A maize calcineurin B‐like interacting protein kinase ZmCIPK42 confers salt stress tolerance

Calcineurin B‐like (CBL) and CBL‐interacting protein kinase (CIPK) play a crucial role in biotic and abiotic stress responses. However, the roles of different CIPKs in biotic and abiotic stress responses are less well characterized. In this study, we identified a mutation leading to an early protein termination of the maize CIPK gene ZmCIPK42 that undergoes a G to A mutation at the coding region via searching for genes involved in salt stress tolerance and ion homeostasis from maize with querying the EMS mutant library of maize B73. The mutant zmcipk42 plants have less branched tassel and impaired salt stress tolerance at the seedling stage. Quantitative real‐time PCR analysis revealed that ZmCIPK42was expressed in diverse tissues and was induced by NaCl stress. A yeast two‐hybrid screen identified a proteinase inhibitor (ZmMPI) as well as calcineurin B‐like protein 1 and protein 4 (ZmCBL1, ZmCBL4) as interaction partners of ZmCIPK42. These interactions were further confirmed by bimolecular fluorescence complementation in plant cells. Moreover, over‐expressing ZmCIPK42 resulted in enhanced tolerance to high salinity in both maize and Arabidopsis. These findings suggest that ZmCIPK42 is a positive regulator of salt stress tolerance and is a promising candidate gene to improve salt stress tolerance in maize through genetic manipulation.     

Overexpression of a putative maize calcineurin B-like protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> confers salt tolerance

The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plants. Although extensive studies and remarkable progress have been made in Arabidopsis (Arabidopsis thaliana) CBLs, their functions in other plant species are still quite limited. Here, we report the cloning and functional characterization of ZmCBL4, a novel CBL gene from maize (Zea mays). ZmCBL4 encodes a putative homolog of the Arabidopsis CBL4/SOS3 protein, with novel properties. ZmCBL4 has one copy in maize genome and harbors seven introns in its coding region. ZmCBL4 expressed differentially in various organs of the maize plants at a low level under normal condition, and its expression was regulated by NaCl, LiCl, ABA and PEG treatments. Expression of 35S::ZmCBL4 not only complemented the salt hypersensitivity in Arabidopsis sos3 mutant, but also enhanced the salt tolerance in Arabidopsis wild type at the germination and seedling stages. Moreover, the LiCl tolerance in all of the ZmCBL4-expressing lines increased more significantly as compared with the NaCl tolerance, and in consistent with this, it was found that the expression of Arabidopsis AtNHX8, a putative plasma membrane Li⁺/H⁺ antiporter gene identified recently, was induced in these transgenic lines under LiCl stress. The ZmCBL4-expressing Arabidopsis lines accumulated less Na⁺ and Li⁺ as compared with the control plants. This study has identified a putative maize CBL gene which functions in the salt stress-elicited calcium signaling and thus in the tolerance to salinity. Database accession number: EF405963.     

Expression of a novel <Emphasis Type="Italic">OSPGYRP</Emphasis> (rice proline-, glycine- and tyrosine-rich protein) gene,which is involved in vesicle trafficking,enhanced cold tolerance in <Emphasis Type="Italic">E. coli</Emphasis>

A novel OSPGYRP gene encoding a rice proline-, glycine- and tyrosine-rich protein was isolated from cold-stress treated rice seedlings using suppression subtractive hybridization. Both amino acid sequence analysis and subcellular localization confirm that OsPGYRP is a novel protein involved in vesicle trafficking. The expression of the OSPGYRP gene was induced by cold, salt, and osmotic stress. In addition, expression of the OSPGYRP gene in E. coli increased the resistance to cold stress. These results show that OsPGYRP is a novel protein involved in vesicle trafficking and plays an important role in plant adaptation to stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

Insulinrecruits glucose transporter 4 (GLUT-4) vesicles from intracellularstores to the plasma membrane in muscle and adipose tissue by specificinteractions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein targetreceptor (SNARE) protein VAMP-2 and the target membrane SNARE proteinsyntaxin 4. Although GLUT-4 vesicle trafficking has been intenselystudied, few have focused on the mechanism by which the SNAREsthemselves localize to specific membrane compartments. We therefore setout to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4;cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporterconstructs. In contrast, the longer 25-amino acid transmembrane domainof syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a completeconversion to cis-Golgi compartmentalization that wasindistinguishable from syntaxin 5. These data support a model whereinshort transmembrane domains (17 amino acids) direct thecis-Golgi localization of syntaxins, whereas longtransmembrane domains (23 amino acids) direct plasma membrane localization.

     

Maize ZmVPP5 is a truncated Vacuole H+‐PPase that confers hypersensitivity to salt stress

In plants, Vacuole H⁺‐PPases (VPPs) are important proton pumps and encoded by multiple genes. In addition to full‐length VPPs, several truncated forms are expressed, but their biological functions are unknown. In this study, we functionally characterized maize vacuole H⁺‐PPase 5 (ZmVPP5), a truncated VPP in the maize genome. Although ZmVPP5 shares high sequence similarity with ZmVPP1, ZmVPP5 lacks the complete structure of the conserved proton transport and the inorganic pyrophosphatase‐related domain. Phylogenetic analysis suggests that ZmVPP5 might be derived from an incomplete gene duplication event. ZmVPP5 is expressed in multiple tissues, and ZmVPP5 was detected in the plasma membrane, vacuole membrane and nuclei of maize cells. The overexpression of ZmVPP5 in yeast cells caused a hypersensitivity to salt stress. Transgenic maize lines with overexpressed ZmVPP5 also exhibited the salt hypersensitivity phenotype. A yeast two‐hybrid analysis identified the ZmBag6‐like protein as a putative ZmVPP5‐interacting protein. The results of bimolecular luminescence complementation (BiLC) assay suggest an interaction between ZmBag6‐like protein and ZmVPP5 in vivo. Overall, this study suggests that ZmVPP5 might act as a VPP antagonist and participate in the cellular response to salt stress. Our study of ZmVPP5 has expanded the understanding of the origin and functions of truncated forms of plant VPPs.     

Cloning and characterization of the SnRK2 gene family from <Emphasis Type="Italic">Zea mays</Emphasis>

The SnRK2 gene family is a group of plant-specific protein kinases that has been implicated in ABA and abiotic stress signaling. We found 11 SnRK2s in maize, assigned names from ZmSnRK2.1 to ZmSnRK2.11 and cloned ten of them. By analyzing the gene structure of all the SnRK2s from Arabidopsis, rice, and maize, we found seven exons that were conserved in length among most of the SnRK2s. Although the C-terminus was divergent, we found seven conserved motifs. Of these, motif 1 was common to all of the SnRK2 genes. Based on phylogenetic analysis using the kinase domain and motif 1, the SnRK2s were divided into three groups. Motifs 4 and 5 were found specifically in group I, and many genes of this group have been confirmed to be induced by ABA. This result suggests that these two motifs mediate the ABA response. The expression patterns of ZmSnRK2 genes were characterized by using quantitative real-time RCR, which revealed that ZmSnRK2 genes were induced by one or more abiotic stress treatments and therefore may play important roles in maize stress responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Huai and M. Wang have contributed equally to this paper.     

Characterization of eight CBL genes expressions in maize early seeding development

Calcium (Ca²⁺) has been firmly established as ubiquitous second messengers functioning in plant growth development and response to environmental conditions. Calcineurin B-like (CBL) proteins, a unique group of calcium sensors, play a key role in plant response to various abiotic stresses. Here, eight ZmCBLs genes were retrieved. In terms of the gene structure, maize CBL gene had greater variability compared with rice and Arabidopsis CBLs. Phylogenetic analysis revealed that ZmCBL proteins display a close relation to OsCBLs and a little far relation to AtCBLs. Expression analysis indicated that all the eight ZmCBLs expressions were regulated by low potassium and in a tissue-dependent manner. In general, the ZmCBLs expressions in roots were more sensitive under low potassium environment, especially for ZmCBL5, 6 and 8. In leaves, only ZmCBL3, 8 and 10 expressions were upregulated. Moreover, the expression patterns of the ZmCBLs in tissues of germinated seeds and seedlings were also analyzed. The results showed that all the expressions of ZmCBLs were tissue specific except for ZmCBL6, suggesting that they may involve in seed germination and seedlings’ early growth.     

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

Hormone‐ and stress‐induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone‐induced ubiquitination plays a crucial role to determine the half‐life of key negative regulators of hormone signaling. For ABA signaling, the degradation of clade‐A PP 2Cs, such as PP 2 CA or ABI 1, is a complementary mechanism to PYR / PYL / RCAR ‐mediated inhibition of PP 2C activity. ABA promotes the degradation of PP 2 CA through the RGLG 1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG 1 with PP 2 CA given that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG 1 and promotes nuclear interaction with PP 2 CA . We found RGLG 1 is myristoylated in vivo , which facilitates its attachment to the plasma membrane. ABA inhibits the myristoylation of RGLG 1 through the downregulation of N‐myristoyltransferase 1 ( NMT 1 ) and promotes nuclear translocation of RGLG 1 in a cycloheximide‐insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP 2 CA protein levels and the formation of RGLG 1–receptor–phosphatase complexes. We show that RGLG 1 ^Gly2Ala mutated at the N‐terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABA and salt or osmotic stress. RGLG 1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG 1– PP 2 CA – PYL 8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG 1– PP 2 CA interaction and hence PP 2 CA degradation.     

ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions

In maize (Zea mays), the mitogen‐activated protein kinase ZmMPK5 has been shown to be involved in abscisic acid (ABA)‐induced antioxidant defence and to enhance the tolerance of plants to drought, salt stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, using ZmMPK5 as bait in yeast two‐hybrid screening, a protein interacting with ZmMPK5 named ZmABA2, which belongs to a member of the short‐chain dehydrogenase/reductase family, was identified. Pull‐down assay and bimolecular fluorescence complementation analysis and co‐immunoprecipitation test confirmed that ZmMPK5 interacts with ZmABA2 in vitro and in vivo. Phosphorylation of Ser173 in ZmABA2 by ZmMPK5 was shown to increase the activity of ZmABA2 and the protein stability. Various abiotic stimuli induced the expression of ZmABA2 in leaves of maize plants. Pharmacological, biochemical and molecular biology and genetic analyses showed that both ZmMPK5 and ZmABA2 coordinately regulate the content of ABA. Overexpression of ZmABA2 in tobacco plants was found to elevate the content of ABA, regulate seed germination and root growth under drought and salt stress and enhance the tolerance of tobacco plants to drought and salt stress. These results suggest that ZmABA2 is a direct target of ZmMPK5 and is involved in ABA biosynthesis and functions.     

Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1

Summary Plasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase, the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADH-oxidase also inhibited WST-1 reduction, but the impermeative SH-blocking reagentpara-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils.Abbreviations WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt - MTT 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium, monosodium salt - MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymemoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt - TTFA thenoyltrifluoroacetone - pCMBS p-chloromercuriphenylsul-fonic acid - SOD Superoxide dismutase - PMOR plasma membrane - NADH oxidoreductase - PMS phenazine methosulfate - PMA phorbol myristate acetate     

Pepper pathogenesis‐related protein 4c is a plasma membrane‐localized cysteine protease inhibitor that is required for plant cell death and defense signaling

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis‐related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT‐triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease‐inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H₂O₂ and significant induction of some defense‐response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT‐triggered resistance responses, and enabled Xcv proliferation in infected leaves. H₂O₂ accumulation, cell‐death induction, and defense‐response gene expression were distinctly reduced in CaPR4c‐silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.     

Genome-wide identification,characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize,and functional analysis of its role in salt tolerance

Na⁺, K⁺ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na⁺/H⁺ exchangers (ZmNHXs), 16 cation/H⁺ exchanger (ZmCHXs), and 6 K⁺ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and most of them were localized to plasma membrane or tonoplast. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analysis of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding.     

非生物逆境胁迫下ZmCIPK10基因表达分析

干旱、高盐、低温、高温等非生物逆境严重影响植物的生长发育,研究参与逆境胁迫应答基因具有重要的理论意义和应用价值。从玉米中克隆了一个CIPK蛋白激酶基因,暂时命名为ZmCIPK10。该基因全长2 730 bp,转录长度2 100 bp,编码438个氨基酸。顺式元件分析发现在基因启动子区域存在ABRE、HSE、TC-rich repeats等推测的逆境顺势元件。荧光实时定量PCR结果表明ZmCIPK10在干旱、低温、盐胁迫下表达量上调,在ABA、高温胁迫下表达量下调。研究结果初步证实ZmCIPK10基因响应非生物逆境胁迫,为ZmCIPK10参与植物逆境信号途径及其功能研究提供理论依据。     

A teosinte-derived allele of an HKT1 family sodium transporter improves salt tolerance in maize

The sodium cation (Na⁺) is the predominant cation with deleterious effects on crops in salt-affected agricultural areas. Salt tolerance of crop can be improved by increasing shoot Na⁺ exclusion. Therefore, it is crucial to identify and use genetic variants of various crops that promote shoot Na⁺ exclusion. Here, we show that a HKT1 family gene ZmNC3 (Zea mays L. Na⁺ Content 3; designated ZmHKT1;2) confers natural variability in shoot-Na⁺ accumulation and salt tolerance in maize. ZmHKT1;2 encodes a Na⁺-preferential transporter localized in the plasma membrane, which mediates shoot Na⁺ exclusion, likely by withdrawing Na⁺ from the root xylem flow. A naturally occurring nonsynonymous SNP (SNP947-G) increases the Na⁺ transport activity of ZmHKT1;2, promoting shoot Na⁺ exclusion and salt tolerance in maize. SNP947-G first occurred in the wild grass teosinte (at a allele frequency of 43%) and has become a minor allele in the maize population (allele frequency 6.1%), suggesting that SNP947-G is derived from teosinte and that the genomic region flanking SNP947 likely has undergone selection during domestication or post-domestication dispersal of maize. Moreover, we demonstrate that introgression of the SNP947-G ZmHKT1;2 allele into elite maize germplasms reduces shoot Na⁺ content by up to 80% and promotes salt tolerance. Taken together, ZmNC3/ZmHKT1;2 was identified as an important QTL promoting shoot Na⁺ exclusion, and its favourable allele provides an effective tool for developing salt-tolerant maize varieties.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Case study of a biological control: <Emphasis Type="Italic">Geobacillus caldoxylosilyticus</Emphasis> (IRD) contributes to alleviate salt stress in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants

The inevitable exposure of crop plants to salt stress is a major environmental problem emerged from the presence of excess NaCl radicals in the soil. Handling the problem in maize plants using a biological agent was the main interest of the present study. The non-pathogenic, halophytic, facultative aerobic bacterium Geobacillus caldoxylosilyticus IRD that was isolated from Marakopara pond in the Atoll Tikehau (French Polynesian, 2005) and found tolerant to salt stress until 3.5% NaCl (w/v). An artificial symbiosis was achieved by inoculating Geobacillus sp. into 5-day-old maize cultivars of triple hybrids (321 and 310) and singlet hybrids (10 and 162). Thereafter, maize seedlings were exposed to 350 mmol NaCl for 10 days. The data revealed that Geobacillus sp. had interacted with salinized maize and improved maize overall growth, dry weight and relative water content. Na⁺ accumulation was six times less and Cl⁻ accumulation was 13 times less in the tips of salinized maize seedlings upon Geobacillus sp. inoculation. Salinized maize without Geobacillus viewed decayed cortical cells of seedlings. In addition, proline content was two times higher in salinized seedlings lacking Geobacillus. Photosynthetic pigments and antioxidant enzymes were significantly regulated upon inoculation. Beyond this study, we presented a novel insight into a possible role of Geobacillus caldoxylosilyticus bacteria in controlling/protecting maize plants against high salt stress.