text; Electronic Dissertation The University of Arizona.

Transcription

1 Effect of Nutrient Solution Electrical Conductivity Levels on Lycopene Concentration, Sugar Composition and Concentration of Tomato (Lycopersicon esculentum Mill.) Item type Authors Publisher Rights text; Electronic Dissertation Wu, Min The University of Arizona. Copyright is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Downloaded 18-Sep :38:07 Link to item

2 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

3 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)

4 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

5 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.

6 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.

7 6 TABLE OF CONTENTS LIST OF FIGURES... 8 ABSTRACT... 9 INTRODUCTION LITERATURE REVIEW PRESENT STUDY REFERENCES APPENDIX A: EFFECTS OF ELECTRICAL CONDUCTIVITY OF HYDROPONIC NUTRIENT SOLUTION ON LEAF GAS EXCHANGE OF FIVE GREENHOUSE TOMATO CULTIVARS Abstract Materials and Methods Results and discussion Conclusions Literature Cited APPENDIX B: EFFECTS OF NUTRIENT SOLUTION EC, PLANT MICROCLIMATE AND CULTIVARS ON FRUIT QUALITY AND YIELD OF HYDROPONIC TOMATOES Abstract Introduction Material and methods Results and discussion Conclusions APPENDIX C: EFFECTS OF HIGH ELECTRICAL CONDUCTIVITY OF NUTRIENT SOLUTION AND ITS APPLICATION TIMING ON LYCOPENE, CHLOROPHYLL AND SUGAR CONCENTRATION OF HYDROPONIC TOMATO... 87

8 7 TABLE OF CONTENTS-Continued Abstract Materials and Methods Results and discussion Conclusions

9 8 LIST OF FIGURES FIGURE 1: The isoprenoid biosynthetic pathway.24 FIGURE 2: Phytoene formation and desaturation reactions to form lycopene.25

10 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

11 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.

12 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

13 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

14 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

15 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.

16 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).

17 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

18 (De Pascale et al., 2001). The effect of environmental factors on lycopene concentration in tomato fruit is reviewed later in this chapter. 17 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.

19 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

20 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 20 o C) (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

21 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 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 Ca 2+ 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).

22 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

23 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

24 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) GGPP (geranylgeranyl pyrophosphate) Phytoene lycopene β- carotene

25 24 Fig1. The isoprenoid biosynthetic pathway (Bramley, 2002). [With permission of the Oxford University Press]

26 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), ζ-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

27 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-β-cyclase, a cyclase to convert lycopene to β-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

28 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 β-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.

29 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

30 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 o C (Leoni, 1992). Air temperature below 12 o C or above 32 o C 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 o C (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

31 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).

32 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 ds m -1 to about or 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.

33 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 (g l ) and transpiration rate (TR) by 28 % and 29 %, respectively, compared to low EC treatment. High and medium EC treatment

34 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 g l 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

35 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 20 o C) 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 CaCl 2 after the first fruit truss was set. In all cultivars, TSS, lycopene concentration of fruits increased by % and %, 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 Effects of nutrient solution EC, plant microclimate and cultivars on fruit quality and yield of hydroponic tomatoes. Acta Hort. 659:

36 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

37 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. Even though the mechanism of lycopene enhancement by increased EC of nutrient solution is still not clear, we learned that, not like soluble sugars, high EC effects on lycopene concentration was not dependent on the time of application during green fruit development. The three studies are relevant to each other and the latter study was designed based on the preliminary studies. From those three studies, we found that we can improve fruit quality including both nutritional value and flavor while sustaining plant growth and yield by manipulating EC of nutrient solution.

38 37 REFERENCES Adams, P 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: Adams, P., and L.C. Ho Effect of constant and fluctuating salinity on the yield, quality and calcium status of tomatoes. J. Hort. Sci.64: Adegoroye A.S. and P.A. Jolliffe Some inhibitory effects of radiation stress on tomato fruit ripening. J. Sci. Food Agric. 39: Alba, R., M.M. Cordonnier-Pratt, and L.H. Pratt Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato. Plant Physiol. 123: Baqar, M.R., and T.H. Lee Interaction of CPTA and high temperature on carotenoid systhesis in tomato fruit. Z. Planzenphysiol. 88: Bartley, G.E., P.V. Viitanen, I. Pecker, D. Chamovitz, J. Hirschberg, and P.A. Scolnik Molecular cloning and expression in photosynthetic bacteria of a soybean cdna coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci. 88: Binzel M.I., F.D. Hess, R.A. Bressa, and P.M. Hasegawa Intracellular compartmentation of ions in salt adapted tobacco cells. Plant Physiol. 86: Bolarin, M.C., M.T. Estan, M. Caro, R.Romero-Aranda, and J.Cuartero Relationship between tomato fruit growth and fruit osmotic potential under salinity. Plant Sci. 160: Bramley P.M Regulation of carotenoid formation during tomato fruit ripening and development. J. Exp. Bot. 53: Causse, M., V. Saliba-Colombani, L. Lecomte., P. Duffe, P. Rousselle, and M. Buret QTL analysis of fruit quality in fresh market tomato : a few chromosome regions control the variation of sensory and instrumental traits. J. Exp. Bot. 53: Cheung A.Y., T. McNellis, and B. Piekos Maintenance of chloroplast components during chromoplasts differentiation in the tomato mutant Green Flesh. Plant Physiol., 101:

39 38 Cook, R. and L. Calvin Greenhouse tomatoes change the dynamics of the North American fresh tomato industry. USDA Economic Research Report No. 2 ( Cornish, P.S Use of high electrical conductivity of nutrients solution to improve the quality of salad tomatoes grown in hydroponic culture. J. Expt. Agr. 32: Cuartero, J., and F.M. Rafael Tomato and salinity. Scientia Hort., 78: Davies, J.N., and G.E. Hobson The constituents of tomato fruit-the influence of environment, nutrition and genotype. Critical Rev. of Food Sci. and Nutrition. 15: Davies, J.N., and R.J. Kempton Changes in the individual sugars of tomato fruit during ripening. J. Sci. Food Agric. 26: De Pascale, S., A. Maggio, V. Fogliano, P. Ambrosino, and A. Ritieni Irrigation with saline water improves carotenoids content and antioxidant activity of tomato. J. Hort. Sci. Biotechnol. 76: Di Mascio, P., S. Kaiser, and H. Sies Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys, 274: Dumas, Y, M. Dadomo, G. Di Lucca, and P. Grolier Effects of environmental factors and agricultural techniques on antioxidant content of tomatoes. J. Sci. Food Agric. 83: Ehret D.L., and L.C. Ho Effects of osmotic potential in nutrient solution on diurnal growth of tomato fruit. J. Exp. Bot. 37, Erdei L., and E. Taleisnik Changes in water relation parameters under osmotic and salt stresses in maize and sorghum, Physiol. Plant. 89: Flores P., M.A., Botella, V. Martinez, and A. Cerda Response to salinity of tomato seedlings with a split-root system: nitrate uptake and reduction. J. Plant Nutrition, 25: Fraser P.D., J.W. Kiano, M.R. Truesdale, W. Schuch, and P.M.Bramley Phytoene synthase-2 enzymes activity in tomato does not contribute to carotenoid synthesis in ripening fruit. Plant Mol. Biol. 40, Fraser, P.D, and P.M.Bramley The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res. 43: