CPK12: A Ca2+-dependent protein kinase balancer in abscisic acid signaling

Ca²⁺ is believed to be a critical second messenger in ABA signal transduction. Ca²⁺-dependent protein kinases (CDPKs) are the best characterized Ca²⁺ sensors in plants. Recently, we identified an Arabidopsis CDPK member CPK12 as a negative regulator of ABA signaling in seed germination and post-germination growth, which reveals that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction. We observed that both RNA interference and overexpression of CPK12 gene resulted in ABA-hypersensitive phenotypes in seed germination and post-germination growth, suggesting a high complexity of the CPK12-mediated ABA signaling pathway. CPK12 stimulates a negative ABA-signaling regulator (ABI2) and phosphorylates two positive ABA-signaling regulators (ABF1 and ABF4), which may partly explain the ABA hypersensitivity induced by both downregulation and upregulation of CPK12 expression. Our data indicate that CPK12 appears to function as a balancer in ABA signal transduction in Arabidopsis.     

A novel rice calmodulin-like gene, <Emphasis Type="Italic">OsMSR2,</Emphasis> enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis

Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca²⁺ signals are perceived by different Ca²⁺ sensors, and calmodulin (CaM) is one of the best-characterized Ca²⁺ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei’ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca²⁺ in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.     

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca²⁺-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca²⁺-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca²⁺-binding loops. We examined Ca²⁺ binding of the protein by a Ca²⁺-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca²⁺-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.     

GhMPK17, a Cotton Mitogen-Activated Protein Kinase,Is Involved in Plant Response to High Salinity and Osmotic Stresses and ABA Signaling

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H₂O₂ level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling.     

Ectopic expression of pigeonpea cold and drought regulatory protein (CcCDR) in yeast and tobacco affords multiple abiotic stress tolerance

Pigeonpea (Cajanus cajan L.) cold and drought regulatory protein encoding gene (CcCDR) has been introduced into yeast and tobacco for its functional validation. In yeast, expression of CcCDR imparted marked tolerance against abiotic stresses exerted by PEG and NaCl. Transgenic tobacco lines, expressing CcCDR under the control of CaMV35S and rd29A promoters, when subjected to mannitol, NaCl and cold (4 °C) stress, developed into healthy plants with profuse root system, increased biomass, root length and chlorophyll content in contrast to the weak-stunted wild-type plants. Transgenic plants also showed increased levels of proline, reducing sugars and endogenous abscisic acid (ABA) content. Exogenous ABA treatment resulted in increased hypersensitivity and decreased stomatal aperture size of transgenic plants compared to wild type. Localization studies confirmed that CcCDR could enter the nucleus as revealed by intense fluorescence, indicating its plausible interaction with various nuclear proteins. The overall results amply demonstrate the intrinsic effect of CcCDR in bestowing multiple abiotic stress tolerance at cellular and whole plant levels. Accordingly, the multipotent CcCDR seems promising as a prime candidate gene to fortify crop plants with abiotic stress tolerance.     

Ectopic expression of a maize calreticulin mitigates calcium deficiency-like disorders in sCAX1-expressing tobacco and tomato

Deregulated expression of an Arabidopsis H⁺/Ca²⁺ antiporter (sCAX1) in agricultural crops increases total calcium (Ca²⁺) but may result in yield losses due to Ca²⁺ deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca²⁺ binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca²⁺ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca²⁺ transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca²⁺ within the plant matrix.     

Regulatory function of AMP1 in ABA biosynthesis and drought resistance in arabidopsis

In this study we reported the isolation of a mutant in which the reporter pVP14-LUC was highly expressed in Arabidopsis. The gene expression of maize VP14 is closely correlated with the endogenous ABA levels, and the Arabidopsis homolog of VP14, AtNCED1, encoding an enzyme of ABA biosynthesis, was up-regulated, and high ABA level was detected in the mutant. Map-based cloning revealed that the mutated gene is a novel allele of the AMP1 (Altered Meristem Program 1) which encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis. We found that the mutant displayed obvious drought tolerance, being with more lateral roots, high seed germination under mannitol, increased ABA accumulation, and highly induced gene expression of RD29A. Using the approaches of artificial microRNA gene silencing in transgenic plants, three AMP1 down-regulated lines were obtained. The AMP1 down-regulated plants exhibited a low rate of water loss, decreased stomatal aperture, and enhanced drought tolerance. These results provide evidence demonstrating the regulatory function of AMP1 in plant drought tolerance and stress responsive gene expression.     

GhCAX3 Gene,a Novel Ca2+/H+ Exchanger from Cotton,Confers Regulation of Cold Response and ABA Induced Signal Transduction

As a second messenger, Ca²⁺ plays a major role in cold induced transduction via stimulus-specific increases in [Ca²⁺]_cyt, which is called calcium signature. During this process, CAXs (Ca²⁺/H⁺ exchangers) play critical role. For the first time, a putative Ca²⁺/H⁺ exchanger GhCAX3 gene from upland cotton (Gossypium hirsutum cv. ‘YZ-1′) was isolated and characterized. It was highly expressed in all tissues of cotton except roots and fibers. This gene may act as a regulator in cotton’s response to abiotic stresses as it could be up-regulated by Ca²⁺, NaCl, ABA and cold stress. Similar to other CAXs, it was proved that GhCAX3 also had Ca²⁺ transport activity and the N-terminal regulatory region (NRR) through yeast complementation assay. Over-expression of GhCAX3 in tobacco showed less sensitivity to ABA during seed germination and seedling stages, and the phenotypic difference between wild type (WT) and transgenic plants was more significant when the NRR was truncated. Furthermore, GhCAX3 conferred cold tolerance in yeast as well as in tobacco seedlings based on physiological and molecular studies. However, transgenic plant seeds showed more sensitivity to cold stress compared to WT during seed germination, especially when expressed in N-terminal truncated version. Finally, the extent of sensitivity in transgenic lines was more severe than that in WT line under sodium tungstate treatment (an ABA repressor), indicating that ABA could alleviate cold sensitivity of GhCAX3 seeds, especially in short of its NRR. Meanwhile, we also found that overexpression of GhCAX3 could enhance some cold and ABA responsive marker genes. Taken together, these results suggested that GhCAX3 plays important roles in the cross-talk of ABA and cold signal transduction, and compared to full-length of GhCAX3, the absence of NRR could enhance the tolerance or sensitivity to cold stress, depending on seedling’s developmental stages.     

Osmotic stress and abscisic acid reduce cytosolic calcium activities in roots of Arabidopsis thaliana*

Cytosolic Ca²⁺· ([Ca²⁺]_i, and elongation growth were measured in the roots of Arabidopsis thaliana. Exposure of plant tissues to high NaCl and abscisic acid (ABA) concentrations results in a reduction in the rate of growth, but the mechanism by which growth is inhibited is not understood. Both NaCl and ABA treatments are known to influence [Ca²⁺]_i, and in this study we measured the effects of salinity and ABA on [Ca²⁺]_i in cells from the meristematic region of Arabidopsis roots. The Ca²⁺-sensitive dye Fura-2 and ratiometric techniques were used to measure [Ca²⁺]_i in cells of the root meristem region. Resting [Ca²⁺]_i was found to be between 100 and 200 μmol m^?3 in roots of untreated plants. Resting [Ca²⁺]_i changed in response to changes in the [Ca²⁺] surrounding growing roots. An increase of external [Ca²⁺] increased [Ca²⁺]_i; conversely, a decrease of external [Ca²⁺] decreased [Ca²⁺]_i. Exposure of roots to NaCl caused a rapid reduction of [Ca²⁺]_i, a response that was proportional to the external NaCl concentration. Thus, as the NaCl concentration was increased, [Ca²⁺]_i in root meristematic cells decreased. Root elongation was also inhibited in proportion to the external NaCl concentration, with maximal inhibition occurring at 120 mol m^?3 NaCl. The [Ca²⁺]_i of root meristem cells also changed in response to ABA, and the magnitude of the effect of ABA was dependent upon ABA concentration. Treatment with 0.2 mmol m^?3 ABA caused a momentary increase in [Ca²⁺]_i followed by a decrease after 15 min, but 10 mmol m^?3 ABA caused an immediate decline in [Ca²⁺]_i. There was a strong positive correlation between [Ca²⁺]_i and root elongation rates. Experiments with the ABA-deficient Arabidopsis mutant aba-3 indicated that the reduction in [Ca²⁺]_i brought about by NaCl was unlikely to be mediated via changes in endogenous ABA. Experiments with solutes such as sorbitol, KCl and NaNO₃ indicated that the effects of NaCl could be mimicked by other solutes and was not specific for NaCl.     

Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis

Comparative proteomic analysis of the Arabidopsis thaliana root microsomal fraction was performed to identify novel components of salt stress signaling. Among the salt-responsive microsomal proteins, two spots that increased upon salt treatment on a two-dimensional gel were identified as the same protein, designated annexin 1 (AnnAt1). Annexins comprise a multigene family of Ca²⁺-dependent membrane binding proteins and have been extensively studied in animal cells. AnnAt1 is strongly expressed in root but rarely in flower tissue. In this study, the results suggest that salt stress induces translocation from the cytosol to the membrane and potential turnover of existing protein. This process is blocked by EGTA treatment, implying that AnnAt1 functions in stress response are tightly associated with Ca²⁺. T-DNA insertion mutants of annAt1 and a different isoform, annAt4, displayed hypersensitivity to osmotic stress and abscisic acid (ABA) during germination and early seedling growth. The results collectively suggest that AnnAt1 and AnnAt4 play important roles in osmotic stress and ABA signaling in a Ca²⁺-dependent manner.     

Root to shoot communication and abscisic acid in calreticulin (CR) gene expression and salt-stress tolerance in grafted diploid potato clones

Potato is an important world crop but its cultivation is relatively limited by its sensitivity to salt-stress. Auto- and hetero-grafting was used to examine the effect of rootstock and abscisic acid (ABA) on expression of the Ca²⁺-storage protein calreticulin (CR) and salt-stress tolerance in potato. Sibling-selected diploid clones of potato (S. tuberosum) were utilized that are distinguished by differential root Na⁺ absorption; including type: late-maturing, LM and excluding type, early-maturing, EM under salt treatment; salt-stress sensitivity (S/T, sensitive or tolerant); and abscisic acid production (AD/AN, ABA-deficient or-normal sibling lines). CR expression, osmotic potential (OP) and leaf Ca²⁺ were measured at the end of a 5 days NaCl stress treatment applied at tuber initiation. Increased CR expression was induced by NaCl stress and associated with salt tolerance in early-maturing tolerant (EMT) and late-maturing tolerant (LMT) clones with higher levels of CR in LMT compared to the EMT clone. Early-maturing sensitive (EMS) clone salt tolerance increased when grafted onto LMT but not onto EMT rootstocks. EMS scions maintained less negative leaf OP when grafted onto LMT rootstocks than grafting onto the EMT rootstock. Exogenous ABA application induced a less negative upper leaf OP in the salt-stress sensitive AD clone but not in the AN clone. AD clones were characterized by low CR levels, which did not increase after stress. However, grafting the AD clone onto LMT increased CR expression in the AD portion of the graft combination. Salt-stress induced CR expression and is positively associated with the presence of ABA and the salt-stress tolerant phenotypes. Both, elevation in CR expression and salt tolerance in the tolerant rootstocks, were translocated to sensitive scions although highest permeation depended on the LM type. Calreticulin expression appears to be involved in ABA-induced salt tolerance and both salt-stress tolerance and CR expression appear to be regulated by the roots.     

Novel calcium-binding GTPase (AtCBG) involved in ABA-mediated salt stress signaling in Arabidopsis

We have identified a novel Ca²⁺-signal sensing GTPase (643 amino acid residues with an estimated molecular mass of 79 kDa) from the Arabidopsis genome database. This protein contains a RHO-like GTPase domain at the N-terminus (15–184 amino acids) and two calcium-binding EF-hand motifs (199–227 and 319–347 amino acids, respectively). It has the capability to bind calcium and hydrolyze GTP; in addition, its GTPase activity is regulated by changes in Ca²⁺ concentration. The expression of this gene was induced by ABA and salt stresses, and specific knock-out mutants were highly sensitive to ABA and salt treatments. These findings suggest that this protein is a novel ABA- and salt stress-related Ca²⁺ signal transducer.     

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca2+ oscillation in guard cells of Arabidopsis thaliana

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca²⁺]_cyt) in guard cells. While abscisic acid-induced [Ca²⁺]_cyt oscillation has been extensively studied, MeJA-induced [Ca²⁺]_cyt oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca²⁺]_cyt oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca²⁺ reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca²⁺]_cyt oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca²⁺]_cyt oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.     

OsCIPK31, a CBL-interacting protein kinase is involved in germination and seedling growth under abiotic stress conditions in rice plants

Calcineurin B-like protein-interacting protein kinases (CIPKs) are a group of typical Ser/Thr protein kinases that mediate calcium signals. Extensive studies using Arabidopsis plants have demonstrated that many calcium signatures that activate CIPKs originate from abiotic stresses. However, there are few reports on the functional demonstration of CIPKs in other plants, especially in grasses. In this study, we used a loss-of-function mutation to characterize the function of the rice CIPK gene OsCIPK31. Exposure to high concentrations of NaCl or mannitol effected a rapid and transient enhancement of OsCIPK31 expression. These findings were observed only in the light. However, longer exposure to most stresses resulted in downregulation of OsCIPK31 expression in both the presence and absence of light. To determine the physiological roles of OsCIPK31 in rice plants, the sensitivity of oscipk31::Ds, which is a transposon Ds insertion mutant, to abiotic stresses was examined during germination and seedling stages. oscipk31::Ds mutants exhibited hypersensitive phenotypes to ABA, salt, mannitol, and glucose. Compared with wild-type rice plants, mutants exhibited retarded germination and slow seedling growth. In addition, oscipk31::Ds seedlings exhibited enhanced expression of several stress-responsive genes after exposure to these abiotic stresses. However, the expression of ABA metabolic genes and the endogenous levels of ABA were not altered significantly in the oscipk31::Ds mutant. This study demonstrated that rice plants use OsCIPK31 to modulate responses to abiotic stresses during the seed germination and seedling stages and to modulate the expression of stress-responsive genes.     

Rice Gene <Emphasis Type="Italic">OsDSR-1</Emphasis> Promotes Lateral Root Development in <Emphasis Type="Italic">Arabidopsis</Emphasis> Under High-Potassium Conditions

Rice gene Oryza sativa Drought Stress Response-1 (OsDSR-1) was one of the genes identified to be responsive to drought stress in the panicle of rice at booting and heading stages by both microarray and quantitative real-time PCR analyses. OsDSR-1 encodes a putative calcium-binding protein, and its overexpression in Arabidopsis rendered transgenic plants to produce much shorter lateral roots (LRs) than wild-type (WT) plants in the medium supplemented with abscisic acid (ABA), suggesting that OsDSR-1 may act as a positive regulator during the process of ABA inhibition of LR development. No significant difference was observed in the total LR length between WT and transgenic plants in the media with the increase of only osmotic stress caused by NaCl, LiCl, and mannitol, while transgenic Arabidopsis seedlings appeared to produce larger root systems with longer total LR lengths under high-potassium conditions than WT seedlings. Further analysis showed that external Ca²⁺ was required for the production of larger root systems, indicating that the promotion by OsDSR-1 of the LR development of transgenic Arabidopsis seemed to occur in a Ca²⁺-dependent manner under high-potassium conditions. We propose that OsDSR-1 may function as a calcium sensor of the signal transduction pathway controlling the LR development under high-potassium conditions.     

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。