Overexpression of two chrysanthemum DgDREB1 group genes causing delayed flowering or dwarfism in Arabidopsis

We isolated 13 DREB1 (dehydration responsive element binding factor 1) genes from chrysanthemum and further divided them into three groups, DgDREB1A, DgDREB1B and DgDREB1C, based on the phylogenetic analysis. Each group showed their unique expression patterns under cold, dehydration and salt stress conditions. Arabidopsis plants overexpressing DgDREB1A (1A plants) exhibited significantly stronger tolerance to freezing and drought than those overexpressing DgDREB1B (1B plants) and the control plants. In addition, 1A plants showed delayed flowering, but not dwarfism; while 1B plants showed dwarfism, but not delayed flowering. In 1A plants, the expression of three stress-related DREB1-downstream genes, COR47, COR15A, and RD29A, was strongly induced while the expression of CO and FT, two photoperiod responsive flowering-time genes, was inhibited. In 1B plants, the expression of GA2ox7, a GA-deactivation enzyme gene, was dramatically enhanced. The results above strongly suggest that members from different DgDREB1 groups may have distinct effects on plant development: DgDREB1A may be involved in photoperiod-related flowering-time determination and DgDREB1B in GA-mediated plant development.     

CaPUB1, a Hot Pepper U-box E3 Ubiquitin Ligase,Confers Enhanced Cold Stress Tolerance and Decreased Drought Stress Tolerance in Transgenic Rice (Oryza sativa L.)

Abiotic stresses such as drought and low temperature critically restrict plant growth, reproduction, and productivity. Higher plants have developed various defense strategies against these unfavorable conditions. CaPUB1 (Capsicum annuum Putative U-box protein 1) is a hot pepper U-box E3 Ub ligase. Transgenic Arabidopsis plants that constitutively expressed CaPUB1 exhibited drought-sensitive phenotypes, suggesting that it functions as a negative regulator of the drought stress response. In this study, CaPUB1 was over-expressed in rice (Oryza sativa L.), and the phenotypic properties of transgenic rice plants were examined in terms of their drought and cold stress tolerance. Ubi:CaPUB1 T3 transgenic rice plants displayed phenotypes hypersensitive to dehydration, suggesting that its role in the negative regulation of drought stress response is conserved in dicot Arabidopsis and monocot rice plants. In contrast, Ubi:CaPUB1 progeny exhibited phenotypes markedly tolerant to prolonged low temperature (4°C) treatment, compared to those of wild-type plants, as determined by survival rates, electrolyte leakage, and total chlorophyll content. Cold stress-induced marker genes, including DREB1A, DREB1B, DREB1C, and Cytochrome P450, were more up-regulated by cold treatment in Ubi:CaPUB1 plants than in wild-type plants. These results suggest that CaPUB1 serves as both a negative regulator of the drought stress response and a positive regulator of the cold stress response in transgenic rice plants. This raises the possibility that CaPUB1 participates in the cross-talk between drought and low-temperature signaling pathways.     

Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis

Plants need to adapt to various stress factors originating from the environment. Signal transduction pathways connecting the recognition of environmental cues and the initiation of appropriate downstream responses in plants often involve intracellular Ca²⁺ concentration changes. These changes must be deciphered into specific cellular signals. Calmodulin-like proteins, CMLs, act as Ca²⁺ sensors in plants and are known to be involved in various stress reactions. Here, we show that in Arabidopsis 2 different CMLs, AtCML37 and AtCML42 are antagonistically involved in drought stress response. Whereas a CML37 knock-out line, cml37, was highly susceptible to drought stress, CML42 knockout line, cml42, showed no obvious effect compared to wild type (WT) plants. Accordingly, the analysis of the phytohormone abscisic acid (ABA) revealed a significant reduction of ABA upon drought stress in cml37 plants, while in cml42 plants an increase of ABA was detected. Summarizing, our results show that both CML37 and CML42 are involved in drought stress response but show antagonistic effects.     

盐芥ThDREB2B基因克隆及功能分析

利用RACE技术从抗逆模式植物盐芥中克隆获得了1个DREB(dehydration responsive element binding)类转录因子基因,命名为ThDREB2B(NCBI登录号EF653377)。结果表明:(1)ThDREB2B基因cDNA全长1 486bp,包含1个954bp的开放阅读框,编码316个氨基酸;推测编码的蛋白质分子量约36.0kD,等电点为4.81,第76～135位氨基酸构成1个AP2结构域。(2)系统进化分析表明,ThDREB2B属于DREB亚家族的A-2亚组,与拟南芥AtDREB2B基因遗传距离最近。(3)半定量RT-PCR检测显示,ThDREB2B基因在正常生长条件下低丰度表达,在低温、干旱或高盐胁迫下上调表达。(4)酵母单杂交结果表明,ThDREB2B蛋白能与DRE元件特异结合,但转录激活能力弱。推测ThDREB2B蛋白可能需要翻译后修饰以获得转录激活功能。     

A novel calcium-dependent protein kinase gene from Populus euphratica, confers both drought and cold stress tolerance

Populus species are the most important timber trees over the Northern hemisphere. Most of them are cold- and drought-sensitive except the Populus euphratica Oliv. Here, a calcium-dependent protein kinase (CDPK) gene cloned from P. euphratica, designated as PeCPK10, was rapidly induced by salt, cold, and drought stresses. The protein encoded by PeCPK10 was localized within the nucleus and cytosol, which may be important for its specific regulation in cellular functions. To elucidate the physiological functions of PeCPK10, we generated transgenic Arabidopsis plants overexpressing PeCPK10. The results showed that PeCPK10-transgenic lines experienced better growth than vector control plants when treated with drought. Stronger abscisic acid-induced promotion of stomatal closing has been showed in transgenic lines. Particularly, overexpression of PeCPK10 showed enhanced freezing tolerance. Constitutive expression of PeCPK10 enhanced the expression of several abscisic acid-responsive genes and multiple abiotic stress-responsive genes such as RD29B and COR15A. Accordingly, a positive regulator responsive to cold and drought stresses in P. euphratica is proposed.     

Accelerated Growth Rate and Increased Drought Stress Resilience of the Model Grass Brachypodium distachyon Colonized by Bacillus subtilis B26

Plant growth-promoting bacteria (PGB) induce positive effects in plants, for instance, increased growth and reduced abiotic stresses susceptibility. The mechanisms by which these bacteria impact the host plant are numerous, diverse and often specific. Here, we studied the agronomical, molecular and biochemical effects of the endophytic PGB Bacillus subtilis B26 on the full life cycle of Brachypodium distachyon Bd21, an established model species for functional genomics in cereal crops and temperate grasses. Inoculation of Brachypodium with B. subtilis strain B26 increased root and shoot weights, accelerated growth rate and seed yield as compared to control plants. B. subtilis strain B26 efficiently colonized the plant and was recovered from roots, stems and blades as well as seeds of Brachypodium, indicating that the bacterium is able to migrate, spread systemically inside the plant, establish itself in the aerial plant tissues and organs, and is vertically transmitted to seeds. The presence of B. subtilis strain B26 in the seed led to systemic colonization of the next generation of Brachypodium plants. Inoculated Brachypodium seedlings and mature plants exposed to acute and chronic drought stress minimized the phenotypic effect of drought compared to plants not harbouring the bacterium. Protection from the inhibitory effects of drought by the bacterium was linked to upregulation of the drought-response genes, DREB2B-like, DHN3-like and LEA-14-A-like and modulation of the DNA methylation genes, MET1B-like, CMT3-like and DRM2-like, that regulate the process. Additionally, total soluble sugars and starch contents increased in stressed inoculated plants, a biochemical indication of drought tolerance. In conclusion, we show a single inoculation of Brachypodium with a PGB affected the whole growth cycle of the plant, accelerating its growth rates, shortening its vegetative period, and alleviating drought stress effects. These effects are relevant to grasses and cereal crops.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

Plant responsiveness to root-root communication of stress cues

Background and Aims

Phenotypic plasticity is based on the organism''s ability to perceive, integrate and respond to multiple signals and cues informative of environmental opportunities and perils. A growing body of evidence demonstrates that plants are able to adapt to imminent threats by perceiving cues emitted from their damaged neighbours. Here, the hypothesis was tested that unstressed plants are able to perceive and respond to stress cues emitted from their drought- and osmotically stressed neighbours and to induce stress responses in additional unstressed plants.

Methods

Split-root Pisum sativum, Cynodon dactylon, Digitaria sanguinalis and Stenotaphrum secundatum plants were subjected to osmotic stress or drought while sharing one of their rooting volumes with an unstressed neighbour, which in turn shared its other rooting volume with additional unstressed neighbours. Following the kinetics of stomatal aperture allowed testing for stress responses in both the stressed plants and their unstressed neighbours.

Key Results

In both P. sativum plants and the three wild clonal grasses, infliction of osmotic stress or drought caused stomatal closure in both the stressed plants and in their unstressed neighbours. While both continuous osmotic stress and drought induced prolonged stomatal closure and limited acclimation in stressed plants, their unstressed neighbours habituated to the stress cues and opened their stomata 3–24 h after the beginning of stress induction.

Conclusions

The results demonstrate a novel type of plant communication, by which plants might be able to increase their readiness to probable future osmotic and drought stresses. Further work is underway to decipher the identity and mode of operation of the involved communication vectors and to assess the potential ecological costs and benefits of emitting and perceiving drought and osmotic stress cues under various ecological scenarios.     

摩西球囊霉对三叶鬼针草保护酶活性的影响

旱地农田入侵杂草三叶鬼针草(Bidens pilosa L.)与摩西球囊霉(Glomus mosseae)(AM真菌)经常形成长效的共生体,该霉菌对三叶鬼针草的入侵能力起到促进作用,但机理并不清楚。盆栽试验对正常浇水、中度干旱和重度干旱条件下接种AM真菌的三叶鬼针草植株与未接种植株之间叶片丙二醛(MDA)含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸氧化酶(ASP)和过氧化物酶(POD)等保护酶活性进行了比较研究。结果表明,干旱胁迫导致三叶鬼针草叶片内MDA含量升高,SOD、CAT、ASP和POD的活性升高;正常浇水条件下,接种G. mosseae 对MDA含量,SOD、ASP和CAT活性影响不显著;中度干旱条件下,接种没有显著影响ASP活性,但对SOD和CAT活性影响显著;在处理前期(7,14,21d)POD活性影响不显著,在处理后期(28,35d)接种植株显著低于未接种植株;重度干旱条件下,未接种植株MDA含量、CAT活性显著高于接种植株,POD活性差异不显著。ASP活性在21d前差异不显著,之后,未接种植株显著高于接种植株。因此,AM真菌G. mosseae 有效地降低了干旱胁迫对三叶鬼针草的伤害程度,随着土壤含水量的严重亏缺和胁迫时间的延长,摩西球囊霉对三叶鬼针草的保护作用逐渐减弱。由于三叶鬼针草和AM真菌之间普遍存在着共生关系,该共生关系可能是三叶鬼针草入侵能力强的关键生物因子之一。     

Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses.Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment.We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.     

Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress.     

Overexpression of CaDSR6 increases tolerance to drought and salt stresses in transgenic Arabidopsis plants

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as a drought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to drought stress tolerance was unknown. In this report, full-length CaDSR6 cDNA was isolated. The deduced CaDSR6 protein was composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in its central region. This Asp-rich domain was highly conserved in all plant DSR6 homologs identified and shared a sequence identity with the N-terminal regions of yeast p23^fyp and human hTCTP, which contain Rab protein binding sites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, as identified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFP leaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehydration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild type plants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is associated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype in transgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response to drought and salt stresses.     

异育银鲫GABAB受体1亚基cDNA部分序列的克隆及表达分析

为了确定γ-氨基丁酸B受体（gamma-aminobutyric acid B receptor,GABA_BR）基因在异育银鲫（Carassius auratus gibelio）不同组织中的表达,本实验分别对异育银鲫不同组织中GABA_BR1基因进行RT-PCR扩增,并进行了克隆和测序,在与GenBank基因库中已知GABA_BR1序列进行同源性比对的基础上采用邻接法构建系统发育树,并进一步分析其在异育银鲫不同组织内的表达水平。经克隆获得异育银鲫GABA_BR1基因CDS区序列383 bp,编码127个氨基酸。荧光定量PCR结果显示,GABA_BR1基因在异育银鲫脑、肝、肾、心、肠、鳔、鳃、肌、尾鳍、脾、卵巢、精巢组织中均有表达,且在不同组织中的表达水平由高到低依次是：脑>尾鳍>精巢>心、肠、鳔>卵巢、脾、鳃、肌>肝、肾。本研究证实了GABA_BR1基因在异育银鲫各组织中表达的广泛性,且有明显的组织特异性。