EFFECT OF NUTRIENT SOLUTION ELECTRICAL CONDUCTIVITY LEVELS ON LYCOPENE CONCENTRATION, SUGAR COMPOSITION AND CONCENTRATION OF TOMATO (Lycopersicon esculentum Mill.) by Min Wu ________________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF PLANT SCIENCES In Partial Fulfillment of the Requirements For the degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2006 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by (Min Wu) entitled (Effect of Electrical Conductivity of Nutrient Solution on Plant Physiological Responses and Fruit Quality of Tomato (Lycopersicon esculentum Mill.) ) and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of (Doctor of Philosophy) _______________________________________________________________________ Date: (08/30/2006) (Chieri Kubota) _______________________________________________________________________ Date: (08/30/2006) (Gene A. Giacomelli) _______________________________________________________________________ Date: (08/30/2006) (Joel Cuello) _______________________________________________________________________ Date: (08/30/2006) (Judith K. Brown) _______________________________________________________________________ Date: (08/30/2006) (Ursula Schuch) Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: (08/30/2006) Dissertation Director: (Chieri Kubota) 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interest of scholarship. In all other instances, however, permission must be obtained from the author. Min Wu 4 ACKNOWLEDGEMENTS Special thanks to my advisor Dr. Chieri Kubota for all the help, encouragement, and guidance. Thanks to the members of my committee Drs. Gene Giacomelli, Joel Cuello, Judith Brown, Ursula Schuch, Jiankang Zhu and Peter Waller for their guidance and direction. I would like to express gratitude to Mark Kroggel and for his suggestions and help in the greenhouse. Thanks to Brandon and Cody for their helps in greenhouse work. Thanks to the other members of CEAC- Dr Merle Jensen, Dr. Patricia Rorabaugh, Dr. Roger Huber, Dr. Anita Hayden and Paula Costa, Nadia Sabeh, Efren Fitz-Rodriguez, Wanwiwat Lovichit, Jason Licamele and Pedro Romero. Thanks to the Plant Sciences Faculty and Staff who were always willing to help. Finally, I would like to thank my parents for their unconditional love and encouragement. And, I would like to thank my husband Yuyi for his unbelievable support and understanding through my PhD study. 5 DEDICATION This dissertation is dedicated to my husband Yuyi Chen for all of his love, help in the lab and the greenhouse, and support. This dissertation is also dedicated to my parents Minghua Luo and Yinhua Wu who gave me the encouragement and confidence to accomplish great things. 6 TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................ 8 ABSTRACT........................................................................................................................ 9 INTRODUCTION ............................................................................................................ 11 LITERATURE REVIEW ................................................................................................. 15 PRESENT STUDY........................................................................................................... 32 REFERENCES ................................................................................................................. 37 APPENDIX A: EFFECTS OF ELECTRICAL CONDUCTIVITY OF HYDROPONIC NUTRIENT SOLUTION ON LEAF GAS EXCHANGE OF FIVE GREENHOUSE TOMATO CULTIVARS .................................................................................................. 45 Abstract ......................................................................................................................... 47 Materials and Methods.................................................................................................. 52 Results and discussion .................................................................................................. 55 Conclusions................................................................................................................... 60 Literature Cited ............................................................................................................. 62 APPENDIX B: EFFECTS OF NUTRIENT SOLUTION EC, PLANT MICROCLIMATE AND CULTIVARS ON FRUIT QUALITY AND YIELD OF HYDROPONIC TOMATOES..................................................................................................................... 70 Abstract ......................................................................................................................... 72 Introduction................................................................................................................... 73 Material and methods.................................................................................................... 74 Results and discussion .................................................................................................. 76 Conclusions................................................................................................................... 81 APPENDIX C: EFFECTS OF HIGH ELECTRICAL CONDUCTIVITY OF NUTRIENT SOLUTION AND ITS APPLICATION TIMING ON LYCOPENE, CHLOROPHYLL AND SUGAR CONCENTRATION OF HYDROPONIC TOMATO............................. 87 7 TABLE OF CONTENTS-Continued Abstract ......................................................................................................................... 89 Materials and Methods.................................................................................................. 94 Results and discussion .................................................................................................. 98 Conclusions................................................................................................................. 104 8 LIST OF FIGURES FIGURE 1: The isoprenoid biosynthetic pathway……………………………………….24 FIGURE 2: Phytoene formation and desaturation reactions to form lycopene………….25 9 ABSTRACT Tomato is an important commodity in fresh vegetable market. Recently, there is great interest for North American hydroponic growers to improve the fruit quality by introducing better cultivation methods. Manipulation of electrical conductivity (EC) of nutrient solution is a well-known technique to increase sugar concentrations of tomato; however, the potential risk of lower yield is the drawback of introducing this technique. Therefore to find a range of EC that can enhance the fruit quality while maintaining overall yield was the goal of this research. For this purpose, plant physiological responses such as transpirational and photosynthetic characteristics and fruit quality attributes including sugars and lycopene were investigated for selected cultivars under different EC. Regardless of cultivar, tomato plants showed a greater net photosynthetic rate at the reproductive growth stage compared to the vegetative growth stage. An increase of EC of influx nutrient solution up to 4.8 dS m-1 did not reduce the leaf photosynthesis, which supported a hypothesis that there is an optimum EC range for enhancing fruit quality without significant yield loss. A following experiment showed that the tomato fruit quality could be significantly enhanced when plants were grown under around 4.5 dS m-1 EC, in terms of total soluble solids (TSS) and lycopene concentration with no significant yield loss. Last experiment was conducted to quantitatively understand the accumulation of lycopene and sugars in fruits as affected by EC and its application timing relative to the fruit ripeness stages. High EC treatment of 4.5 dS m-1, regardless of its application timing, enhanced TSS and sugar concentration in the juice and lycopene concentrations of the fruit. However, the delayed high EC 10 treatment (application of high EC after 4 weeks of anthesis) showed less enhancement for TSS and sugar concentration. Lycopene concentration of the fruit in the delayed EC treatment reached the same level as that in the standard high EC treatment (application since anthesis), which suggests that enhanced lycopene development under high EC is more related to an abiotic stress response during the fruit maturation, rather than fruit mass balance altered by the limited water flux to the fruit. 11 INTRODUCTION Tomato (Lycopersicon esculentum Mill.) is considered one of the main horticultural commodities cultivated in the world (Rick, 1995). It is also one of the most common and oldest crops grown hydroponically in the greenhouse (Jones, 1999). Tomato plants are grown under either greenhouse or field conditions. However, the production of greenhouse tomato in North America is showing substantial increases in recent years (Cook and Calvin, 2005). From 1998 to 2003, the U.S. greenhouse area has been increased from 257 to 330 hectares whereas the total production increased from 106,594 to 159,664 metric tons, but the field grown tomato production only increased from 1,492,591 to 1,594,241 metric tons (Cook and Calvin, 2005). All the large commercial greenhouse growers in the U.S. and Canada use high technologies such as hydroponics and active climate control, to maintain a consistent supply of high quality tomatoes. U.S. greenhouse tomato production had an average greenhouse tomato yield of 484 metric tons/hectare, which was much higher than the average field tomato yield of 32 metric tons/hectare (Cook and Calvin, 2005). Since 1985, the consumption of fresh tomato in the U.S. has increased about 30 %, with annual per capita consumption level of 8.8 kilograms estimated in 2003 (Cook and Calvin, 2005). At the same time, the percentage of greenhouse tomato at U.S. retail sales increased dramatically since early 1990s and now accounts for 37% including produce imported from Mexico and Canada (Cook and Calvin, 2005). However, the greenhouse industry is facing a competitive market as the expanding supply outpacing the rising demand. U.S. greenhouse growers are moving towards the production of high value 12 cultivars such as smaller cluster tomatoes including cocktail type tomatoes or roma type tomatoes instead of more common beefsteak type tomatoes. Producing value-added high quality tomato, such as fruit with a high lycopene concentration or better flavor balanced with acidity and sweetness, would be a marketing strategy for most greenhouse tomato growers, yet feasible cultivation techniques need to be developed. Tomato fruit quality improvement can be achieved by plant breeding, modern plant biotechnology, and horticultural techniques such as environmental control or the combination of these approaches. Some horticultural techniques showed that specific quality traits of tomato fruit can be improved by controlling the tomato plant root-zone and ambient environments. This study aims to develop a practical strategy for improving tomato fruit quality by increasing sugar, other soluble solids and lycopene concentrations through EC manipulation under controlled environments. The following studies have been conducted during this research. APPENDIX A presents the leaf photosynthetic and transpiration responses of five tomato cultivars as affected by different electrical conductivity (EC) of nutrient solution in both vegetative and reproductive developmental stages. Plant physiological responses such as net photosynthetic and transpiration rates under high EC of nutrient solution were expected to serve as a reference for selecting cultivars and the range of EC of nutrient solution for producing tomato fruits with enhanced total soluble solids (TSS) but with little or no yield reduction. The results of the experiment indicated that a moderate salt stress around 4.8 dS m-1 EC might increase the quality of tomato fruits without negatively 13 affecting the photosynthesis and transpiration, and therefore possibly fruit yield for all five cultivars examined. APPENDIX B presents the effects of a moderately high EC (4.5 dS m-1) of nutrient solution and greenhouse micro environmental conditions on TSS and lycopene concentrations in the fruit, and yield of five tomato cultivars. The objective of this study was to produce high quality tomato without yield reduction by manipulating the EC of nutrient solution. The results showed that, regardless of cultivar, the TSS and lycopene concentrations can be significantly enhanced when plants were grown under the high EC conditions, with no significant yield loss. The magnitude of increase in TSS and lycopene concentration by applying the high EC was cultivar-specific. This experiment also addressed the need to better understand the mechanisms of enhancing lycopene under high EC. APPENDIX C presents the change of TSS and sugar (sucrose, fructose and glucose) in the juice and lycopene and chlorophyll concentrations in the fruit during fruit development and maturation in response to high/low EC of nutrient solution and the application timing of high EC relative to the fruit development stage. The objective of this study was to better understand the lycopene accumulation and sugar concentration as affected by high EC in the nutrient solution. The results showed that high EC treatment, regardless of its application timing, enhanced TSS and lycopene concentrations of tomato fruit. However, the delayed high EC treatment (application of high EC starting 4 weeks after anthesis) showed less enhancement for TSS and sugar concentration, which confirmed that the accumulation of TSS was a result of water status during the entire fruit 14 development stages. Lycopene concentration of the red tomato fruit in the delayed EC treatment reached the same level as that in the standard high EC treatment (application since anthesis), suggesting that enhanced lycopene development under high EC is an abiotic stress response during the fruit maturation. 15 LITERATURE REVIEW Although there is no standard measure to define the quality factors of tomato fruit, Jones (1999) concluded that the ideal tomato fruit should be full size, vine ripened, unblemished and near or at the red-ripe stage. There are three main factors for evaluating the quality of tomato fruit: overall appearance, firmness and flavor. Causse et al. (2002) determined that the overall quality characteristics included physical traits such as fruit size, weight, color, firmness, and elasticity; and chemical traits such as sugars, titratable acidity, pH, dry matter, lycopene, other carotenoids and aroma volatiles. Stevens et al. (1977) simply stated that sugar and organic acids were the major determinants of tomato quality. Postharvest durability and fruit safety are also important fruit quality criteria for tomato marketing (Grierson and Kader, 1986). In the present dissertation I focus on soluble solid concentration (commonly measured as Brix), sugar concentration and lycopene concentration of the fruit as affected by horticultural techniques and environmental conditions in the greenhouse. I define that fruit flavor and nutritional concentrations are two major factors of tomato fruit quality, unless otherwise stated specifically. The following summarizes the current understanding and research status on environmental factors affecting tomato quality. Tomato Fruit Flavor Sugar and organic acids are important components to determine tomato flavor (Peet, 1996). Glucose and fructose are two major sugars, which account for about 95% of total sugars in tomato (Davies and Kempton, 1975; Haila et al., 1992; Young et al., 1993). 16 Sucrose and sugar alcohol myo-inositol are detected in low or trace amounts in fruit (Davies and Kempton, 1975; Haila et al., 1992). More than 95% of the organic acids in tomato are citrate and L-malate, whereas low amounts of quinate, isocitrate and succinate have been reported (Davies and Hobson, 1981). Sugars and acids of tomato are influenced by genotypes and the environmental conditions. Sugars and acids could be enhanced by traditional breeding, which can produce new cultivars with improved flavor (Stevens et al., 1979; Jones and Scott, 1984). Transforming tomato with a gene that increases the fructose to glucose ratio is another approach for flavor improvement since fructose is almost twice as sweet as glucose (Levin et al., 2000). The effect of environmental factors on total soluble solid concentration in tomato fruit is reviewed later in this chapter. Tomato Nutritional Contents Tomato contains secondary plant metabolites such as carotenoids, vitamins and phenolic compounds, which play an important role in human health and nutrition. Ripe tomato contains various vitamins including vitamine A, B1, B2, B3, B6, C and E, niacin, folic acid, and biotin (Davies and Hobson, 1981). Tomato has traditionally been considered as one of the best sources of antioxidants, with its role as a principal dietary source of lycopene. Concentration of lycopene in tomato fruit was reportedly altered by light quantity and quality (Alba et al., 2000; Dumas et al., 2003), air temperature (Baqar and Lee, 1978) and level of salinity in the nutrient solution 17 (De Pascale et al., 2001). The effect of environmental factors on lycopene concentration in tomato fruit is reviewed later in this chapter. Effects of Electrical Conductivity of Nutrient Solution on Tomato Fruit As a result of the presence of positively and negatively charged ions, a fertilizer solution can conduct an electric current. The EC (electrical conductivity) is a measure of the total ion concentrations in the solution and is expressed in desi-Siemens per meter of solution (dS m-1). The more charged ions there are the more current the solution can conduct and the higher the EC. Through an accurate measurement of the EC in the solution, the grower will know the strength of the nutrient solution. However, EC does not tell the specific ions or its concentration in the solution. Monitoring and manipulation of EC of nutrient solution is a common cultivation technique in hydroponics for enhancing yield or improving fruit quality. The EC of nutrient solution can be increased either by increasing the overall strength of nutrient solution or by the addition of NaCl to the basal nutrients. The latter seems to be preferred as it is more practical for growers, because addition of NaCl to the nutrient solution does not increase the costs. It should be noted that, under high EC, plants may be affected by water deficit from the low water potential of the nutrient solution caused by the decreased osmotic potential of the solution, an excessive ion uptake (e.g. Na+, Cl-) by greater availability of such ions in the solution (Greenway and Munns, 1980), or both. Therefore, reported results on plant responses to increased EC may contain both types of effects. 18 Effect of EC of nutrient solution on tomato yield and flavor Tomato fruit growth is closely linked to the movement of water to the fruit (Johnson et al., 1992). Tomato fruit is mainly composed of water, carbohydrates and salts, with water accounting for about 94% of fresh weight of tomato fruits (Jones, 1999). Transpiration plays a major role in water transportation in plants because the transpiration reduces the water potential of leaves and causes the water transport through the xylem. However, it seems that phloem serves as the main route for water transportation to fruits. More than 90% water enters into tomato fruits through the phloem (Ho et al., 1987; Lee, 1989). In fruit development, the xylem discontinuity in the pedicle was observed in tomato fruits (Lee, 1989) from anatomical studies. This may cause the restricted water transportation to tomato fruit through the xylem. However, the apoplasmic water potential gradients within the plant have a direct effect on phloem translocation. During fruit development, the lowered rate of xylem flow reduced the phloem turgor, thus reduced the phloem flow into the fruit (Lang and Thorpe, 1986; Johnson et al., 1992; Van de Sanden and Uittien, et al., 1995). The optimum EC of the nutrient solution to provide the maximum tomato fruit yield may be affected by cultivar, environmental conditions and cultural practice. Excessive water stress from high EC nutrient solution may cause a significant yield reduction, which is a result of smaller fruit size and/or less number of fruits harvested (Adams, 1991). Adam (1991) reported that compared to the standard EC of 3.0 dS m-1, an EC of about 8 dS m-1 decreased the tomato yield from 4% to 5% per 1-dS m-1 increase, whereas further increase of EC from 8 dS m-1 to 12 dS m-1 decreased the tomato yield from 6% to 8% per 1-dS m-1 19 increase, where both high EC were achieved by adding more NaCl to the basal nutrients. There was no difference in yield between 2.7 and 4.5 dS m-1, respectively; however, the yield was reduced significantly and linearly when the EC was increased from 4.5 to either 6.0, 7.4 or 8.6 dS m-1 by addition NaCl to the nutrient solution (Leonardi et al., 2004). The main effect of high EC on tomato fruit yield reduction was due to water stress causing less water transport to the fruit, and dry weight of the fruit was not affected by high EC (Ehret and Ho, 1986; Adam and Ho, 1989; Li et al., 2004). Under 15 dS m-1 EC, achieved by adding NaCl to the basal nutrients, the fruit set on the upper trusses was reduced, which in turn caused the reduction of fruit yield (Adam and Ho., 1992). Although there is a risk of reducing fruit yield and size, increasing EC of nutrient solution is a well known technique to grow sweeter tomatoes because the restricted water transport to fruits can increase the total soluble solid concentration (TSS, % Brix at 20oC) (Adams, 1991; Mitchell et al., 1991; Cornish, 1992; Lin and Glass, 1999). TSS is the most common index associated directly with sugar and organic acid concentrations in tomato juice (Stevens et al., 1977; Young et al., 1993). Leonardi et al. (2004) found that the TSS of tomato was increased linearly from 4.2 to 6.2 % per every increase of EC by 1 dS m-1 within the range of 2.7 to 8.6 dS m-1. Cuartero and Fernandez-Munoz (1999) found that TSS of two commercial tomato cultivars increased 10.5 % per dS m-1 when EC of nutrient solution was increased from 2.5 to 8.0 dS m-1 by adding NaCl to the nutrient solution. There are inconsistent results regarding whether the source of increasing EC affects fruit yield and flavor, either by adding NaCl or increasing the whole strength of nutrient solution. Adams (1991) found no difference of tomato fruit yield between the addition of 20 major ions and the addition of NaCl to achieve 12.0 dS m-1 in his study, whereas Willumsen et al (1996) found the increased EC to 4.7-4.8 dS m-1 by NaCl only decreased the yield and fruit size more than did the increased EC by adding other ions including K, N, Ca, Mg, P, and S. The increased EC by adding NaCl reduces K and N concentrations in fruit but increases Na and Ca concentratoins ( Adams and Ho, 1989; Adams,1991). The type of nutrient ions used to increase EC seems to have little effects on most components in tomato fruits (Ehret and Ho, 1986; Peterson et al, 1998). Effect of EC of nutrient solution on tomato plant growth, development and other physiological responses Plant growth is severely inhibited when exposed to solution containing excessively low osmotic potential or high ion concentrations (Zhu, 2002). These osmotic and ionic stresses affect the plant physiological status individually (Lefèvre et al., 2001; Ueda et al., 2003). The osmotic stress in the root zone led to a reduction of leaf turgor which reduced leaf expansion (Erdei and Taleisnik, 1993; Huang and Redmann, 1995). The toxic accumulation of Na+ and Cl- in the leaves was correlated with stomatal closure or reduction of total chlorophyll concentration of leaves (Seemann and Critchley, 1985; Romero-Aranda and Syvertsen, 1996). In addition, the influx of Na+ impairs the transport of other ions such as K+ and Ca2+ and results in ionic imbalance (Binzel et al., 1988). During the early phase of salt stress (root zone exposure to high salt and low osmotic potential environments), osmotic stress was dominant in inhibiting plant growth (Zhu, 2002). 21 The water uptake by tomato plants grown in the open field decreased with increasing salt concentration in the irrigation water (Soria and Cuartero, 1997; Soria et al., 2001). For tomato plant, water and nitrate uptake were reduced for tomato seedlings under salinity level of 75 mM NaCl (Flores et al., 2002). The plant dry matter, plant height, number of leaves, total plant leaf and leaf stomatal density were reduced in proportion to the salinity level in the nutrient solution when the NaCl concentration was increased from 0 to 70 mM (Romero-Aranda et al., 2001). Under high EC, the tomato plant may be affected by water stress from the low water potential of the nutrient solution, which is caused by the decreased osmotic potential of the solution, or by excessive ion uptake due to greater ion concentrations in solution (Greenway and Munns, 1980). There was no difference in Na+ uptake for tomato plants under different EC of 1.9, 4.7, 7.1 and 9.1 dS m-1, in which EC was increased by adding NaCl; however, the total plant water uptake was reduced with increasing salinity for all the four cultivars tested (Reina-Sanchez et al., 2005). Therefore, the decreased osmotic potential of the solution of high EC might be the dominant factor affecting tomato plant growth and development. Photosynthesis, transpiration and stomatal conductance under high EC were affected by limited irrigation and/or increased salt concentrations in nutrient solution (Romero-Aranda et al., 2001). Xu et al. (1995) studied the effects of EC of hydroponic nutrient solution, growth medium (substrate) and irrigation frequency on tomato plant photosynthetic response and found that the maximum leaf photosynthetic rate was increased by 15.4% and 14.1% when EC was increased from 2.5 to 4.0 dS m-1 for plants 22 grown in a nutrient film technique (NFT) and rockwool systems, respectively, in a greenhouse. But a greater increase of EC to 5.5 dS m-1 resulted in a 10 % lower maximum photosynthetic rate compared to an EC of 4.0 dS m-1 in a rockwool system. Schwarz et al. (2002) found that an increase of EC from 1.25 dS m-1 up to 8.75 dS m-1 did not reduce the leaf net photosynthetic rate of tomato. In both experiments reported by Xu et al. (1995) and Schwarz et al. (2002), the EC was enhanced by increasing the overall strength of nutrient solutions. Lycopene as a Quality Attribute for Fresh Tomato and Its Biosynthesis The tomato fruit is the principal dietary source of lycopene. At least 85% of the dietary lycopene comes from tomato fruit and tomato-based food products, with a smaller source of lycopene in other fruits such as guava, watermelon, papaya and pink grapefruit (Scott and Hart, 1994). Lycopene is a powerful antioxidant, which can prevent the initiation or propagation of oxidizing chain reaction (Di Mascio et al., 1989; Nguyen and Schwartz, 1999; Riso et al., 1999). Lycopene has been reported to have important roles to prevent disease and promote health in humans, usually associated with reducing the risk of cancer and cardiovascular disease (Gerster, 1997; Stahl and Sies, 1997; Giovanucci, 1999). Several studies reported that the incidence of prostate cancer was reduced for men who consumed more servings of tomato products (Rehman, et al., 1999; Giovannucci et al., 2002). The ripening of tomato fruit is accompanied by the chlorophyll degradation and lycopene synthesis, as chloroplasts are converted into chromoplasts (Rhodes, 1980; Fraser 23 et al., 1994). Lycopene is synthesized in the plastid in higher plants. It involves a great change in plastid structure and several plastid proteins were involved in the chloroplast-chromoplast transition (Susan et al., 1997). There is much information on lycopene biosynthesis and its pathway. However information seems to be limited with regard to how the physical environmental factors affect lycopene synthesis. Lycopene biosynthesis in tomato fruit development Lycopene is a major carotenoid responsible for the red color in ripe tomato fruit. The ripening of tomato fruit is accompanied by the dramatic increase in the carotenoid content, especially a massive accumulation of lycopene (Rhodes, 1980; Fraser et al., 1994). Lycopene biosynthesis can be summarized as the following pathway from the recent studies (Bramley, 1997, 2002; Fraser et al., 2002): IPP (isopentenyl diphosphate) lycopene - carotene GGPP (geranylgeranyl pyrophosphate) Phytoene 24 Fig1. The isoprenoid biosynthetic pathway (Bramley, 2002). [With permission of the Oxford University Press] 25 Fig 2. Phytoene formation and desaturation reactions to form lycopene (Bramley, 2002) [With permission of the Oxford University Press] The carotenoid biosynthetic pathway of plants is described in the Fig 1 and Fig 2 (Bramley, 2002). The major enzymes involved in this process include Geranylgeranyl diphosphate synthase (GGPS), phytoene synthase (PSY), phytoene desaturase (PDS), -carotene desaturase (ZDS) and cyclases. The tomato contains two genes, Psy-1 and Psy-2 taking key roles in the lycopene biosynthesis. The Psy-1 encodes the 26 fruit-ripening-specific isoform, whilst Psy-2 predominates in green tissues (Fraser et al., 1999) A pool of GGPP is a prerequisite for lycopene accumulation. Several tomato non-ripen fruits mutants are related to the mutation of genes involved in GGPP formation pathway (Yen et al., 1997; Fraser et al., 2001). However, in a colorless-non-ripening (cnr) tomato fruit mutant caused by a lesion of single gene in tomato chromosome 2, GGPP was found still accumulated whereas the lycopene was at a non-detectable level (Fraser et. al., 2001). Recent studies suggest that alternative pathway of synthesizing GGPP exist in tomato that supplies the pool of GGPP in plants (Fraser et. al., 2001). Even the GGPP formation from IPP was blocked; there was still a stable GGPP concentration in plants (Fraser et. al., 2001). Therefore, GGPP concentration is not a restriction for lycopene biosynthesis. During maturation of tomato fruits, lycopene concentration in the fruit is significantly increased due to the increased activity of PSY-1 and reduced activity of LCYB (lycopene- -cyclase, a cyclase to convert lycopene to -carotene) (Bartley et. al., 1991; Bramley, 2002). The regulation of PSY-1 is regarded as the most influential step for lycopene synthesis in tomato. Psy-1 is not expressed or down-regulated in several colorless tomato fruits mutants (Fray and Grierson, 1993). In a high pigment (hp) tomato ripening mutant, the level of lycopene was twice that in normal fruits, but the activity of PSY-1 was at similar level (Yen et. al., 1997). The increased lycopene level in hp mutants was caused by the increased plastid number, in which the lycopene synthesis occurs. (Yen et. al., 1997). The hp gene maps to the same chromosome 2 as the cnr gene. However, no 27 gene related to lycopene synthesis has been found in the same chromosome. The regulation of hp and cnr genes to lycopene formation is poorly understood. As for the family of cyclases that convert the lycopene to carotene, their expression is reduced during normal fruit ripening (Pecker et al., 1996). Their activity is very low when the tomato fruits reach the ripening. Tomato fruits of tangerine mutants, which up-regulate the expression of cyclases could produce a yellow-flesh tomato fruits with high level of -carotene (Isaacson et al., 2002). The study for regulation of lycopene biosynthesis at the gene and enzyme level is very limited (Fraser and Bramley, 2004). No regulatory genes in lycopene biosynthesis pathway have been isolated yet. IPP and GGPP can be synthesized from multiple pathways, which are impossible to be regulated in a single process. Lycopene concentration in tomato fruit as affected by environmental factors Kuti and Konuru (2005) studied the effects of cultivation environment (greenhouse Vs. field) on lycopene content in red-ripe tomatoes. They found that the greenhouse-grown cultivars had 20% higher lycopene concentration compared to field-grown tomatoes for all the cluster and round-type cultivars tested in their experiments. The environmental difference, such as temperature, light and nutrient solution, between greenhouse and field may account for the difference of lycopene concentration in fruit in this study; however, the greenhouse and field environmental parameters were not reported in the experiment. 28 Increase of lycopene concentration in tomato fruit under salt or water stress has been reported for studies conducted both in open fields and in greenhouse. Lycopene concentration in tomato grown in the open field was reportedly increased by high EC fertigation treatment (4.4 dS m-1) (De Pascale et al., 2001) or reducing the amount of irrigation water (Matsuzoe et al., 1998; Zushi and Matsuzoe, 1998). When the EC of nutrient solution was increased from 0.5 to 4.4 dS m-1 by adding NaCl, the lycopene concentration in tomato fruit increased by 74%. Similarly, when the EC of nutrient solution was increased from 2.3 (close to the EC used in commercial tomato greenhouse) to 4.4 dS m-1, the lycopene concentration in tomato fruit increased by 23 % (De Pascale et al., 2001). However, the lycopene concentration decreased when the EC was increased from 4.4 to 15.7 dS m-1 (De Pascale et al., 2001). The reduction of lycopene concentration in tomato fruit at high EC (> 4.4 dS m-1) might be caused by the high temperature, since most fruits of stressed plants were exposed directly to high solar radiation due to the smaller leaf area developed under high EC (De Pascale et al., 2001). The lycopene concentration in both cherry and beefsteak-type tomatoes grown in the field were enhanced, when the amount of irrigation water was reduced by 47% compared to the control (Matsuzoe et al., 1998; Zushi and Matsuzoe, 1998). Among limited research conducted under greenhouse conditions, salt stress provided by a high concentration of NaCl in the nutrient solution reportedly affected fruit color. More intense red color was obtained at 60 mM NaCl than at 0 mM NaCl in the nutrient solution for tomato plants grown hydroponically in perlite bags in greenhouse (Botella et al., 2000). Although the more intense red color suggests increase in lycopene 29 concentration under high NaCl treatment, lycopene concentration in tomato fruit was not quantified in their experiment. Sakamoto et al. (1999) applied EC of 5.0 dS m-1 or 8.0 dS m-1 at two fruit ripening stages (the green stage and the breaker stage) for hydroponically grown tomato plants. These ECs were achieved by adding NaCl to the standard solution (2.4 dS m-1). The concentration of lycopene in fresh fruit was increased by the early application of EC of nutrient solution at 5 dS m-1, but the absolute amount of lycopene per fruit was decreased or not affected. The early application of EC of nutrient solution at 8 dS m-1 and the late application of two ECs at 5 dS m-1 or 8 dS m-1 did not affect the concentration of lycopene in fresh fruit. Air temperature and light are well studied environmental factors affecting lycopene concentration. The formation of lycopene was reportedly optimal at the air temperature range of 12-32 oC (Leoni, 1992). Air temperature below 12 oC or above 32 oC reduced the lycopene concentration in fresh tomato fruits, mainly by inhibiting lycopene synthesis (Baqar and Lee, 1978). Both light intensity and light quality affected lycopene accumulation. It was shown that high light intensity increased the lycopene synthesis under favorable temperatures between 22-25 oC (Dumas et al., 2003); however, solar radiation higher than 650 W m-2 inhibited lycopene accumulation, which might be explained by the overheating of fruit caused by the intense radiation (Adegoroye and Jolliffe, 1987). Phytochrome is considered to be involved in regulating lycopene accumulation (Alba et al., 2000). Red-light treatment increased lycopene concentration by 230% during fruit development, and the effects were reversed by the far-red light treatment (Alba et al., 2000). The increase of vapor pressure deficit (VPD) had no effect on 30 enhancing the fruit color (Leonardi et al., 2000). However, the VPD effect on lycopene concentration remains unknown. From the literature review, we learned that limited amount of research has been done to investigate how to enhance the lycopene concentration in tomato fruit by controlling environmental conditions. Compared to open field condition, a controlled environmental greenhouse is advantageous in quantifying the environmental factors and considered as an ideal system to conduct research on the effects of environmental factors on tomato fruit lycopene concentration. Chlorophyll degradation in tomato fruit development During tomato fruit ripening, there is a dramatic change in chlorophyll concentration as well as lycopene concentration. Chlorophyll concentration starts to decline in the breaker stage and then disappears or declines to a trace level in the ripe fruit. The degradation of chlorophyll was shown during the transformation of fully differentiated chloroplasts into chromoplasts by electron micrographic analysis (Thelander et al., 1986). In fully differentiated chloroplasts, the expression of genes encoding photosynthetic proteins is suppressed and therefore the photosynthetic proteins are very low in chloroplasts of mature green tomato fruit. The transcription of plastid-targeted photosynthetic proteins stops 5 to 10 days before the fruit reaches full size (Piechulla et al., 1986). The amount of stroma thylakoids, stacking, and photosynthetic capacity were decreased in chloroplasts of mature tomato fruit (Piechulla et al., 1987). 31 The chloroplasts senescence, chlorophyll degradation and chromoplasts development are independent pathways during tomato ripening. For the tomato gf mutant (chlorophyll degradation defective mutant), in which the chlorophyll synthesis proceeds throughout fruit ripening, the chromoplasts can still be formed (Cheung et al., 1993). Therefore, chlorophyll degradation is not a prerequisite for chromoplasts formation even though the chloroplasts have been observed to being transformed into chromoplasts during fruit development. When EC of the nutrient solution was increased, increase in leaf chlorophyll concentration is often observed. For example, increase of EC in the nutrient solution from 1.8-2.0 dS m-1 to about 4.0-5.4 or 8.1-9.2 dS m-1 increased chlorophyll concentration per unit of leaf area significantly (Romero-Aranda et al., 2001). Little information is available for chlorophyll concentration in tomato fruit as affected by cultural practice and environmental conditions. Sakamoto et al. (1999) reported that chlorophyll a concentration in fresh fruit was enhanced by EC of nutrient solution at 5.0 dS m-1 and 8.0 dS m-1 when applied at the green stage, compared to control at 2.4 dS m-1. The same EC treatments at the breakers stage did not affect the chlorophyll a concentration in fresh fruit. However, the chlorophyll b concentration in fresh fruit was not affected by both EC treatments, applied in either green or breakers stages. Little is known about the regulation of chlorophyll degradation during tomato ripening. It is not clear that whether the decrease of photosynthetic proteins synthesis or increases in degradative enzymes which are involved in the process causes the breakdown of chloroplast structure. 32 PRESENT STUDY The research for this dissertation is presented as three manuscripts, each in a separate appendix. Included in each manuscript are brief introductions, methods, results, discussion, and conclusions. The following are a summary of the most important findings from each of the manuscripts. EFFECTS OF ELECTRICAL CONDUCTIVITY OF HYDROPONIC NUTRIENT SOLUTION ON LEAF GAS EXCHANGE OF FIVE GREENHOUSE TOMATO CULTIVARS (APPENDIX A) To optimize tomato fruit quality and yield by manipulating EC of nutrient solution, it is import to understand the plant photosynthetic and transpirational responses of selected cultivars with local importance. The objective of this study was to evaluate the effects of electrical conductivity (EC) (2.3, 4.8 or 8.4 dS m-1) of nutrient solution on tomato plant leaf photosynthesis, transpiration and stomatal conductance and their interactions with cultivars and plant developmental stages. Five cultivars (Blitz, Mariachi, Quest, Rapsodie and Trust) of tomato were grown hydroponically inside the greenhouse and their physiological responses were measured. Leaf photosynthetic light response curves were measured in both vegetative (Mariachi and Rapsodie) and reproductive (all cultivars) stages. The leaf transpiration rate and stomatal conductance were measured for all five cultivars in both vegetative and reproductive stages. During the vegetative stage, high EC treatment of 8.4 dS m-1 reduced leaf conductance (gl) and transpiration rate (TR) by 28 % and 29 %, respectively, compared to low EC treatment. High and medium EC treatment 33 (4.8 and 8.4 dS m-1) reduced 35 % and 15 %, respectively, of the maximum photosynthetic rate for Mariachi, compared to low EC treatment. For Rapsodie, however, high EC did not affect the maximum photosynthetic rate whereas medium EC treatment increased 17% of the maximum photosynthetic rate than that of low EC treatment. High EC treatment reduced the initial slope and increased light compensation point for both Mariachi and Rapsodie, compared to low EC treatment. During reproductive stage, high EC treatment reduced gl by 15% compared to low EC treatment; but leaf TR was not affected regardless of cultivar. High EC treatment reduced the maximum photosynthetic rate of Blitz and Mariachi about 34% and 23%, compared to low EC treatment. Medium EC treatment increased 26% of the maximum photosynthetic rate for Rapsodie, compared to low EC treatment. Plant physiological response to EC treatments was cultivar and growth-stage specific. It provided reference information in selecting greenhouse tomato cultivars and EC level of nutrient solution that may improve the fruit quality when plants are grown under high EC while sustaining plant growth and yield. EFFECTS OF NUTRIENT SOLUTION EC, PLANT MICROCLIMATE AND CULTIVARS ON FRUIT QUALITY AND YIELD OF HYDROPONIC TOMATOES (APPENDIX B) The aim of our study was to find the effects of EC of nutrient solution and environmental conditions on fruit quality as well as fruit yield. In the present experiment, four tomato cultivars (Blitz, Mariachi, Quest and Rapsodie) were grown hydroponically in two different microclimate conditions inside the greenhouse. The effects of electrical 34 conductivity (EC) of nutrient solution (2.6 or 4.5 dS m-1) and plant microclimates in greenhouse on tomato fruit total soluble solids (TSS, %Brix at 20oC) concentration, lycopene concentration, and yield were examined. Four cultivars of tomato were grown hydroponically on rockwool in two microclimates inside the greenhouse under two EC levels, adjusted by adding NaCl and CaCl2 after the first fruit truss was set. In all cultivars, TSS, lycopene concentration of fruits increased by 12-22 % and 34-85 %, respectively, with increasing EC level. Fruits harvested from the east side of the greenhouse had higher TSS than those from the west side, due to the different plant microclimate such as daily photosynthetic active radiation and vapor pressure deficit. However, lycopene concentration in fruits was not significantly affected by plant microclimate regardless of cultivars or EC. The cultivar of Mariachi showed the strongest effect in response to EC levels regarding both TSS and lycopene concentration among the cultivars examined. The cumulative yield at 7 weeks showed no significant differences between EC treatments and between different greenhouse microclimates, regardless of cultivars. The result indicated that value added tomato fruits could be produced by manipulating EC and plant microclimate in the greenhouse without causing yield reduction. This study was published as: Wu, M., J.S. Buck, and C. Kubota. 2004. Effects of nutrient solution EC, plant microclimate and cultivars on fruit quality and yield of hydroponic tomatoes. Acta Hort. 659:541-547. 35 EFFECTS OF HIGH ELECTRICAL CONDUCTIVITY OF NUTRIENT SOLUTION AND ITS APPLICATION TIMING ON LYCOPENE, CHLOROPHYLL AND SOLUBLE SUGAR CONCENTRATIONS OF HYDROPONIC TOMATO (APPENDIX C) Manipulation of EC of nutrient solution has been studied as an effective way to enhance flavor and nutritional values of tomato fruits. The objective of this research was to quantitatively understand the accumulation of lycopene and sugars and the degradation of chlorophyll in fruits as affected by EC and its application timing relative to the fruit ripeness stages. Tomato (cv Durinta) was grown hydroponically inside the greenhouse under two EC (2.3 and 4.5 dS m-1). The high EC treatment of 4.5 dS m-1 was started either after anthesis (high EC treatment) or four weeks after anthesis (delayed high EC treatment). Fruits were harvested weekly beginning two weeks after anthesis, until all fruits reached the red stage. The chlorophyll concentration in tomato fruits showed no difference between three EC treatments when compared at the same ripeness stages. Lycopene concentration of the red tomato fruit in the delayed EC treatment reached the same level as that in the standard high EC treatment, and 35 % greater than that in the low EC treatment. TSS of red ripe tomato fruits in the high EC treatment was 6.2 ± 0.2 %, which was greater than those grown in the delayed high EC treatment (5.8 ± 0.2 %) or low EC treatment (5.2 ± 0.3 %). The total soluble sugar concentration (glucose and fructose) of red ripe tomato fruits under high EC treatment was 57% greater than that of low EC treatment; the high EC treatment had a greater enhancement for glucose and fructose of red ripe tomato fruits than those grown under delayed high EC treatment (37%). The fruit ripeness was enhanced under the 36 high EC, regardless of the timing of treatment. The results indicated that lycopene synthesis was driven by, but chlorophyll degradation was independent from, the osmotic and/or salt stress caused by the high EC. The study provides valuable information to better understand mechanisms of lycopene and sugar accumulation of tomato fruits under high EC treatment. Tomato physiological parameters of photosynthesis and transpiration are closely related to plant growth as well as fruit yield and quality. Yield and fruit quality of seven weeks of harvest showed that electrical conductivities suggested by plant responses in leaf gas exchanges were effective in producing high quality tomatoes without reducing overall yield. In the same experiment, lycopene concentration was more sensitive to high EC than the total soluble solid concentrations. 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Effects of soil water deficit on vitamin C, sugar, organic acid and carotene contents of large-fruited tomatoes. J. Jpn. Soc. Hort. Sci. 67: 927-933. 45 APPENDIX A: EFFECTS OF ELECTRICAL CONDUCTIVITY OF HYDROPONIC NUTRIENT SOLUTION ON LEAF GAS EXCHANGE OF FIVE GREENHOUSE TOMATO CULTIVARS 46 Effects of Electrical Conductivity of Hydroponic Nutrient Solution on Leaf Gas Exchange of five Greenhouse Tomato Cultivars Min Wu* and Chieri Kubota Department of Plant Sciences, The University of Arizona, Tucson, AZ 85721 This paper includes results funded by the Controlled Environment Agriculture Program at the University of Arizona. *To whom reprints should be addressed. Email address: [email protected]. 47 Effects of Electrical Conductivity of Hydroponic Nutrient Solution on Leaf Gas Exchange of Five Greenhouse Tomato Cultivars Additional index words. Fruit quality, photosynthesis, reproductive stage, stomatal conductance, total soluble solids, transpiration, vegetative stage. Abstract. Five cultivars (Blitz, Mariachi, Quest, Rapsodie and Trust) of tomato (Lycopersicon esculentum Mill.) were grown hydroponically in a greenhouse to determine photosynthetic and transpirational responses to three electrical conductivities (EC) (2.3 (control), 4.8 and 8.4 dS m-1) of influx nutrient solution. Leaf photosynthetic light response curves were measured during the early vegetative growth stage for cultivars Mariachi and Rapsodie and during the reproductive growth stage for all five cultivars. Leaf transpiration rate and leaf conductance were measured for all five cultivars in both stages. During the vegetative growth stage, high EC treatment of 8.4/14.3 dS m-1 influx/efflux solution reduced leaf conductance and transpiration rate by 28% and 29%, respectively, compared to low EC treatment (2.3/5.9 dS m-1) regardless of cultivar. For Mariachi, the medium EC treatment (4.8/8.7 dS m-1) and high EC treatments in the vegetative growth stage reduced the maximum photosynthetic rate by 49%, compared to the low EC treatment. However, for Rapsodie, the medium EC treatment increased the maximum photosynthetic rate during the vegetative stage by 8% and 47%, compared to low and high EC treatments, respectively. During reproductive growth stage, EC treatment did not significantly affect the transpiration rate, but high EC treatment still reduced the leaf conductance by 15%, 48 regardless of cultivar. Medium EC treatment did not significantly affect the leaf photosynthetic rate compared to low EC treatment, in all cultivars except Rapsodie, which showed the greatest maximum photosynthetic rate in the medium EC treatment. The results showed that the plant physiological response under elevated EC was cultivar and growth-stage specific, and increasing the influx EC to the moderate level of around 4.8 dS m-1 would not negatively impact photosynthesis, transpiration and stomatal conductance of tomato plants during the reproductive growth stage, for all cultivars tested in the present experiment. 49 Tomato has been an important horticultural crop in the U.S. market (Rick, 1995; Jones, 1999). For fresh tomato production, 159,664 and 1,594,241 tons of tomatoes were produced in the greenhouse and field, respectively, in the U.S. in 2003 (Cook and Calvin, 2005). Since 1985, the consumption of fresh tomato in the U.S. increased about 30 %, with annual per capita consumption level estimated at 8.8 kg in 2003 (Cook and Calvin, 2005). In 2003, greenhouse tomato production reportedly accounted for only 9% of total fresh tomato production in the U.S. (Cook and Calvin, 2005). However, the percentage of greenhouse tomato available in the U.S. retail markets has increased dramatically during the past decade and accounts for 37% of the weekly quantity of tomatoes sold in the average U.S. supermarket in 2003 (Cook and Calvin, 2005), including produce imported from Mexico and Canada. This suggests that greenhouse tomatoes are preferred in retail sales, likely due to the consistent supply and generally higher quality than field grown tomatoes. This trend seems to drive neighboring countries into more greenhouse production. Canada produced 89% of fresh tomato in greenhouses (Cook and Calvin, 2005). In Mexico, the total area of greenhouse used for production of vegetables is increasing rapidly, reportedly as high as 30% annually (Steta, 2004). Due to this competition, U.S. greenhouse growers are shifting toward high quality tomato production in order to compete with Canadian and Mexican growers. One such example is to pursue a better flavor of the fruit; and another is a reduced use of pesticides. Sugar and organic acids are the major attributes of tomato flavor (Stevens et al., 1977). Total soluble solid concentration (TSS, % Brix at 20oC) is the most common index for overall flavor of tomato fruit associated directly with sugar and organic acid 50 concentrations in tomato juice (Stevens et al., 1977; Young et al., 1993). In hydroponic tomato production, increasing electrical conductivity (EC) of nutrient solution is a well known technique to increase TSS of tomato fruit, because the decreased osmotic potential of nutrient solution restricts the water transport to fruits, resulting in higher concentrations of soluble solids (Adams, 1991; Mitchell et al., 1991; Cornish, 1992; Lin and Glass, 1999; Dorais et al., 2000). Electrical conductivity can be increased either by increasing overall strength (total concentration) of the nutrient solution or adding NaCl, but the latter is more widely accepted by commercial growers as it is economically feasible. One of the disadvantages of increasing TSS by high EC treatment is reduction in fruit size by reducing water content in fresh fruit (Adams and Ho, 1989). The EC of nutrient solution used for commercial hydroponic tomato production generally ranges between 1.6-5.0 dS m-1 (Voogt et al., 1997). Dorais et al. (2000) examined the effects of EC on tomato fruit yield and found that tomato yield was not reduced when EC ranged from 2.1 to 5.1 dS m-1. Adam (1991) reported that, compared to the control treatment of 3.0 dS m-1 EC, application of 8 dS m-1 EC decreased tomato yield by 4% to 5% per dS m-1, whereas 12 dS m-1 EC decreased tomato yield by 6% to 8% per dS m-1, where both high EC treatments were achieved by adding NaCl to the nutrient solution. Another report showed that there was no significant difference in yield between plants grown under 2.7 and 4.5 dS m-1; however, the yield was reduced linearly when the EC was increased from 4.5 to 6.0, 7.4 or 8.6 dS m-1 (Leonardi et al., 2004). These results suggest that when EC was increased moderately to around 5 dS m-1, TSS of fruits could be enhanced without yield reduction. 51 Under high EC, the tomato plant may be affected by a water stress from the low water potential of the nutrient solution, which is caused by the decreased osmotic potential of the solution, or by excessive ion uptake due to greater ion concentrations in solution (Greenway and Munns, 1980). Photosynthesis, transpiration and stomatal conductance under high EC were affected by limited irrigation and/or increased salt concentrations in nutrient solution (Romero-Aranda et al., 2001). These physiological parameters are closely related to plant growth as well as fruit yield and quality. Xu et al. (1995) studied the effects of EC of hydroponic nutrient solution, growth medium (substrate) and irrigation frequency on tomato plant photosynthetic response and found that the maximum leaf photosynthetic rate was increased by 15.4% and 14.1% when EC was increased from 2.5 to 4.0 dS m-1 for plants grown in nutrient film technique (NFT) and rockwool systems, respectively. But a further increase of EC to 5.5 dS m-1 resulted in a 10 % lower maximum photosynthetic rate compared to that under 4.0 dS m-1 EC when plants were grown in a rockwool system. Schwarz et al. (2002) found that an increase of EC from 1.25 dS m-1 up to 8.75 dS m-1 did not reduce the leaf photosynthetic rate of tomato. In both experiments reported by Xu et al. (1995) and Schwarz et al. (2002), the EC was enhanced by increasing the overall strength of nutrient solutions. Romero-Aranda et al. (2001) showed that the leaf net photosynthetic rate of tomato plants was reduced proportionally as increasing NaCl concentration in the nutrient solution (0, 35, and 70 mM NaCl), and stated that the decrease might have resulted from the reduction in stomatal conductance and stomatal density (Romero-Aranda et al., 2001). The nutrient solution examined in their experiment had 4.0-5.4 and 8.1-9.2 dS m-1 EC for 35 52 and 70 mM NaCl treatments, and 1.8-2.0 dS m-1 for the control (0 mM NaCl) (Romero-Aranda, personal communication). The decrease in net photosynthetic rate observed at EC of 4.0 dS m-1 or greater may be due to cumulated sodium in the plant tissue. To optimize tomato fruit quality and yield by manipulating EC of nutrient solution, plant photosynthetic and transpirational responses to varied EC of selected cultivars with local importance would provide critical information to be used for a reference study or further long term investigation in greenhouse production. The objective of this study is to evaluate the effects of EC of nutrient solution on tomato plant leaf photosynthetic response, transpiration rate and leaf conductance and its interaction with cultivars and plant developmental stages. Materials and Methods Plant material and growth conditions. Five greenhouse cultivars of tomatoes, Blitz (DeRuiter Seeds Inc., Columbus, OH), Mariachi (Rijk Zwaan Seeds Ltd., De Lier, The Netherlands), Rapsodie (Rogers Seeds Inc., Boise, ID), Trust (DeRuiter Seeds Inc.), and Quest (DeRuiter Seeds Inc.) were selected based on the trials conducted at the University of Arizona in previous years (Rorabaugh et al., 2002, unpublished). Seeds were sown into 4×4 cm rockwool cubes covered with a thin layer of vermiculite on September 16th, 2002 and germinated under frequent water mist in the greenhouse. After the cotyledons were fully unfolded (21 days after seeding), the seedlings were transplanted to 10×10 cm rockwool cubes and sub-irrigated with full strength of modified Hoagland nutrient solution (EC 2.3 dS m-1, pH 6.0) once a day. When all plants had more than 4 fully expanded true 53 leaves (35 days after seeding), 15 uniform seedlings were selected from each cultivar and subjected to one of three EC treatments (2.3, 4.8 or 8.4 dS m-1) (sub-irrigation once a day). The three EC levels were achieved by increasing the strength of the nutrient solution. The full strength nutrient solution (2.3 dS m-1) contained the following elements in mg L-1: 142 N, 65 P, 374 K, 150 Ca, 50 Mg, 2 Fe, 0.6 Mn, 0.3 Zn, 0.05 Cu, 0.4 B, and 0.05 Mo (Jensen and Rorabaugh, 2002, unpublished). Forty two days after seeding (7 days after start of EC treatments), plants were transplanted to 3.8-liter black plastic pots filled with a mixture of vermiculite, perlite and peatmoss (v: v: v=1:1:1). Drip irrigation tubing was provided to each pot and irrigation was applied at 8:00 AM, 11:00 AM and 2:00 PM daily with about 300-400 mL nutrient solution supplied per irrigation event. The experiment was conducted in a 7.3 m X 14.6 m compartment in a gutter connected multi-span greenhouse located at the University of Arizona Campus Agriculture Center in Tucson, AZ. The greenhouse was equipped with an evaporative cooling system with the exhaust fans located at the south end and the wet pads located at the north end of the greenhouse. The experimental plants were grown on benches located in the middle of the greenhouse compartment. Measurements. The EC, pH and volume of the inflow nutrient solution were recorded daily. The EC, pH and volume of the efflux nutrient solution were collected and recorded weekly. The temperature and relative humidity at the plant canopy were continuously recorded. The second fully open leaf below the shoot tip was used for measurements of leaf conductance (gl), transpiration rate (TR) and net photosynthetic rate 54 (NPR) in both vegetative and reproductive growth stages. The measurement for gl and TR during the vegetative growth stage was conducted 36 days after seeding, which was 1 day after the start of EC treatments. The NPR in the plant vegetative and reproductive growth stages were measured 37 to 40 days after seeding (2 to 5 days after the start of EC treatments) and 68 to 75 days after seeding, during which period first small fruit settings were observed on the first truss. The gl and TR were measured using a portable photosynthesis measurement system (CIRAS2, PP Systems, Co., Amesbury, MA) set at a 1000 µmol m-2 s-1 PPF, 400 µmol mol-1 CO2 concentration and 200 mL min-1 internal flow rate. Using the same system, NPR were measured under 6 PPF levels (0, 250, 500, 1000, and 2000 µmol m-2 s-1) at 400 µmol mol-1 CO2 concentration under 200 mL min-1 internal flow rate. The gl, TR and NPR were measured during morning hours (no later than 1 PM), before plants were experienced with mid-day high temperature and radiation. The NPR at varied PPF were fitted with a common photosynthetic model: NPR = k1 × [ 1 Exp( k2 × (PPF k1 k3 ) ] where parameters k1, k2 and k3 represent the maximum photosynthetic rate, initial slope and light compensation point, respectively, and were estimated by nonlinear regression. Treatment and cultivar significances on these parameters were examined statistically using the JMP software (version 5.1, SAS Institute, Cary, NC). Plants were grown on three 2.7 m x 1.9 m benches in the greenhouse each with three blocks of EC treatments arranged according to the Latin square design. Five cultivars were randomly distributed within each block. The total number of plants was 45 (3 plants per 55 cultivar per EC treatment). Treatment significances and significance among individual treatment levels were analyzed by analysis of variance (ANOVA) and Tukey HSD test, respectively using JMP software. Results and discussion Greenhouse temperature and relative humidity. Average day (6:00 AM-6:00 PM) and night (6:00 PM – 6:00 AM) air temperatures inside the greenhouse during the experiment were 21.5 ± 0.9 °C and 13.3 ± 0.8 °C , respectively. Average day and night relative humidity inside the greenhouse were 50.6 ± 9.6 % and 85.8 ± 3.6 %, respectively. EC of nutrient solution and pH. Average EC and pH of the inflow solution during the experiment were 2.3±0.4 dS m-1 and 6.3±0.2 in the low EC treatment, 4.8±0.9 dS m-1 and 6.3±0.3 in the medium EC treatment, and 8.4±0.7 dS m-1 and 6.2±0.3 in the high EC treatment. Average EC and pH of the efflux solution during the experiment were 5.9±1.3 dS m-1 and 7.1±0.5 in the low EC treatment, 8.7±2.1 dS m-1 and 7.6±0.5 in the medium EC treatment, and 14.3±2.5 dS m-1 and 7.4±0.6 in the high EC treatment. Average efflux percentage over the inflow nutrient solution was 35% for the low EC treatment, 31% for the medium EC treatment and 37% for high EC treatment, all of which are within the conventional range recommended in commercial hydroponic tomato production. The relatively high EC of the efflux solution was due to the infrequent irrigation (three times a day), despite the conventional efflux percentage. Although we could not measure the EC in the rootzone directly in this experiment, the efflux EC suggest that there were relatively large diurnal fluctuations in EC between successive irrigation events. 56 Leaf conductance and transpiration. The TR and gl as affected by EC and cultivar are shown in Table 1. When measured 1 day after the EC application during the vegetative growth stage, the high EC treatment reduced TR and gl by 29 % and 28 %, respectively, compared to the low EC control, suggesting that the reduction in TR was associated with the decreased gl. However, there was no significant difference in TR and gl between the medium EC treatment and the low or high EC treatment. A similar correlation between TR and gl under varied EC levels was reported by Romero-Aranda et al. (2001). When measured after first fruit set (the reproductive growth stage), the high EC treatment significantly reduced gl by 15%, compared to low EC. However, there was no difference in gl between the medium EC treatment and the low or high EC treatment. The TR in reproductive growth stage was not significantly affected by the EC treatments. The smaller reduction in TR and gl by high EC treatment in the reproductive growth stage than in the vegetative growth stage might be due to the plant osmotic adjustment after prolonged exposure to high EC. Osmotic adjustment in plant is an important adaptation to water stress by decreasing the leaf water potential to compensate the lowering of water potential in the nutrient solution (Guerrier, 1996; Shannon et al., 1987). Among the five cultivars, Trust exhibited lower TR and gl than Blitz, Mariachi, or Rapsodie when measured 1 day after the start of EC treatment during the vegetative growth stage (Table 1). Quest had a TR similar to Raposodie, and a gl similar to Rapsodie or Trust. The relatively low TR and gl in Trust may be due to the fact that Trust is a cultivar well adapted to northern climates compared to the other cultivars. In fact, Trust is widely cultivated in Northern Europe such as Denmark and is known as sensitive to high light 57 environment (De Ruiter Seeds, 2006). Blitz performs well under high light and high temperature conditions and the plant grows more vegetatively than reproductively (De Ruiter Seeds, 2006). Mariachi and Rapsodie reportedly had a better adaptation to heat than Quest and Blitz according to the trials conducted in a semiarid greenhouse (Rorabaugh and Jensen, 2002). During the reproductive growth stage, all cultivars had greater TR than those measured 1 day after EC treatment during vegetative growth stage, but there was no difference in TR among cultivars. The gl for Rapsodie and Trust were similar to Blitz and Quest or greater than Mariachi during the reproductive growth stage. Photosynthetic light response. The photosynthetic light response curves during the vegetative growth stage for Mariachi and Rapsodie are shown in Fig. 1. The photosynthetic light response curves obtained for all five cultivars during the reproductive growth stage are shown in Fig. 2. The parameters of photosynthetic response curves, maximum photosynthetic rate (k1), initial slope (k2) and light compensation point (k3), as affected by cultivar and EC are shown in Tables 2 and 3. During the vegetative growth stage, the high EC and medium EC treatments reduced an average of 48.8 % of the maximum photosynthetic rate for Mariachi, compared to low EC treatment. For Rapsodie, the medium EC treatment had the greatest maximum photosynthetic rate out of the three EC treatments. Initial slope decreased, and light compensation point increased with increasing EC for both Mariachi and Rapsodie. Lower initial slope in the higher EC treatments indicates a lower efficiency of photosynthesis under low light conditions, and the greater light compensation point in the higher EC treatments indicates a greater respiration rate. The plant vegetative growth (stem length and leaf size) was significantly reduced in 58 medium and high EC treatments (data not shown), which may be attributed to lower photosynthetic efficiency and greater respiration under the EC treatments. During the reproductive growth stage, there was no significant difference in maximum photosynthetic rate between low and medium EC treatments for all cultivars except Rapsodie. Rapsodie had a 26% greater maximum photosynthetic rate for the medium EC than low EC treatment. The maximum photosynthetic rate in the high EC treatment was significantly lower than that in the low EC treatment by 34% and 23% for Blitz and Mariachi, respectively, while no significant difference was observed in the maximum photosynthetic rate between high and low EC treatments for Rapsodie, Trust, and Quest. There were neither significant effects of EC treatment nor cultivar in the initial slope of the photosynthetic light response curve. The EC treatment did not significantly affect the light compensation point for all cultivars except Rapsodie, where high EC treatment caused a greater light compensation point than low or medium EC treatment. The rather unique response of Rapsodie photosynthetic parameters to EC treatment compared to other cultivars may attribute to its genotype, as Rapsodie is reportedly suitable for cultivation under relatively mild climate and greater radiation at latitude of 36 degrees or less (Roger Seeds, 2006). It is also known that Rapsodie is relatively tolerant to water stress (Costa, 2006, personal communication). Noticeably, Rapsodie showed the highest NPR under medium EC treatment compared to low and high EC treatments, regardless of the plant growth stage. Xu et al. (1995) found that increased EC at 4.0 dS m-1 could increase the tomato leaf NPR compared to the control of 2.0 dS m-1 in both nutrient film technique (NFT) and 59 rockwool systems. Schwarz et al. (2002) found that an increase of EC up from 1.25 to 8.75 dS m-1 did not reduce the leaf net photosynthetic rate of tomato plant. Romero-Aranda et al. (2001) observed cultivar specific responses in leaf gas exchange characteristics in response to different NaCl concentrations. For ‘Daniela’ tomato plants, an increase of NaCl from 0 mM (EC 1.8 – 2.0 dS m-1) to 35 mM (4.0-5.4 dS m-1) or 70 mM (8.1-9.2 dS m-1) reduced the leaf stomatal conductance (gs) by 40 % or 68 %, respectively. For ‘Moneymaker’, both high NaCl treatments of 35 mM or 70 mM reduced the leaf gs by 52 %, compared to the control at 0 mM of NaCl in the nutrient solution. The leaf transpiration rate decreased by about 27% and 60% for ‘Daniela’ and by 52% and 50% for ‘Moneymaker’ under 35 and 70 mM NaCl treatments, respectively, compared to the control. In our results, the effect of medium to high EC (4.8 to 8.4 dS m-1 for inflow solution) of nutrient solution on photosynthetic characteristics was also cultivar specific. The different results observed in leaf photosynthetic responses as affected by EC in these experiments might be attributed to the differences in plant genotypes and/or plant growth environments such as light levels used in the measurements in the both reports. In Xu et al. (1995), the leaf net photosynthetic rate was measured under 1000 µmol m-2 s-1 photosynthetic photon flux (PPF), whereas it was measured under 400 and 625 µmol m-2 s-1 PPF of two controlled environment chambers in Schwarz et al. (2002). Therefore, the lower PPF in Schwarz et al. (2002) might have mitigated the effect of high EC on the photosynthesis, and resulted in a higher EC that sustained the leaf photosynthetic rate than those in Xu et al. (1995). There were somewhat different responses of photosynthetic characteristics to medium or high EC between vegetative and reproductive growth stages for Mariachi and 60 Rapsodie in the present experiment. The maximum photosynthetic rate was relatively greater in the reproductive growth stage than in the vegetative growth stage within the same EC treatment. The initial slope of the photosynthetic response curve decreased with increasing EC for the vegetative growth stage, while there was no significant difference between EC treatments for the reproductive growth stage. The changes observed between two stages may be caused by the plant acclimation to the increased EC levels as observed for TR and gl (Table 1). Although stomatal conductance seems to be the main cause of photosynthesis decrease (Kaiser, 1987), another possible reason of causing greater maximum photosynthetic rate and initial slope is the increased sink strength of plant in the reproductive growth stage than the vegetative growth stage, a phenomenon associated with a sink-source relationship (Ho, 1988). The reduction in sink activity caused an increase in sucrose in source leaves and led to a decrease in photosynthetic rate by feedback inhibition (Stitt, 1991). Conclusions The photosynthetic and transpirational responses of tomato plants were affected by cultivar, EC of nutrient solution and plant growth stages. Photosynthesis and transpiration are closely related to plant growth as well as fruit yield and quality. The relatively sensitive response of leaf gas exchange and its related parameters to EC observed during the vegetative growth stage suggests that the application timing of high EC should not be too early in the vegetative stage, as it could possibility stunt the overall plant growth by reducing photosynthetic and transpiration rates. During the reproductive growth stage, 61 photosynthesis and transpiration were less affected by higher EC in the nutrient solution. The results obtained in the experiment presented provides a reference for selecting greenhouse tomato cultivars and EC levels of nutrient solution that may improve the fruit quality while sustaining plant growth and yield. 62 Literature Cited Adams, P. 1991. Effects of increasing the salinity of the nutrients solution with major nutrients or sodium chloride on the yield, quality and composition of tomato grown in rockwool. J. Hort. Sci. 66:201-207. Adams, P., and L.C. Ho. 1989. Effect of constant and fluctuating salinity on the yield, quality and calcium status of tomatoes. J. Hort. Sci. 64:725-732. Cook, R. and L. Calvin. 2005. Greenhouse tomatoes change the dynamics of the North American Fresh Tomato Industry. USDA Economic Research Report No. 2 Available on line at: <www.ers.usda.gov> (Accessed on March 22, 2006). Cornish, P.S. 1992. Use of high electrical conductivity of nutrients solution to improve the quality of salad tomatoes grown in hydroponic culture. J. Expt. 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Gronwald, and M. Tal, 1987. Effects of salinity on growth and accumulation of organic and inorganic ions in cultivated and wild tomato species. J. Amer. Soc. Hort. Sci. 112:416–423. Steta, M. 2004. Mexico as the new major player in the vegetable greenhouse industry. Acta Horticulturae. 259:31-36. Stevens, M.A., A.A. Kader, and M. Albright-Houlton. 1977. Intercultivar variation in composition of locular and pericarp portions of fresh market tomatoes. J. Amer. Soc. Hort. Sci. 102:689-692. 64 Stitt, M. 1991. Rising CO2 levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ. 14:741-762. Xu, H.L., L. Gauthier, and A. Gosselin. 1995. Effects of fertigation management on growth and photosynthesis of tomato plant grown in peat, rockwool and NFT. Scientia Hort. 63:11-20. Young, T.E., J.A. Juvik, and J.G. Sullivan, 1993. Accumulation of the components of total solids in ripening fruits of tomato. J. Amer. Soc. Hort. Sci. 118:286-292. 65 Table 1. Transpiration rate (TR) and leaf conductance (gl) of tomato plant as affected by electrical conductivity (EC) of the nutrient solution and cultivar, measured 1 day after start of EC treatment during the vegetative growth stage and the reproductive stage (after first fruit set). Factor EC Low Medium High Cultivar Blitz Mariachi Rapsodie Trust Quest ANOVA(P = 0.05) EC Cultivar Cultivar X EC Vegetative growth stage TR (mmol m-2 s-1) gl (mmol m-2 s-1) Reproductive growth stage TR (mmol m-2 s-1) gl (mmol m-2 s-1) 4.0ab 3.6ab 2.8b 188.9a 183.1ab 135.6b 6.3 6.4 5.8 445.9a 438.0ab 380.6b 3.9a 4.6a 3.7ab 2.3c 2.8b 204.9a 241.4a 183.2ab 108.6c 135.0bc 6.1 5.8 6.6 6.4 6.0 411.7ab 380.2b 460.6a 473.0a 391.1ab * * NS * * NS NS NS NS * * NS Means followed by the same letters within the column are not significantly different according to a Tukey HSD test at P =0.05. 66 Table 2. Effects of electrical conductivity (EC) of the nutrient solution and cultivar (Mariachi and Rapsodie) on maximum photosynthetic rate, initial slope and light compensation point of tomato plants during the vegetative growth stage (1 day after start of EC treatment). Factor Cultivar x ECZ Mariachi Rapsodie Maximum photosynthetic rateY (µ mol m-2 s-1) Initial slopeY Light compensation pointY (µ mol m-2 s-1) 16.7a 8.7e 8.4e 14.6c 15.7b 10.7d 0.052b 0.046bc 0.022d 0.057a 0.044c 0.017e 45.6c 45.2c 97.9a 46.1c 57.0b 128.7a Low Medium High Low Medium High Z Treatment significances determined by ANOVA at P=0.05. Y Means followed by the same letters within the column are not significantly different according to a Tukey HSD test at P=0.05. 67 Table 3. Effects of electrical conductivity (EC) of nutrient solution and cultivar (Blitz, Mariachi, Rapsodie, Trust, and Quest) on maximum photosynthetic rate, initial slope and light compensation point of tomato plants during the reproductive growth stage. Factor Cultivar x ECZ Blitz Mariachi Rapsodie Trust Quest Maximum photosynthetic rateY (µ mol m-2 s-1) Low Medium High Low Medium High Low Medium High Low Medium High Low Medium High 22.9a 24.0a 14.1f 21.4abc 23.6a 16.4ef 18.5bcde 23.3a 16.8def 24.5a 21.4abc 24.7a 20.7abcd 18.2cdef 22.4ab Initial slopeY Light compensation pointY (µ mol m-2 s-1) 0.053 0.054 0.052 0.053 0.054 0.061 0.054 0.054 0.046 0.056 0.056 0.055 0.056 0.058 0.052 44.6bc 55.5ab 46.5bc 50.6bc 56.7ab 50.0bc 35.5c 53.1bc 72.8a 49.5bc 47.6bc 41.6bc 44.4bc 51.8bc 39.0bc Z Treatment significances determined by ANOVA at P =0.05. Y Means followed by the same letters within the column are not significantly different according to a Tukey HSD test at P =0.05. 68 NPR (µmol m-2 s-1) 40 'Mariachi' Low EC Medium EC High EC 30 20 10 0 -10 0 500 1000 1500 2000 PPF (µmol m-2 s-1) NPR (µmol m-2 s-1) 40 Low EC Medium EC High EC 30 'Rapsodie' 20 10 0 -10 0 500 1000 1500 2000 PPF (µmol m-2 s-1) Fig. 1. Leaf photosynthetic light response curve of ‘Mariachi’ and ‘Rapsodie’ in the vegetative stage as affected by EC treatments. The data were obtained during plant vegetative stage, and were fitted with the model NPR=k1 [1-Exp{-k2(PPF-k3)/k1}] as , k1, k2 and k3 represent the maximum photosynthetic rate, initial slope and light compensation point, respectively. 69 40 Low EC Medium EC High EC PPF vs Photosynthetic response at high EC 20 10 0 -10 0 500 1000 1500 2000 20 10 0 -10 0 500 PPF (µmol m-2 s-1) 1000 1500 2000 PPF (µmol m-2 s-1) 40 40 Low EC Medium EC High EC 30 'Rapsodie' NPR (µmol m-2 s-1) NPR (µmol m-2 s-1) 'Mariachi' Low EC Medium EC High EC 30 NPR (µmol m-2 s-1) NPR (µmol m-2 s-1) 30 40 'Blitz' 20 10 0 -10 'Trust' Low EC Medium EC High EC 30 20 10 0 -10 0 500 1000 1500 2000 PPF (µmol m-2 s-1) 0 500 1000 1500 2000 PPF (µmol m-2 s-1) NPR (µmol m-2 s-1) 40 Low EC Medium EC High EC 30 'Quest' Fig. 2. Leaf photosynthetic light response curve of ‘Rapsodie’, ‘Blitz’, ‘Mariachi’, ‘Trust’ and ‘Quest’ in the reproductive stage as affected by EC treatments. The data were obtained during plant reproductive stage, and were fitted with the model NPR=k1 [1-Exp{-k2(PPF-k3)/k1}] as , k1, k2 and k3 represent the maximum photosynthetic rate, initial slope and light compensation point, respectively. 20 10 0 -10 0 500 1000 PPF (µmol m-2 s-1) 1500 2000 70 APPENDIX B: EFFECTS OF NUTRIENT SOLUTION EC, PLANT MICROCLIMATE AND CULTIVARS ON FRUIT QUALITY AND YIELD OF HYDROPONIC TOMATOES 71 Effects of nutrient solution EC, plant microclimate and cultivars on fruit quality and yield of hydroponic tomatoes (Lycopersicon esculentum) Min Wu*, Johann S. Buck, and Chieri Kubota Controlled Environment Agriculture Program (CEAC), Department of Plant Sciences, The University of Arizona, Tucson, AZ, USA 72 Keywords: cultivar, gas exchange, greenhouse, fruit yield, harvest, lycopene, total soluble solid, vapor pressure deficit Abstract Four cultivars (Blitz, Mariachi, Quest and Rapsodie) of tomato were grown hydroponically on rockwool in two microclimates (east and west) inside the greenhouse (Tucson, AZ) under two nutrient solution electrical conductivity (EC) levels (2.6 or 4.5 dS m-1), adjusted by adding NaCl and CaCl2 after the setting of first fruit truss. In all cultivars, total soluble solid (TSS, %Brix at 20oC) and lycopene concentration of fruits increased by 12-22 % and 34-85 %, respectively, with increasing EC level. Fruits harvested from the east side of the greenhouse had higher TSS than those from the west side, due to the different plant microclimate varying by daily PPF (photosynthetic photon flux) and VPD (vapor pressure deficit). However, lycopene concentration in fruits was not significantly affected by plant microclimate regardless of cultivars or EC. The cultivar of Mariachi showed the strongest effect in response to nutrient solution EC levels regarding both TSS and lycopene concentration among the cultivars examined. The cumulative yield at 7 weeks showed no significant differences between nutrient solution EC and locations, regardless of cultivars. The result indicated that value added tomato fruits could be produced by manipulating EC and plant microclimate in the greenhouse without causing yield reduction. 73 Introduction Tomato is an important crop in fresh vegetable market around the world. Recently, there is great interest for growers to improve the fruit quality by introducing better cultivation methods. Total soluble solid concentration (TSS, measured as %Brix) of tomato fruits is one of the important variables that determines the fruit flavor and quality because TSS is the most common index associated directly with sugars and organic acids concentrations in the juice (Stevens et al., 1977; Young et al., 1993). Manipulation of electrical conductivity (EC, dS m-1) is a well known technique to grow flavor-enhanced tomato because the elevated salinity in nutrient solution restricts the water transport to fruits and thus increase the TSS (Adams, 1991; Mitchell et al., 1991; Cornish, 1992; Lin and Glass, 1999). However, too severe water stress from high EC nutrient solution may cause a significant yield reduction (Adams, 1991). The aerial environmental factors influence fruit growth and quality, and they also interact with the nutrient solution EC effects on the plant. It was reported that increasing photosynthetic photon flux (PPF) significantly increased the leaf net photosynthetic rate and total carbon fixed by tomato plants (Schwarz et al., 2002). When the PPF was lower than 200 µmol m-2 s-1, there was no effect on plant growth up to a nutrient solution EC of 8 dS m-1; however, when the plants were grown at a higher light intensity (1000 µmol m-2 s-1), the tomato plant growth was reduced by the same EC treatments (Xu et al., 1995). Vapor pressure deficit (VPD) in the greenhouse influences tomato plant growth and fruit quality and yield as well. Lowering VPD in the greenhouse by use of a mist system increased the leaf stomatal conductance, plant growth and yield for tomato plant grown under salinity stress condition (Romero-Aranda et al., 2002). Increasing VPD contributes to the significant 74 reduction of fresh fruit weight and fruit water content, but increased TSS of tomato fruits (Leonardi et al., 2000). The regulation of lycopene formation in crop plants has become an area of considerable interest due to its antioxidant activity. The tomato fruit is the principle dietary source of lycopene; the ripening of tomato fruit is accompanied by a dramatic increase in the carotenoid content, especially a massive accumulation of lycopene (Fraser et al., 1994). In addition, red color development in fruits is due to carotenoid pigments, particularly lycopene, which is a major quality index for marketing. However, enhancing lycopene concentration in tomato fruits by crop management is rarely reported. The aim of our study is to find the effects of nutrient solution EC and environmental conditions on fruit quality as well as fruit yield. In the present experiment, four tomato cultivars were grown hydroponically in two different microclimate conditions inside the greenhouse. The effects of EC level and plant microclimates in greenhouse on tomato fruit TSS, lycopene concentration, and yield were studied. Material and methods Four cultivars of tomatoes were used (Blitz (DeRuiter Seeds, USA), Mariachi (Rijk Zwann Seeds, Netherlands), Quest (DeRuiter Seeds) and Rapsodie (Roger's Seeds, USA)). Seeds were sown into 4×4 cm rockwool cubes covered with a thin layer of vermiculite on January 23, 2003 and germinated/grown under frequent water mist in the greenhouse. After the cotyledons were fully unfolded, the seedlings were sub-irrigated once a day with one-half 75 strength modified Hoagland nutrient solution (EC 1.2 dS m-1, pH 6.0). When four true leaves emerged, the nutrient solution EC was increased to about 2.4 dS m-1. The seedlings were transplanted to 10×10 cm rockwool cubes 3 weeks after seeding. Nine weeks after seeding, uniform seedlings were selected and transplanted in the rockwool hydroponic systems at the east and west sides of the greenhouse (North-south orientation, with pad and fan cooling system) located in the University of Arizona Campus Agriculture Center (Tucson, AZ). Nutrient solution (2.4 dS m-1 EC) was supplied using a drip irrigation system. When the fruits at the first truss became visible, half of the plants were supplied with an EC about 4.6 dS m-1 which was achieved by adding NaCl and CaCl2. Leaf net photosynthetic and transpiration rates were measured 2 weeks after the EC treatment using a portable photosynthesis measurement system (CIRAS2, PPSystems Co., USA) at a 1500 µmol m-2 s-1 PPF and 400 µmol mol-1 CO2. We harvested and weighed fruits twice a week when the fruits reached the red ripening stage. The TSS of the harvested fruit juice was determined by hand refractometer (Atago N-20E) and the measured values (Brix) were converted to a standard temperature condition of 20oC. Lycopene concentrations of tomato fruits were measured spectrophotometrically using a modified method based on Fish et al. (2002). Each sample was assayed in triplicate. Lycopene analysis was performed only for the last harvest of 10 weeks after EC treatment. EC and pH levels of the inflow nutrient solution and drainage were recorded daily. The air temperatures of east and west sides of the greenhouse were monitored using a 0.5 mm Type-T thermocouple, connected to a datalogger (CR-10X, Campbell Scientific, USA) throughout the experiment. The thermocouple sensors were kept at the plant canopy height. 76 Photosynthetic active radiation (PAR) and VPD of the east and west sides of the greenhouse were monitored during limited time in the experiment. There were 4 blocks at each side of the greenhouse, each of which consisted of 9 plants growing in three 20×91 cm rockwool slabs. Cultivars were randomly distributed within each block. The EC treatment was alternately distributed within four blocks placed at each side of the greenhouse. Data obtained from the experiment were analyzed by JMP software (version 4.0.4 SAS Institute, USA). Treatment significances and significance among individual treatment levels were analyzed by analysis of variance (ANOVA) and Tukey HSD test, respectively. Results and discussion Nutrient solution EC/pH and greenhouse microclimates The nutrient solution (input solution) of the low EC treatments had 2.6±0.2 dS m-1 EC with 6.7±0.2 pH throughout the experiment, while that of the high EC treatments had 4.5±0.8 dS m-1 EC with 6.7±0.2 pH. The drainage solution EC was 5.5±2.0 dS m-1 at pH 7.4±0.6, and 9.0±3.2 dS m-1 at pH 7.3±0.5 for the low and high EC treatments, respectively. Average drainage percentage was 29% for the low EC treatments and 33% for high EC treatments. The average day and night temperatures were 24.1±2.4 and 20.7±3.1, respectively, for the east and 23.7±2.2 and 20.8±2.9, respectively, for the west. Although the measurements were not complete throughout the experiment, the microclimate differences between the east and west locations seemed to be more pronounced in PAR and VPD, rather than air temperature. The PAR, monitored for May 16th-May 27th, 2003 (4-6 weeks after the start of 77 EC treatment) and June 12th-June 21st, 2003 (8-9 weeks after the start of EC treatments), showed that the east location had a higher daily PAR (35.1±5.2 mol m2) than that of the west location (32.9±6.1 mol m2) during the day. The VPD, monitored from June 5th to June 11th, 2003 (7 weeks after the start of EC treatments) showed that the VPD during the day in the east location was also higher in the east (1.1±0.3 kPa) than in the west location (0.9±0.3 kPa) of the greenhouse. Generally, the higher daily PAR is responsible for a higher plant growth and net photosynthesis rate. VPD had reportedly small effects on the physiology and development of horticultural crops when it is ranged between 0.2 kPa to 1 kPa, but a higher VPD induced leaf water stress (Grange and Hand, 1987). An increase of VPD from 1.6 to 2.2 kPa increased the TSS in tomato fruits but reduced fruit fresh weight by about 10% (Leonardi et al., 2000).. In this experiment, the difference of PAR and VPD in different location in the greenhouse influenced the fruit quality. Leaf gas exchange of the plants Both microclimate and cultivar did not have significant effects on leaf gas exchange rates measured 2 weeks after the start of EC treatments. The leaf net photosynthetic rate, transpiration rate and stomatal conductance were in a range between 19.4±6.6 to 27.2±6.7 µmol m-2 s-1, 4.8±0.2 to 7.2±0.4 mmol m-2 s-1, and 411±45 to 506±122 mmol m-2 s-1, respectively. The high EC level of 4.5 dS m-1 did not significantly reduce leaf net photosynthetic rate (P = 0.89) and leaf stomatal conductance (P = 0.40). However, there was a tendency suggested that transpiration rates of the plants decreased under high EC compared to under low EC (P = 0.07). Different results were reported regarding the nutrient solution 78 EC effect on leaf gas exchange of plants. Xu et al (1995) tested the leaf photosynthetic rate of tomato plants grown both using a nutrient film technique (NFT) and a rockwool system at three nutrient solution EC levels (2.0, 4.0 and 5.5 dS m-1), where they found EC could increase the photosynthetic rate in both growing systems. Schwarz et al., (2002) found that an increase of EC up to 8.75 dS m-1 had no effect on leaf net photosynthetic rate; however, the whole plant photosynthesis was decreased due to the decreased leaf area. Those inconsistent findings may be caused by the interaction between the aerial environmental and cultural factors. Total soluble solid concentration and yield of fruits as affected by nutrient solution EC and greenhouse microclimates For all cultivars, fruits grown under high EC had a higher TSS than those grown under low EC level during 7 weeks of harvests (Table 1, Fig. 1). Plant microclimate of greenhouse also had an effect on TSS and the East location, which had a higher PAR and VPD, induced higher TSS. Mariachi cultivar had the highest TSS for plants grown under high nutrient EC at the east location of greenhouse (Fig. 1). The increase of TSS of all cultivars in response to nutrient solution EC was between 12-23 % whereas Rapsodie has the lowest increase and Mariachi has the highest. In the present experiment, TSS of tomato fruits grown hydroponically on rockwool system in greenhouse increased in response to the increased EC level (2.6 - 4.5 dS m-1), which generally agreed with the previous results reported by Adams (1991), Mitchell et al. (1991), Cornish (1992) and Lin and Glass (1999). Cuartero and Fernandez-Munoz (1999) 79 found that TSS of two commercial tomato cultivars increased at a 10.5 % rate per dS m-1 when nutrient solution EC is increased from 2.5 to 8.0 dS m-1. The cumulative yield per plant for the 7 weeks of harvest ranged between 4.0 kg to 6.5 kg per plant. However, there was no significant difference for fruit yield between EC levels, cultivars and greenhouse microclimates. The results showed that an increase of nutrient solution EC up to 4.5 dS m-1 improved the fruit quality regarding TSS level without reducing the total fruit yield. For effects of the nutrient solution EC on the yield, Adam (1991) reported that an EC about 8 dS m-1 decreased the tomato yield from 4% to 5% per 1 dS m-1, whereas the EC about 12 dS m-1 decreased the tomato yield from 6% to 8% per 1 dS m-1. The results indicated that a further increase of EC could decrease the fruit yield significantly. Water accounts for more than 90% of the total weight of ripe tomato fruit. Therefore, the water uptake of fruits is very important to both fruit quality and yield. In previous research, it was shown that the higher nutrient solution EC increased the phloem sap concentration and the ratio of phloem water to xylem water (Ho et al., 1987). In this experiment, the EC level up to 4.5 dS m-1 increased the TSS of tomato fruit with no significant reduction of yield. The transpiration rate of tomato plants also showed no significant difference under increased nutrient solution EC. In addition, little or no incidence of blossom-end rot was observed during the experimental period, which is related to calcium deficiency cause by the reduced amount of water flow to the fruit by xylem. All these data supported that the water uptake in tomato fruit grown under an increased nutrient solution EC of 4.5 dS m-1 was not greatly reduced. Increasing nutrient solution EC to a moderate level 80 might contribute to the increased concentration in phloem sap and thus improve the total soluble solid in fruit without yield reduction. The relatively higher VPD and PAR inside the greenhouse increased the TSS of most cultivars tested in the experiment. It suggested that fruit quality could be enhanced by controlling the aerial environments in addition to nutrient solution EC. The higher VPD could also provide a water stress to the plants (Grange and Hand, 1987); therefore this method is effective for enhancing the TSS of fruits. Lycopene concentration of fruits as affected by nutrient solution EC Nutrient solution EC had a significant effect on lycopene concentration of tomato fruits. For all cultivars, the lycopene concentration increased significantly for fruits grown under high nutrient EC; however, greenhouse microclimate had no effect (Table 1). The increase of lycopene concentration for all cultivars was between 34-85 %, ranging between 31.4 to 73.7 mg kg-1 FW (Fig. 2). The reported fresh tomato quantified lycopene concentration is between 31 to 77 mg kg-1 FW from several cultivars quantified by a reversed-phase HPLC (Nguyen and Schwartz, 1999). Nutrient solution EC manipulation is a technique to grow sweet tomato fruits. Our experiment showed that the lycopene concentrations could be also enhanced by increasing the nutrient solution EC in the experiment. Enhancing lycopene concentration in the fruits has been a major interest in plant breeding using both conventional method and genetic engineering; however, there is little research to increase lycopene levels by plant production management as far as we know. The increase of VPD had no effect on enhancing the fruit 81 color (Leonardi et al., 2000). It indicates that the VPD has no effect on lycopene concentration in tomato fruit since lycopene is the major pigment in the ripening tomato fruit. This may also indicate that lycopene enhancement observed in high EC treatments was not associated with altered water balance (less water) of the fruits, but with salinity level induced under high EC and related metabolisms in the fruit, which remained unclear in the present experiment. Lycopene is a major carotenoid present in the human diet, in which tomato and tomato products are the predominant sources. It is an effective antioxidant, twice as effective as -carotene, associated with reducing the risk of cancer and cardiovascular disease (Gerster, 1997; Stahl and Sies, 1997; Giovanucci, 1999). Lycopene can not be synthesized de novo in the human body and must be acquired from the diet. The lycopene-enriched tomato has an important nutritional value. Conclusions From the overall results, a crop management technique by nutrient solution EC manipulation is a potential method to grow high quality tomatoes rich in sugar and lycopene. The results showed that the fruit quality can be significantly enhanced when plants were grown under moderate water stress conditions, in terms of sugar and lycopene in the fruit with no significant yield loss. The greenhouse microclimate could influence the TSS of fruit but not lycopene. The enhancement of lycopene and TSS contribute to the overall improvement of tomato quality, either in nutritional values or flavor. 82 AKNOWLEDGEMENTS CEAC Paper #P-125929-14-04. Support provided by the Controlled Environment Agriculture Center (CEAC), College of Agriculture and Life Sciences, The University of Arizona, Tucson, AZ. Authors would like to thank Merle Jensen, Pat Rorabaugh, and Mark Kroggel for the assistance and support to conduct the analyses presented in this paper. 83 Literature Cited Adams P. 1991. Effects of increasing the salinity of the nutrients solution with major nutrients or sodium chloride on the yield, quality and composition of tomato grown in rockwool. J. Hort. Sci., 66:201-207. Cornish, P.S., 1992. Use of high electrical conductivity of nutrients solution to improve the quality of salad tomatoes grown in hydroponic culture. J. Expt. Agr. 32:513-520. Cuartero, J. and Rafael, F.M. 1999. Tomato and salinity. Scientia Hort., 78: 83-125. Fish, W.W., Perkins-Veaziea, P. and Collins, J.K. 2002. A Quantitative Assay for Lycopene That Utilizes Reduced Volumes of Organic Solvent. J. Food Composition and Analysis, 15:309-317. Fraser, P.D., Truesdale, M.R., Bird, C.R., Schuch, W. and Bramley, P. M. 1994. Carotenoid biosynthesis during tomato fruit development. Plant Physiol. 105:405-413. Gerster, H. 1997. The potential role of lycopene for human health. J. Am. Coll. Nutr. 16:109-126. Giovannucci, E. 1999. Tomatoes, tomato-based products, lycopene, and prostate cancer: review of the epidemiologic literatures. J. Natl Cancer Inst, 91:317-331. Grange, R.I., and Hand, D.W. 1987. A review of the effects of atmospheric humidity on the growth of horticultural crops. J. Hort. Sci. 62:125-134. Ho, L.C., Grange, R.I. and Picken A.J. 1987. An analysis of the accumulation of water and dry matter in tomato fruit. Plant, Cell and Environment, 10:157-162. Leonardi, C., Soraya, G.. and Nadia, B. 2000. High vapour pressure deficit influences growth, transpiration and quality of tomato fruits. Scientia Hort., 84:285-296. Lin, W.C. and Glass, A.D.M. 1999. The effects of NaCl addition and macronutrient concentration on fruit quality and flavor volatiles of greenhouse tomatoes. Acta Hort. 481:487-491. Mitchell, J.P., Shennan, C. and Grattan, S.R. 1991. Developmental changes in tomato fruit composition in response to water deficit and salinity. Physiol Planta. 83:177-185. Nguyen, M.L. and Schwartz, S.J. 1999. Lycopene: Chemical and biological properties. Food Technology, 53: 38-45. 84 Romero-Aranda, R., Soria, T. and Cuartero, J. 2002. Greenhouse mist inproves yield of tomato plants grown under saline conditions. J. Amer. Soc. Hort. Sci. 127:644-648. Schwarz D., Klaring, H.P., Van Iersel, M.W. and Ingram, I.T. 2002. Growth and photosynthetic response of tomato to nutrient solution concentration at two light levels. J. Amer. Soc. Hort. Sci. 127:984-990. Stahl, W., Sies, H. 1996. Lycopene: a biologically important carotenoid for humans? Arch. Biochem. Biophy. 336:1-9. Stevens, M.A., Kader, A.A. and Albright-Houlton, M. 1977. Intercultivar variation in composition of locular and pericarp portions of fresh market tomatoes. J. Amer. Soc. Hort. Sci. 102:689-692. Xu, H.L., Gauthier, L. and Gosselin, A. 1995. Effects of fertigation management on growth and photosynthesis of tomato plant grown in peat, rockwool and NFT. Scientia Hort. 63:11-20. Young, T.E., Juvik, J.A. and Sullivan, J.G. 1993. Accumulation of the components of total solids in ripening fruits of tomato. J. Amer. Soc. Hort. Sci. 118:286-292. 85 Table1. Main effects of EC, cultivar and plant microclimate (East and West) in the greenhouse on the total soluble solid concentration (TSS, %Brix at 20oC) of fruits. The TSS was the average of 7 weeks of harvest; the first harvest was on 4th week after start of EC treatment (approximately 2.6 and 4.5 dS m-1 for low and high EC, respectively). Factor TSS (%Brix) Nutrient solution EC High EC 5.8 Low EC 4.9 ANOVA * Cultivar Blitz 5.2c Mariachi 5.6a Quest 5.4b Rapsodie 5.2c ANOVA * Greenhouse microclimates East 5.6 West 5.2 ANOVA * Significant differences were determined by ANOVA at P=0.05. Means with the same letters are not significantly different according to a Tukey HSD test at P=0.05. 86 9 TSS (%) 8 7 6 5 4 MARIACHI BLITZ 3 9 1 2 3 4 5 6 7 1 2 3 5 6 7 LOW EC at EAST HIGH EC at EAST LOW EC at WEST HIGH EC at WEST 8 TSS (%) 4 7 6 5 4 QUEST RAPSODIE 3 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Weeks of harvest Weeks of harvest Lycopene concentration (mg/kg) Fig. 1. Weekly changes in the total soluble solid concentrations (TSS, % Brix at 20oC) of tomato fruits of four cultivars grown in the greenhouse as affected by nutrient solution EC (2.6 and 4.5 dS m-1) and plant microenvironments of different locations (west and east sides) inside the greenhouse. The data were obtained from 7 weeks harvests with the first harvest on 4th week after start of EC treatment. Means are shown with standard deviations. 80 a HIGH EC LOW EC 60 b,c b,c b,c,d d,e 40 d,e e e 20 0 Blitz Mariachi Quest Rapsodie Fig. 2. The lycopene concentration (mg/kg FW) of fruits of four cultivars grown in the greenhouse under a high nutrient solution EC (approximately 4.5 dS m-1) treatment. The data were obtained from the 10th week’s harvest after start of EC treatment. Means with the same letters are not significantly different according to a Tukey HSD test at P=0.05. 87 APPENDIX C: EFFECTS OF HIGH ELECTRICAL CONDUCTIVITY OF NUTRIENT SOLUTION AND ITS APPLICATION TIMING ON LYCOPENE, CHLOROPHYLL AND SUGAR CONCENTRATION OF HYDROPONIC TOMATO 88 Effects of High Electrical Conductivity of Nutrient Solution and Its Application Timing on Lycopene, Chlorophyll and Sugar Concentrations of Hydroponic Tomato Min Wu* and Chieri Kubota Department of Plant Sciences, The University of Arizona, Tucson, AZ 85721 This paper includes results funded by the Controlled Environment Agriculture Program at the University of Arizona. *To whom reprints should be addressed. Email address: [email protected]. 89 Effects of High Electrical Conductivity of Nutrient Solution and Its Application Timing on Lycopene, Chlorophyll and Sugar Concentrations of Hydroponic Tomato Additional index words. Brix, controlled environment, greenhouse, fructose, glucose, Lycopersicon esculentum, total soluble solids Abstract. Tomato (cv. Durinta) plants were grown hydroponically under two EC (2.3 and 4.5 dS m-1) nutrient solutions inside a greenhouse. The application of high EC treatment (4.5 dS m-1) was initiated either immediately after anthesis (high EC treatment) or four weeks after anthesis (delayed high EC treatment). Fruits were harvested weekly beginning two weeks after anthesis, until all fruits reached the red stage (8 weeks after anthesis). Lycopene, chlorophyll, sugar and total soluble solids (TSS) concentrations of fruits were measured every week for all harvested tomatoes from the different ripeness stage. The results showed that lycopene concentration, fructose and glucose concentrations and TSS of red ripe tomato fruits were enhanced by both high EC and delayed high EC treatments compared to low EC treatment. The lycopene concentration of red ripen tomato fruits in the high EC and the delayed high EC treatments showed an increase of 35 % compared to those grown in the low EC treatment; however, there was no significant difference in the lycopene concentration between the high EC and delayed high EC treatments. TSS of red ripe tomato fruits grown in the high EC treatment was greater than those grown in the delayed high EC treatment. Weekly change in lycopene concentration indicated that lycopene synthesis was enhanced by the high EC treatment, regardless of the application timing. Regardless of EC treatment, chlorophyll concentration in fruit declined linearly during fruit development and ripening 90 and reached non-detectable levels 7 weeks after anthesis. Our results indicated that: 1) accumulation of sugars and TSS in fruit was due to reduced water flux to the fruit under high EC, and 2) lycopene synthesis was driven by, but chlorophyll degradation was independent from, the osmotic and/or salt stress caused by the high EC. 91 Over the past decade, approximately 30% of Americans have expressed a significant interest in diet and nutrition to improve their health and are actively making food choices to improve their health (ADA data, 2002). Along with such a trend, the nutritional value of tomato, such as high lycopene, has been emphasized as a marketing strategy in addition to overall quality and flavor. Increasing daily consumption of fruits and vegetables has been a major public health focus for many years with minimal success. Given a general resistance of Americans to increase daily fruit and vegetable intake to recommended daily consumption levels, it seems opportunistic to develop fresh produce containing a greater concentration of phytochemicals to improve health (Kubota et al., 2006). Manipulation of target compounds in plants such as phytochemicals and antioxidants by controlling environmental conditions has been recognized as a research area attracting applied scientists’ interest (e.g., Afreen et al., 2006; Kubota et al., 2006; Zobayed et al., 2006). Lycopene is a powerful antioxidant, which can prevent the initiation or propagation of oxidizing chain reaction (Di Mascio et al., 1989; Nguyen and Schwartz, 1999; Riso et al., 1999). Lycopene has been reported to have important roles to prevent disease and promote health in humans, usually associated with reducing the risk of cancer and cardiovascular disease (Gerster, 1997; Stahl and Sies, 1997; Giovanucci, 1999). Enhancing lycopene concentration in tomato fruit by manipulating plant growth environments has been reported. For example, lycopene concentration in tomato fruit was reportedly altered by light quantity and intensity (Alba et al., 2000; Dumas et al., 2003), air temperature (Baqar and Lee, 1978) and level of salinity in the nutrient solution (De Pascale et al., 2001). Our previous research showed that the lycopene concentration in tomato fruit was enhanced when plants were 92 grown hydroponically using a nutrient solution with a high electrical conductivity (EC) of 4.8 dS m-1 compared to those grown under the standard low EC of 2.4 dS m-1 (Wu et al., 2004). The increase of lycopene observed for five cultivars tested under high EC was 34-85%, compared to the low EC, while increase of total soluble solids was only 12-22%, suggesting that lycopene increase might be due to a plant stress response to osmotic and/or salt stress, rather than the result of concentration caused by reduced water content of the fruit. However, the exact mechanism of enhancing lycopene concentration under high EC of nutrient solution remains unknown. Tomato has six ripeness stages characterized by fruit color development: green, breakers, turning, pink, light red and red (USDA, 1975). Ripening of tomato fruit is accompanied by the chlorophyll degradation and lycopene synthesis, as chloroplasts are converted into chromoplasts (Rhodes, 1980; Fraser et al., 1994). As soon as lycopene development becomes visually noticeable, the fruit is classified in the breakers stage. The concentrations of lycopene and chlorophyll determine the redness and greenness of tomato fruit, respectively. Little information is known on the degradation of chlorophyll and the accumulation of lycopene over time of fruit development and ripening, as affected by environmental conditions. Total soluble solid concentration (TSS, commonly measured as %Brix using a refractometer) is the most common flavor index associated directly with sugar and organic acid concentrations dissolved in the juice (Stevens et al., 1977; Young et al., 1993). Much research has been conducted for enhancing TSS under high EC (e.g., Adams, 1991; Mitchell 93 et al., 1991; Cornish, 1992; Lin and Glass, 1999; Wu et al., 2004). The enhancement of TSS under high EC is known as the result of restricted water transport to the fruit. Glucose and fructose are two major sugars, which account for about 95% of total sugars in tomato (Davies and Kempton, 1975; Haila et al., 1992; Young et al., 1993), whereas sucrose is detected in low or trace amounts in tomato fruit (Davies and Kempton, 1975; Haila et al., 1992). In addition, the sugar composition of tomato fruit is critical because fructose is almost twice as sweet as glucose. For example, transforming tomato with a gene that increases the fructose to glucose ratio is another approach for flavor improvement (Levin et al., 2000). Peterson et al. (1998) found that the fructose and glucose concentrations in tomato fruit were linearly correlated to the EC of nutrient solution at a range of 3 to 10 dS m-1. However there are relatively limited amounts of data presented regarding sugar concentration of tomato fruit under high EC conditions. A study on the changes in chlorophyll, lycopene, TSS, and sugar concentrations during tomato development and ripeness stages in response to high EC of the nutrient solution, initiated at different timings relative to fruit development, would help better understand the role of water and salinity stress on the degradation and synthesis of these compounds important for tomato fruit quality. In the present experiment, EC of the nutrient solution was increased at either immediately (0 weeks) or 4 weeks after anthesis, when all the fruits were still green. 94 Materials and Methods Plant material and growth conditions. Durinta tomato seeds (Western Seed Americas Inc., Westport, CT) were sown into 4×4 cm rockwool cubes (Grodan, Roermond, Netherlands) covered with a thin layer of vermiculite on April 17, 2004 and germinated under frequent mist in the greenhouse. The cultivar Durinta was selected because: 1) it is a cluster variety which has relatively simultaneous anthesis and fruit development within the same truss; and 2) it is a tomato cultivar well adopted and commercially produced in Arizona. After cotyledons were fully unfolded, the seedlings were sub-irrigated once a day with a half strength modified Hoagland nutrient solution (EC 1.2 dS m-1 and pH 6.0). When four true leaves emerged, the full strength of nutrient solution (EC 2.3 dS m-1 and pH 6.0) was applied by sub-irrigation. The basal composition of the nutrient solution contained the following elements in mg/ L: 189 N, 39 P, 341 K, 170 Ca, 48 Mg, 2 Fe, 0.6 Mn, 0.3 Zn, 0.05 Cu, 0.4 B, and 0.05 Mo. Five weeks after seeding, 240 seedlings were transplanted to 3.8-L pots filled with a commercial substrate (Sunshine Mix #1, Sun Gro Horticulture Inc., Bellevue, Wash.) and grown hydroponically with a high-wire system in a 9.8 m X 28.5 m north-south oriented single-span greenhouse at The University of Arizona Campus Agriculture Center (Tucson, AZ). The greenhouse had an arc roof covered with air-inflated double-polyethylene glazing. In order to maintain the optimum temperature, the greenhouse was shaded manually from 8 AM to 5 PM during sunny days. The height from the ground to the roof peak and gutter are 6.3 m and 4.0 m, respectively. A fan-and-pad evaporative cooling system was used to regulate 95 air temperature of the greenhouse. Day time temperature was set to 24°C and night time air temperature was set to 18°C. The nutrient solution was supplied using a drip irrigation system with a feeding rate of 100 mL per plant every 15 to 18 min (adjusted according to the transpiration demand) starting from 6 AM until 7 PM. This irrigation procedure is common in aggregate hydroponics so as to maintain 30-40 % efflux of the nutrient solution (Jensen, personal communication). Common greenhouse plant maintenance, including leaf pruning and removing side shoots was conducted on a weekly basis. Fruits were pruned to four per truss. Pests were controlled biologically, periodically introducing parasitic wasps (Encarsia formosa Gohan) for whitefly (Trialeurodes vaporariorum Westwood) and applying sulfur dust for russet mites (Aculops lycopersici Massee). Flowering trusses were vibrated mechanically for one second every other day using an electric devise to promote pollination. A high EC of 4.5 dS m-1 was achieved by adding 957 mg/L NaCl and 80 mg/L CaCl2 to the solution. Application of the high EC treatment (4.5 dS m-1) began immediately after anthesis (the high EC treatment) or 4 weeks after anthesis (the delayed high EC treatment). More specifically, after 11 weeks of transplanting, we selected well developed trusses with at least 4 fully opened flowers for each plant and marked them as experimental trusses. Immediately after, the irrigation system for one third of the tomato plants was switched to high EC nutrient solution (high EC treatment). After 4 weeks of starting the high EC treatment, the irrigation system for another one third of tomato plants was switched to the high EC nutrient solution (delayed high EC treatment). The other plants remained under the standard low EC (2.3 dS m-1) (low EC control). 96 Measurements. We harvested fruit trusses weekly from 2nd week up to 8th week after the start of high EC treatment. The fruit fresh weight and ripeness stage (based on the USDA fresh tomato fruit standard color chart) of each fruit were recorded immediately after each harvest. Then fruits were transported to the laboratory within 2 hours from the harvest and stored at -20 °C for 24 hours before the lycopene and TSS analysis. After pureeing the fresh tomato sample, TSS in juice was determined by a hand refractometer (N-20E, ATAGO USA, Inc., Bellevue, WA) and the measured values were corrected to a standard temperature condition of 20°C. Lycopene of tomato puree was extracted using solvent mixture of hexane/acetone/ethanol (2:1:1, v:v:v), and the lycopene concentrations in hexane layer were determined spectrophotometrically, according to the method described by Fish et al. (2002) with minor modifications. Chlorophyll of tomato fruit puree was extracted over night in N,N-Dimethylformamide, and the concentration was measured spectrophotometrically using a modified method of Moran (1982). Lycopene and chlorophyll concentrations were then converted to a dry weight basis using fruit dry/fresh weight ratio obtained each week. The remaining tomato juice of the puree was stored at -80°C for sugar analysis using HPLC (LC-10AD, Shimadzu, Kyoto, Japan). Pinnacle ⅡAmino column (250 x 4.6 mm, 5 µm particle; Restek, Bellefonte, PA) was used to separate glucose, fructose and sucrose of the sample, detected by refractive index detector (RID-10A, Shimadzu, Columbia, MD) with a temperature setting at 35 °C. The mobile phase was water/acetonitrile (25:75, v:v) with a flow rate of 1.0 mL/min. The sample injection amount was 20 µL. In the greenhouse, EC, pH, volume of the influx and efflux nutrient solutions were recorded daily throughout the experiment. The EC and pH were measured using a hand held 97 EC (HI 98312, Hanna Instruments, Inc., Woonsocket, RI) and pH meter (Hanna HI 98127, Hanna Instruments, Inc., Woonsocket, RI). Air temperature, relative humidity, and photosynthetic photon flux were measured using 0.5 mm Type-T thermocouples, a relative humidity sensor (CS500-L, Campbell Scientific, Inc., Logan, , UT) and a photosynthetic active radiation (PAR) sensor (QSO-A, Apogee Instruments Inc., Logan, UT) placed at the plant canopy level at the center of the greenhouse. All sensors were connected to a CR-10X datalogger (Campbell Scientific, Logan, UT) and the averages of every 15 min were recorded throughout the experiment. Atmospheric vapor pressure deficit (VPD) was computed from the air temperature and relative humidity. There were four blocks (replications) each consisting of 60 tomato plants. Three EC treatments were randomly distributed within each block (20 plants per treatment per each block). Each week, one experimental truss was randomly sampled from each EC treatment within the block (total of four plants sampled per week per treatment). The plant density was 3.0 plants per m2. Data obtained from the experiment were analyzed by JMP software (version 4.0.4, SAS Institute, Cary, NC). Treatment significances and significance among individual treatment levels were analyzed by analysis of variance (ANOVA) and Tukey HSD test, respectively. 98 Results and discussion Greenhouse microenvironment. Average day and night temperatures inside the greenhouse during the experiment were 26.2±1.8 and 20.1±3.7°C, respectively. Average VPD during the day was 0.7±0.3 kPa. Average daily photosynthetic active radiation (PAR) was 19.1±5.2 mol m-2 above the plant canopy. Nutrient solution EC and pH. The influx nutrient solution had 2.3±0.3 dS m-1 EC with 6.3±0.1 pH in the low EC treatment, and 4.5±0.5 dS m-1 EC with 6.4±0.2 pH in the high EC treatment. The efflux solution had 3.1±0.4 dS m-1 EC with 6.9±0.2 pH, and 5.8±0.6 dS m-1 EC with 6.9±0.1 pH for the low and high EC treatments, respectively. Average efflux percentage over the inflow nutrient solution was 48± 7% for the low EC treatment and 43±6 % for high EC treatment, which indicates little difference in plant transpiration rate. This agrees with our previous study (Wu and Kubota, 2006, unpulished), showing that the tomato leaf transpiration rate was not affected by the EC ranging from 2.4 to 4.8 dS m-2 during the reproductive growth stage. Fruit development and fruit fresh weight. The fruit fresh weight increased over time after anthesis (Fig. 1) and reached a maximum of 106 g per fruit after 7 weeks of anthesis. High EC treatment did not significantly affect the fresh weight of fruit, regardless of EC application timing in this study. Total soluble solids concentration and sugar concentrations in juice. Fruits harvested 2 to 5 weeks after anthesis were all in the green stage, while those harvested 8 weeks were all in the red stage. Fruits harvested after 6 and 7 weeks contained mixed stages of ripeness, and 99 therefore the data on TSS, sugar, lycopene, and chlorophyll concentrations are presented relative to the fruit ripeness stage or weeks after anthesis. Table 1 shows TSS and sugar concentrations in juice measured for tomatoes of different ripeness stages as affected by EC treatment. High EC treatments increased TSS of fruits at all stages except breakers and turning stages. For fruits at the red stage, TSS under the high EC treatment was 15 % and 7 % greater than that of the low EC treatment and the delayed high EC treatment, respectively. Weekly changes of TSS remained at almost the same level in the low EC treatment, while a gradual increase with increasing weeks after anthesis was observed for the high EC treatment (Fig. 2). In the delayed high EC treatment, within one week after the application of high EC, significant increase of TSS was observed. Eight weeks after the anthesis, TSS was the highest in the high EC treatment, followed by the delayed high EC treatment and the low EC treatment. This confirmed that increase in TSS was a cumulative effect over time of the fruit development and ripening, since the increased TSS is the result of restricted water flux to the fruits due to the osmotic effect of high EC. A similar effect of delayed high EC on TSS is reported by Sakamoto et al. (1999), where TSS was the highest in the high EC treatment applied at earlier stage. Sucrose was not detected in juice of tomato fruits throughout the fruit development and ripening. The high EC treatment enhanced both fructose and glucose concentrations in green and red ripe fruit juice, compared to the low EC treatment (Table 1). The delayed EC treatment enhanced the fructose and glucose concentrations in red ripe fruit juice, compared to the low EC treatment. Glucose concentration in red ripe fruit juice was the highest in the high EC treatment. Consequently, the total sugar (glucose and fructose) concentration in red 100 ripe tomato juice was the greatest in the high EC treatment (38.0 g/L), followed by the delayed EC treatment (33.2 g/L) and low EC treatment (24.2 g/L). However, EC of nutrient solution had no effects on the glucose and fructose concentrations in the fruit juice at the breakers/turning and pink/light red stages. Four weeks after anthesis, fruit juice in the high EC treatment showed a weak significance (P=0.08) of higher total sugar concentration than the low EC treatment (Fig. 3). After 5 weeks and onward, the high EC treatment increased sugar concentration in juice, compared to the low EC treatment. Significance of the delayed high EC treatment in enhancing sugar concentration was first observed 7 weeks after anthesis or 3 weeks after its initiation of the high EC. Since high EC treatment reportedly increased the titratable acidity of tomato fruit juice in addition to sugars (Sakamoto et al., 1999), the early response in TSS observed immediately after increase of EC in the present experiment, may be due to the increase of organic acids in the juice. Lycopene and chlorophyll concentrations. Lycopene was not detected in green tomato fruits (Table 2) and it increased 12-20 fold as fruits developed from breaker/turning stages to red stage. The high EC treatment and the delayed high EC treatment significantly increased the lycopene concentration of tomato fruit at pink, light red and red stages. At the red stage, the lycopene concentration of the high EC treatment and the delayed high EC treatment was 35 % greater than that of the low EC treatment. Similarly, lycopene concentration in tomato were reportedly increased by applying high EC of nutrient solution (De Pascale et al., 2001), where increase in lycopene concentration was 74% and 23%, when the nutrient solution EC was increased from 0.5 to 4.4 dS m-1 and from 2.3 to 4.4 dS m-1, respectively. Although, 101 lycopene concentration was not quantified, Botella et al. (2000) also found more intense red color in tomatoes grown at 60 mM NaCl than those at 0 mM NaCl in the nutrient solution. Lycopene concentration of the fruit started increasing faster in the high EC and the delayed high EC treatments than in the low EC treatment (Fig. 4). After 5th week, the tomato fruits grown under both high EC treatments had a consistently higher lycopene concentration than those under low EC treatment. The lycopene concentration in the delayed high EC treatment was at the same level as that in the high EC treatment throughout the experiment except week 8, suggesting that the application timing of high EC treatment did not affect the lycopene synthesis. Similarly, Sakamoto et al. (1999) reported the effect of high EC increased by adding NaCl, CaCl2 and KNO3 to the solution applied at different fruit ripeness stages: the early application of 8 dS m-1 EC at the green stage and the late application of two ECs at 5 dS m-1 or 8 dS m-1 at the breaker stage equally increased the lycopene concentration in tomato fruit, compared to their low EC control (2.4 dS m-1). These results, together with our finding, indicate that the ostmotic stress and/or salt stress caused by the high EC accelerated the synthesis of lycopene in the fruit. Tomato is known as a climacteric fruit and the fruit ripening process begins with an increase in respiration and ethylene (C2H4) synthesis. Therefore, slowing or inhibition of tomato fruit ripening was observed in ethylene-suppressed transgenic plants (Oeller et al., 1991; Theologis et al., 1993). Application of exogenous ethylene triggers ripening tomato fruits and the technique has been applied commercially to green-harvested tomatoes from the open field. Development of red color (primarily contributed to the presence of lycopene) in fruit begins after this initial increase in ethylene. The lycopene concentration increases 102 rapidly during this maturation process. High Na and Cl in the nutrient solution, and thereby high EC nutrient solution, reportedly induced ethylene emission across several tomato cultivars (Mizrahi, 1982). Botella et al. (2000) showed that irrigation of tomato plants with 40 to 60 mM NaCl increases red color, ethylene production and total ACC (1-aminocyclopropane-1-carboxylic acid). This suggests that ethylene production induced by osmotic and/or salt stress enhanced the concentration of lycopene in the tomatoes. Thus, under osmotic and salt stress, tomatoes mature earlier and accumulate more lycopene during the time before harvest. Our previous study showed that the light compensation point of tomato leaves was increased under high EC treatment, which means that tomato plants had a higher respiration rate under high EC treatment (Wu and Kubota, unpublished). This may suggest that overall enhancement of metabolisms under high EC treatment resulted in the increased ethylene production and lycopene in the fruit ripening process. Although the exact biological mechanisms that contribute to the enhancement of lycopene concentrations under high EC stress are not known, the above evidences suggest that ethylene synthesis triggered by osmotic and/or salt stress could be central to the increase in lycopene deposition within the flesh of the tomatoes. The chlorophyll in fruit was being degraded during fruit development (Fig 5). During tomato fruit ripening, there is a dramatic change in plastid structure and function. Chlorophyll concentration starts to decline in the breaker stage and then disappear or declines to a trace level in the ripe fruit. The degradation of chlorophyll was shown during the transformation of fully differentiated chloroplasts into chromoplasts by electron micrographic analysis (Thelander et al., 1986). 103 There were no significant effects of EC on fruit chlorophyll concentration in all developmental stages (Table 2). Increase in leaf chlorophyll concentration or dark color of tomato leaves has been reported for tomato plants grown under high EC. For example, when EC of the nutrient solution was increased from 1.8-2.0 dS m-1 to about 4.0-5.4 and 8.1-9.2 dS m-1, chlorophyll concentration per unit of leaf area increased by 14 % (Romero-Aranda et al., 2001). Since the ripe tomato fruit contains trace amount of chlorophyll concentration, limited information is available for chlorophyll concentration in tomato fruits under high EC. Sakamoto et al. (1999) found that chlorophyll a concentration in fresh fruit was enhanced by applying nutrient solution with 5.0 dS m-1 or 8.0 dS m-1 EC, when applied at the green stage, compared to control at 2.4 dS m-1. The same EC treatments at the breakers stage did not affect the chlorophyll a concentration in fresh fruit. However, the chlorophyll b concentration in fresh fruit was not affected by both EC treatments, regardless of application timing. Overall, little is known about the regulation of chlorophyll degradation during tomato ripening. It is not clear whether the decrease of photosynthetic protein synthesis or increase in degradative enzymes which are involved in the process causes the breakdown of chloroplast structure. The results of the present experiment showed that the degradation of chlorophyll was not affected by the high EC treatment, showing that the degradation process was independent from lycopene synthesis. In fact, the chloroplasts senescence, chlorophyll degradation and chromoplasts development are independent pathways during tomato ripening. Chlorophyll degradation is not a prerequisite for chromoplasts formation. For the tomato gf mutant (chlorophyll degradation defective mutant), in which the chlorophyll 104 synthesis proceeds throughout fruit ripening, the chromoplasts can still be formed (Cheung et al., 1993). Conclusions Our results showed that tomato plants grown with nutrient solution containing high EC of 4.5 dS m-1 can mature earlier and accumulate more lycopene, TSS and sugars than low EC of 2.3 dS m-1. The timing of high EC application did not affect the lycopene concentration in tomato fruits but delaying application lowered the TSS and sugar concentrations. Fruit size increase and chlorophyll degradation were not affected by the EC treatments. Although further investigation is necessary to find the mechanisms of enhanced lycopene synthesis under high EC of the nutrient solution, the reduced water flux to the fruits under the high EC treatment was indicated as the major cause of increase in TSS, while osmotic and/or salt stress at the time of ripening contributed to enhanced lycopene synthesis. Examining a much shorter application of high EC during ripening may be of practical interest for enhancing lycopene in fruit without affecting overall growth or yield, and should be investigated together with biochemical and enzymatic analyses for revealing the mechanism. 105 Literature Cited Adams, P. 1991. Effects of increasing the salinity of the nutrients solution with major nutrients or sodium chloride on the yield, quality and composition of tomato grown in rockwool. J. Hort. Sci., 66:201-207. Afreen, F., S.M.A. Zobayed, and T. Kozai. 2006. 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Use of a tomato mutant constructed with reverse genetics to study fruit ripening, a complex developmental process. Developmental Genetics. 14, 282–295. USDA. 1976. United State standards for grade of fresh tomatoes. US Dept. Agric., Mktg., Serv., Washington DC, PP10. Wu, M., J.S. Buck, and C. Kubota. 2004. Effects of nutrient solution EC, plant microclimate and cultivars on fruit quality and yield of hydroponic tomatoes. Acta Hort, 659: 541-547. 108 Young, T.E., J.A. Juvik, and J.G. Sullivan, 1993. Accumulation of the components of total solids in ripening fruits of tomato. J. Amer. Soc. Hort. Sci. 118:286-292. Zobayed, S.M.A., F. Afreen, and T. Kozai. 2006. Phytochemical and physiological changes in the leaves of St. John’s wort plants under a water stress condition. Environ. Experiment. Bot. (in press). 109 Table 1. Effects of EC, application timing of EC on the total soluble solid concentration (TSS, %Brix at 20oC), fructose and glucose concentrations of tomato fruits at different repeness stages. The six fruit ripeness stages characterized by color development, which include green (G), breakers (B), turning (T), pink (P), light red (LR) and red (R) (USDA, 1975). Low and high EC were 2.3 and 4.5 dS m-1, respectively. The high EC and the delayed high EC treatments were applied immediately after anthesis and 4 weeks after anthesis, respectively. TSS Treatment G High EC 5.6a Delayed high EC 5.4b Low EC 5.3b ANOVA (P=0.05) * B&T 6.0 5.4 5.0 NS P&LR R 5.9a 6.1a 5.6b 5.7b 5.2c 5.3c * * Fructose concentration (g/L) G B&T P&LR R 15.8a 13.2 13.6 18.8a 13.7ab 12.9 12.0 16.1b 13.5b 12.8 11.7 12.1c * NS NS * Glucose concentration (g/L) G B&T P&LR R 16.5a 12.9 13.8 19.2a 13.9b 12.6 12.1 17.1a 13.8b 13.0 12.4 12.1b * NS NS * Means within the column followed by the same letters are not significantly different according to a Tukey HSD test at P=0.05. 110 Table 2. Effects of EC, application timing of EC on lycopene and chlorophyll concentration of tomato fruits at different ripeness stages. The six fruit ripeness stages characterized by color development, which include green (G), breakers (B), turning (T), pink (P), light red (LR) and red (R) (USDA, 1975). Low and high EC were 2.3 and 4.5 dS m-1, respectively. The high EC and the delayed high EC treatments were applied immediately after anthesis and 4 weeks after anthesis, respectively. Treatment High EC Delayed high EC Low EC ANOVA (P=0.05) G ND ND ND — Lycopene conc. (mg/g DW) B&T P&LR R 0.07 0.39a 1.39a 0.10 0.32b 1.29a 0.08 0.25c 0.99b NS * * G 0.47 0.47 0.44 NS Chlorophyll conc. (mg/g DW) B&T P&LR 0.11 ND 0.06 ND 0.10 ND NS — Means with the same letters are not significantly different according to a Tukey HSD test at P=0.05. ND: not detected. R ND ND ND — 111 160 Fruit fresh weight (g) 140 120 100 80 60 40 20 0 2 3 4 5 6 7 8 Weeks after anthesis Fig 1. Weekly change of fruit fresh weight (g per fruit). Average weight of three treatments of high EC, delayed high EC and low EC are shown with S.D. Total soluble solids (Brix % at 20 oC) 112 6.6 6.4 High EC Delayed high EC Low EC 6.2 a 6.0 a a b a 5.8 b 5.6 NS b 5.4 a a a b b 4 5 5.2 b b c c 5.0 4.8 2 3 6 7 8 Weeks after anthesis Fig 2. Effect of high EC treatment and its application timing on TSS of tomato fruit juice during fruit development. Low and high EC were 2.3 and 4.5 dS m-1. The high EC and the delayed high EC treatments were applied immediately after anthesis and 4 weeks after anthesis, respectively. Means with the same letters for each week are not significantly different according to a Tukey HSD test at P=0.05, followed by ANOVA. Total sugar concentrations in fruit juice (g L-1) 113 50 High EC Delayed high EC Low EC 40 a a a 30 ab NS NS P=0.08 NS 20 a a b NS P=0.06 b b 10 0 2 3 4 5 6 7 8 Weeks after anthesis Fig 3. Effect of high EC treatment and its application timing on total sugar (glucose and fructose) concentrations in tomato fruit juice during fruit development. Low EC and high EC treatments 2.3 dS m-1 and 4.5 dS m-1. The high EC and the delayed high EC treatments were applied immediately after anthesis and 4 weeks after anthesis, respectively. Means with the same letters for each week are not significantly different according to a Tukey HSD test at P=0.05, followed by ANOVA. Lycopene concentration (mg g-1 dry weight) 114 1.6 a 1.4 High EC Delayed high EC Low EC 1.2 a b a 1.0 c 0.8 a b 0.6 ab 0.4 0.2 b 0.0 2 3 4 5 6 7 8 Weeks after anthesis Fig 4. Effect of high EC treatment and its application timing on lycopene concentration of tomato fruits during fruit development. Low and high EC were 2.3 and 4.5 dS m-1. The high EC and the delayed high EC treatments were applied immediately after anthesis and 4 weeks after anthesis, respectively. Means with the same letters for each week are not significantly different according to a Tukey HSD test at P=0.05, followed by ANOVA. Chlorophyll concentration (mg g-1 dry weight) 115 1.0 0.8 0.6 0.4 0.2 0.0 2 3 4 5 6 7 Weeks after anthesis Fig 5. Weekly change of chlorophyll concentration of tomato fruits during fruit development. Average of three treatments of high EC, delayed high EC and low EC are shown with S.D.. 8
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