Characterization of watermelon fruitlet development 1

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1 Characterization of watermelon fruitlet development 1 A. Salman-Minkov *, and T. Trebitsh Department of Life sciences Ben-Gurion University of the Negev, P.O.B 653, Beer-Sheva 84105, Israel * Corresponding author salmanay@bgu.ac.il Keywords: 1-Aminocyclopropane-1-carcoxylate synthase (ACS), aminoethoxyvinylglycine (AVG), Citrullus lanatus Abstract Watermelon (Citrullus lanatus var. lanatus) fruit produce low amounts of ethylene during ripening and is therefore considered as a non-climacteric fruit. Here we examined the involvement of ethylene in early stages of fruit development. Ethylene production was high in ovary and young fruitlets but decreased at later stages of development. Increased fruit set was observed in plants treated with ethylene biosynthesis inhibitor. To identify genes involved in the process we isolated genes encoding for the rate-limiting enzyme in ethylene biosynthesis pathway, 1- aminocyclopropane-1-carcoxylate synthase (ACC synthase, ACS). The expression pattern during early fruit development of watermelon ACS genes (CitACS) was analyzed. High CitACS1 transcript level was detected in the early stages of fruit set but it decreased at later stages of fruit development. CitACS1 transcript accumulation was in direct correlation with ethylene evolution at the corresponding stages. INTRODUCTION In watermelon, fruit set per plant is low and only one to two fruits develop. In order to improve this important crop yield we need to extend our knowledge of watermelon fruit set and development. Fruits can be divided into two groups, climacteric or non- climacteric. Climacteric fruits are characterized by an increased respiration rate, along with dramatic increase of ethylene production at the onset of ripening. Ethylene is needed for climacteric ripening-related processes (Giovannoni 2004). In past few years, studies in non-climacteric fruits such as orange (Citrus sinensis) and strawberry (Fragaria x ananasa), imply a role for ethylene in nonclimacteric fruits development and ripening. Orange fruitlets evolve high ethylene levels, in contrary to the mature fruit (Katz et al. 2004). In strawberry fruits ethylene related genes are expressed concomitantly with ethylene production (Trainotti et al. 2005). Previous studies in watermelon indicate that it is a non-climacteric fruit. It was shown that respiratory rate of preripe and ripe fruits is low and ethylene treatments do not induce autocatalytic ethylene production (Elkashif et al. 1989). In a recent study, ethylene related genes were detected in an EST library of watermelon flesh (Levi et al. 2006). This finding suggests ethylene involvement in watermelon fruit 1 Cucurbitaceae 2008, Proceedings of the IX th EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May th,

2 development. The goal of this work was to investigate the role of ethylene during early stages of watermelon fruitlet development. MATERIALS AND METHODS Plant material and treatments Seeds of cultivated watermelon line (C. lanatus var. lanatus Sugar Baby ) were obtained from Hazera Genetics Ltd. (Berurim M.P. Shikmim, Israel). Plants were grown in 3.5 L pots at 27±4 C under a photoperiod of 16 hours daylight. Plants were sprayed with or without a solution containing 50 ppm aminoethoxyvinylglycine (AVG, 'Retain') and % ABG-7044 (Valent Biosciences CO., Libertyville, USA). The plants were treated three times at 2 days intervals starting at the stage of two fully expanded leaves followed by weekly treatment. The plants floral sex phenotype and fruit set were measured up to 30 nodes along the main shoot. CitACS sequences isolation Genes encoding for ACS were isolated from genomic DNA by PCR, using degenerate primers designed against conserved regions in amino acid sequences of known ACS as previously (Trebitsh et al. 1997). Ethylene measurement Fruitlets at early stages of development were collected from C. lanatus var. lanatus Sugar Baby. Each fruitlet was sealed in a separate jar for six hours. Two ml air sample was taken out with a syringe and injected into a gas chromatograph (Varian 3300) with a flame ionization detector (FID) at 120 C. RNA blot hybridization analysis Total RNA was extracted from fruitlets by EZ-RNA extraction kit according to the manufacturer s instructions (Biological Industries, Beit Haemek, Israel). RNA blot hybridization and CitACS1 probe preparation were performed as previously (Trebitsh et al. 1997; Barak and Trebitsh 2007). RESULTS AND DISCUSSION Ethylene production and CitACS1 expression in fruitlets To study the role of ethylene during early stages of watermelon fruitlet development we first measured ethylene evolution. Fruitlets from different developmental stages were collected and ethylene evolution was determined. Ethylene production was highest in the youngest fruitlets, cm in diameter, and decreased thereafter as the fruitlet increased in size (Fig. 1). A ten-fold decrease in ethylene production was observed starting from fruitlet of 4.5 cm and onwards. These results are in agreement with the high ethylene production observed in young orange fruits (Katz et al. 2004). In young fruitlets ethylene may control the number of fruits per plant, by increasing fruitlets abscission. On the other hand, it might be required in early developmental processes that lead to fruit set. ACC synthase (ACS) is considered as the key regulatory enzyme in ethylene biosynthesis pathway. We isolated genes encoding for ACS in watermelon in order to analyze their expression in fruitlets. Generally in plants, ACS genes form a gene 610

3 family whose members are differentially expressed in tissues, developmental stages and in response to environmental changes. We isolated four ACS encoding genes from watermelon, designated as CitACS1-4 (GeneBank accession numbers EF EF154458, respectively). Of the four genes only CitACS1 transcript was detected in fruitlets. In young fruitlets CitACS1 expression is high and then decreases as the fruitlet develops. In 3 cm fruitlets CitACS1 expression is bearly detectable (Fig. 1). CitACS1 transcript accumulation is in correlation with the ethylene production levels. The results suggest that of the ACS genes in watermelon, CitACS1 is specifically involved in ethylene production in fruitlet development. CitACS1 expression pattern resembles that of CsACS1 from orange that was detected in young fruitlets and its expression correlated with ethylene production during early orange fruit development (Katz et al. 2004). Figure 1. Ethylene evolution and CitACS1 expression in watermelon young fruitlets. RNA blot hybridization analysis of CitACS1. A: Watermelon fruitlets were sealed in a jar for six hours and ethylene evolution was determined; B: Values are means ± SE; n=4. AVG foliar treatment increases fruit set It was previously shown that ethylene is involved in watermelon floral sex determination, possibly as a masculinazing hormone (Christopher and Loy 1982; Arora et al. 1985; McArdle 1990; Sugiyama 1998). Plant treatments with ethylene inhibitors increased the number of perfect flowers. Thus, perfect flowers developed on monoecious plants normally producing male and female flowers (Christopher and Loy 1982). We found that the number of perfect flowers increased in plants sprayed with an ethylene biosynthesis inhibitor (data not shown). In addition, fruit set was increased in treated plants, relatively to control, resulting on average 1.5 fruits per plant (Fig. 2). This could be the result of self-pollination of perfect flowers and/or the 611

4 result of reduced ethylene leading to reduced fruitlet abscission and increased number of surviving fruitlets. Further research is needed to elucidate the role of ethylene in watermelon fruit set and development. Figure 2. Effect of AVG on fruit set in the monoecious watermelon 'Sugar Baby'. Histogram shows the number of fruits developed per plant when treated with AVG (50 ppm) (right) or untreated (left). Values are means ± SE; n=10. White arrows mark fruits. Literature cited Arora SK, Pandita ML, Partap PS, Sidhu AS (1985) Effect of ethephon, gibberellic acid, and maleic hydrazide on vegetative growth, flowering, and fruiting of cucurbitaceous crops. J Amer Soc Hort Sci 110: Barak M, Trebitsh T (2007) A developmentally regulated GTP binding tyrosine phosphorylated protein A-like cdna in cucumber (Cucumis sativus L.). Plant Mol Biol 65: Christopher DA, Loy BJ (1982) Influence of foliarly applied growth regulators on sex expression in watermelon. J Amer Soc Hort Sci 107: Elkashif ME, Huber DJ, Brecht JK (1989) Respiration and ethylene production in harvested watermelon fruit: evidence for nonclimacteric respiratory behavior. J Am Soc Hort Sci 114: Giovannoni JJ (2004) Genetic regulation of fruit development and ripening. Plant Cell 16: S170-S

5 Katz E, Lagunes PM, Riov J, Weiss D, Goldschmidt EE (2004) Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in nonclimacteric citrus fruit. Planta 219: Levi A, Davis A, Hernandez A, Wechter P, Thimmapuram J, Trebitsh T, Tadmor Y, Katzir N, Portnoy V, King S (2006) Genes expressed during the development and ripening of watermelon fruit. Plant Cell Rep 25: McArdle RN (1990) Effect of growth regulator regimes and cultivar on femaleness and compactness of watermelon plants in the greenhouse. PGRSA Quart 18: Sugiyama K (1998) Varietal differences in female flower bearing ability and evaluation method in watermelon. Jarq-Japan Agric Res Quart 32: Trainotti L, Pavanello A, Casadoro G (2005) Different ethylene receptors show an increased expression during the ripening of strawberries: does such an increment imply a role for ethylene in the ripening of these non-climacteric fruits? J Exp Bot 56: Trebitsh T, Staub JE, O'Neill SD (1997) Identification of a 1-aminocyclopropane-1-carboxylic acid synthase gene linked to the Female (F) locus that enhances female sex expression in cucumber. Plant Physiol 113:

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