Chill-induced decrease in capacity of RuBP carboxylation and associated H2O2 accumulation in cucumber leaves are alleviated by grafting onto figleaf gourd

BACKGROUND AND AIMS: Chilling results in a significant decrease in Rubisco content and increased generation of reactive oxygen species (ROS) in cucumber (Cucumis sativus), a chilling-sensitive species. The role of roots in the regulation of the tolerance is unknown. Here, cucumber plants grafted onto figleaf gourd (Cucurbita ficifolia), a chilling-tolerant species were used to study the role of roots in the regulation of shoot functioning and the associated root-to-shoot communication. METHODS: Gas exchange and chlorophyll fluorescence were measured using an infrared gas analyser combined with a pulse amplitude fluorimeter during chilling at 14 degrees C or 7 degrees C and subsequent recovery. At the same time, Rubisco content and activity and ROS generation were spectrophotometrically assayed. Abscisic acid and cytokinin concentrations in xylem sap were also determined by enzyme-linked immunosorbent assay. KEY RESULTS AND CONCLUSIONS: Grafted plants showed a significantly higher light-saturated rate of CO(2) assimilation (A(sat)) than own-rooted plants when roots were gradually cooled, but no differences were detected when shoots were cooled. Chill at 7 degrees C irreversibly reduced A(sat), and significantly decreased maximum carboxylation activity, Rubisco content and initial Rubisco activity. However, grafted plants showed weaker inhibition, together with decreased electron flux in the water-water cycle. Higher activity of antioxidant enzymes with less ROS production was found in grafted plants. In addition, ABA concentration increased by 48.4-fold whilst cytokinin concentration decreased by 91.5% in the xylem sap of own-rooted plants after exposure to a 7 degrees C chill. In comparison, ABA and cytokinin concentrations increased by 10.5-fold and 36.9%, respectively, for the grafted plants. Improved plant growth was also observed in grafted plants after the chill. These results suggest that some signals coming from chilling-resistant roots (i.e. ABA and cytokinins) protect leaf photosynthesis in shoots of chilling-sensitive plants.     

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β‐N‐oxalyl‐L ‐α,β‐diaminopropionic acid (β‐ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed‐specific expression of an oxalate‐degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β‐ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC‐expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate‐degrading enzyme.     

Development of EST‐SSRs in Cucumis sativus from sequence database

Simple sequence repeat markers derived from expressed sequence tags (EST‐SSR) are potentially valuable tools for plant breeding and germplasm collection conservation, and increasingly, efforts have been made for developing this type of marker. We have identified 20 polymorphic SSR markers from cucumber ESTs deposited in public sequence database. The average allele number was 3.3 per locus, ranging from two to six alleles during screening 20 cucumber genotypes with the mean expected heterozygosity of 0.477. Amplification products were also detected by 13 pairs of primer in Cucumis melo. These informative EST‐SSR markers can be used in cucumber genetic improvement projects.     

The effect of cytokinins on in vitro cultured inbred lines of Raphanus sativus var. radicula Pers. with genetically determined tumorigenesis

The higher plant tumors are convenient models for studying the genetic control mechanism of plant cell division. There are two types of tumors: induced by the pathogenic factor and genetically determined. The development of both tumor types was related to the changes in cytokinin metabolism and/or signal transduction. In this work, the effect of synthetic cytokinins on the in vitro morphogenesis of cotyledon explants and isolated apices of radish seedlings was studied in several inbred radish lines (Raphanus sativus var. radicula Pers.) that differed in their in vivo tumorigenic properties. It was noted that root formation was stronger affected by kinetin while the treatment with thidiazuron tended to induce active callus formation in cotyledon explants of all inbred lines, except IIa. Growing with benzyladenine produced an intermediate effect as regards all morphogenetic responses. Cytokinin treatment of tumorigenic lines enhanced necrotic development in cotyledon explants. Culturing isolated apices of regenerated plants produced tumors anatomically and morphologically similar to those developing in vivo. Some of the lines nontumorigenic in vivo with enhanced formation of calli on cotyledon explants also developed tumors on apical explants in vitro when treated with cytokinins. These data suggest that different mechanisms for tumor formation operate in various radish lines. The radish lines are classified into three types: (1) necrotic lines with tumor formation putatively related to endogenous cytokinin level, (2) callus-forming lines with cell division enhanced in response to cytokinins, and (3) necrosis-and callus-forming lines with both mechanisms of tumor formation involved.     

黄瓜无菌苗雌花诱导体系的建立

研究Spm和IAA对无菌黄瓜苗雌花诱导的协同作用,及不同外植体、培养基中琼脂含量和KH2PO4含量对雌花诱导的影响,由此建立了有效的雌花诱导体系。黄瓜去根苗接种在MS培养基上,单独添加Spm、IAA时的雌花诱导率、雌花数偏低或为0,同时添加12mg.L-1Spm与0.01mg.L-1IAA时达21%、29枚,对照组未见雌花,说明Spm和IAA对雌花诱导的协同作用显著。实验证明,在全苗、去根苗、去根去顶苗、顶芽四种外植体中,及在0.5%～0.9%琼脂含量和1.0～2.0mmol.L-1KH2PO4含量的培养条件下,采用0.7%琼脂含量和1.75mmol.L-1KH2PO4含量培养黄瓜去根去顶苗的雌花诱导效果最好,其诱导率和雌花数分别达46%、54枚。     

山黧豆叶片蛋白质双向电泳技术的建立

以山黧豆叶片为材料,比较分析了蛋白质的不同提取方法,在此基础上着重于样品制备。对IPG胶条的选择,第一向等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳的电泳程序及参数、染色方法等相关技术进行了比较和条件优化。结果显示：采用TCA-丙酮沉淀法提取蛋白质,裂解液中加入Tris-base作为蛋白酶抑制剂,等电聚焦电泳时延长低电压的电泳时间（30V、12h,500V、1h,1000V、2h）以促进盐离子泳出的方法对山黧豆叶片蛋白质进行双向电泳,并用考马斯亮蓝和银染复合染色法进行凝胶染色,能够获得蛋白点清晰的双向电泳图谱,说明用优化后的方法建立起的山黧豆叶片蛋白质双向电泳技术,蛋白质样品制备质量好,电泳分辨率高,完全适合于进一步的蛋白质组学研究。     

外源多胺对低氧胁迫下黄瓜幼苗光合特性和膜脂过氧化的影响

以抗低氧能力不同的2个黄瓜（Cucumis sativus）品种为试材,研究了外源多胺对黄瓜幼苗植株生长、光合特性和膜脂过氧化的影响.结果表明,外源多胺能显著提高低氧胁迫下黄瓜幼苗叶片的净光合速率和水分利用率,降低叶片中丙二醛含量和质膜透性,使幼苗鲜重和干重明显增加.因此在低氧胁迫下,外源喷施多胺能提高幼苗叶片的净光合速率,促进植株生长,缓解胁迫对黄瓜幼苗的伤害.此外,与抗低氧能力较强的品种绿霸春4号相比,外源多胺对抗低氧能力较弱的品种中农8号的影响更明显.     

黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因克隆及序列分析

根据6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6PGDH)基因的保守氨基酸序列设计简并引物,应用RT-PCR技术从黄瓜栽培种品种'北京截头'(Cucumis sativus 'Beijingjietou')叶片中获得了640 bp的特异片段,以该序列在EST数据库进行同源检索筛选,发现甜瓜EST序列AM715537.2与之高度一致,据此设计引物经RT-PCR扩增、分子克隆和序列拼接,获得了黄瓜6-磷酸葡萄糖酸脱氢酶基因全长序列,命名为Cs6PGDH(GenBank登录号FJ610345).序列分析表明,该基因全长1 829 bp,其中开放读码框(ORF)长1 488 bp编码495个氨基酸组成的多肽,编码区内无内含子存在,5'、3'端非翻译区长度各为70 bp和271 bp.Blast同源性分析显示该基因编码的氨基酸序列与拟南芥、大豆、水稻、玉米、菠菜等物种6PGDH 基因有74%以上的一致性.由于与其他物种胞质6PGDH相类似氨基酸N端都缺少长度约为40aa的转运肽,推断Cs6PGDH为黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因.