Purification and Properties of Cytosolic Copper,Zinc Superoxide Dismutase from Watermelon (Citrullus vulgaris Schrad.) Cotyledons

Cytosolic copperzinc-superoxide dismutase (CuZn-SOD I; EC 1.15.1.1) was purified to homogeneity from watermelon (Citrullus vulgaris Schrad.) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography, gel-filtration column chromatography, and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3, 540 units per milligram protein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recognized by the antibody against peroxisomal CuZn-SOD from watermelon cotyledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2–4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equal isoforms with isoelectric points of 4.63 and 4.66.     

Purification and Properties of Cytosolic Copper, Zinc Superoxide Dismutase from Watermelon (Citrullus vulgaris Schrad.) Cotyledons

Cytosolic copperzinc-superoxide dismutase (CuZn-SOD I; EC 1.15.1.1) was purified to homogeneity from watermelon (Citrullus vulgaris Schrad.) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography, gel-filtration column chromatography, and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3, 540 units per milligram protein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recognized by the antibody against peroxisomal CuZn-SOD from watermelon cotyledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2-4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equal isoforms with isoelectric points of 4.63 and 4.66.     

西瓜抗枯萎病育种分子标辅助选择的研究

将西瓜野生种质ＰＩ２９６３４１抗枯萎病生理小种１的抗性基因连锁的ＲＡＰＤ标记ＯＰＰ０１．７００进行克隆、测序,Ｓｏｕｔｈｅｒｎ杂交证明此标记为１个单拷贝,并转化为ＳＣＡＲ标记,简化了ＳＣＡＲ扩增产物的检测技术。上述技术在抗病转育后代造反中得到了很好的应用,初步建立了西瓜抗枯萎病育种分子标记辅助选择技术系统。     

Control of Pyrophosphated-Fructose-6-Phosphate 1-Phosphotransferase Activity in the Cotyledons of Citrullus lanatus

After initiation of radicle elongation, the pyrophosphate:d-fructose-6-phosphate 1-phosphotransferase (PFP) activity sharply increases in the cotyledons of Citrullus lanatus. Removal of the radicle early during incubation prevents the increase in PFP activity in the cotyledons evident in the control. Removal of the radicle at any stage after germination results in a decrease in PFP activity in the cotyledons. Application of kinetin (0.5 micromolar) or 2-chlorophosphonic acid (0.1 micromolar) to isolated cotyledons replaces the effect of the radicle. Gibberellic acid (0.09 micromolar GA₃) also partially mimics the presence of the radicle. Anaerobic conditions, as well as cycloheximide application (0.18 micromolar) to intact embryos or to kinetin and ethrel treated isolated cotyledons prevent the increase in PFP activity evident in the control.     

Purification and Properties of Glyoxysomal Cuprozinc Superoxide Dismutase from Watermelon Cotyledons (Citrullus vulgaris Schrad)

A glyoxysomal copper,zinc-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity, for the first time, from watermelon cotyledons (Citrullus vulgaris Schrad.). The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography and gel-filtration column chromatography. Pure copper,zinc-superoxide dismutase (Cu,Zn-SOD II) had a specific activity of 1211 units per milligram protein and was purified 400-fold, with a yield of 8 micrograms enzyme per gram cotyledon. The glyoxysomal Cu,Zn-SOD had a relative molecular weight of about 33,000 and was composed of two equal subunits of 16,500 Daltons. Metal analysis showed that the enzyme, unlike other Cu,Zn-SODs, contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. No iron and manganese were detected. Ultraviolet and visible absorption spectra were reminiscent of other copper,zinc-superoxide dismutases.     

Light Affects the Chloroplast Ultrastructure and Post-Storage Photosynthetic Performance of Watermelon (Citrullus lanatus) Plug Seedlings

Watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] plug seedlings were stored at 15°C in the light at a photosynthetic photon flux density of 15 µmol·m⁻²·s⁻¹ or in darkness for 6 days, to evaluate their chloroplast ultrastructure, and associated photosynthetic characteristics. Storage in the dark caused swelling, disordered granal arrangement, and starch grain disappearance in the chloroplasts. In contrast, the chloroplasts stored in the light were relatively normal. As a result, the light-stored seedlings had a significantly higher chlorophyll content, Fv/Fm, and Pn than did dark-stored seedlings. Regardless of whether the seedlings were stored in light or darkness, the Gs and Ls of the seedlings significantly decreased, while the Ci obviously increased when the Pn decreased after 6 days of storage. This result suggests that the decreased Pn is not solely a stomatal effect, as the effects on the chloroplasts contributed to this photosynthetic inhibition. Six days after transplanting, seedlings that were stored in the light or darkness for 2 or 4 days showed complete recovery of chloroplast ultrastructure, chlorophyll content, Fv/Fm, Gs and Pn. When the storage period increased to 6 days, the dark-stored seedlings had a significantly lower Fv/Fm and Pn than the light-stored and control seedlings 6 days after transplanting, which was mainly ascribed to incomplete recovery of chloroplast ultrastructure. Furthermore, the light-stored seedlings exhibited a significantly higher shoot dry weight during storage and a higher percentage dry weight increase after transplanting than the dark-stored seedlings. These effects were enhanced by prolonged storage (4 to 6 days). This study demonstrated that dim light during storage is beneficial for maintaining chloroplast ultrastructure as well as photosynthetic efficiency in watermelon seedlings, thus contributing to the rapid recovery of post-storage photosynthetic performance, which ensures the transplant quality of the seedlings after removal from storage.     

Some Effects of Light Intensity, Daylength and Temperature on Growth of Fruiting and Non-fruiting Watermelon (Citrullus lanatus)

Plants of watermelon [Citrullus lanatus(Thunb.) Matsum. &Nakai, cv. Early Yates] were grown for up to 3 months aftergermination in controlled environment cabinets, and variousaspects of vegetative growth and fruit development were measured.Effects of light intensity were studied by comparing growthat 8, 16 and 32 klx at constant temperature and daylength (25^°C, 14 h). Effects of daylength were studied by comparing8, 14 and 24 h at constant light intensity and temperature (32klx, 25 ^°C), and effects of tem perature were studied bycomparing 20^°, 25^°, 30^°, 35^° and 40 ^°C atconstant light intensity and day- length (32 klx, 14 h). Withincreasing light intensity and daylength lateral growth waspromoted whereas main shoots were less affected. Increase intemperature above 25 ^°C resulted in longer main shoots andprolific lateral growth, due both to more and to longer laterals.Environmental differences had little effect on internode lengthbut did affect the size of basal leaves. However, an increasein total leaf area at higher temperatures or with Continuouslight was mainly due to more leaves rather than larger leaves.The presence of developing fruit greatly reduced vegetativegrowth of plants. Main shoot length, lateral growth, numberof leaves, and even size of individual leaves, were all reduced.This reduction did not apply to d. wt of whole plants. Fruitingplants were very efficient, on a leaf area basis, in accumulatingd. wt. At 25 ^°C at the two higher light intensities with14 h days the presence of one developing fruit was inhibitoryto the setting of any subsequent fruit. With short days or lowlight, more fruits were set but they were small. With continuouslight or high temperature more than one fruit could developand they were large.     

Identification of a RAPD Marker Linked to Fusarium Wilt Resistant Gene in Wild Watermelon Germplasm (Citrullus lanatus var. citroides

Random amplified polymorphic DNA(RAPD) was employed to detect a molecular marker linked to Fusarium wilt resistant gene in the wild watermelon ( Citrullus lanatus (Thunb.) Mansfeld var. citroides ) germplasm P1296341. The resistance to race 1 Fusarium wilt of PI296341 was controlled by one dominant gene. A RAPD marker OPPO1/700 was proved to be linked to the resistant gene. The genetic distance is 3.0 cM (centimorgan). This work has provided a solid basis for molecular marker-assisted selection (MAS) for disease resistance, and made location and cloning of disease resistant genes possible.     

西瓜甜瓜质量性状的分子标记与定位研究进展

分子标记在作物育种、资源遗传多样性分析、遗传图谱构建等方面有着广泛的应用,常见的分子标记方法主要有RFLP、RAPD、SSR、AFLP、SCAR、CAPS、SNP等.迄今为止,国内外学者已经利用分子标记技术对西瓜和甜瓜的20多个质量性状进行了标记和定位.本文主要对西瓜和甜瓜质量性状的分子标记和定位研究进展进行了综述,发现RAPD标记使用最多,而更具优势的SSR标记使用较少;对抗病虫等育种相关的基因研究较多,且多数标记遗传距离偏大,达不到图位克隆的要求,而对西瓜和甜瓜遗传基础相关的基因研究较少.     

Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon (Citrullus lanatus)

Phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway, plays a critical role in plant growth, development, and adaptation. PAL enzymes are encoded by a gene family in plants. Here, we report a genome-wide search for PAL genes in watermelon. A total of 12 PAL genes, designated ClPAL1-12, are identified . Nine are arranged in tandem in two duplication blocks located on chromosomes 4 and 7, and the other three ClPAL genes are distributed as single copies on chromosomes 2, 3, and 8. Both the cDNA and protein sequences of ClPALs share an overall high identity with each other. A phylogenetic analysis places 11 of the ClPALs into a separate cucurbit subclade, whereas ClPAL2, which belongs to neither monocots nor dicots, may serve as an ancestral PAL in plants. In the cucurbit subclade, seven ClPALs form homologous pairs with their counterparts from cucumber. Expression profiling reveals that 11 of the ClPAL genes are expressed and show preferential expression in the stems and male and female flowers. Six of the 12 ClPALs are moderately or strongly expressed in the fruits, particularly in the pulp, suggesting the potential roles of PAL in the development of fruit color and flavor. A promoter motif analysis of the ClPAL genes implies redundant but distinctive cis-regulatory structures for stress responsiveness. Finally, duplication events during the evolution and expansion of the ClPAL gene family are discussed, and the relationships between the ClPAL genes and their cucumber orthologs are estimated.     

Some Effects of Light Intensity, Daylength and Temperature on Flowering and Pollen Tube Growth in the Watermelon (Citrullus lanatus)

The effects of light and temperature on flowering and pollentube growth were studied in watermelon [Citrullus lanatus(Thunb.)Matsum. and Nakai, cv. Early Yates] plants grown in controlledenvironment cabinets. All female flowers were pollinated inone group of plants; none was pollinated in the other group. Temperature increase from 25 ^°C to 35 ^°C with daylengthof 14 h and light intensity of 32 klx caused increase in flowernumber per plant, proportion of male flowers, ovary length anddiameter, ovule number per ovary, rate of pollen tube growthand percentage of penetrated ovules at 24 hand 48 h after pollination.Very few flowers were produced at 40 ^°C, but there was ahigh proportion of male flowers. Increase in daylength from14 h to 24 h at 25 ^°C with light intensity of 32 klx alsoincreased number of flowers per plant, ovary length and diameterand number of ovules per ovary but sex expression and rate ofpollen tube growth were unaffected. Reduction in daylength from14 h to 8 hat 25 °C and light intensity of 32 klx and reductionin light intensity from 32 klx to 8 klx at 25 ^°C and 14h daylength both produced an increase in the percentage of immatureovules. The presence of fruit on the vine resulted in fewerflowers per plant and in reduced ovary legnth and diameter underall conditions tested. The results are discussed in relation to the fruiting responseof the plant.     

Enzymic hydrolysis of placental cell wall pectins and cell separation in watermelon (Citrullus lanatus) fruits exposed to ethylene

Watermelon [ Citrullus lanatus (Thunb.) Matsum and Nakai, cv. Charleston Gray] fruits were examined to determine the effect of ethylene on cell wall hydrolases. pectin degradation, and cell wall ultrastructure. Enzymic studies showed that activity of polygalacturonase (EC 3.2.1.15) increased in placental tissue following 1 day of ethylene treatment and was 10 times higher after 6 days of treatment. The increase in polygalacturonase activity was accompanied by the appearance in ethanol powders of low-molecular-weight pectic polymers and a decrease in total pectin. The enhanced enzyme activity and decrease in total pectins were observed only in fruits exposed to ethylene. Ultrastructural studies of ethylene-treated tissue revealed an early disintegration of the middle lamella. The onset of wall separation coincided with the first notable increase in polygalacturonase activity. Cell wall of untreated fruit showed no evidence of structural changes. The results indicate that initiation of enzymic activity and cell wall separation in response to ethylene are not characteristic phenomena of normal ripening and senescence in watermelon fruit.     

Grafting of watermelon (Citrullus lanatus cv. Mahbubi) onto different squash rootstocks as a means to minimize cadmium toxicity

To test the possibility that using appropriate rootstocks could improve the tolerance of watermelon to cadmium (Cd) toxicity, a greenhouse experiment was conducted to determine growth and antioxidant activities of watermelons, either nongrafted or grafted onto summer squash and winter squash. We provided nutrient solutions having four levels (0, 50, 100, and 200 μM) of cadmium to treat the plants. Shoot and root biomass reduction were significantly lower in summer squash rootstock-grafted watermelon than winter squash rootstock-grafted and nongrafted watermelons. Cadmium induced a smaller decrease in leaf area index in grafted watermelons compared with nongrafted plants. The Cd- related reductions in chlorophyll content and efficiency of photosynthesis were more severe in nongrafted watermelons compared with dose grafted onto summer squash. Cd accumulation in shoot at the highest dose (200 µM) of CdCl₂ was significantly lower (19.76 mg/kg) in summer squash rootstock-grafted watermelon compared with winter squash rootstock-grafted (37.58 mg/kg) and nongrafted watermelon (72.12 mg/kg). H₂O₂, MDA production and electrolyte leakage of summer squash rootstock-grafted watermelon showed less increase, which was associated with a significant increase in the activities of antioxidant. The improved crop performance of grafted watermelons was attributed to their strong capacity to inhibit Cd accumulation in the aerial parts.     

Cell wall-degrading enzymes and pectin solubility and depolymerization in immature and ripe watermelon (Citrullus lanatus) fruit in response to exogenous ethylene

Watermelon fruit have been shown to be extremely sensitive to exogenous ethylene, exhibiting acute symptoms of whole-fruit softening and placental-tissue water-soaking following short periods of exposure to the gas. This study addressed the firmness, specific activities of cell wall hydrolases, and solubility and molecular mass properties of polyuronides in placental tissue in response to treatment of intact fruit with ethylene. Watermelon fruit were harvested at the immature and full-ripe stages and exposed to 50 µl l⁻¹ ethylene for 6 days at 20°C. The firmness of placental tissue from ethylene-treated ripe and immature fruit decreased nearly 80% during 6 days of ethylene exposure, whereas the firmness of placental tissue from fruit maintained in air remained relatively constant up to day 3 and then decreased slightly (12%) during the following 3 days of storage. Although ethylene treatment in some cases influenced the levels of extractable placental-tissue polygalacturonase (EC 3.2.1.15), pectinmethylesterase (EC 3.2.1.11), and α -(EC 3.2.1.22) and β -galactosidase (EC 3.2.1.23) specific activities, these effects were not observed for fruit of both developmental stages and appeared not to be directly involved in the water–soaking syndrome. Symptoms of water-soaking were accompanied by increases in the levels of water- and CDTA ( trans -1,2-cyclohexanediamine- N,N,N',N' -tetraacetic acid)-soluble polyuronides and significant molecular mass downshifts in polyuronides in both immature and ripe watermelon fruit. Polyuronide depolymerization in ethylene-treated ripe fruit was extensive. The parallel trends of enzyme changes in ethylene- compared with air-treated fruit indicate that extractable enzyme levels are not associated with development of the water-soaking disorder. The potential involvement of membrane dysfunction in the water-soaking phenomenon is discussed.     

西瓜含有JmjC结构域的组蛋白去甲基化酶家族的生物信息学分析

利用生物信息学方法,对西瓜（Citrullus lanatus（Thunb.）Matsum.&Nakai）JmjC基因家族的成员进行鉴定,对该基因家族的染色体定位、基因结构、蛋白结构域、选择压力和酶活位点进行分析,并对该基因家族与其它物种的系统进化及共线性关系进行研究。结果显示：西瓜全基因组含有17个JmjC候选基因,核苷酸序列长度为1209~5541 bp;这些基因均含有JmjC结构域,分别位于9条染色体上,归属8个亚族。系统进化、选择压力以及共线性分析结果表明,西瓜与黄瓜（Cucumis sativus L.）亲缘关系较近,JmjC家族基因数量相同,其中14个成员呈现一对一的共线性关系;而西瓜与拟南芥（Arabidopsis thaliana（L.）Heynh）亲缘关系较远,但西瓜和拟南芥同一亚族中JmjC基因间K_a/K_s的比值均小于1,推测西瓜各个亚族成员的编码蛋白功能与同一亚族的拟南芥成员功能极为相似。酶活位点分析结果表明西瓜JmjC基因家族中有10个成员具有潜在的组蛋白去甲基化酶活性。     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

Establishment of a transgenic hairy root system in wild and domesticated watermelon (Citrullus lanatus) for studying root vigor under drought

Root vigor is an important trait for the growth of terrestrial plants, especially in water-deficit environments. Although deserts plants are known for their highly developed root architecture, the molecular mechanism responsible for this trait has not been determined. Here we established an efficient protocol for the genetic manipulation of two varieties of watermelon plants: a desert-grown wild watermelon that shows vigorous root growth under drought, and a domesticated cultivar showing retardation of root growth under drought stress. Agrobacterium rhizogenes-mediated transgenic hairy roots were efficiently induced and selected from the hypocotyls of these plants. Transgenic GUS expression was detected in the roots by RT-PCR and histochemical GUS staining. Moreover, a liquid culture system for evaluating their root growth was also established. Interestingly, growth of the hairy roots derived from domesticated variety of watermelon strongly inhibited under high osmotic condition, whereas the hairy roots derived from wild variety of watermelon retained substantial growth rates under the stress condition. The new protocol presented here offers a powerful tool for the comparative study of the molecular mechanism underlying drought-induced root growth in desert plants.