L-Citrulline Levels in Watermelon Cultigens Tested in Two Environments

Producers of fresh fruits and vegetables face increasing production costs and international market competition. Growers who can offer high-quality watermelons [Citrullus lanatus (Thumb.) Matsum. & Nakai] that are also highly nutritious will have better market opportunities. To accomplish that, germplasm must be identified that has enhanced phytonutrient levels. Surprisingly, there is little information on the genetics of nutritional quality in watermelon. The present study was performed on 56 watermelon cultivars, breeding lines, and PI accessions (hereafter collectively referred to as cultigens) to determine the importance of genotype and environmental effects on L-citrulline concentration in fruit, an amino acid that helps regulate blood pressure. Our results demonstrated that L-citrulline concentration was affected by environment and the amount of environmental effect varies among cultigens. The mean of fruit tested in Lane, OK, was 3.10 mg g fresh weight and in College Station, TX, it was 1.67 mg g fresh weight. All cultigens had a higher mean L-citrulline concentration when grown in Lane, OK, instead of College Station, TX. Additionally, the L-citrulline concentration varied considerably within cultigens; i.e., ‘Congo’ had a 1.26 to 7.21 mg g fresh sample deviation. The cultigen ‘AU-Jubilant’ had the most stable L-citrulline concentration (2.23 to 4.03 mg g fresh deviation) when tested from one location. Environment did not significantly increase within-genotype variation (average SE of 10 cultigens tested at each location was ± 35.3% for College Station, TX, and ± 32.9% for Lane, OK). L-citrulline concentration did not correlate with watermelon type (open-pollinated or F1 hybrid) or flesh color (red, orange, salmon yellow, or white). Differences among cultigens for L-citrulline were large (1.09 to 4.52 mg g fresh sample). The cultigens with the highest L-citrulline concentration were ‘Tom Watson’, PI 306364, and ‘Jubilee’. These could be used to develop cultivars having a high concentration of L-citrulline. Watermelon is the number two fresh vegetable crop in the world in terms of area harvested and total production (FAOSTAT, 2009). Recently, it has been identified as a healthy food because it is high in the carotenoid, lycopene. Although watermelon is the leading fruit and vegetable source of lycopene, it also contains other antioxidants and amino acids that have health-promoting activities. Fruits and vegetables contain many compounds that work synergistically. Boileau et al. (2003) indicated that lycopene alone is not responsible for reducing prostate cancer associated with increased tomato intake. Because tomatoes contain the glycoalkaloid tomatine, a demonstrated anticarcinogen, as well as several well-known phenolic compounds such as quercitin, this finding is not unexpected and demonstrates the importance of maintaining the other nutritional compounds in fruits and vegetables. Amino acids have well-established individual roles in disease prevention. Arginine, an essential amino acid, functions as one of the 20 building blocks of proteins and in free form as a physiological amino acid. L-citrulline is a physiological amino acid endogenous to most living systems. These amino acids are directly involved in clearing excess metabolic ammonia from the human body and are indirectly involved in cardiovascular function, immunostimulation, and protein metabolism (Curtis et al., 2005). Ingested arginine is cleared by hepatic cells, but L-citrulline is not and can serve as an arginine source in other parts of the body. Watermelon is rich in citrulline (Tedesco et al., 1984) but differences among cultivars have not been adequately studied nor have effects of production environments. Fish and Bruton (2010) reported that one cultivar grown at two locations had little variation in flesh concentration of L-citrulline. Tarazona-Dı́az et al. (2011) reported values on five lines (four of them triploid seedless cultivars) grown at one location with a mean citrulline concentration in watermelon flesh of 2.33 mg g; they also demonstrated that the seeded cultivar had the least L-citrulline concentration in flesh tissue. In an earlier report, 14 watermelon cultivars had 0.5 to 3.6 mg g fresh weight of citrulline with an average concentration of 2.4 mg g (Rimando and Perkins-Veazie, 2005). Those authors reported less L-citrulline in red-fleshed fruit than in yellow or orange, but only a small sample size (three fruit for each variety) was used, and because the fruit tested were grown at multiple locations, it is difficult to assess the genotype and environmental effect on L-citrulline concentration in those fruits. Liu et al. (2010) reported that greenhouse-grown watermelon fruit of nine induced autotriploid hybrids had higher Lcitrulline values than their diploid and induced autotetraploid parents. Fish and Bruton (2010) and Liu et al. (2010) reported that the concentration of L-citrulline was highest at peak ripeness. Humans can effectively absorb L-citrulline from watermelon, which also increases plasma arginine levels (Collins et al., 2007; Mandel et al., 2005). Recently, subjects consuming watermelon or synthetic citrulline as a drink, combined with exercise, had reduced arterial blood pressure compared with a placebo (Figueroa et al., 2011). The heightened importance of watermelon as a source of bioactive compounds such as L-citrulline highlights the need for a better understanding of the genetic control of this amino acid and other phytonutrients in watermelon. The current study expands on a preliminary trial by Davis et al. (2010) and was designed to measure effects of genotype and environment on L-citrulline accumulation in watermelon. A diverse collection of watermelon germplasm was screened to identify cultigens having a high concentration of L-citrulline that was stable over environments. Materials and Methods Plant material. Fifty-six (Table 1) watermelon cultigens (open-pollinated and hybrids) grown in Lane, OK (Bernow fine-loamy, siliceous, thermic, Glossic Paleudalf soil) were arranged in a randomized complete block design with four replications. Plant management Received for publication 1 Aug. 2011. Accepted for publication 8 Sept. 2011. This project was partially funded by the National Watermelon Promotion Board and the National Watermelon Association. We thank Amy Helms and Cody Sheffield for technical help. To whom reprint requests should be addressed; e-mail [email protected]. 1572 HORTSCIENCE VOL. 46(12) DECEMBER 2011 provided was as outlined in Motes and Cuperus (1995). Plants were transplanted onto bare, raised beds, were irrigated as needed (at the first signs of water stress), and fertigated bimonthly. To assess environmental effects, 10 watermelon cultigens (Table 2) were also grown at College Station, TX [Zack very fine sandy loam (fine, smectitic, thermic Udertic Paleustalfs)] using the same planting scheme. Pre-plant fertilizer was added according to soil test results and plant care provided as outlined in Masabni et al. (2011). The plants were transplanted on 21 May onto black plastic mulched beds that were drip-irrigated using a timer to irrigate daily based on evapotranspiration rates. All plants were grown during the summer of 2010 and were harvested throughout the season. Flesh from all fruit harvested from Lane, OK, and from a representative collection of fruit from College Station, TX, were collected from ripe fruit only. Maturity was assessed by external and internal characteristics (i.e., waxiness, tendril death, Brix, firmness, seed maturity). A digital refractometer was used to determine Brix. Samples were collected from the center of each fruit, pureed for 3 min with a Polytron PT 10-35 grinder (Kinematica AB, Lucerne, Switzerland) set at medium speed and stored at –80 C until analyzed for L-citrulline. Citrulline quantification by thin-layer chromatography plates. L-citrulline was analyzed using a thin-layer chromatography (TLC) plate method, which was a slight modification of a Brenner and Niederwieser (1960) method. L-citrulline was quantified on the basis of an L-citrulline standard (Sigma-Aldrich, St. Louis, MO). Briefly, 40-g samples were pureed using a Brinkmann Polytron Homogenizer (Brinkmann Instruments, Inc., Westbury, NY) with a 20 mm O.D. blade. One milliliter of the liquid puree was centrifuged at 15,800 gn for 10 min to remove debris. Supernatants were diluted to make a 10% and a 20% solution in deionized water. Ten microliters of the diluents were loaded on a 20 · 20-cm silica gel matrix (200-mm layer thickness, 5to 17-mm particle size) TLC plate (SigmaAldrich). The spots were air-dried and amino acids were resolved using a solvent (2:1:1 n-butanol:acetic acid:deionized water). Plates were developed with 0.2% ninhydrin in ethanol by baking at 95 C for 5 to 10 min. Densitometric scans of the citrulline spots were visualized and calculated against standards using a Kodak Image station (Model 440CF; Eastman Kodak, Rochester, NY). Data are in mg g fresh weight, because 1 mL of watermelon puree is very nearly and consistently 1 g. Citrulline quantification by highperformance liquid chromatography. Frozen tissue was thawed and then centrifuged at 10,000 gn for 10 min at 15 C to remove insoluble components. L-citrulline was quantified by a method first reported in Chang et al. (1983) and subsequently modified by Krause et al. (1995) and by Sethuraman et al. (2004) for the determination of physiological amino acids and biogenic amines by reversed-phase high-performance liquid chromatography (HPLC) separation of their dabsyl derivatives. The Sethuraman et al. (2004) method was recently modified by Fish and Bruton (2010) for extraction and quantification of physiological amino acids from cucurbits. The gradient program of Sethuraman et al. (2004) was used. Separation/quantification was performed on a Varian ProStar tenary solvent HPLC system equipped with an autosampler and diode array detector (Varian, Walnut Creek, CA). A 250 mm · 4 mm, 5-mm Luna C18 reversed-phase column was used (Phenomenex, Torrance, CA) at 50 C and at a flow rate of 1.0 mL min. The dabsyl derivatives eluting from the column were monitored by absorbance at 468 nm. Dabsyl chloride [4-(4-dimethylaminophenylazo) benzenesulfonyl chloride] was purchased from Pierce (Pierce Biotechnology, Rockford, IL), and the amino acids used for calibration of the method were purchased from SigmaAldrich as a commercial 18 amino acid calibration mixture. The physiological amino Table 1. L-citrulline concentrations averaged by cultigen across replications at Lane, OK. Cultigen Mean (mg L-citrulline/g fresh wt) SD Plant type Flesh color Number of fruit tested Tom Watson 4.52a 0.72 OP R 2 PI 306364 3.70 abc 0.25 OP R 2 Jubilee 3.57 abcd 1.06 OP R 26 Charleston Gray 3.56 abcde 0.93 OP R 50 Graybelle 3.55 abcde 1.09 OP R 11 Early Canada 3.47 abcde 1.09 OP R 14 Congo 3.47 abcde 1.47 OP R 30 King & Queen 3.46 abcde 1.13 OP R 36 Early Arizona 3.42 abcde 1.41 OP R 19 Navajo Sweet 3.40 abcde 1.20 OP R 13 Starbrite F1 3. abcde 35 0.94 Hy R 36 Sangria F1 3.30 abcde 0.86 Hy R 24 Legacy F1 3.30 abcde 0.99 Hy R 45 Sugar Baby 3.29 abcde 1.13 OP R 26 Crimson Sweet 3.23 abcde 0.96 OP R 25 Regency F1 3.22 abcde 1.12 Hy R 37 Yellow Crimson 3.22 abcde 1.37 OP CY 26 Sweet Princess 3.21 abcde 1.00 OP R 25 Big Crimson 3.17 abcde 0.99 OP R 43 Carolina Cross #183 3.17 abcde 1.27 OP R 13 Stars-N-Strips F1 3.16 abcde 0.98 Hy R 40 Black Diamond 3.13 abcde 0.87 OP R 11 Quetzali 3.13 bcde 0.86 OP R 32 Mountain Hoosier 3.10 bcde 1.01 OP R 20 Mickylee 3.10 bcde 0.91 OP R 9 Allsweet 3.09 bcde 0.97 OP R 18 Calhoun Gray 3.08 bcde 1.09 OP R 41 Georgia Rattlesnake 2.95 bcdef 0.95 OP R 9 AU-Jubilant 2.94 bcdef 0.53 OP R 12 Stone Mountain 2.90 bcdef 1.03 OP R 36 Fiesta F1 2.89 bcdef 0.94 Hy R 42 Hopi Red Flesh 2.88 bcdef 1.22 OP R 23 Tendersweet Orange Flesh 2.87 bcdef 0.98 OP O 16 Sugarlee 2.83 bcdefg 1.02 OP R 29 Desert King 2.83 bcdefg 1.29 OP SY 20 Calsweet 2.78 bcdefg 0.98 OP R 49 North Carolina Giant 2.67 bcdefgh 0.97 OP R 30 Minilee 2.53 bcdefgh 0.87 OP R 34 PI 385963 2.53 bcdefgh — OP R 1 PI 225557 2.52 bcdefgh 0.87 OP R 2 Royal Flush F1 2.49 bcdefgh 1.06 Hy R 33 PI 306367 2.46 bcdefghi — OP R 1 Peacock WR-60 2.42 bcdefghi 1.13 OP R 29 Golden Midget 2.39 bcdefghi 0.81 OP R 19 PI 186974 2.34 cdefghi 1.35 OP R 6 PI 255139 2.26 defghi — OP R 1 PI 189317 2.18 efghi 0.62 OP W 3 PI 161373 1.65 fghi 0.24 OP R 2 PI 164993 1.46 ghi — OP R 1 PI 181742 1.38 hi 0.62 OP R 6 PI 162667 1.35 hi 0.35 OP R 10 Low Sugar Watermelon 194 1.22 i 1.02 OP R 7 PI 164992 1.21 i — OP R 1 Low Sugar Watermelon 177 1.09 i 0.52 OP R 3 Cultigens shaded in gray were grown at two locations and appear in Table 2. The values were estimated using a thin-layer chromatography plate method. Any two means within a column not followed by the same letter are significantly different at P # 0.05. Type of watermelon: OP = open-pollinated line, Hy = hybrid line. Flesh color for each cultigen: R = red or pink, SY = salmon yellow, CY = canary yellow, O = orange, and W = white fruit. Indicate SD was not calculated because only one fruit was tested. HORTSCIENCE VOL. 46(12) DECEMBER 2011 1573 | BREEDING, CULTIVARS, ROOTSTOCKS, AND GERMPLASM RESOURCES