换锦花和中国石蒜对干旱胁迫的生理响应

以2种春出叶石蒜属植物中国石蒜和换锦花为材料,通过盆栽控水试验,以适宜水分(最大持水量的75%~80%)为对照,设置干旱胁迫(最大持水量的35%~40%)处理,研究干旱胁迫对其幼苗生理生化指标的影响,以明确2种植物的耐旱特性。结果显示:(1)换锦花和中国石蒜幼苗叶片相对含水量(RWC)和叶绿素a、b含量均随着干旱胁迫时间的延长而降低。(2)换锦花可溶性糖含量和脯氨酸含量均随着干旱时间的延长表现出持续增加的趋势,而中国石蒜则表现出先升高后降低的趋势。(3)换锦花和中国石蒜幼苗叶片TBARS含量和相对电导率总体上呈增大趋势,并在干旱末期达到最大值;超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均呈现出先上升后下降趋势。(4)换锦花和中国石蒜幼苗叶片净光合速率(Pn)、胞内二氧化碳浓度(Ci)和蒸腾速率(Tr)随着干旱胁迫时间延长均有不同程度下降。研究表明,在土壤干旱胁迫条件下,换锦花和中国石蒜幼苗叶片在水分生理、光合特性、渗透调节物质和抗氧化酶活性等方面表现出一定的差异,其中换锦花较中国石蒜表现出较强的耐旱性,且具有明显的优势。     

Overexpression of TaLEA Gene from Tamarix androssowii Improves Salt and Drought Tolerance in Transgenic Poplar (Populus simonii × P. nigra)

Late embryogenesis abundant (LEA) genes were confirmed to confer resistance to drought and water deficiency. An LEA gene from Tamarix androssowii (named TaLEA) was transformed into Xiaohei poplar ( Populus simonii × P. nigra) via Agrobacterium . Twenty-five independent transgenic lines were obtained that were resistant to kanamycin, and 11 transgenic lines were randomly selected for further analysis. The polymerase chain reaction (PCR) and ribonucleic acid (RNA) gel blot indicated that the TaLEA gene had been integrated into the poplar genome. The height growth rate, malondialdehyde (MDA) content, relative electrolyte leakage and damages due to salt or drought to transgenic and non-transgenic plants were compared under salt and drought stress conditions. The results showed that the constitutive expression of the TaLEA gene in transgenic poplars could induce an increase in height growth rate and a decrease in number and severity of wilted leaves under the salt and drought stresses. The MDA content and relative electrolyte leakage in transgenic lines under salt and drought stresses were significantly lower compared to those in non-transgenic plants, indicating that the TaLEA gene may enhance salt and drought tolerance by protecting cell membranes from damage. Moreover, amongst the lines analyzed for stress tolerance, the transgenic line 11 (T11) showed the highest tolerance levels under both salinity and drought stress conditions. These results indicated that the TaLEA gene could be a salt and drought tolerance candidate gene and could confer a broad spectrum of tolerance under abiotic stresses in poplars.     

Improvement of cold tolerance of the half-high bush Northland blueberry by transformation with the <Emphasis Type="Italic">LEA</Emphasis> gene from <Emphasis Type="Italic">Tamarix androssowii</Emphasis>

Previous studies have shown that the late embryogenesis abundant (LEA) gene of Tamarix androssowii can enhance the drought tolerance of transgenic tobacco. In this study, the cloned LEA gene was transformed into half-high bush Northland blueberry in order to enhance its ability to tolerate cold stress. The cephalosporin antibiotics ceftriaxone, cefotaxime and cefazolin were used to optimize transformation of leaf explants, and kanamycin sulfate was used to select for transgenic shoots. PCR and Southern blot analysis confirmed 8 transformants with LEA gene copy numbers ranging from 1 to 7. The LEA chimeric gene was found to be normally transcribed in 6 transgenic lines by RT-PCR. The 8 transgenic lines were tested for cold tolerance by measuring the activities of peroxidase (POD) and superoxide dismutase (SOD), malondialdehyde (MDA) content and relative electrolyte leakage (REL). Overexpression of the LEA gene enhanced the activity of both POD and SOD under low temperature stress conditions. Lipid peroxidation in the transgenic lines was significantly lower than in non-transgenic plants after cold stress, as determined by MDA content and REL. Thus, our findings indicate that the LEA gene confers increased cold tolerance in the Northland blueberry and implicate the metabolic pathways through which it exerts this effect.     

Over-expression of a <Emphasis Type="Italic">LEA</Emphasis> gene in rice improves drought resistance under the field conditions

Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. In this report, a LEA protein gene OsLEA3-1 was identified and over-expressed in rice to test the drought resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought, salt and abscisic acid (ABA), but not by cold stress. The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibits strong activity under drought- and salt-stress conditions. Three expression constructs consisting of the full-length cDNA driven by the drought-inducible promoter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed into the drought-sensitive japonica rice Zhonghua 11. Drought resistance pre-screening of T₁ families at anthesis stage revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had significantly higher relative yield (yield under drought stress treatment/yield under normal growth conditions) than the wild type under drought stress conditions, although a yield penalty existed in T₁ families under normal growth conditions. Nine homozygous families, exhibiting over-expression of a single-copy of the transgene and relatively low yield penalty in the T₁ generation, were tested in the field for drought resistance in the T₂ and T₃ generations and in the PVC pipes for drought tolerance in the T₂ generation. Except for two families (transformed with OsLEA3-A), all the other families (transformed with OsLEA3-S and OsLEA3-H constructs) had higher grain yield than the wild type under drought stress in both the field and the PVC pipes conditions. No significant yield penalty was detected for these T₂ and T₃ families. These results indicate that transgenic rice with significantly enhanced drought resistance and without yield penalty can be generated by over-expressing OsLEA3-1 gene with appropriate promoters and following a bipartite (stress and non-stress) in-field screening protocol.     

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na⁺ and K⁺ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.     

Seasonal Dynamics in Resource Partitioning to Growth and Storage in Response to Drought in a Perennial Rhizomatous Grass, <Emphasis Type="Italic">Leymus chinensis</Emphasis>

A natural grassland in northeastern China dominated by Leymus chinensis (Trin.) was subject to drought treatments to determine the seasonal dynamics in resource partitioning to shoot and storage organs in response to drought. The growing season was divided into six stages according to the phenology of L. chinensis. Plant samples of L. chinensis were collected at each stage to determine biomass, gross calorific value, relative water content, and key mineral contents of plant parts, including rhizomes, roots, leaves, and stems. Resource partitioning to shoot and storage organs as measured by biomass, gross calorific value, and N, K⁺, and Na⁺ contents varied significantly among phenologic stages. Drought treatment (natural precipitation, 50–60 % of field capacity) significantly reduced biomass, gross calorific value, relative water content of shoot, and N and K⁺ contents in both shoot and storage organs, but it enhanced rhizome : shoot ratio and Na⁺ content. A negative correlation in biomass, gross calorific value, and K⁺ and Na⁺ contents between shoot and storage organs was found throughout the growing season, which may have been accentuated by drought when soil moisture was limited. Our results indicate that resource partitioning to storage plays an important role in regulating plant growth of L. chinensis, especially under drought conditions.     

Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene

In the present work, the bacterial mannitol-1-phosphodehydrogenase(mtlD) gene was introduced into eggplant(Solanummelongena L.) by Agrobacteriumtumefaciens-mediated transformation. Several transformants weregenerated and the transgene integration was confirmed by PCR, dot blot andSouthern blot analysis. Transgenic lines of T₀ and T₁generations were examined for tolerance to NaCl-induced salt stress,polyethylene glycol-mediated drought and chilling stress under bothinvitro and in vivo growth conditions. Aconsiderable proportions of transgenic seeds germinated and seedlings grew wellon 200 mM salt-amended MS basal medium, whereas seeds ofuntransformed control plants failed to germinate. Further, leaf explants fromthe transgenics could grow and showed signs of shoot regeneration onsalt-amended MS regeneration medium, whereas wild type did not respond, and infact the explants showed necrosis and loss of chlorophyll after about one week.The transgenic leaves could also withstand desiccation, and transgenics couldgrow well under chilling stress, and hydroponic conditions with salt stress ascompared to wild type plants. Thus, the transgenic lines were found to betolerant against osmotic stress induced by salt, drought and chilling stress.The morphology of the transgenic plants was normal as controls, but thechlorophyll content was higher in some of the lines. These observations suggestthat mtlD gene can impart abiotic stress tolerance ineggplant.     

异源表达CkLEA1基因增强了拟南芥对非生物胁迫的耐受性

植物在生长过程中会受到各种非生物胁迫的伤害,导致生长发育和产量受到严重影响,胚胎晚期丰富蛋白（late embryogenesis abundant proteins,LEA蛋白）在植物抵抗非生物胁迫过程中起着重要的保护作用。在前期的研究基础上,将受多种胁迫诱导的柠条锦鸡儿CkLEA1（GenBank登录号KC309408）基因转入野生型拟南芥,通过实时荧光定量PCR从7株T₃代纯合体中筛选出3个转基因株系做进一步研究。种子萌发率实验发现,在200 mmol/L NaCl和400 mmol/L甘露醇处理下,转基因株系萌发率均高于野生型拟南芥。干旱处理2周大的幼苗后,转基因株系明显比野生型更抗旱,存活率高于野生型,并且失水率低于野生型。同时,转基因株系积累了较少的丙二醛（MDA）,超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）含量也高于野生型。这些结果表明,柠条锦鸡儿CkLEA1基因在种子萌发阶段提高了拟南芥对盐和渗透胁迫的耐受性,并且提高了转基因拟南芥幼苗生长阶段对干旱胁迫的抵抗能力。     

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.     

Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus

A protocol for producing transgenic radish (Raphanus sativus) was obtained by using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 contained the binary vector pBI121-LEA (late embyogenesis abundant), which carried a Group 3 LEA gene, from Brassica napus. Among six combinations, Agrobacterium-mediated transformation assisted by a combination of 5-min sonication with 5-min vacuum infiltration resulted in the highest transformation frequency. The existence, integration and expression of transferred LEA gene in transgenic T₁ plants were confirmed by PCR, genomic Southern and Western blot analysis. Transgenic radish demonstrated better growth performance than non-transformed control plants under osmotic and salt stress conditions. Accumulation of Group 3 LEA protein in the vegetative tissue of transgenic radish conferred increased tolerance to water deficit and salt stress.     

Variation of physiological and antioxidative responses in tea cultivars subjected to elevated water stress followed by rehydration recovery

Water stress is a major limitation for plant survival and growth. Several physiological and antioxidative mechanisms are involved in the adaptation to water stress by plants. In this experiment, tea cultivars (TV-1, TV-20, TV-29 and TV-30) were subjected to drought stress by withholding water for 20 days followed by rehydration. An experiment was thus performed to test and compare the effect of dehydration and rehydration in growing seedlings of tea cultivars. The effect of drought stress and post stress rehydration was measured by studying the reactive oxygen species (ROS) metabolism in tea. Water stress decreased nonenzymic antioxidants like ascorbate and glutathione contents with differential responses of enzymic antioxidants in selected clones of Camellia sinensis indicating an oxidative stress situation. This was also apparent from increased lipid peroxidation, O₂ ⁻ and H₂O₂ content in water stress imposed plants. But the oxidative damage was not permanent as the plants recovered after rehydration. Comparatively less decrease in antioxidants, higher activities of POX, GR, CAT with higher phenolic contents suggested better drought tolerance of TV-1, which was also visible from the recovery study, where it showed lower ROS level and higher recovery of antioxidant property in response to rehydration, thus proving its better recovery potential. On the other hand, highest H₂O₂ and lipid peroxidation with decrease in phenolic content during stress in TV-29 suggested its sensitivity to drought. The antioxidant efficiency and biochemical tolerance in response to drought stress thus observed in the tested clones of Camellia sinensis can be arranged in the order as TV-30 > TV-1 > TV-29 > TV-20.     

Overexpression of barley <Emphasis Type="Italic">hva1</Emphasis> gene in creeping bentgrass for improving drought tolerance

The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T₀ generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T₀ transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.     

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。     

Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines

Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T₃ generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l⁻¹ NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.     

Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non‐transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.