Silencing ZmPP2C-A10 with a foxtail mosaic virus (FoMV) derived vector benefits maize growth and development following water limitation

• Global climate change is causing more frequent and severe droughts, which can have negative impacts on plant growth and crop productivity. Under drought conditions, plants produce the hormone ABA (abscisic acid), which regulates adaptive responses, such as stomatal closure and root elongation. Plant viruses have been used in the lab to convey new traits to plants and could also be used to increase production of ABA or to enhance downstream plant drought resistance responses.
• In this study, foxtail mosaic virus (FoMV) was used to silence ZmPP2C-A10, a negative regulator of ABA signalling, in maize (Zea mays L.). Both silenced and control plants were exposed to an 8-day drought treatment, followed by a 30-day period of rewatering, after which indicators of drought resistance were measured.
• After drought treatment, we observed a nearly twofold increase in expression of a stress-mitigation gene, ZmRAB17, reduced chlorophyll fluorescence changes (indicator of stress), and increased plant biomass and development in the ZmPP2C-A10-silenced maize compared to controls.
• These results demonstrate that the FoMV system can be used to silence endogenous expression of ZmPP2C-A10 and increase maize tolerance to drought. This could offer a useful tool to improve crop traits and reduce yield loss during the growing season.

     

Interaction between abscisic acid receptor PYL3 and protein phosphatase type 2C in response to ABA signaling in maize

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively.     

Interaction network of core ABA signaling components in maize

Key message

We defined a comprehensive core ABA signaling network in monocot maize, including the gene expression, subcellular localization and interaction network of ZmPYLs, ZmPP2Cs, ZmSnRK2s and the putative substrates.

Abstract

The phytohormone abscisic acid (ABA) plays an important role in plant developmental processes and abiotic stress responses. In Arabidopsis, ABA is sensed by the PYL ABA receptors, which leads to binding of the PP2C protein phosphatase and activation of the SnRK2 protein kinases. These components functioning diversely and redundantly in ABA signaling are little known in maize. Using Arabidopsis pyl112458 and snrk2.2/3/6 mutants, we identified several ABA-responsive ZmPYLs and ZmSnRK2s, and also ZmPP2Cs. We showed the gene expression, subcellular localization and interaction network of ZmPYLs, ZmPP2Cs, and ZmSnRK2s, and the isolation of putative ZmSnRK2 substrates by mass spectrometry in monocot maize. We found that the ABA dependency of PYL-PP2C interactions is contingent on the identity of the PP2Cs. Among 238 candidate substrates for ABA-activated protein kinases, 69 are putative ZmSnRK2 substrates. Besides homologs of previously reported putative AtSnRK2 substrates, 23 phosphoproteins have not been discovered in the dicot Arabidopsis. Thus, we have defined a comprehensive core ABA signaling network in monocot maize and shed new light on ABA signaling.

     

Expression profiles and genomic organisation of group A protein phosphatase 2C genes in Brassica oleracea

We investigated the expression profiles and genomic organisation of the ABA‐responsive genes encoding protein phosphatases 2C (PP2C, group A members) in Brassica oleracea to better understand their functional and genetic relations. Gene expression profiling of drought responsive genes in B. oleracea and Arabidopsis thaliana revealed significant differences in the gene expression pattern of a key regulator of ABA signalling—ABI1 PP2C. This finding prompted us to study genetic relations within the PP2Cs group A in the Brassica species. Twenty homologous B. oleracea sequences were identified and characterised as putative PP2C group A members. Phylogenetic analysis revealed that the B. oleracea homologues were closely related to the particular members of the A. thaliana PP2C. The genetic analysis corroborated the presence of two to three gene copies in B. oleracea in comparison to the nine unique PP2C genes in the A. thaliana genome. Gene expression analyses showed significant differences in PP2C gene expression pattern in B. oleracea. Our results indicate that PP2C‐based drought stress signalling in B. oleracea has evolved distinctly. Different reactions of particular B. oleracea PP2C genes to drought stress and ABA treatment indicate low conservation of gene expression patterns and functional divergence between B. oleracea and A. thaliana homologous genes.     

玉米ZmSOD基因克隆及其在干旱胁迫下的表达

为探讨超氧化物歧化酶(superoxide dismutase，SOD)基因在玉米抗逆反应中的作用，研究选用2个抗旱性有明显差别的玉米品种为试验材料，采用RT-PCR、实时荧光定量PCR(qRT-PCR)及二维凝胶电泳(2-DE)耦联的基质辅助激光解吸电离/飞行时间质谱(MALDI-TOF)法，对ZmSOD基因的序列结构及其在干旱胁迫下的表达特性进行分析。结果表明：(1)从干旱敏感品种‘登海605’(DH605)和抗旱品种‘蠡玉35’(LY35)玉米叶片中分别成功克隆出ZmSOD1和ZmSOD2基因。DH605中ZmSOD1开放阅读框(ORF)全长456 bp，编码151个氨基酸，编码的蛋白等电点为5.76，分子量为15.05 kD。LY35中ZmSOD2ORF全长459 bp，编码152个氨基酸，编码的蛋白等电点为5.65，分子量为15.11 kD；ZmSOD1和ZmSOD2是亲水性稳定蛋白，都含有Cu/Zn-SOD结构域，蛋白序列N端无信号肽及无跨膜结构域。同源性和系统进化分析表明，玉米ZmSOD和谷子Cu/Zn-SOD2的亲缘关系最近，相似性高达96.05%。(2)在干旱条件下，ZmSOD1在DH605中转录水平明显降低，LY35中ZmSOD2转录水平显著升高；2-DE耦联的质谱分析显示：ZmSOD1在DH605中表达量明显降低，LY35中ZmSOD2表达量无显著性变化，却检测到Mn-SOD蛋白表达量显著增加。(3)相关分析显示，干旱条件下，DH605叶片中ZmSOD1转录水平和其蛋白丰度及SOD酶活性呈极显著性正相关，LY35叶片中ZmSOD2转录水平与SOD酶活性呈显著性正相关。研究结果为进一步探索SOD基因在调节玉米抗性以及逆境胁迫应答过程中的作用机制提供了基础。     

Maize protein phosphatase gene family: identification and molecular characterization

Background

Protein phosphatases (PPs) play critical roles in various cellular processes through the reversible protein phosphorylation that dictates many signal transduction pathways among organisms. Recently, PPs in Arabidopsis and rice have been identified, while the whole complement of PPs in maize is yet to be reported.

Results

In this study, we have identified 159 PP-encoding genes in the maize genome. Phylogenetic analyses categorized the ZmPP gene family into 3 classes (PP2C, PTP, and PP2A) with considerable conservation among classes. Similar intron/exon structural patterns were observed in the same classes. Moreover, detailed gene structures and duplicative events were then researched. The expression profiles of ZmPPs under different developmental stages and abiotic stresses (including salt, drought, and cold) were analyzed using microarray and RNA-seq data. A total of 152 members were detected in 18 different tissues representing distinct stages of maize plant developments. Under salt stress, one gene was significantly up-expressed in seed root (SR) and one gene was down-expressed in primary root (PR) and crown root (CR), respectively. As for drought stress condition, 13 genes were found to be differentially expressed in leaf, out of which 10 were up-regulated and 3 exhibited down-regulation. Additionally, 13 up-regulated and 3 down-regulated genes were found in cold-tolerant line ETH-DH7. Furthermore, real-time PCR was used to confirm the expression patterns of ZmPPs.

Conclusions

Our results provide new insights into the phylogenetic relationships and characteristic functions of maize PPs and will be useful in studies aimed at revealing the global regulatory network in maize abiotic stress responses, thereby contributing to the maize molecular breeding with enhanced quality traits.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-773) contains supplementary material, which is available to authorized users.     

拟南芥类受体蛋白激酶基因CRK45对外源钙离子的响应

以拟南芥野生型和类受体蛋白激酶基因CRK45的T-DNA插入突变体crk45为材料,采用差异基因表达筛选技术检测ABA处理后野生型和crk45中基因表达的差异。结果显示:(1)crk45突变体中有1个基因的表达比野生型高约4倍。(2)NCBI数据库检索表明,该基因编码的蛋白具有EF手型结构,蛋白序列全长为130个氨基酸,是典型的Ca2+结合蛋白,故命名为CRK45抑制的钙离子结合蛋白(CICBP)。(3)Northern blotting分析结果显示,ABA处理后crk45突变体中CICBP的表达明显升高,证明CICBP基因的确受ABA诱导,且其表达受CRK45的抑制。(4)外源75mmol/L的Ca2+处理后,crk45突变体的萌发率(30.8%)显著高于野生型(17.16%),说明在Ca2+介导下CRK45的功能是抑制种子萌发。(5)qRT-PCR检测显示,野生型中CRK45的表达受Ca2+诱导明显升高,而crk45突变体中的表达一直保持很低,说明crk45突变体是一个基因敲除突变体;Ca2+处理后crk45突变体中CICBP基因表达上调,而野生型中CICBP的表达反而降低,说明Ca2+处理下CRK45抑制CICBP基因的表达。研究表明,ABA或Ca2+处理后,CRK45通过负调控CICBP基因的表达,从而抑制拟南芥种子萌发。     

旱麦草EtAP2基因的克隆及其对干旱胁迫的响应表达

以旱麦草(Eremopyrum triticeum)为实验材料,利用RT-PCR技术从旱麦草叶片中克隆了1个AP2/ERF家族基因,命名为EtAP2(GenBank登录号KX622583)。EtAP2基因含有1 128bp开放阅读框,编码375个氨基酸,相对分子质量40.87kD,等电点为5.36。多序列比对和进化树分析表明,该基因编码蛋白具有2个AP2保守结构域,与小麦AP2/ERF家族蛋白具有较近的亲缘关系。实时荧光定量PCR分析表明,15%PEG 6000模拟干旱胁迫可诱导EtAP2基因在根和叶中表达,且在根中对干旱胁迫的响应大于叶片。研究表明,EtAP2可能参与旱麦草对干旱逆境胁迫应答的调节。     

沙鞭PvDREB基因克隆与表达特异性分析

基于沙鞭的三代转录组数据,该研究利用PCR技术克隆DREB基因,并对其进行生物信息学分析;采用实时荧光定量PCR分析该基因的表达模式以及在20%PEG-6000模拟干旱胁迫处理下的表达特征,以探讨干旱胁迫下沙鞭DREB转录因子的功能和作用,为揭示PvDREB基因响应沙鞭的耐旱分子机制奠定基础。结果表明：(1)成功克隆获得一个沙鞭DREB基因,命名为PvDREB;PvDREB基因编码区长度831 bp、编码276个氨基酸,含有典型的AP2转录因子保守结构域;PvDREB蛋白是亲水性蛋白,不具有信号肽结构,存在跨膜结构和可能的糖基化及磷酸化位点。(2)系统进化分析显示,PvDREB基因与毛竹的DREB亲缘关系较近。(3)亚细胞定位预测表明,PvDREB蛋白定位于线粒体和细胞核中。(4)qRT-PCR显示,沙鞭根、茎和叶中PvDREB均可诱导表达但差异较大,且在茎中表达量最高,叶中次之,根中最低,具有明显的组织特异性;20%PEG-6000模拟干旱下,PvDREB基因在叶中的表达量随干旱胁迫时间增加而增加,12 h时达到最高,之后逐渐下降。研究推测,沙鞭PvDREB基因受干旱胁迫诱导表达,且...     

Isolation and expression of a maize type 1 protein phosphatase

The dephosphorylation of phosphoproteins by protein phosphatases represents an important mechanism for regulating specific cellular processes in eukaryotic cells. The aim of the present study was to examine the structural and biochemical characteristics of a specific class of protein Ser/Thr phosphatases (type 1 protein phosphatases) which have received very little attention in higher plants. A cDNA clone (ZmPP1) was isolated from a maize (Zea mays L.) cDNA library. The deduced amino acid sequence is 80% identical with a 292-amino acid core region of rabbit and yeast type 1 protein phosphatase catalytic subunit. Southern blot analysis indicates that ZmPP1 may belong to a family of related genes in maize. ZmPP1 RNA was present in all maize tissues examined, indicating that it may play a fundamental role in cellular homeostasis. To demonstrate that ZmPP1 encodes an active protein phosphatase and, in an effort to characterize this gene product biochemically, high levels of ZmPP1 were expressed in Escherichia coli. Active ZmPP1 enzyme dephosphorylates rabbit phosphorylase a and is strongly inhibited by okadaic acid and by the mammalian inhibitor-2. These data show that ZmPP1 is structurally and biochemically very similar to the corresponding enzyme in animal cells. These results also suggest that the function and regulation of the higher plant type 1 protein phosphatases may be similar to the mammalian protein phosphatases.     

Impairment of NtAQP1 gene expression in tobacco plants does not affect root colonisation pattern by arbuscular mycorrhizal fungi but decreases their symbiotic efficiency under drought

We investigated in two tobacco (Nicotiana tabacum) plant lines (wildtype or antisense mutant) whether impairment in expression of the plasma membrane aquaporin gene (NtAQP1) affects the arbuscular mycorrhizal (AM) fungal colonisation pattern or the symbiotic efficiency of AM fungi. These two objectives were investigated under well-watered and drought stress conditions. Both plant lines had a similar pattern of root colonisation under well-watered and drought stress conditions. In contrast, under drought stress, AM wildtype plants grew faster than mycorrhizal antisense plants. Plant gas exchange also appeared to depend on the expression of NtAQP1 and parallelled the determined growth increments. The implications of enhanced symplastic water transport via NtAQP1 for the efficiency of the AM symbiosis under drought stress conditions are further discussed.     

中间锦鸡儿CiMYB68基因克隆及表达分析

该研究以中间锦鸡儿(Caragana intermedia)为材料,利用RACE技术克隆了CiMYB68基因的全长序列。对CiMYB68的基因组DNA和cDNA全长分析显示,CiMYB68基因无内含子,开放阅读框为852bp,编码284个氨基酸。预测CiMYB68基因编码的蛋白质等电点为8.95,分子量约为31 459.4Da。序列比对和系统进化分析表明,该蛋白和大豆的GmMYB68一致性最高,达到67%。构建了CiMYB68基因与GFP融合表达质粒,激光共聚焦显微镜观察发现,融合蛋白定位于细胞核。用实时荧光定量PCR技术对在不同胁迫条件下CiMYB68基因的表达检测结果表明,在干旱和低温处理下CiMYB68均受到不同程度的诱导,暗示CiMYB68基因可能与中间锦鸡儿响应逆境胁迫有关。     

Microspore-derived cell suspension cultures of oilseed rape as a system for studying gene expression

Abiotic stress, such as extreme temperature, drought, or excessive salinity, is one of the leading causes of crop loss worldwide. Microspore-derived (MD) cell suspension cultures of Brassica napus L. cv. Jet Neuf have been shown to be a useful system for studying the biochemistry of developing oilseeds. In the present study, we describe the application of MD cell suspension cultures of B. napus as a system for studying gene expression in response to abiotic stress, and demonstrate emybryogenic competence in cultures that have been continuously subcultured for more than 20 years. MD cell suspension cultures of B. napus L. cv Jet Neuf were exposed to low temperature or osmotic stress and the expression profile of known stress responsive genes was evaluated. The gene expression profile of BN115, a known cold-responsive gene in B. napus, was similar to that described for intact cold-acclimated plants. Likewise, two late embryogenesis abundant (Lea) genes were shown to be up-regulated in response to low temperature or osmotic stress. The results demonstrate that B. napus MD cell suspension cultures are a useful system for the investigation of changes in gene expression in plants brought about by abiotic stress.     

An analysis of the polymorphisms in a gene for being involved in drought tolerance in maize

Plant invertases catalyze the conversion of sucrose to glucose and fructose, which are distinct signals of widely varied stress-tolerance processes, including the biosynthesis and detection of hormones under water deficit. In the invertase gene family, the candidate gene ivr2 encoding plant acid-soluble invertase plays a vital role in drought tolerance. In this study, a putative genomic sequence of ivr2 including three exons and two introns was acquired and genetically analyzed using bioinformatics and statistics, based on a partial ivr2 gene sequence of the GenBank library. The ivr2 genomic sequence data from 106 maize inbred lines were obtained using five nested primer pairs. Further analysis showed that the detected polymorphic sites were mainly located in exon-1, intron-1 and exon-2 regions; High linkage disequilibrium level and low nucleotide diversity were identified at this ivr2 locus. Association mapping combined the genotypic and phenotypic data, and a total of 48 associations showed high contributions to the variations in grain yield and its components under well-watered and water-stressed conditions over 2 years of experiments. This suggested that functional polymorphisms in ivr2 were possibly associated with maize drought tolerance. This provides reference information for efficient marker-assisted selection of superior alleles in drought tolerance breeding programs.