ZmMKK4 regulates osmotic stress through reactive oxygen species scavenging in transgenic tobacco

Mitogen-activated protein kinase kinase (MAPKKs) are important components of MAPK cascades, which are universal signal transduction modules and play important role in regulating both plant development and biotic or abiotic stress responses. In this study, we identified the group C MAPKK gene, ZmMKK4, in maize (Zea mays L.). Overexpression of ZmMKK4 in tobacco enhanced tolerance to osmotic stress by increased proline content and antioxidant enzyme (POD) activities compared with wild-type plants. RT-PCR revealed that one peroxidase (POX) gene, NtPOX1, was higher in ZmMKK4-overexpressing plants than in the wild-type plants. In addition, the accumulation of reactive oxygen species (ROS) in ZmMKK4-overexpressing plants is much less than that of wild-type plants. These results suggest that ZmMKK4 may be involved in ROS signaling. Taken together, these results indicate that ZmMKK4 is a positive regulator of osmotic stress by regulating scavenging of ROS in plants.     

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants.     

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants

A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.     

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.     

Overexpression of cotton GhMKK4 enhances disease susceptibility and affects abscisic acid,gibberellin and hydrogen peroxide signalling in transgenic Nicotiana benthamiana

Mitogen‐activated protein kinase (MAPK) cascades are involved in plant development, stress responses and hormonal signal transduction. MAPK kinases (MAPKKs), as the key nodes in these cascades, link MAPKs and MAPKK kinases (MAPKKKs). In this study, GhMKK4, a novel group C MAPKK gene from cotton (Gossypium hirsutum), was isolated and identified. Its expression can be induced by various stresses and signalling molecules. The overexpression of GhMKK4 in Nicotiana benthamiana enhanced its susceptibility to bacterial and fungal pathogens, but had no significant effects on salt or drought tolerance. Notably, the overexpressing plants showed increased sensitivity to abscisic acid (ABA) and gibberellin A3 (GA3), and ABA and gibberellin (GA) signalling were affected on infection with Ralstonia solanacearum bacteria. Furthermore, the overexpressing plants showed more reactive oxygen species (ROS) accumulation and stronger inhibition of catalase (CAT), a ROS‐scavenging enzyme, than control plants after salicylic acid (SA) treatment. Interestingly, two genes encoding ornithine decarboxylase (ODC) and S‐adenosylmethionine decarboxylase (SAMDC), the key enzymes in polyamine synthesis, exhibited reduced R. solanacearum‐induced expression in overexpressing plants. These findings broaden our knowledge about the functions of MAPKKs in diverse signalling pathways and the negative regulation of disease resistance in the cotton crop.     

A cotton group C MAP kinase gene, GhMPK2, positively regulates salt and drought tolerance in tobacco

Mitogen-activated protein kinase (MAPK) cascades play important roles in mediating biotic and abiotic stress responses. In plants, MAPKs are classified into four major groups (A-D) according to their sequence homology and conserved phosphorylation motifs. Compared with well-studied MAPKs in groups A and B, little is known about group C. In this study, we functionally characterised a stress-responsive group C MAPK gene (GhMPK2) from cotton (Gossypium hirsutum). Northern blot analysis indicated that GhMPK2 was induced by abscisic acid (ABA) and abiotic stresses, such as NaCl, PEG, and dehydration. Subcellular localization analysis suggested that GhMPK2 may activate its specific targets in the nucleus. Constitutive overexpression of GhMPK2 in tobacco (Nicotiana tabacum) conferred reduced sensitivity to ABA during both seed germination and vegetative growth. Interestingly, transgenic plants had a decreased rate of water loss and exhibited enhanced drought and salt tolerance. Additionally, transgenic plants showed improved osmotic adjustment capacity, elevated proline accumulation and up-regulated expression of several stress-related genes, including DIN1, Osmotin and NtLEA5. β-glucuronidase (GUS) expression driven by the GhMPK2 promoter was clearly enhanced by treatment with NaCl, PEG, and ABA. These results strongly suggest that GhMPK2 positively regulates salt and drought tolerance in transgenic plants.     

Biochemical identification of the OsMKK6-OsMPK3 signalling pathway for chilling stress tolerance in rice

MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice.     

Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.