A zinc finger-containing glycine-rich RNA-binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress conditions

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to changing environmental conditions, the reports demonstrating their biological roles are severely limited. Here, we examined the functional roles of a zinc finger-containing GRP, designated atRZ-1a, in Arabidopsis thaliana under drought or salt stress conditions. Transgenic Arabidopsis plants overexpressing atRZ-1a displayed retarded germination and seedling growth compared with the wild-type plants under salt or dehydration stress conditions. In contrast, the loss-of-function mutants of atRZ-1a germinated earlier and grew faster than the wild-type plants under the same stress conditions. Germination of the transgenic plants and mutant lines was influenced by the addition of ABA or glucose, implying that atRZ-1a affects germination in an ABA-dependent way. H(2)O(2) was accumulated at higher levels in the transgenic plants compared with the wild-type plants under stress conditions. The expression of several germination-responsive genes was modulated by atRZ-1a, and proteome analysis revealed that the expression of different classes of genes, including those involved in reactive oxygen species homeostasis and functions, was affected by atRZ-1a under dehydration or salt stress conditions. Taken together, these results suggest that atRZ-1a has a negative impact on seed germination and seedling growth of Arabidopsis under salt or dehydration stress conditions, and imply that atRZ-1a exerts its function by modulating the expression of several genes under stress conditions.     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative Functional Analysis of Wheat (Triticum aestivum) Zinc Finger-Containing Glycine-Rich RNA-Binding Proteins in Response to Abiotic Stresses

Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including Arabidopsis thaliana and rice (Oryza sativa), the physiological functions of RZs in wheat (Triticum aestivum) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic Arabidopsis plants under various abiotic stress conditions. Expression of TaRZs was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic Arabidopsis seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic Arabidopsis seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in Arabidopsis. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.     

Three zinc‐finger RNA‐binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions

Despite the increasing understanding of the stress‐responsive roles of zinc‐finger RNA‐binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2‐ or BrRZ3‐expressing Arabidopsis seeds was delayed compared with that of wild‐type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3‐expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ‐expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.     

Glycine-rich RNA-binding protein 7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, their physiological functions and mechanisms of action in stress responses remain largely unknown. Here, we assessed the functional roles of GRP7, one of the eight GRP family members in Arabidopsis thaliana , on seed germination, seedling growth, and stress tolerance under high salinity, drought, or cold stress conditions. The transgenic Arabidopsis plants overexpressing GRP7 under the control of the cauliflower mosaic virus 35S promoter displayed retarded germination and poorer seedling growth compared with the wild-type plants and T-DNA insertional mutant lines under high salinity or dehydration stress conditions. By contrast, GRP7 overexpression conferred freezing tolerance in Arabidopsis plants. GRP7 is expressed abundantly in the guard cells, and has been shown to influence the opening and closing of the stomata, in accordance with the prevailing stress conditions. GRP7 is localized to both the nucleus and the cytoplasm, and is involved in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions. Collectively, these results provide compelling evidence that GRP7 affects the growth and stress tolerance of Arabidopsis plants under high salt and dehydration stress conditions, and also confers freezing tolerance, particularly via the regulation of stomatal opening and closing in the guard cells.     

The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis

The molecular mechanism governing the response of plants to salinity stress, one of the most significant limiting factors for agriculture worldwide, has just started to be revealed. Here, we report AtSZF1 and AtSZF2, two closely related CCCH-type zinc finger proteins, involved in salt stress responses in Arabidopsis. The expression of AtSZF1 and AtSZF2 is quickly and transiently induced by NaCl treatment. Mutants disrupted in the expression of AtSZF1 or AtSZF2 exhibit increased expression of a group of salt stress-responsive genes in response to high salt. Significantly, the atszf1-1/atszf2-1 double mutant displays more sensitive responses to salt stress than the atszf1-1 or atszf2-1 single mutants and wild-type plants. On the other hand, transgenic plants overexpressing AtSZF1 show reduced induction of salt stress-responsive genes and are more tolerant to salt stress. We also showed that AtSZF1 is localized in the nucleus. Taken together, these results demonstrated that AtSZF1 and AtSZF2 negatively regulate the expression of salt-responsive genes and play important roles in modulating the tolerance of Arabidopsis plants to salt stress.     

拟南芥高迁移率族蛋白B（AtHMGB）族基因启动子的转录激活功能分析

为了解高迁移率族蛋白B族（high mobility group protein B,HMGB）基因调控植物响应低温、高盐和干旱等外源胁迫的表达调控方式, 本文克隆了拟南芥AtHMGB前5个家族成员的启动子区域（PAtHMGB1,PAtHMGB2,PAtHMGB3,PAtHMGB4和PAtHMGB5）.运用基因重组技术将其分别替换表达载体上35S启动子区域获得重组表达载体,利用农杆菌介导法侵染烟草获得稳定表达的转基因烟草. 运用实时定量PCR检测上述5种启动子的转基因烟草,观察在外源胁迫（低温、高盐和干旱）处理前后gusA基因的表达差异,同时检测转基因烟草种子在不同外源胁迫条件下的萌发状况. 检测结果证实,在低温胁迫下,PAtHMGB2,PAtHMGB3和PAtHMGB4正调控gusA基因的表达,而在干旱或盐胁迫下,gusA基因的表达被PAtHMGB2和PAtHMGB3负调控. 种子萌发结果表明,在干旱胁迫下,PAtHMGB2调控下的转基因烟草比野生型烟草萌发及生长迟缓|在低温胁迫下,PAtHMGB2调控的转基因烟草长势明显强于野生型. 本研究克隆了拟南芥AtHMGB家族前5个成员启动子,分析其生物学功能发现,PAtHMGB2在响应低温和干旱胁迫方面效果尤为显著.     

小拟南芥ApZFP基因异源超表达促进拟南芥开花并提高耐逆性

锌指蛋白(ZFP)是一类重要的转录因子, 广泛参与植物的生长发育和非生物胁迫应答。新疆小拟南芥(Arabidopsispumila)又名无苞芥, 是十字花科短命植物, 具有高光效、繁殖力强和适应干旱等生物学特征, 而且比模式植物拟南芥(A.thaliana)更耐高盐胁迫。将前期克隆的小拟南芥锌指蛋白基因ApZFP通过花滴法转化到哥伦比亚生态型拟南芥(Col-0)中,获得了独立表达的转基因株系。表型观察发现, 过量表达ApZFP基因可促使拟南芥在长短日照下均提前开花。实时荧光定量PCR结果显示, 转基因拟南芥株系中, 光周期途径中的CO基因和年龄途径中的SPL基因表达上调; 春化、环境温度和自主途径中的FLC基因表达下调; 编码成花素的基因FT及下游开花相关基因AP1和LFY的表达量均升高。进一步通过盐、干旱和ABA胁迫处理ApZFP转基因株系的种子和幼苗, 发现在胁迫处理下, 与对照相比, 转基因拟南芥种子萌发率较高, 幼苗主根较长。因此推测, ApZFP在植物发育过程中具有多种功能, 可能既参与植物的开花转变过程, 又同其它植物的锌指蛋白基因一样, 参与植物的耐逆过程。     

过量表达棉花GaSus3基因增强拟南芥的耐盐性

构建了植物过量表达载体p35S：：GaSus3,通过花序浸染法成功获得转GaSus3基因拟南芥植株。利用NaCl模拟盐胁迫处理,证实转基因拟南芥与野生型相比耐盐性明显增强。在盐胁迫下,转基因拟南芥受到的影响较小,而野生型则受盐害影响严重：转基因拟南芥具有更好的萌发率和主根长度,以保证植株正常生长;盐胁迫下转基因拟南芥能保持较多的绿色叶片,而野生型则过早黄化死亡。研究还发现,转基因拟南芥的过氧化氢酶活性在胁迫前后都高于野生型,这说明转GaSus3基因能够提高拟南芥抗氧化胁迫的能力。研究结果为进一步探讨GaSus3基因在棉花耐盐方面的功能奠定了基础。     

Overexpression of three orthologous glutathione S-transferases from Populus increased salt and drought resistance in Arabidopsis

Glutathione S-transferases (GSTs) are multifunctional proteins and play a role in detoxification of xenobiotics as well as prevention of oxidative damage. This study exogenously overexpressed PtGSTF4 from Populus trichocarpa and its two orthologs from Populus yatungensis and Populus euphratica in Arabidopsis thaliana, respectively. To elucidate the function of three GSTF4 proteins in stress response, we compared germination and seedling growth in transgenic Arabidopsis with salt and drought treatments. All three Populus GSTF4 genes overexpressed Arabidopsis showed enhanced resistance to salt stress and drought. GSTF4 transgenic plants accumulated less hydrogen peroxide and more chlorophylls and decreased levels of lipid peroxidation under salt stress and drought comparing to the mock control plants. The difference observed by GSH and GSSG measurements indicated GSTF4 proteins may involve in glutathione-dependent peroxide scavenging which lead to reduced oxidative damage. The Arabidopsis transformed with the GSTF4 gene form P. euphratica showed higher germination rate and different performance of affecting GSSG contents comparing with the other two orthologous GST genes under NaCl treatment. These results suggested three Populus GSTF4 orthologs may have functional divergence in stress responding. This study provides insights into molecular mechanisms that underlie salt and drought stress tolerance of Phi GSTs and gives evidence for the functional divergence among orthologs in vivo.     

Molecular and functional profiling of Arabidopsis pathogenesis-related genes: insights into their roles in salt response of seed germination

Pathogenesis-related (PR) proteins are a group of heterogeneous proteins encoded by genes that are rapidly induced by pathogenic infections and by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). They are widely used as molecular markers for resistance response to pathogens and systemic acquired response (SAR). However, recent studies have shown that the PR genes are also regulated by environmental factors, including light and abiotic stresses, and by developmental cues, suggesting that they also play a role in certain stress responses and developmental processes. In this work, we systematically examined the expression patterns of Arabidopsis PR genes. We also investigated the effects of environmental stresses and growth hormones on the expression of PR genes. We found that individual PR genes are temporally and spatially regulated in distinct patterns. In addition, they are differentially regulated by plant growth hormones, including SA, ABA, JA, ET and brassinosteroid (BR), and by diverse abiotic stresses, supporting the contention that the PR proteins play a role in plant developmental processes other than disease resistance response. Interestingly, PR-3 was induced significantly by high salt in an ABA-dependent manner. Consistent with this, a T-DNA insertional knockout plant with disruption of the PR-3 gene showed a significantly reduced rate of seed germination in the presence of high salt. It is thus proposed that PR-3 mediates ABA-dependent salt stress signals that affect seed germination in Arabidopsis. PR-4 and PR-5 also contributed to salt regulation of seed germination, although their effects were not as evident as those of PR-3.     

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。