A rice (Oryza sativa L.) MAP kinase gene, OsMAPK44, is involved in response to abiotic stresses

We have isolated and characterized a putative rice MAPK gene (designated OsMAPK44) encoding for a protein of 593 amino acids that has the MAPK family signature and phosphorylation activation motif, TDY. Alignment of the predicted amino acid sequences of OsMAPK44 showed high homology with other rice MAPKs. Under normal conditions, the OsMAPK44 gene is highly expressed in root tissues, but relatively less in leaf and stem tissues of the japonica type rice plant (O. sativa L. Donggin). mRNA expression of the gene is highly inducible by salt and drought treatment, but not by cold treatment. Moreover, the mRNA level of the OsMAPK44 is up-regulated by exogenously applied Abscisic acid (ABA) and H₂O₂. When we compared the OsMAPK44 gene expression level between a salt sensitive indica cultivar (IR64) and a salt resistant indica cultivar (Pokkali), they showed some difference in expression kinetics with the salt treatment. OsMAPK44 gene expression in Pokkali was slightly up-regulated within 30 min and then disappeared rapidly, while IR64 maintained its expression for 1 h following down-regulation. Under the salinity stress, OsMAPK44 overexpression transgenic rice plants showed less damage and greater ratio of potassium and sodium than OsMAPK44 suppressed transgenic lines did, suggesting that OsMAPK44 may have a role to prevent damages due to working for favorable ion balance in the presence of salinity.     

Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.)

Mitogen-activated protein kinases (MAPK) signalling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signalling in plants, aMAPK cDNA clone, OsMAPK33, was isolated from rice. The gene is mainly induced by drought stress. In phylogenetic analysis, OsMAPK33 (Os02g0148100) showed approximately 47-93% identity at the amino acid level with other plant MAPKs. It was found to exhibit organ-specific expression with relatively higher expression in leaves as compared with roots or stems, and to exist as a single copy in the rice genome. To investigate the biological functions of OsMAPK33 in rice MAPK signalling, transgenic rice plants that either overexpressed or suppressed OsMAPK33 were made. Under dehydration conditions, the suppressed lines showed lower osmotic potential compared with that of wild-type plants, suggesting a role of OsMAPK33 in osmotic homeostasis. Nonetheless, the suppressed lines did not display any significant difference in drought tolerance compared with their wild-type plants. With increased salinity, there was still no difference in salt tolerance between OsMAPK33-suppressed lines and their wild-type plants. However, the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+/Na+ ratio inside the cell than that in the wild-type plants and OsMAPK33-suppressed lines. These results suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. Gene expression profiling of OsMAPK33 transgenic lines through rice DNA chip analysis showed that OsMAPK33 altered expression of genes involved in ion transport. Further characterization of downstream components will elucidate various biological functions of this novel rice MAPK.     

水稻愈伤组织形成过程中甲基化对OsMAPK2的表达调控

利用MSAP方法从经过5-azaC处理和未处理的水稻愈伤组织中获得了1个存在甲基化位点的片段。测序后比对分析表明,该片段为水稻MAPK家族OsMAPK2基因的5′端区域。该基因5′端区域有一个CpG岛,与拟南芥AtMAPK12基因序列高度相似。利用实时定量PCR和HpaII-McrBC PCR分析了在水稻芽段受生长素刺激后形成愈伤组织过程中OsMAPK2基因的表达与甲基化的关系。结果表明:该基因表达量与基因5′端甲基化水平相对应,甲基化调控基因的表达。在愈伤组织形成过程中2.0mg/L的2,4-D诱导该基因5′端区域去甲基化,使基因表达,而长时间(100h)的诱导后或导致重新甲基化,基因表达量降低;低浓度的2,4-D(0.5和1.0mg/L)也可以产生同样的趋势,但是基因的表达量低于2.0mg/L的2,4-D的诱导;高浓度的2,4-D(5.0mg/L)可以在较短的时间内完成对基因的诱导和抑制。     

OsMAPK6, a mitogen‐activated protein kinase,influences rice grain size and biomass production

Grain size is an important agronomic trait in determining grain yield. However, the molecular mechanisms that determine the final grain size are not well understood. Here, we report the functional analysis of a rice (Oryza sativa L.) mutant, dwarf and small grain1 (dsg1), which displays pleiotropic phenotypes, including small grains, dwarfism and erect leaves. Cytological observations revealed that the small grain and dwarfism of dsg1 were mainly caused by the inhibition of cell proliferation. Map‐based cloning revealed that DSG1 encoded a mitogen‐activated protein kinase (MAPK), OsMAPK6. OsMAPK6 was mainly located in the nucleus and cytoplasm, and was ubiquitously distributed in various organs, predominately in spikelets and spikelet hulls, consistent with its role in grain size and biomass production. As a functional kinase, OsMAPK6 interacts strongly with OsMKK4, indicating that OsMKK4 is likely to be the upstream MAPK kinase of OsMAPK6 in rice. In addition, hormone sensitivity tests indicated that the dsg1 mutant was less sensitive to brassinosteroids (BRs). The endogenous BR levels were reduced in dsg1, and the expression of several BR signaling pathway genes and feedback‐inhibited genes was altered in the dsg1 mutant, with or without exogenous BRs, indicating that OsMAPK6 may contribute to influence BR homeostasis and signaling. Thus, OsMAPK6, a MAPK, plays a pivotal role in grain size in rice, via cell proliferation, and BR signaling and homeostasis.     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced in response to cold and gibberellin

Ca²⁺-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca²⁺-regulated serine/threonine protein kinase in plants and their genes are encoded by a multigene family. CDPKs are important components in signal transduction, but the precise role of each individual CDPK is still largely unknown. A CDPK gene designated as OsCDPK13 was cloned from rice seedlings and it showed a high level of sequence similarities to rice and other plant CDPK genes. OsCDPK13 contains all conserved regions found in CDPKs. It was a single copy gene and was highly expressed in root and leaf sheath tissues of rice seedlings. OsCDPK13 expression was increased in leaf sheath segments treated with gibberellin or subjected to cold stress. The results in this investigation, together with our previous studies, suggest that OsCDPK13 may be an important signaling component in rice seedlings under cold stress condition and in response to gibberellin.     

rHsp90 gene expression in response to several environmental stresses in rice (Oryza sativa L.).

In this study, the gene for a rice (Oryza sativa L.) 90 kDa heat shock protein (rHsp90, GenBank accession no. AB037681) was identified by screening rice root cDNAs that were up-regulated under carbonate (NaHCO(3)) stress using the method of differential display, and cloned. The open-reading-frame of rHsp90-cDNA was predicted to encode a protein containing 810 amino acids, which showed high similarity to proteins in Hordeum vulgare (accession no. X67960) and Catharathus roseus (accession no. L14594). Further studies showed that rHsp90 mRNA accumulated following exposure to several abiotic stresses, including salts (NaCl, NaHCO(3) and Na(2)CO(3)), desiccation (using polyethylene glycol), high pH (8.0 and 11.0) and high temperature (42 and 50 degrees C). Yeast (Saccharomyces cerevisiae) over-expressing rHsp90 exhibited greater tolerance to NaCl, Na(2)CO(3) and NaHCO(3) and tobacco seedlings over-expressing rHsp90 could tolerate salt concentrations as high as 200 mM NaCl, whereas untransformed control seedlings couldn't. These results suggest that rHsp90 plays an important role in multiple environmental stresses.     

Role of the JIP4 scaffold protein in the regulation of mitogen-activated protein kinase signaling pathways

The c-Jun NH2-terminal kinase (JNK)-interacting protein (JIP) group of scaffold proteins (JIP1, JIP2, and JIP3) can interact with components of the JNK signaling pathway and potently activate JNK. Here we describe the identification of a fourth member of the JIP family. The primary sequence of JIP4 is most closely related to that of JIP3. Like other members of the JIP family of scaffold proteins, JIP4 binds JNK and also the light chain of the microtubule motor protein kinesin-1. However, the function of JIP4 appears to be markedly different from other JIP proteins. Specifically, JIP4 does not activate JNK signaling. In contrast, JIP4 serves as an activator of the p38 mitogen-activated protein (MAP) kinase pathway by a mechanism that requires the MAP kinase kinases MKK3 and MKK6. The JIP4 scaffold protein therefore appears to be a new component of the p38 MAP kinase signaling pathway.     

Two novel protein kinase genes,OsMSRPK1 andOsMSURPK2, are regulated by diverse environmental stresses in rice

Two novel rice (Oryza sativa L.) protein kinase (PK) genes have been isolated.OsMSRPK1 andOsMSURPK2, which most likely exist as single-copy genes in the rice genome, encode 693 and S03 amino acids polypeptide, respectively, and have the serine/threonine kinase domain of cyclin dependent protein kinase (OsMSRPK1), or the serine/threonine kinase domain and NAF domain (OsMSURPK2). Steady-state mRNA analyses of these PKs, with constitutive expression in the leaves of two-week-old seedlings, revealed thatOsMSRPK1 is up-regulated upon exposure to environmental stresses, whereasOsMSVRPK2 is down-regulated by these same stresses. Furthermore, the two PKs are developmentally regulated in both young and mature rice plants, including in the panicles. These results strongly suggest that the genes have roles in both plant development and in their defense/stress-signaling pathways.