greater than the acclimated plants during the first experiment (Table 4 1). Because the control plants were constantly exposed to favorable temperatures for growth, the total increase of mean leaf area for the control plants was 2671 cm2 more than the acclimated plants. Results showed that at the end of the 1st experiment the increase in mean leaf area of the cold acclimated plants were 65% less than the control. This reduction in growth of plants due to low temperature condition is a direct result of the cold acclimation process. Cold accl imation and the induction of freezing tolerance in temperate perennials have been associated with the slowing or cessation of growth during the gradual transition from hot to cold temperature (Guy, 1990). When temperatures are low, water uptake by citrus d ecreased because hydraulic conductivity of the citrus roots is greatly controlled by soil temperature. Bialoglowski (1936) found for rooted leafy lemon cuttings that when root temperature is the only variable, transpiration during the day was greatest at 2 5 oC, and declines

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44 at temperatures less than 25 oC or greater than 40 oC. Size of leaves is also affected by cold temperatures. Levy et al. (1978) suggested that water stress reduces final leaf size of grapefruit. Leaf area of the leaves trimmed from the c ontrol plants during the first week of the 2nd experiment (39.03 cm2) was subtracted from the leaf area at the end of the 1st experiment. This resultant value was used as the leaf area for the control plants at the beginning of the 2nd experiment. During t he 2nd experiment, total increase in mean leaf area for the acclimated plants was greater than the total increase in the control plants (Table 4 2). Unlike the 1st experiment leaf growth of the control plants was 31% less than the acclimated plants in the alternate high and low temperature This response was likely caused by deacclimation mechanism. Plants started deacclimating by increasing transpiration and conductance, experiencing greater water uptake and reduced root resistance during the period of hi gher temperatures compared to the period of lower temperatures. Plants exposed to prolonged lower temperatures followed by warmer temperatures grew faster than those plants that experienced constant temperatures. In addition to that, the root growths of the control plants were probably restricted in the pot because the same plants and soils were used throughout the two experiments. The restricted root growth probably led to reduced shoot growth and thus reduced leaf area. Besides, the sugars produced and/or concentrated during acclimation are quickly available to cells when additional water via increasing temperatures is made available. In effect, the anti -freeze becomes an energy source. Stem Water P otential Figure 4 8 illustrates a decline in stem water po tential ( stem) with reduced temperatures, while stem for the control plants remained nearly constant. The results are similar with studies carried out on C. unshiu that showed a decrease in leaf water potential during cold acclimation (Yamada et al., 198 5). Yelenosky (1982) reported that decreasing leaf water content with cooler temperatures and apparent non -water -stressed field conditions suggested changes not only in

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45 stomatal behavior but also in hydraulic resistances and water uptake through citrus roo ts. In contrast, Vu and Yelenosky (1987) found leaf water potential of cold acclimated citrus to be higher than unacclimated controls. The most probable reason for this different effect of cold acclimation as explained by Dansereau (2007) was due to the us e of higher light and dark period temperatures for both acclimated and control treatments. Vu and Yelenosky (1987) used substantially higher light period temperatures for acclimated (15.6 oC) and control (32.2 oC) treatments and higher dark period temperat ures for the acclimated (5.4 oC) and control (21.1 oC) than the temperatures in this study. stem indicates the capacity of a plant to conduct water from soil to atmosphere (Chone, stem has been successfully applied as a water deficit indicator on peach and plum orchards (Garnier and Berger, 1985; MacCutchan and Shackel, 1992). Stem water potential was greater during deacclimation and lower during the period of cold acclimation (Figure 4 9). Stem water potential is the result of whole plant transpiration, and soil and root/soil hydraulic conductivity (Chone et al. 2001). During cold temper ature, the osmotic potential ( ) decreased (Figure 4 stem. Transpiration The transpiration rate of trees in the acclimated chamber decreased significantly from the 1st week through the 9th week (Figure 4 10). The transpiration rate in cold acclimated pl ants sharply declined until the 3rd week, when the chamber temperature was 15oC /10 oC; and then from the 4th week onwards plants transpired at a nearly the same rate. Because the control plants were exposed to constant environmental conditions, transpirat ion was expected to be the same throughout the experimental period. But transpiration initially declined through the 5th week, then increased through the 8th week and then decreased again. One of the reasons for the initial decline in transpiration could b e the acclimation to the growing conditions in the chambers.

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46 Before starting the experiment, the plants were kept outside, where they had been exposed to much higher temperatures than that of the growth chamber. In this higher temperature condition there w as higher leaf conductance as well as transpiration. When plants were brought to the chambers, the temperature was set at 25 oC (day) and 20 oC (night). Probably because of the different environmental conditions, the plants showed lower initial conductance and transpiration. As indicated by the findings, plants took approximately 5 weeks to get acclimatized to the growth chambers. Gradually, after 5th week, the control plants started conducting more water and increased transpiration as the trees acclimated to the new growing conditions. With the alternating high and low temperatures, transpiration rate was also found to increase and decease, respectively (Figure 4 11). Plants evidently take relatively less time to deacclimate to warm temperature than to acclimate to colder temperature. Guy (1990) reported that in cold climates, hardy perennials can undergo partial deacclimation and lose a portion of their freezing tolerance within a few days of unseasonably warm temperature in winter. Stomatal C onductance In figure 4 12 it is evident that leaf conductance of the acclimated plants increased initially, then from the 2nd week onwards conductance declined sharply and then remained almost constant (< 50 mmol/cm2/s) during remainder of the experiment, with the exce ption of the 7th week when conductance found to be spiked up to about 75 mmol/cm2/ s. Towards the end of the experiment, a slight increase in conductance and transpiration was observed, which probably indicates a metabolic adjustment to lower temperatures. A similar trend was observed by Dansereau (2007) for C. unshiu on P. trifoliata

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47 Stomatal conductance increased and decreased along with the increased and decreased temperatures in the second experiment (Figure 4 13). This change was associated with the opening of the stomata during deacclimation with higher temperatures. Osmotic P otential Osmotic adjustment in response to water stress is considered an important physiological mechanism enabling plants to tolerate water deficits (Begg and Turner, 1970). A leaf can increase its resistance to dehydration through a reduction in cel lular osmotic potential by a net accumulation of cellular solutes (Hsiao et al, 1976). Figure 4 decreased significantly as the temperature was reduced, and reached a minimum at a temperature 10 oC /5 oC. However, when temperatures rema increased slightly. Guinchard et al. (1996) reported that the osmotic potential was lower in leaves and stolons of cold acclimated plants subjected to chilling ( 1.5 MPa) than to control conditions ( 1.0 MPa). Accumulation of free proli ne and c (Yelenosky, 1979). Yelenosky and Guy (1977) reported that carbohydrates accumulated rapidly between 15 and 5 oC. Carbohydrates accumulate in the leaves of citrus tress more rapidly th an in the stems at 10 oC (Yelenosky, 1979). In the second experiment, osmotic potential for the acclimated plants fluctuated to higher and lower values with alternate high and low temperatures respectively, but remained nearly constant for the control plants (Figure 4 15). Root R esistance Hydraulic root resistance of the cold acclimated Hamlin orange trees increased greatly during the cold acclimation experiment (Figure 4 16). Results showed that Rroot of the acclimated plants after 9 weeks of cold acclim ation was 5 -fold greater than Rroot in the 1st week of the cold acclimation experiment. Several studies demonstrated that root resistance increases with cold acclimation (Kriedmann and Barrs, 1981; Landsberg and Jones, 1981). However, the exact

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48 reason for the high root resistance is still not clear. The route of water uptake into root systems is most likely controlled by water channel proteins, most of which appear to be down-regulated during cold acclimation (Kriedmann and Barrs, 1981; Landsberg and Jones, 1981). Flow rate is also one of the major criteria to be considered while discussing root resistance. There is a substantial body of evidence that root resistance is indirectly related to flow rate (Kriedmann and Barrs, 1981; Landsberg and Jones, 1981). R egardless of the reason, higher root resistance for acclimated plants hinder water uptake, which may promote water deficits. Figure 4 5 showed that there was a sudden increase in temperature in the acclimated chamber during the 6th week (day 41 and 42). Pr obably because of the increase in temperature in those two days, root resistance for the acclimated plants was lower in the 6th week. It was noticed from the graph that as the experiment progressed, there was also a slight increase in root resistance for the control plants also. The increase in canopy growth of control plants with time indicates that root growth probably also increased with time. As the roots proliferated within the limited volume of soil, some of the soil pore spaces might be filled with roots. In that case hydraulic conductivity would decrease and might tend to increase root s) and soil resistance (Rs) were zero given well watered conditions, might h ave introduced some error to this study. To determine whole plant transpiration, the plants were watered 2 hours before the pots were weighed the first time and transpiration was allowed to occur for 4 hours before the plants were reweighed. This 6 hours p s and an increase in Rs and if these changes are significant, it would increase the hydraulic strain on the plants and stem (Dansereau, 2004).

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49 When plants were exposed to alternate high an d low temperatures, root resistance (Figure 4 17) was found to be higher during cold acclimation period compared to the deacclimation period. Water uptake by a plant is associated with the root resistance and therefore during deacclimation period more wate r use by plants could be expected. Relative Water C ontent Results showed that the relative water content did not vary significantly with temperature changes during the two experiments (Figure 4 18, Figure 4 19). Even though the data obtained from the phys iological parameters (e.g. stem, ) indicated water deficit within the plants, the relative water content data indicated that the whole leaves were not dehydrated. Similar results were reported by Guinchard et al. (1996) for white clover (Trifolium repen s L., cv Huia). The unchanged relative water content may be related to the osmotic adjustment of the cold acclimated plants. The key role of osmotic adjustment is in turgor maintenance during water deficits, which in turn is essential for maintenance of turgor related processes, especially stomatal regulation (Cram, 1976). For any given leaf water potential, a leaf with a lower will have more turgor pressure to expend and can therefore withstand greater dehydration before a critical loss of turgor occurs (ONeill, 1983). Lower cellular osmotic potentials also conserve cellular volume and maintain gradients of water potential f avorable for water influx (ONeill, 1983). The reduction in of the acclimated plants during the experimental period probably increased the turgor pressure of the leaves which helped the plants maintain water inside the cells and to reduce dehydration. S imilar logic is applicable to the plants exposed to the alternating high and low temperatures.

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50 Citrus Water Use And Water Requirement Water U se Results (Figure 4 20) showed that the overall use of water by plants declined when temperatures were gradually decreased and became nearly constant with constant low values of temperatures. Water loss is influenced by several factors such as temperature, stomatal behavior, and root resistance (Ball et al., 1987). During cold acclimation, root resistance of citrus becomes higher, which inhibits water uptake by the plants (Kadoya et al., 1981). This increased root uptake resistance is one reason for the occurrence of reduced water use of citrus during cold acclimation (Kadoya et al., 1981; Kramer, 1969; Nielson, 1974; Syvertsen et al. 1983). Apart from root resistance, stomatal behavior is also responsible for regulating water use and in citrus; stomatal behavior is controlled by temperature and humidity (Kriedemann and Barrs, 1981). In this study, stomates were closed during cold acclimation which led to reduc ed transpiration. The increased root resistance and decreased stomatal conductance resulted in decreased water use during cold temperatures. However, the relative water content data (Figure 4 18, Figure 4 19) indi cated that the plants were still hydrated even during the cold acclimation. Although a constant trend of water use was expected for the control plants, it was found that water use showed an initial decline and then increased (Figure 4 20). This could be t he result of the initial acclimation of the plants from warm greenhouse temperatures to the relatively colder growth chamber temperatures. Figure 420 showed that the acclimated plants used the maximum water in the 6th week. This water use could be correla ted with the rise in temperature in the 6th week (Figure 4 5). Because the temperature was greater in that particular week, water uptake by plants was also high. As expected, plants exposed to alternate high and low temperatures, were found to use more wa ter in high temperatures and less during low temperatures (Figure 4 21). Water use for

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51 the second period of deacclimation was greater than that of the first period of deacclimation, indicating that plants need more than 3 days of warm temperature (>10 oC) to get deacclimated. Relative Water U se The average water use for the control plants was 31.42 cm3/ hour and 31.88 cm3/ hour in the1st and 2nd experiments, respectively. Figure 4 22 illustrates the percent relative water use by the Hamlin sweet orange pl ants in the acclimated chamber in relation to the plants in the control chamber. Relative water use was about 94% for the acclimated plants in the 1st week of study and then it reduced with the reduction in temperatures. Water use by the acclimated plants decreased by nearly 50% and 63% in the 2nd and the 3rd weeks when the temperatures were 20 oC/15 oC and 15 oC/10 oC, respectively (Table 4 3). Average relative water use from the 4th week through the end of the experiment for the plants exposed in 10 oC/5 oC was 35% indicating that during that period, plants used nearly 65% less water than the water used by control plants. Relative water use for the second experiment was reduced by 70% for the acclimated plants and increased and decreased with warmer and c ooler temperatures (Figure 4 23, Table 4 4) respectively The plot for the acclimated plants in Figure 4 23 illustrates that relative water use in each period of deacclimation increased compared with the previous period of deacclimation. During the 3rd pe riod of deacclimation, relative water use was only 8 % lower than the control plants indicating that even though plants were exposed to low temperatures, the influence of the previous deacclimation period carried over, allowing a sequential increase in rel ative water use.

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52 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Day 0 7 14 21 28 35 42 49 56 63 70 Absolute humidity ( gram/m^3) 4 6 8 10 12 14 16 18 20 22 24 Control Acclimated Day 0 7 14 21 28 35 42 49 56 63 70 Dew point ( 0 C) 0 5 10 15 20 25 Control Acclimated Day 0 7 14 21 28 35 42 49 56 63 70 Relative humidity (%) 50 55 60 65 70 75 80 85 90 95 100 Control Acclimated Figure 4 1 Absolute humidity, dew point, and relative humidity in the c ontrol and acclimated chambers during the cold acclimation experiment.

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53 Light 25 10 Dar k 20 5 Day 0 2 4 6 8 10 12 14 16 Absolute humidity (g/m^3) 6 8 10 12 14 16 18 20 Control Acclimated Day 0 2 4 6 8 10 12 14 16 Dew point (degree C) 0 5 10 15 20 25 Control Acclimated Day 0 2 4 6 8 10 12 14 16 Relative humidity (%) 78 80 82 84 86 88 90 Control Acclimated Figure 4 2. Absolute humidity, dew point and relative humidity in control and acclimated chambers during the 1st cycle of the alternate acclimation and deacclimation experiment.

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54 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Week 0 1 2 3 4 5 6 7 8 9 10 Quantum ( moles/m 2 /s) 160 180 200 220 240 260 280 300 320 340 360 Control Acclimated Figure 4 3. Average daily light intensity in the control and acclimated chambers during the cold acclimation experiment Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 36 Quantum ( moles/m 2 /s) 200 220 240 260 280 300 320 340 360 Control Acclimated Figure 4 4. Average daily light intensity in the control and acclimated chambers during the alternate cold acclimation and deac climation experiment.

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55 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Day 0 7 14 21 28 35 42 49 56 63 70 Mean daily temperature (oC) 6 8 10 12 14 16 18 20 22 24 26 Control Acclimated Figure 4 5. Mean daily temperatures in control and acclimated chambers during the cold acclimation experiment. Light 25 10 Dark 20 5 Day 0 2 4 6 8 10 12 14 16 Mean daily temperature (0C) 6 8 10 12 14 16 18 20 22 24 26 Control Acclimated Figure 4 6. Mean daily temperatures in control and acclimated chambers during the 1st cycle of the alternate cold acclimation and deacclimation experiment.

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56 Figure 4 7. Comparison of overall growth of the control and the acclimated plants at the end of the cold acclimation experiment. Control Acclimated

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57 Table 4 1. Leaf area (cm2) at the beginning and at the end of the cold acclimation experiment. Measurement taken Control Acclimated Beginning of experiment 277 6.23 2861.07 End of experiment 6873.83 4287.02 Total increase 4097.60 1425.95 Table 4 2. Leaf area (cm2) at the beginning and at the end of the alternate cold acclimation and deacclimation experiment. Measurement taken Control Acclimated Beginning of experiment 6834.80 4287.02 End of experiment 7163.86 4763.24 Total increase 329.06 476.22

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58 Light25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Week 1 2 3 4 5 6 7 8 9 Stem water potential (MPa) -1.0 -0.8 -0.6 -0.4 -0.2 Control Acclimated Figure 4 8 Stem water potential during the cold acclimation experiment. Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 36 Stem water potential (MPa) -1.0 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 Control Acclimated Figure 4 9. Stem water potential during the alternate cold acclimation and deacclimation experiment

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59 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Week 1 2 3 4 5 6 7 8 9 Transpiration (mmol/m 2 /s) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Control Acclimated Figure 4 10. Transpiration during the cold acclimation experiment Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 Transpiration (mmol/m 2 /s) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Control Acclimated Figure 4 11. Transpiration during the alternate cold acclimation and deacclimation experiment

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60 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Week 1 2 3 4 5 6 7 8 9 Stomatal conductance (mmol/m2/s) 0 50 100 150 200 250 Control Acclimated Figure 4 12. Stomatal conductance duri ng the cold acclimation experiment. Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 Stomatal conductance (mmol/m2/s) 50 100 150 200 250 Control Acclimated Figure 4 13. Stomatal conductance during the alternate cold acclimation and deacclimation experiment.

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61 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Weeks 1 2 3 4 5 6 7 8 9 Osmotic potential (MPa) -6 -5 -4 -3 -2 -1 0 Control Acclimated Figure 4 14. Osmotic potential during the cold acclimation experiment. Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 Osmotic potential (MPa) -5 -4 -3 -2 -1 0 Control Acclimated Figure 4 15. Osmotic potential during the alternate cold acclimation and deacclimation experiment.

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62 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Weeks 0 1 2 3 4 5 6 7 8 9 10 Root resistance (MPa s /m) 0 10000 20000 30000 40000 50000 60000 Control Acclimated Figure 4 16. Root resistance during cold acclimation experiment. Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 36 Root resistance (MPa s/m) 0 10000 20000 30000 40000 50000 60000 Control Acclimated Figure 4 17. Root resistance during alternate cold acclimation and deacclimation experiment.

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63 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Week 0 1 2 3 4 5 6 7 8 9 10 Relative water content (%) 60 65 70 75 80 85 90 95 100 Control Acclimated Figure 4 18. Relative wat er content during the cold acclimation experiment. Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 Relative water content (%) 75 80 85 90 95 100 Control Acclimated Figure 4 19. Relative water content during the alternate cold acclimation and deacclimation experiment.

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64 Light 25 20 15 10 10 10 10 10 10 Dark 20 15 10 5 5 5 5 5 5 Week 0 1 2 3 4 5 6 7 8 9 10 Water use (cm3/hour) 5 10 15 20 25 30 35 40 Control Acclimated Figure 4 20. Hourly water use during the cold acclimation experiment Light 25 10 25 10 25 Dark 20 5 20 5 20 Day 0 3 6 9 12 15 18 21 24 27 30 33 36 Water use (cm3/hour) 5 10 15 20 25 30 35 40 45 Control Acclimated Figure 4 21. Hourly water use during the alternate cold acclimation and deacclimation experiment.

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65 Week 0 1 2 3 4 5 6 7 8 9 10 Relative water use (%) 20 30 40 50 60 70 80 90 100 110 Control Acclimated Figure 4 22. Relative water use between acclimate d and control plants during the cold acclimation experiment. Day 0 3 6 9 12 15 18 21 24 27 30 33 36 Relative water use (%) 30 40 50 60 70 80 90 100 110 Control Acclimated Figure 4 23. Relative water use between acclimated and control plants during the alternate cold acclimation and deacclimation experiment.

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66 Table 4 3. Wate r use and relative water use of the acclimated plants at different air temperatures during the cold acclimation experiment. Week Average water use by the control plants (cm 3 /hour) Water use by acclimated plants (cm 3 /hour) Relative water use (%) 1 31.42 29 .69 94.19 2 31.42 15.63 49.73 3 31.42 11.56 36.79 4 31.42 10.00 31.83 5 31.42 10.00 31.83 6 31.42 12.50 39.78 7 31.42 10.94 34.81 8 31.42 10.63 33.81 9 31.42 11.25 35.81 Table 4 4. Water use and relative water use of the acclimated plants at dif ferent air temperatures during alternating cold acclimation and deacclimation experiment. Day Average water use by the control plants (cm 3 /hour) Water use by acclimated plants (cm 3 /hour) Relative water use (%) 4 31.88 20.63 64.70 14 31.88 11.56 36.27 18 31.88 26.88 84.30 28 31.88 18.13 56.85 32 31.88 29.33 92.01

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67 CHAPTER 5 CONCLUSION In the summer of 2008, two growth chamber experiments were conducted at the University of Florida, Southwest Florida Research and Education Center to evaluate the effect of cold temperatures and alternately increasing and decreasing temperatures on the physiological responses on Hamlin sweet orange trees. Selected trees were exposed to progressively lower temperatures during the first experiment and alternating warm and cold temperatures during the second. Plants treated with lower temperatures were compared with the plants having temperatures > 20 oC with respect to transpiration, stomatal conductance, stem water potential, osmotic potential, relative water content, roo t resistance, and evapotranspiration. Hourly water use was determined and finally water requirement of Hamlin sweet orange trees at different temperature treatments were estimated. Total leaf areas were measured before and at the conclusion of each exper iment to determine the effect of temperatures on tree growth. According to the findings of the two experiments, temperature had a profound affect on the growth, water use, and several physiological processes of citrus trees. Due to low temperatures, plants demonstrated increased root resistance, which led to reduced water uptake. As a consequence, transpiration decreased resulting in a reduction in overall water use and relative water use. Therefore, the average water use during the lowest temperature treat ments was small compared with water use of control plants at higher temperatures. Hence the effect of temperature that promote acclimation on root resistance and reduced water use should be considered for water use planning, irrigation scheduling and/or soil water management. Water is one of the most precious natural resources in the world. Although Florida often has abundant water, excessive withdrawal of groundwater during recent years and drought has resulted in insufficient water. Because water requirement by citrus varies with temperature, the

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68 irrigation need are most critical during periods of high plant water demand during late spring and summer months when rainfall is insufficient. Determining water demand during winter months when a long period of time with no rainfall occurs is also important for proper tree growth, flower induction, and fruit maturation. Recently in Florida, microsprinkler irrigation system has become the most popular method of irrigation because of its dual importance of reduced energy cost and protection against frost. S cheduling irrigation based on seasonal variations of water use is one of the major concerns regarding citrus irrigation. Most of the irrigation practices are scheduled on the basis of ETo and Kc. However, determining ETo in field conditions require lots of information, skill and time. Therefore, using relative water use in winter season over the summer season would be helpful to estimate Kc and subsequently to schedule need-based irrigation. Effective irrigation sc heduling based on crop demand as it is affected by cold acclimating temperatures could save considerable quantities of water in Florida while providing adequate water for maintenance of citrus yields and quality for which Florida has become renowned.

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69 LI ST OF REFERENCES Allen, R.G., L.S. Pereira, D. Raes, and M. Smith. 1998. Crop evapotranspiration Guidelines for computing crop water requirements. FAO Irrigation and Drainage Paper No. 56. FAO, Rome. Aloni, B., and G. Rosenshtein. 1982. Effect of flooding on tomato cultivars the relationship between proline accumulation and other morphological and physiological changes. Physiol. Plant. 56: 513517. Ball, J. T., I. E. Woodrow, and J. A. Berry. 1987. A model predicting stomatal conductance and its contribution to the control photosynthesis under different environmental conditions. Preogress in Photo. Res. 4:221228. Bialoglowski, J. 1937. Effect of extent and temperature of root on transpiration of rooted lemon cuttings. Proc. Amer. Soc. Hort. Sci. 34: 96 102. Boman, B.J. 1994. Evapotranspiration by young Florida flatwoods citrus trees. J. Irrig. Drain. Eng. 120:80 88. Boman, B. J. 2002. Water and Florida citrus: use, regulation, irrigation, systems and management. IFAS. Univ. of Florida, Gainesville. Boma n, B., N. Morris, and M. Wade. 2002. Overview of grove design and development. Water and Florida citrus: use, regulation, irrigation, systems and management. Chapter 2: 9 19. Boman, B. and Parsons, L. 2002. Water and Florida citrus: use, regulation, irriga tion, systems, and management. Univ. of Florida, IFAS, Chapter 15: 148 162. Boman, B. J., S. Smith, and B. Tullos. 2008. Control and automation in citrus microirrigation systems. Circular 1413. IFAS, Fla. Coop. Ext. Serv., Univ. of Florida, Gainesville. Begg, J.E., N. C. Turner. 1970. Water potential gradients in field tobacco. Plant Physiol 46: 343346. Boyer, J.S. 1 982. Plant productivity and environment. Sci. 218: 443448. Bryla, D.R., T.J. Bouma, U. Hartmond, and D.M. Eissenstat. 2001. Influence of tem perature and soil drying on respiration of individual roots in citrus: integrating greenhouse observations into a predictive model for the field. Plant, Cell & Environment 24: 781 790. Castel, J.C. 1978. Some aspects of gas exchange and water relations in orange cuttings. M.S. Thesis, University of New South Wales. Castel, J.R., and A. Buj. 1992.Growth and evapotranspiration of young, dripirrigated Clementine trees. Proc. Int. Citriculture 651 656.

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70 Chone, X, C.V. Leeuwen, D. Dubourdieu, and J.P. Gaudiller e. 2001. Stem water potential is a sensitive indicator of grapevine water status. Annals of Botany. 87: 477483. Cohen, Y., M. Fuchs, and S. Cohen. 1983. Resistance to water uptake in a mature citrus tree. Oxford Journals. Journal of Experimental Botany. Volume 34 (4): 451 460. Cram, W. J. 1976. Negative feedback regulation of transport in cells: the mainte nance of turgor volume and nutrient supply. In U Luttge, MG Pitman (eds). Encyclopedia of Plant Physiology. 2: 284316. Dansereau, K. N. 2007. The role of plant water deficits on cold tolerance during cold acclimation of a cold tolerant ( Poncitrus Trifoliata ) and cold sensitive ( Citrus Unshiu ) species. M.S. thesis. Auburn University. Eaks, I. L. 1960. Physiological studies of chilling injury in citrus fru its. Plant physiol. 35: 632636. Ebel, R. C., B.L. Cambell, M.L. Nesbitt, W.A. Dozier, J.K. Lindsey, and B.S. Wilkins. 2005. A temperature index model to estimate long-term freeze risk of Satsuma mandarins grown on the northern coast of the Gulf of Mexico. J. Amer. Soc. Hort. Sci. 130: 500-507. Erickson, L.C. 1968. The general physiology of citrus. In The Citrus Industry, W. Reuther, L.D. Batchelor, and H. J. Webber, eds., Vol. II, Div. Agric. Sci., University of California, Berkeley, pp. 54 63. Fares, A., and A.K. Alva. 1999. Estimation of citrus evapotranspiration by soil water mass balance. Soil Sci. 164: 302 310. Fischbach, F. E. and Schleusener, P. E. 1961. Tensiometers, a tool to help control. Univ. Nebr. Ext. Serv., Publ. No. E.C. 61 716. Fuchigami, L. H. 1996. Cold hardiness. F. B. Salisbury (ed.) Units, symbols, and terminology for plant physiology: A reference for presentation of results in plant sciences, Oxford Univ. Press, New York: 146 -Sanchez, F., and J.P. Syvertsen. 2006. Salinity tolerance of Cleopatra mandarin and Carrizo citrange citrus rootstock seedlings is affected by CO2 enrichment during growth. J. Am. Soc. Hort. Sci. 131: 2431. Garnier, E. and A. Berger. 1985. Testi ng water potential in peach trees as an indicator of water stress. J. Horti. Sc.. 60: 47 56. Gilmour, S. J., N. Artus, and M. F. Thomashow. 1992. cDNA sequence analysis and expression of two cold regulated genes of Arabidopsis thaliana. Plant Mol. Biol. 1 8: 13 21. Guinchard, M. P., C. Robin, P. Grew, and A. Guckert. 1996. Cold acclimation in white clover subjected to chilling and frost: changes in water and carbohydrate status. European Journal of Agronomy. 6: 225 233.

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77 BIOGRAPHICAL SKETCH Smita Barkataky was born in a North -e astern state of India called Assam in 1973. The youngest of the three children, s he grew up and completed her elementary, high sc hool and early college education in Guwahati, India. She earned her BS and MS degrees, majoring in Soil Science from Assam Agricultural University in 1996 and 1998, respectively. Her MS research was on agro hydrological characterization of lowland rice eco system. She was married to Debashish Goswami in 1999 and in the same year, she joined the State Institute of Rural Development, Assam as Junior Lecturer and she continued working there for two years. During that period, her job responsibilities were to org anize training programs and to give classes related to agriculture and production for educating unemployed rural youths for self employment. She moved to North Carolina, US in December, 2001 to join her husband who was completing his MS there. Her son, Sam prit was born in Illinois in May 2006. She was admitted for MS program in Soil and Water Science department at the University of Florida in 2008 and currently she is working as a graduate research assistant at University of Florida.