Maternal inheritance of plant variegation in cowpea, Vigna unguiculata (L.) Walp.

A chimeric plant was observed in the F₂ generation of a cross between a mutant cultivar, Ife BPC, and a germplasm line, TVu 2, in cowpea, Vigna unguiculata (L.) Walp. The chimeric plant had four lateral branches, one of which was intensely variegated, while the others were mostly green with few white sectors. F₃ progeny from the intensely variegated branch of this plant were all variegated, while seed derived from the mostly green branches produced only green progeny. In subsequent generations, the descendants of the variegated branch bred true for the variegated trait, while those of the mostly green branches were also true-breeding for green colour. No pure-green or pure-white shoots were observed in any of the variegated plants examined in this study. Consequently, no pure-green or pure-white seedlings were produced from seeds harvested from the variegated plants. The results of reciprocal crosses between variegated and normal green plants indicate that variegation is inherited in a strictly uniparental maternal fashion. This is the first report of a cytoplasmically inherited mutation affecting foliage colour in cowpea. Received: 10 March 2000 / Accepted: 16 May 2000     

Interactive effects of high temperature and elevated carbon dioxide concentration on cowpea [Vigna unguiculata (L.) Walp.]

Limitations in carbohydrate supplies have been implicated as a factor responsible for reproductive failure under heat stress. Heat stress affects two stages of reproductive development in cowpea [Vigna unguiculata (L.) Walp.], and genotypes are available with tolerance and sensitivity to heat during these different stages. The objectives of this study were to determine the responses of these cowpea lines to ambient and elevated [CO₂], under heat stress and optimal temperature, and test whether differences in carbohydrate supplies due to genotypes, CO₂ enrichment and heat stress are associated with differences in sensitivity to heat during reproductive development. Plants were grown in reach-in growth chambers and subjected to day/night temperatures of either 33/20 or 33/30°C, and [CO₂] levels of either 350 or 700 μmol mol^-1. Under intermediate night temperature (33/20°C), all lines set substantial numbers of pods. Under high night temperature (33/30°C) with either ambient or elevated [CO₂], one heat-sensitive line produced no flowers and the other set no pods, whereas the heat-tolerant line abundantly set pods. High night temperature reduced the overall carbohydrate content of the plants, especially peduncle sugars, and caused decreases in photosynthetic rates. The high pod set of the heat-tolerant line, under high night temperature, was associated with higher levels of sugars in peduncles compared with the heat-sensitive lines. The heat-tolerant line accumulated substantial shoot biomass, exhibited less accumulation of starch in leaves, and possibly had less down-regulation of photosynthesis in response to CO₂ enrichment and heat stress than the heat-sensitive lines. Elevated [CO₂] resulted in higher overall carbohydrate levels in heat-sensitive lines (starch in leaves, stems and peduncles), but it did not increase their heat tolerance with respect to flower production or pod set. Heat-induced damage to floral buds and anthers in the sensitive lines was associated with low sugars levels in peduncles, indicating that heat had greater effects on assimilate demand than on leaf assimilate supply. The heat-tolerant line was the most responsive genotype to elevated [CO₂] with respect to pod production under either high or intermediate temperatures.     

High-level constitutive expression in Pichia pastoris and one-step purification of phospholipase D from cowpea (Vigna unguiculata L. Walp)

Phospholipase D (PLD) is one of the main enzymes involved in signal transduction, vesicle trafficking and membrane metabolism processes. Here we describe the heterologous high-yield expression in the yeast Pichia pastoris, one-step purification and characterization of catalytically active PLDalpha from cowpea (Vigna unguiculata L. Walp). Immunoblotting experiments showed that recombinant PLDalpha is recognized by a polyclonal antibody raised against native soybean PLDalpha. A single calcium-dependent octyl-Sepharose chromatography step was used to obtain a highly purified recombinant PLDalpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry data. From 1L of yeast culture medium, about 8 mg of pure recombinant PLDalpha was obtained and the specific activity measured on phosphatidylcholine was 27 micromol/min/mg. Contrary to what was observed previously with Vigna unguiculata PLDalpha expressed in insect cells, no proteolytic degradation of the N-terminal calcium-dependent C2 lipid binding domain was observed here. This functional recombinant PLDalpha should provide a valuable tool for performing detailed studies on the molecular characterization of enzymes as well as structural studies.     

Gene expression and activity of antioxidant enzymes in barley (Hordeum vulgare L.) under controlled severe drought

The objective of the present study was to determine the activity of antioxidant enzymes: superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) and the expression of their genes in two barley genotypes under controlled severe drought. To fulfill this objective, 21-day-old barley plants of two genotypes: Rum and Yarmouk were exposed to controlled severe drought (25% field capacity) for 2, 9, and 16 days. The activity of SOD was significantly high in Rum genotype after 2 days of drought treatment. In Yarmouk genotype, the activity of APX was significantly high after 2 and 9 days of drought treatment. In Rum genotype, CAT2 was upregulated after 9 days of drought treatment and SOD and APX were upregulated after 16 days of drought treatment, whereas CAT2, SOD, and APX were upregulated in Yarmouk genotype after 2 days of drought treatment. The results indicate a unique pattern of activity and gene expression of the antioxidant enzymes in the two barley genotypes under controlled severe drought. Moreover, the data suggest that each genotype utilizes different molecular and biochemical responses under the same drought conditions.     

不同抗旱性花生品种的根系形态发育及其对干旱胁迫的响应

为明确不同抗旱性花生品种的根系形态发育特征,探讨其根系形态发育特征对不同土壤水分状况的响应机制,在防雨棚旱池内进行土柱栽培试验,研究抗旱型品种“花育22号”、“唐科8号”和干旱敏感型品种“花育23号”3个不同抗旱性花生品种根系形态发育特征及其对干旱胁迫的响应.结果表明:抗旱型品种根系较发达,具有较大的根系生物量、总根长、总根系表面积.干旱胁迫使抗旱型品种根系总表面积和体积增加,而干旱敏感型品种则相反.干旱胁迫显著增加抗旱型品种“花育22号”20 cm以下土层内根长密度分布比例及根系表面积和体积,但“唐科8号”相应根系性状仅在20-40 cm土层内增加;干旱胁迫使干旱敏感型品种“花育23号”40 cm以下土层内各根系性状升高,但未达显著水平且其深层土壤内各根系性状增加幅度小于“花育22号”.花生根系总长、总表面积及0-20 cm土层内根系性状与产量间呈显著或极显著正相关.土壤水分亏缺条件下,花生主要通过增加深层土壤内根长、根系表面积和体积等形态特性,优化空间分布构型,以调节植株对水分的利用.     

Comprehensive expression analysis of rice phospholipase D gene family during abiotic stresses and development

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.     

缺磷胁迫下不同长豇豆品种幼苗的解剖结构

研究了不同长豇豆[Vigna unquiculata W. ssp. sesquipedalis (L.) Verd]品种幼苗在缺磷胁迫下的形态结构变化.结果表明:缺磷胁迫下,耐缺磷品种‘芦花白'的叶片和海绵组织厚度增幅较大,栅栏组织厚度与海绵组织厚度的比值减小,气孔密度增幅较小;茎和茎导管直径增大,且比不耐缺磷的品种‘二芦白'大;根直径变小,根量增加,这使其在缺磷胁迫下能保持较强的养分和水分吸收、输导能力和光合能力.缺磷胁迫下,长豇豆叶表皮气孔密度增大,气孔蒸腾加强,促进了水分和磷从根部向上运转及磷的被动吸收.不耐缺磷品种‘二芦白'的气孔密度增加幅度较大,促进磷吸收运转的强度较大,为避免过多失水,栅栏组织厚度及栅栏组织与海绵组织厚度的比值增加较大,减弱了非气孔蒸腾的强度.     

Regulation of rhizosphere acidification by photosynthetic activity in cowpea (Vigna unguiculata L. walp.) seedlings

In contrast to cereals or other crops, legumes are known to acidify the rhizosphere even when supplied with nitrates. This phenomenon has been attributed to N2 fixation allowing excess uptake of cations over anions; however, as we have found previously, the exposure of the shoot to illumination can cause rhizosphere acidification in the absence of N2 fixation in cowpea (Vigna unguiculata L. Walp). In this study, we examined whether the light-induced acidification can relate to photosynthetic activity and corresponding alterations in cation-anion uptake ratios. The changes of rhizosphere pH along the root axis were visualized using a pH indicator agar gel. The intensity of pH changes (alkalization/acidification) in the rhizosphere was expressed in proton fluxes, which were obtained by processing the images of the pH indicator agar gel. The uptake of cations and anions was measured in nutrient solution. The rhizosphere was alkalinized in the dark but acidified with exposure of the shoots to light. The extent of light-induced acidification was increased with leaf size and intensity of illumination on the shoot, and completely stopped with the application of photosynthesis inhibitor. Although the uptake of cations was significantly lower than that of anions, the rhizosphere was acidified by light exposure. Proton pump inhibitors N,N'-dicyclohexyl carbodimide and vanadate could not stop the light-induced acidification. The results indicate that light-induced acidification in cowpea seedlings is regulated by photosynthetic activity, but is not due to excess uptake of cations.     

Functional expression in insect cells, one-step purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp)

Phospholipase D (PLD) is an important enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. In this study, large amounts of a recombinant plant PLD alpha were secreted into the culture medium of baculovirus-infected insect cells and purified to homogeneity in the form of a fully active enzyme. The transient production of recombinant PLD alpha yielded a protein (rPLD alpha a, 88 kDa) together with a shorter form (rPLD alpha b, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLD alpha a and rPLD alpha b showed that rPLD alpha b resulted from proteolytic cleavage at Gly8-Ile9. Immunoblotting showed that both rPLD alpha a and rPLD alpha b are recognized by a polyclonal antibody previously raised against native soybean PLD alpha. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLD alpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLD alpha is homologous with the C2 domains which are present in a number of enzymes known to be involved in signal transduction and/or phospholipid metabolism. The truncated rPLD alpha b lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLD alpha b was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLD alpha a was eluted after chelating calcium ions with EDTA. The purified rPLD alpha yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLD alpha constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.     

植物体内干旱信号的传递与基因表达

干旱是严重影响植物生长发育的重要环境胁迫因子之一。干旱能影响植物的水分状态,使植物缺水遭受伤害。近年来,相继从拟南芥等植物中克隆出了一些受干旱诱导的基因,如蛋白激酶基因、光合基因、渗透调节基因、功能蛋白基因（如LEA基因）等。干旱等胁迫信号经历一系列的传递过程,最后诱导这些特定基因的表达。在植物体中,可能存在依赖ABA型和不依赖ABA型两条干旱信号的传递途径。近年来从高等植物中分离出一系列调控干旱相关基因表达的转录因子,通过转录因子之间以及与其它相关蛋白之间的相互作用,激活或抑制干旱等胁迫因子诱导的基因表达。     

花后高温对不同耐热性小麦品种籽粒淀粉形成的影响

以耐热性不同的2个小麦品种济麦20和鲁麦21为材料,分别于花后5～9d（T1）和15～19d（T2）进行高温处理,研究了小麦花后不同阶段高温对籽粒淀粉积累、淀粉粒分布及相关酶活性的影响。结果表明,花后高温显著降低籽粒淀粉积累量;显著降低籽粒淀粉及支链淀粉含量,但提高直链淀粉含量、直／支链淀粉比例。处理间比较,他处理对籽粒淀粉积累的影响程度较T1处理大。品种间比较,高温对济麦20的影响程度较鲁麦21大。高温使A型淀粉粒的体积、数量和表面积比例显著增加,B型淀粉粒的体积、数量和表面积比例显著降低。T1处理后,两品种籽粒蔗糖含量、蔗糖合酶（SS）和腺苷二磷酸葡萄糖焦磷酸化酶（AGPP）、可溶性淀粉合酶（SSS）、束缚态淀粉合酶（GBSS）和淀粉分支酶（SBE）活性均略高于对照;但济麦20、鲁麦21上述指标分别于花后15、20d开始低于对照。他处理后,两品种籽粒蔗糖含量、SS、AGPP、SSS、GBSS和SBE活性显著低于对照,济麦20上述指标的降幅较鲁麦21大。与其它淀粉合成相关酶相比,高温对籽粒GBSS活性的影响程度较小。两品种处理间籽粒蔗糖含量、SS、AGPP、SSS、GBSS及SBE活性的变化趋势,与淀粉积累量的变化趋势基本一致。说明灌浆期高温使籽粒淀粉积累量降低,一方面是由于籽粒蔗糖供应较低引起糖源不足;另一方面则是由于灌浆中后期淀粉合成相关酶活性下降使淀粉合成受抑所致。     

荧光定量PCR检测干旱胁迫下长春花Crlea基因的相对表达

首次从长春花中克隆了Crlea（Crlea for Catharanthus roseus late embryogenesis abundant）的全长基因,采用荧光定量PCR方法对干旱胁迫下长春花叶片和根部Crlea基因的表达模式进行监测,结果表明,在0.5～8 h的胁迫时间中,叶片和根部的Crlea基因表现出相似的积累模式。长春花Crlea基因的表达随着胁迫时间的延长而表达增强。在叶片中,在6 h和8 h的干旱处理后,Crlea基因表达显著提高,分别是未处理材料的9.984和20.431倍。在根部, 在8 h的处理后,Crlea基因的表达量显著提高（2.831倍于对照)。初步结果表明Crlea基因的表达没有组织特异性,并且为干旱胁迫正调控。     

Influence of silicon on antioxidant mechanisms and lipid peroxidation in chickpea (Cicer arietinum L.) cultivars under drought stress

Abstract

The effects of exogenous silicon (Si) on leaf relative water content (RWC), and the growth, Si concentrations, lipid peroxidation (MDA), lipoxygenase (LOX) activity, proline and H₂O₂ accumulation, non-enzymatic antioxidant activity (AA) and the activity of some antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX) in shoots of ten chickpea cultivars grown under drought were investigated. Drought stress decreased the growth of all the cultivars while applied Si improved the growth at least five of the 10 chickpea cultivars. Silicon applied to the soil at 100 mg kg^?1 significantly increased Si concentrations of the cultivars and counteracted the deleterious effects of drought in 5 of the ten chickpea cultivars by increasing their RWC. In most cultivars tested H₂O₂, proline and MDA content and LOX activity were increased by drought whereas application of Si decreased their levels. APX activity was increased by drought but it was depressed by Si. In general, SOD and CAT activities of the cultivars were decreased by drought. Depending on cultivars, the CAT activity was decreased, and increased or unchanged in response to applied Si, while the SOD activity of the cultivars increased or unchanged by Si. The non-enzymatic antioxidant activity of the cultivars was also increased by Si. These observations implied an essential role for Si in minimizing drought stress-induced limitation of the growth and oxidative membrane damage in chickpea plants.     

Effects of silicon supply on apoplastic manganese concentrations in leaves and their relation to manganese tolerance in cowpea (Vigna unguiculata (L.) Walp.)

The effects of silicon (Si) supply on manganese (Mn) toxicity symptoms and Mn and Si concentrations in the leaf apoplast in a Mn-sensitive cowpea cultivar (Vigna unguiculata (L.) Walp. cv. TVu 91) were investigated in solution culture experiments. When 1.44 mM Si was supplied concurrently with 50 M Mn, the Mn toxicity symptoms were clearly avoided without decreasing the total Mn concentration. On the other hand, the symptoms were not completely alleviated when the plants were pretreated with 1.44 mM Si and then exposed to 50 M Mn without concurrent Si supply. Plants of both of these treatments exhibited lower Mn concentrations in the apoplastic washing fluids but higher amounts of adsorbed Mn on the cell walls than the plants treated with 50 M Mn without Si supply. However, the difference in Mn concentration between plants with continuous and interrupted Si supply was not significant. Moreover, the Mn concentration in the apoplastic washing fluids of the plants with continuous supply of 1.44 mM Si and 50 M Mn and not showing Mn toxicity symptoms was higher than that of the plants grown at 10 M Mn without Si supply which showed distinct Mn toxicity symptoms. These results show that Si supply alleviates Mn toxicity not only by decreasing the concentration of soluble apoplastic Mn through the enhanced adsorption of Mn on the cell walls. A role of the soluble Si in the apoplast in the detoxicification of apoplastic Mn is indicated.