Comprehensive structural,interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice

Calcium ion is involved in diverse physiological and developmental pathways. One of the important roles of calcium is a signaling messenger, which regulates signal transduction in plants. CBL (calcineurin B-like protein) is one of the calcium sensors that specifically interact with a family of serine–threonine protein kinases designated as CBL-interacting protein kinases (CIPKs). The coordination of these two gene families defines complexity of the signaling networks in several stimulus-response-coupling during various environmental stresses. In Arabidopsis, both of these gene families have been extensively studied. To understand in-depth mechanistic interplay of CBL–CIPK mediated signaling pathways, expression analysis of entire set of CBL and CIPK genes in rice genome under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages) were done using microarray expression data. Interestingly, expression analysis showed that rice CBLs and CIPKs are not only involved in the abiotic stress but their significant role is also speculated in the developmental processes. Chromosomal localization of rice CBL and CIPK genes reveals that only OsCBL7 and OsCBL8 shows tandem duplication among CBLs whereas CIPKs were evolved by many tandem as well as segmental duplications. Duplicated OsCIPK genes showed variable expression pattern indicating the role of gene duplication in the extension and functional diversification of CIPK gene family in rice. Arabidopsis SOS3/CBL4 related genes in rice (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were employed for interaction studies with rice and Arabidopsis CIPKs. OsCBLs and OsCIPKs are not only found structurally similar but likely to be functionally equivalent to Arabidopsis CBLs and CIPKs genes since SOS3/CBL4 related OsCBLs interact with more or less similarly to rice and Arabidopsis CIPKs and exhibited an interaction pattern comparable with Arabidopsis SOS3/CBL4.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Differential gene expression in response to brown planthopper feeding in rice

Plant responses to herbivores are complex. 108 cDNA clones representing genes relating to plant responses to chewing insect-feeding, pathogen infection, wounding and other stresses were collected. Northern blot and cDNA array analysis were employed to investigate gene expression regulated by piercing-sucking insect, brown planthopper (BPH), Nilaparvata lugens (Homoptera: Dephacidae) on both the resistant and susceptible rice genotypes. After BPH feeding in rice for 72 h, the expression of most tested genes was affected. 14 genes in resistant rice variety B5 and 44 genes in susceptible MH63 were significantly up- or down-regulated. Most of the well-regulated genes were grouped in the categories of signaling pathways, oxidative stress/apoptosis, wound-response, drought-inducible and pathogen-related proteins. Those related to the flavonoid pathway, aromatic metabolidsm and the octadecanoid pathway were mostly kept unchanged or down-regulated. Our results indicate that BPH feeding induces plant responses which would take part in a jasmonic acid-independent pathway and crosstalk with those related to abiotic stress, pathogen invasion and phytohormone signaling pathways.     

Sequence and expression analysis of the thioredoxin protein gene family in rice

Thioredoxin (Trx) proteins play important biological functions in cells by changing redox via thioldisulfied exchange. This system is especially widespread in plants. Through database search, we identified 30 potential Trx protein-encoding genes (OsTrx) in rice (Oryza sativa L.). An analysis of the complete set of OsTrx proteins is presented here, including chromosomal location, conserved motifs, domain duplication, and phylogenetic relationships. Our findings suggest that the expansion of the Trx gene family in rice, in large part, occurred due to gene duplication. A comprehensive expression profile of Trx genes family was investigated by analyzing the signal data of this family extracted from the whole genome microarray analysis of Minghui 63 and Zhenshan 97, two indica parents, and their hybrid Shanyou 63, using 27 different tissues representing the entire life cycle of rice. Results revealed specific expression of some members at germination transition as well as the 3-leaf stage during the vegetative growth phase of rice. OsTrx genes were also found to be differentially up- or down-regulated in rice seedlings subjected to treatments of phytohormones and light/dark conditions. The expression levels of the OsTrx genes in the different tissues and under different treatments were also checked by RT-PCR analysis. The identification of OsTrx genes showing differential expression in specific tissues among different genotypes or in response to different environmental cues could provide a new avenue for functional analyses in rice.     

Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Retrograde regulation of nuclear gene expression in CW-CMS of rice

The CW-cytoplasmic male sterility (CMS) line has the cytoplasm of Oryza rufipogon Griff, and mature pollen is morphologically normal under an optical microscope but lacks the ability to germinate; restorer gene Rf17 has been identified as restoring this ability. The difference between nuclear gene expression in mature anthers was compared for the CW-CMS line, [cms-CW] rf17rf17, and a maintainer line with normal cytoplasm of Oryza sativa L., [normal] rf17rf17. Using a 22-k rice oligoarray we detected 58 genes that were up-regulated more than threefold in the CW-CMS line. Expression in other organs was further investigated for 20 genes using RT-PCR. Five genes, including genes for alternative oxidase, were found to be preferentially expressed in [cms-CW] rf17rf17 but not in [normal] rf17rf17 or [cms-CW] Rf17Rf17. Such [cms-CW] rf17rf17-specific gene expression was only observed in mature anthers but not in leaves, stems, or roots, indicating the presence of anther-specific mitochondrial retrograde regulation of nuclear gene expression, and that Rf17 has a role in restoring the ectopic gene expression. We also used a proteomic approach to discover the retrograde regulated proteins and identified six proteins that were accumulated differently. These results reveal organ-specific induced mitochondrial retrograde pathways affecting nuclear gene expression possibly related to CMS. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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.     

Induction of a polyubiquitin gene promoter by dehydration stresses in transformed rice cells

The expression of the maize polyubiquitin gene promoter UBI1 in rice cells has been used to study the involvement of ubiquitin in cell protection responses to dehydration caused by osmotic, saline or freezing stress. The effect of these stresses on UBI1 activity was investigated by the use of stably transformed rice calli (UBI1:GUS), as well as by transient expression experiments performed with cell lines with high or low tolerance to each type of stress. The theoretical analysis of the UBI1 promoter shows several putative stress-regulated boxes that could account for the stress-related UBI1 induction pattern described in this work. We suggest that the study of the differential UBI1 promoter-driven expression in rice cell lines with different level of tolerance to stress might be useful to elucidate complex signal transduction pathways in response to dehydration stresses in monocots.     

OsITL1 gene encoding an inositol 1,3,4-trisphosphate 5/6-kinase is a negative regulator of osmotic stress signaling

The expression of the gene OsITL1 coding for the inositol 1,3,4-trisphosphate 5/6-kinase protein was induced by 200 mM NaCl or dehydration. The expression of OsITL1 in response to NaCl and dehydration suggests the possible functions of OsITL1 in osmotic stress responses; however, physiological tests indicate that the expression of OsITL1 in tobacco decreases tolerance to NaCl during germination and seedling development. This result was consistent with that obtained on treatment of mature tobacco seedlings with NaCl (200 mM), suggesting that OsITL1 inversely regulates plant responses to osmotic stress.     

水稻根系响应镉胁迫的蛋白质差异表达

为探讨水稻根系对镉胁迫的分子生理响应,以抗镉水稻PI312777和镉敏感水稻IR24为材料,设置Cd~(2+)浓度为0、50和100μmol/L的水培试验,处理7 d后分析了水稻根系的蛋白质差异表达。结果表明,在镉胁迫下水稻PI312777和IR24根系有18个蛋白质发生了差异表达,其中的12个得到MALDI-TOF/MS鉴定。这些鉴定的蛋白功能可分四类:(1)与活性氧(ROS)胁迫相关的过氧化物酶(POD)、蛋氨酸腺苷转移酶(MAT)、类萌发素蛋白前体;(2)与谷胱甘肽(GSH)合成相关的S-腺苷甲硫氨酸合成酶(SAMS)、谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH);(3)与逆境胁迫相关的ABA胁迫诱导蛋白含HVA22域蛋白、ABA-胁迫-成熟诱导蛋白5(ASR5);(4)与细胞分裂调控相关的GTP结合核蛋白Ran-2。镉胁迫下SAMS和GTP结合核蛋白Ran-2在两种水稻根系均发生上调表达;MAT、POD、类萌发素蛋白前体和GS发生下调表达;依赖NADP-GDH、GDH和磷酸甘油酸变位酶在IR24根部均发生下调表达,在PI312777根部仅在100μmol/L Cd~(2+)处理发生下调表达;含HVA22域蛋白在PI312777根部上调表达,在IR24根部发生下调表达;ASR5在PI312777根部上调表达,在IR24根部的表达无显著差异;100μmol/L Cd~(2+)胁迫下60S酸性核糖体蛋白P0在水稻PI312777根部表达下调,在IR24根部表达上调。可见,镉胁迫使水稻根部ROS增加,形成氧化胁迫反应,造成毒害作用,而水稻根通过调节SAMS和GS提高GSH合成降低镉毒害。ASR5和HVA22蛋白等逆境胁迫蛋白的表达差异则是水稻品种间抗性差异的重要原因之一。     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis

MicroRNA398 targets two Cu/Zn superoxide dismutases (CSD1 and CSD2) in higher plants. Previous investigations revealed both decreased miR398 expression during high Cu²⁺ or paraquat stress and increased expression under low Cu²⁺ or high sucrose in the growth medium. Here, we show that additional abiotic stresses such as ozone and salinity also affect miR398 levels. Ozone fumigation decreased miR398 levels that were gradually restored to normal levels after relieved from the stress. Furthermore, miR398 levels decreased in Arabidopsis leaves infiltrated with avirulent strains of Pseudomonas syringae pv. tomato, Pst DC3000 (avrRpm1 or avrRpt2) but not the virulent strain Pst DC3000. To our knowledge, miR398 is the first miRNA shown to be down-regulated in response to biotic stress (P. syringae). CSD1, but not CSD2, mRNA levels were negatively correlated with miR398 levels during ozone, salinity and biotic stress, suggesting that CSD2 regulation is not strictly under miR398 control during diverse stresses. Overall, this study further establishes a link between oxidative stress and miR398 in Arabidopsis.     

水稻OsZAT12基因响应非生物胁迫和植物激素的研究

C2H2锌指蛋白是真核生物体内一类重要的转录因子,在植物生长发育和应对非生物胁迫方面具有重要作用。实验室前期克隆了水稻C2H2锌指蛋白OsZAT12,该基因在水稻根中特异表达,定位于细胞核,异源过表达OsZAT12的拟南芥植株矮小。为进一步研究OsZAT12在水稻中的功能,该文分析了OsZAT12的启动子元件和转录活性,并采用qRT-PCR技术分析OsZAT12在非生物胁迫和植物激素处理下的响应模式。结果表明:(1)OsZAT12含有2个典型的C2H2锌指结构域和1个EAR motif,具有转录抑制活性,该基因的启动子中含有与非生物胁迫和植物激素相关的元件。(2)对野生型水稻进行非生物胁迫和激素处理发现,低温胁迫(4 ℃)和激素脱落酸(ABA)处理显著下调OsZAT12的表达; 而渗透胁迫(20% PEG 6 000)、激素油菜素甾醇(BR)或吲哚-3-乙酸(IAA)处理则显著上调OsZAT12的表达,这说明OsZAT12介导了水稻应对多种非生物胁迫和激素的变化。(3)利用含35S启动子的过表达载体和CRISPR/Cas9基因编辑技术分别得到纯合的OsZAT12过表达植株和OsZAT12敲除植株。(4)对过表达OsZAT12的水稻表型观察发现,相比于野生型,OsZAT12过表达植株在分蘖期、抽穗期和成熟期这3个时期的株高均显著降低; OsZAT12敲除植株的株高与野生型虽无明显差异,但每株穗数和结实率均显著低于野生型,这说明OsZAT12影响了水稻株型、穗型及结实率等农艺性状的建成。(5)实验进一步表明,过表达OsZAT12降低了水稻对外源ABA的敏感性,而OsZAT12敲除植株则相反。因此推测,OsZAT12对植株生长发育的影响可能与该基因响应多种非生物胁迫和激素信号的调控有关,该研究结果为将来利用OsZAT12进行水稻耐逆稳产分子设计育种提供了依据。