Molecular characterization and expression analysis of a rice protein phosphatase 2C gene, OsBIPP2C1, and overexpression in transgenic tobacco conferred enhanced disease resistance and abiotic tolerance

A full-length cDNA of a rice protein phosphatase 2C gene, OsBIPP2C1 , was cloned and identified. OsBIPP2C1 is predicted to encode a 569 amino acid protein that contains phosphatase domain at its C-terminal and a relatively long N-terminal extension. Expression profiles of OsBIPP2C1 in rice seedlings upon treatments with disease resistance inducers, pathogen infection, and mechanical wounding as well as various environmental stress conditions were analyzed. Expression of OsBIPP2C1 was activated upon treatments with benzothiadiazole (BTH), salicylic acid, and hydrogen peroxide, which are signal molecules in plant disease resistance responses, and was induced during the first 48 h after inoculation with Magnaporthe grisea in BTH-treated rice seedlings. OsBIPP2C1 was also upregulated upon mechanical wounding and treatments with abscisic acid, high salt, low temperature, and drought stress. Transgenic tobacco plants overexpressing OsBIPP2C1 gene showed enhanced disease resistance against tobacco mosaic virus and Phytophthora paratisca and increased tolerance against salt and osmotic stresses. These results suggest that OsBIPP2C1 may play important roles in responses to biotic and abiotic stresses.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

The Bt gene <Emphasis Type="Italic">cry2Aa2</Emphasis> driven by a tissue specific ST-LS1 promoter from potato effectively controls <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R₁ transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.     

Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

We employed a comparative genomic approach to understand protein phosphatase 2C (PP2C)-mediated abscisic acid (ABA) signaling in the moss Physcomitrella patens. Ectopic expression of Arabidopsis (Arabidopsis thaliana) abi1-1, a dominant mutant allele of ABI1 encoding a PP2C involved in the negative regulation of ABA signaling, caused ABA insensitivity of P. patens both in gene expression of late embryogenesis abundant (LEA) genes and in ABA-induced protonemal growth inhibition. The transgenic abi1-1 plants showed decreased ABA-induced freezing tolerance, and decreased tolerance to osmotic stress. Analyses of the P. patens genome revealed that only two (PpABI1A and PpABI1B) PP2C genes were related to ABI1. In the ppabi1a null mutants, ABA-induced expression of LEA genes was elevated, and protonemal growth was inhibited with lower ABA concentration compared to the wild type. Moreover, ABA-induced freezing tolerance of the ppabi1a mutants was markedly enhanced. We provide the genetic evidence that PP2C-mediated ABA signaling is evolutionarily conserved between Arabidopsis and P. patens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession Numbers: PpABI1A-AB369256, PpABI1B-AB369255, pphn39k21-AB369257.     

The rice <Emphasis Type="Italic">OsLOL2</Emphasis> gene encodes a zinc finger protein involved in rice growth and disease resistance

Arabidopsis LSD1-related proteins that contain LSD1-like zinc finger domains have been identified to be involved in disease resistance and programmed cell death. To investigate the potential role of LSD1-related gene in rice (Oryza sativa L.), we cloned an LSD1 ortholog, OsLOL2, from the rice cDNA plasmid library. The OsLOL2 gene is predicted to encode a polypeptide of 163 amino acids with two LSD1-like zinc finger domains with 74.5% identity to those of LSD1. Southern blot analysis indicated that OsLOL2 was a single-copy gene in the rice genome. Transgenic rice lines carrying the antisense strand of OsLOL2 with decreased expression of OsLOL2 had dwarf phenotypes, and the dwarfism could be restored by exogenous GA₃ treatment, suggesting that the dwarfism was the result of a deficiency in bioactive gibberellin (GA). In agreement with this possibility, the content of endogenous bioactive GA₁ decreased in the antisense transgenic lines. Expression of OsKS1, one of the genes encoding for GA biosynthetic enzymes, was suppressed in the antisense transgenic lines. Sense transgenic lines with increased expression of OsLOL2 were more resistant to rice bacterial blight, while antisense transgenic lines were less resistant to rice bacterial blight. The OsLOL2-GFP (green fluorescence protein) fusion protein was localized in the nucleus of cells of transgenic BY2 tobacco (Nicotiana tabacum L.). These data suggest that OsLOL2 is involved in rice growth and disease resistance.     

Overexpression of <Emphasis Type="Italic">OsiSAP8</Emphasis>, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt,drought and cold stress in transgenic tobacco and rice

We describe here the isolation and characterization of OsiSAP8, a member of stress Associated protein (SAP) gene family from rice characterized by the presence of A20 and AN1 type Zinc finger domains. OsiSAP8 is a multiple stress inducible gene, induced by various stresses, namely heat, cold, salt, desiccation, submergence, wounding, heavy metals as well as stress hormone Abscisic acid. OsiSAP8 protein fused to GFP was localized towards the periphery of the cells in the epidermal cells of infiltrated Nicotiana benthamiana leaves. Yeast two hybrid analysis revealed that A20 and AN1 type zinc-finger domains of OsiSAP8 interact with each other. Overexpression of the gene in both transgenic tobacco and rice conferred tolerance to salt, drought and cold stress at seed germination/seedling stage as reflected by percentage of germination and gain in fresh weight after stress recovery. Transgenic rice plants were tolerant to salt and drought during anthesis stage without any yield penalty as compared to unstressed transgenic plants. OsiSAP8 is deposited in the Genbank with the Accession number AY345599.     

A serine/threonine protein phosphatase-like protein, CaPTC8, from Candida albicans defines a new PPM subfamily

Protein phosphatase M family (PPM; Mg²⁺-dependent protein phosphatases), which specifically dephosphorylates serine/threonine residues, consists of pyruvate dehydrogenase phosphatases, SpoIIE, adenylate cyclase and protein phosphatase type 2Cs (PP2Cs). To identify Candida albicans PP2Cs, the archetype of the PPM Ser/Thr phosphatases, we thoroughly searched the public C. albicans genome database and identified seven PP2C members. One of the PP2Cs in C. albicans, designated as CaPTC8 gene, represents a new member of PP2C genes. Northern blot analysis showed that the expression of CaPTC8 was positively responsive to high osmolarity, temperature or serum-stimulated filamentous growth. Gene disruption further demonstrated that deletion of CaPTC8 gene caused the defect of hyphal formation. Sequence analysis revealed that two conserved amino acids His and Asn in the prototypical members of the PPM family were substituted by Val and Asp in the PTC8p-like proteins. In addition, posterior analysis for site-specific profile showed that seven more sites are under the selection of functional divergence between these two groups of proteins. Three-dimensional homology modeling illustrated the signatures of the two groups in the conserved catalytic region of the protein phosphatases. Hence, CaPTC8 plays a role in stress responses and is required for the yeast-hyphal transition, and the CaPTC8-related genes are evolutionarily conserved. The phylogenetic relationships of all members of the PPM family strongly support the existence of a distinct and new subfamily of the PP2C-related proteins, PP2CR.     

DIW1 encoding a clade I PP2C phosphatase negatively regulates drought tolerance by de-phosphorylating TaSnRK1.1 in wheat

Drought seriously impacts wheat production (Triticum aestivum L.), while the exploitation and utilization of genes for drought tolerance are insufficient. Leaf wilting is a direct reflection of drought tolerance in plants. Clade A PP2Cs are abscisic acid (ABA) co-receptors playing vital roles in the ABA signaling pathway, regulating drought response. However, the roles of other clade PP2Cs in drought tolerance, especially in wheat, remain largely unknown. Here, we identified a gain-of-function drought-induced wilting 1 (DIW1) gene from the wheat Aikang 58 mutant library by map-based cloning, which encodes a clade I protein phosphatase 2C (TaPP2C158) with enhanced protein phosphatase activity. Phenotypic analysis of overexpression and CRISPR/Cas9 mutant lines demonstrated that DIW1/TaPP2C158 is a negative regulator responsible for drought resistance. We found that TaPP2C158 directly interacts with TaSnRK1.1 and de-phosphorylates it, thus inactivating the TaSnRK1.1–TaAREB3 pathway. TaPP2C158 protein phosphatase activity is negatively correlated with ABA signaling. Association analysis suggested that C-terminal variation of TaPP2C158 changing protein phosphatase activity is highly correlated with the canopy temperature, and seedling survival rate under drought stress. Our data suggest that the favorable allele with lower phosphatase activity of TaPP2C158 has been positively selected in Chinese breeding history. This work benefits us in understanding the molecular mechanism of wheat drought tolerance, and provides elite genetic resources and molecular markers for improving wheat drought tolerance.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。     

Expression of an elicitor-encoding gene from <Emphasis Type="Italic">Magnaporthe grisea</Emphasis> enhances resistance against blast disease in transgenic rice

Elicitors are molecules that stimulate defense responses in plants. Previously, an elicitor-encoding gene, named pemG1, was isolated from Magnaporthe grisea. To assess the function of pemG1 in rice (Oryza sativa L. cv. Nipponbare), the gene was cloned under a constitutive maize ubiquitin promoter and introduced into Nipponbare cultivar. The resultant plants showed stable integration and constitutive expression of the pemG1 gene. The expression of defense-related gene for phenylalanine ammonia-lyase was triggered and proline content was also increased in pemG1-expressing plants. The pemG1-expressing plants showed enhanced resistance against rice blast after inoculation with M. grisea spores, suggesting that the pemG1 expression enhances disease resistance in transgenic rice. DQ and JM contributed equally to this paper.     

植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展

蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,而PP2C磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,是高等植物中最大的蛋白磷酸酶家族,包含76个家族成员,广泛存在于生物体中。迄今为止,在植物体内已经发现了4种PP2C蛋白磷酸酶。蛋白激酶和蛋白磷酸酶协同催化蛋白质可逆磷酸化,在植物体内信号转导和生理代谢中起着重要的调节作用,蛋白质的磷酸化几乎存在于所有的信号转导途径中。大量研究表明,PP2Cs参与多条信号转导途径,包括PP2C参与ABA调控,对干旱、低温、高盐等逆境胁迫的响应,参与植物创伤和种子休眠或萌发等信号途径,其调控机制不同,但酶催化活性都依赖于Mg2+或Mn2+的浓度。植物PP2C蛋白的C端催化结构域高度保守,而N端功能各异。文中还综述了高等植物PP2C的分类、结构、ABA受体与PP2Cs蛋白互作、PP2C基因参与ABA信号途径以及其他逆境信号转导途径的研究进展。     

Heat-inducible C3HC4 type RING zinc finger protein gene from <Emphasis Type="Italic">Capsicum annuum</Emphasis> enhances growth of transgenic tobacco

Capsicum annuum RING Zinc Finger Protein 1 (CaRZFP1) gene is a novel C3HC4-type RING zinc finger protein gene which was previously isolated from a cDNA library for hot pepper plants treated of heat-shock. The CaRZFP1 was inducible to diverse environmental stresses in hot pepper plants. We introduced the CaRZFP1 into the Wisconsin 38 cultivar of tobacco (Nicotiana tabacum) by Agrobacterium mediated transformation under the control of the CaMV 35S promoter. Expression of the transgene in the transformed tobacco plants was demonstrated by RNA blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T₁ lines showed that the transgene segregated in a Mendelian fashion. Transgenic tobacco lines that expressed the CaRZFP1 gene were compared with several different empty vector lines and they exhibited enhanced growth; they have larger primary root, more lateral root, larger hypocotyls and bigger leaf size, resulting in heavier fresh weight. Enhanced growth of transgenic lines accompanied with longer vegetative growth that resulted in bigger plants with higher number of leaves. Microarray analysis revealed the up-regulation of some growth related genes in the transgenic plants which were verified by specific oligomer RNA blot analyses. These results indicate that CaRZFP1 activates and up-regulates some growth related proteins and thereby effectively promoting plant growth. N. Zeba and M. Isbat contributed equally to the work.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.