Identification and characterization of phospholipase D and its association with drought susceptibilities in peanut (Arachis hypogaea)

Preharvest aflatoxin contamination has been identified by the peanut industry as a serious issue in food safety and human health because of the carcinogenic toxicity. Drought stress is the most important environmental factor exacerbating Aspergillus infection and aflatoxin contamination in peanut. The development of drought-tolerant peanut cultivars could reduce aflatoxin contamination and would represent a major advance in the peanut industry. In this study, we identified a novel PLD gene in peanut (Arachis hypogaea), encoding a putative phospholipase D (PLD, EC 3.1.4.4). The completed cDNA sequence was obtained by using the consensus-degenerated hybrid oligonucleotide primer strategy. The deduced amino acid sequence shows high identity with known PLDs, and has similar conserved domains. The PLD gene expression under drought stress has been studied using four peanut lines: Tifton 8 and A13 (both drought tolerant) and Georgia Green (moderate) and PI 196754 (drought sensitive). Northern analysis showed that PLD gene expression was induced faster by drought stress in the drought-sensitive lines than the drought tolerance lines. Southern analysis showed that cultivated peanut has multiple copies (3 to 5 copies) of the PLD gene. These results suggest that peanut PLD may be involved in drought sensitivity and tolerance responses. Peanut PLD gene expression may be useful as a tool in germplasm screening for drought tolerance. The nucleotide sequence, reported in this paper, have been submitted to GenBank under accession number AY274834.     

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 in peanut to improve drought and salt tolerance

The Arabidopsis gene AVP1 encodes an H⁺-pyrophosphatase that functions as a proton pump at the vacuolar membranes, generating a proton gradient across vacuolar membranes, which serves as the driving force for many secondary transporters on vacuolar membranes such as Na⁺/H⁺-antiporters. Overexpression of AVP1 could improve drought tolerance and salt tolerance in transgenic plants, suggesting a possible way in improving drought and salt tolerance in crops. The AVP1 was therefore introduced into peanut by Agrobacterium-mediated transformation. Analysis of AVP1-expressing peanut indicated that AVP1-overexpression in peanut could improve both drought and salt tolerance in greenhouse and growth chamber conditions, as AVP1-overexpressing peanuts produced more biomass and maintained higher photosynthetic rates under both drought and salt conditions. In the field, AVP1-overexpressing peanuts also outperformed wild-type plants by having higher photosynthetic rates and producing higher yields under low irrigation conditions.     

Overexpression of a Pea DNA Helicase (PDH45) in Peanut (Arachis hypogaea L.) Confers Improvement of Cellular Level Tolerance and Productivity Under Drought Stress

Peanut, a major edible oil seed crop globally is predominantly grown under rainfed conditions and suffers yield losses due to drought. Development of drought-tolerant varieties through transgenic technology is a valid approach. Besides superior water relation traits like water mining, intrinsic cellular level tolerance mechanisms are important to sustain the growth under stress. To achieve this objective, the focus of this study was to pyramid drought adaptive traits by overexpressing a stress responsive helicase, PDH45 in the background of a genotype with superior water relations. PCR, Southern, and RT-PCR analyses confirmed stable integration and expression of the PDH45 gene in peanut transgenics. At the end of T₃ generation, eight transgenic events were identified as promising based on stress tolerance and improvement in productivity. Several transgenic lines showed stay-green phenotype and increased chlorophyll stability under stress and reduced chlorophyll retardation under etherel-induced simulated stress conditions. Stress-induced root growth was also substantially higher in the case of transformants. This was reflected in increased WUE (low Δ¹³C) and improved growth rates and productivity. The transgenics showed 17.2 and 26.75 % increase in yield under non-stress and stress conditions over wild type ascertaining the feasibility of trait pyramiding strategy for the development of drought-tolerant peanut.     

Drought stimulation by hypocotyl exposure altered physiological responses to subsequent drought stress in peanut seedlings

Drought stress occurring at the seedling stage of peanut (Arachis hypogaea L.) plants is a limiting factor resulting in considerable reductions in production. Plants can improve their resistance to subsequent stresses after experiencing an initial stress. The aim of this study was to explore the possible role of drought priming by hypocotyl exposure in alleviating subsequent severe drought stress in peanut. Hypocotyl exposure in peanut seedlings as a drought stimulus induced xerophytophysiological regulation, shown by induced osmotic adjustment, activated antioxidant enzymes, anthocyanin accumulation, up-regulation of Gdi-15 and fewer amyloplasts. The seedlings primed by hypocotyl exposure showed improved leaf water retention and reduced proline content when exposed to subsequent drought stress. The alleviated oxidative damage and lower antioxidant enzyme activities indicated rapid acclimation following past hypocotyl exposure and further defenses against subsequent drought stress by retaining ‘memories’ to enable more rapid or stronger physiological responses. The improved leaf photosynthesis and low photosynthetic hysteresis as drought ended indicated a positive effect of drought priming in peanut seedlings. The peanut seedlings ‘remembered’ the xerophytophysiological responses caused by the prior drought stimulation from hypocotyl exposure and displayed quicker and more potent physiological responses to following drought stress. The results showed that hypocotyl exposure could help peanut seedlings survive the severe environments that occurred in the later growth stages.     

Transgenic Agrostis mongolica Roshev. with enhanced tolerance to drought and heat stresses obtained from Agrobacterium-mediated transformation

Efficient procedures for regeneration and Agrobacterium-mediated transformation were established for Agrostis mongolica Roshev. and generated transgenic plants tolerant to drought and heat stresses using a regulatory gene from Arabidopsis, ABF3, which controls the ABA-dependent adaptive responses. The identification and selection of regenerable and reproducible callus type was a key factor for successful transformation. The transformation efficiency was 49.2% and gfp expression was detected in hygromycin-resistant calli and stem of putative transgenic plants. The result of Southern blot analysis showed that the ABF3 transgene was stably integrated into the genome of transgenic plants. Of the five transgenic lines analyzed, single transgene integration was observed in two lines and two copy integration was observed in three transgenic lines. Northern blot analysis confirmed that ubi::ABF3 was expressed in all transgenic lines. Transgenic plants exhibited neither growth inhibition nor visible vegetative phenotypic alternations. However, both transgenic and wild-type plants were highly sterile and did not flower during 3 years of growth period in the open field under subtropical Jeju Island climate. The stomata of the transgenic plants opened less than did stomata of the wild-type plants, and water content of the transgenic leaves remained about 3–4 fold higher than observed for wild-type leaves under drought stress. The transgenic plants showed about 2 fold higher survival rates under drought stress and about 3 fold higher survival rates under heat stress when compared to wild-type plants. Thus, overexpression of the Arabidopsis ABF3 gene results in enhancement of both drought and heat stress tolerance in Agrostis mongolica Roshev.     

Maize root responses to drought stress depend on root class and axial position

In this study we tested the hypotheses that root classes would exhibit distinctive anatomical and architectural responses to drought stress, and that those responses would vary along the root axes. The root systems of four maize (Zea mays L.) sweet corn genotypes designated SC1, SC2, SC3 and SC4 were phenotyped under well-watered and drought treatments in greenhouse mesocosms, permitting increasing stratification of moisture availability as the drought progressed. Anatomical and architectural responses to drought were evaluated for each root class. Lignin distribution was assessed by image processing of UV-illuminated root cross-sections acquired by laser ablation tomography. The two cultivars with less biomass reduction under drought, SC3 and SC4, substantially enhanced lateral root development along the apical segments of axial roots when plants were grown with drought stress. These segments grew into the deeper part of the mesocosm where more moisture was available. Apical segments of the axial and large lateral roots from drought-stressed plants were thicker and had greater theoretical axial water conductance than basal segments, especially in SC3 and SC4. Basal segments of crown roots of SC3 and SC4 showed increased lignification of the stele under drought. Root anatomical and architectural responses to drought are complex and vary among cultivars and root classes, and along root axes. Drought-induced proliferation of lateral roots on apical segments of axial roots would be expected to enhance deep water acquisition, while lignification of axial roots could help preserve axial water transport.

     

Expression of an Arabidopsis sodium/proton antiporter gene (AtNHX1) in peanut to improve salt tolerance

Salinity is a major environmental stress that affects agricultural productivity worldwide. One approach to improving salt tolerance in crops is through high expression of the Arabidopsis gene AtNHX1, which encodes a vacuolar sodium/proton antiporter that sequesters excess sodium ion into the large intracellular vacuole. Sequestering cytosolic sodium into the vacuoles of plant cells leads to a low level of sodium in cytosol, which minimizes the sodium toxicity and injury to important enzymes in cytosol. In the meantime, the accumulation of sodium in vacuoles restores the correct osmolarity to the intracellular milieu, which favors water uptake by plant root cells and improves water retention in tissues under soils that are high in salt. To improve the yield and quality of peanut under high salt conditions, AtNHX1 was introduced into peanut plants through Agrobacterium-mediated transformation. The AtNHX1-expressing peanut plants displayed increased tolerance of salt at levels up to 150 mM NaCl. When compared to wild-type plants, AtNHX1-expressing peanut plants suffered less damage, produced more biomass, contained more chlorophyll, and maintained higher photosynthetic rates under salt conditions. These data indicate that AtNHX1 can be used to enhance salt tolerance in peanut.     

砂藓RcPLD基因的抗旱功能分析

砂藓(Racomitrium canescens)是一种具有极强耐脱水性的苔藓植物,编码磷脂酶D的基因RcPLD能够在砂藓的脱水和复水过程中产生显著的表达响应,它可能参与了砂藓的强耐脱水性功能。该研究使用已克隆的RcPLD编码序列构建拟南芥(Arabidopsis thaliana)过量表达转基因株系rcpld-oe,初步考察过表达株系的干旱胁迫耐受能力及其相关的生理生化指标,分析RcPLD增强拟南芥抗旱性的机制。结果表明:(1)利用已克隆的RcPLD编码序列构建了植物中的过表达载体,成功构建了RcPLD的过表达转基因拟南芥株系rcpld-oe,并获得了多个T_3代rcpld-oe纯合体株系。(2)在正常生长条件下,rcpld-oe株系T_3代纯合体植株比野生型拟南芥植株体积小,但营养生长期较长,抽薹较晚,莲座叶衰老速率较慢;在干旱处理条件下,rcpld-oe株系表现出比野生型拟南芥更强的干旱耐受能力。(3)在干旱胁迫处理过程中,rcpld-oe株系莲座叶的水分散失速率降低,可能在一定程度上降低了干旱对膜完整性的损伤和光合作用的抑制,但其渗透调节物质含量的变化相对较小。研究发现,在干旱胁迫条件下,rcpld-oe植株莲座叶的水分散失速率和光合作用抑制程度显著降低,从而表现出明显强于野生型的干旱耐受能力,这为后续RcPLD功能的深入研究和更多砂藓抗旱功能基因的挖掘奠定了基础。     

Enhanced Expression of <Emphasis Type="Italic">AtNHX1</Emphasis>, in Transgenic Groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) Improves Salt and Drought Tolerence

Salinity and drought are main threat to agriculture productivity, to avoid further losses it is necessary to improve the genetic material of crops against these stresses In this present study, AtNHX1, a vacuolar type Na⁺/H⁺ antiporter gene driven by 35S promoter was introduced into groundnut using Agrobacterium tumefaciens transformation system. The stable integration of the AtNHX1 gene was confirmed by polymerase chain reaction (PCR) and southern blot analysis. It was found that transgenic plants having AtNHX1 gene are more resistant to high concentration of salt and water deprivation than the wild type plants. Salt and proline level in the leaves of the transgenic plants were also much higher than that of wild type plants. The results showed that overexpression of AtNHX1 gene not only improved salt tolerance but also drought tolerance in transgenic groundnut. Our results suggest that these plants could be cultivated in salt and drought-affected soils.