A splice variant of Arabidopsis mitogen-activated protein kinase and its regulatory function in the MKK6–MPK13 pathway

Alternative splicing (AS) generates multiple forms of proteins. A role for AS in the plant mitogen-activated protein kinase (MAPK) cascade has not been clarified. In this study, we analyzed expression of 20 Arabidopsis MAPK genes by RT-PCR and found five that generate splice variants. The MPK13 gene, with six exons and five introns, generates at least three splice variants, one in which complete splicing of five introns occurs (MPK13 Full), and ones in which the 4th and 5th introns are retained (MPK13 I4 and I5). Translation products of the splice variants MPK13 Full and I4, were found in Arabidopsis tissues by Western blot. Yeast two-hybrid analysis and protein kinase assays of recombinant proteins showed that neither I4 nor I5 interacted with upstream MAPKKs, and neither had protein kinase activity. However, MPK13 I4 protein enhanced the MKK6-dependent activation of MPK13 Full, indicating the possibility of an additional mechanism to regulate the MAPK cascade by AS.     

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.     

Expression of the ipomoelin gene from sweet potato is regulated by dephosphorylated proteins, calcium ion and ethylene

A wound‐inducible cDNA, ipomoelin (IPO) was isolated from the subtraction library of sweet potato (Ipomoea batatas cv. Tainung 57) and used as a molecular probe to investigate the transduction pathway of wounding signal within plant cells. Following mechanical wounding of the leaves of sweet potato, IPO mRNA accumulation peaked at 6 h and then continuously declined. However, IPO gene expression in the apical unwounded leaves began at 6 h after wounding and continued for a further 10 h. Besides mechanical wounding, methyl jasmonate (MeJA) was identified as a signal transducer leading to the accumulation of IPO mRNA. Treatment with salicylic acid reduced the production of IPO mRNA, further supporting the involvement of the octadecanoid pathway in the signal transduction of wounding in sweet potato. In addition, ethylene was involved in the signal pathway and induced the expression of the IPO gene. Furthermore, the application of okadaic acid, a protein phosphatase inhibitor, blocked the accumulation of IPO mRNA induced by MeJA or ethylene, indicating that activation of the IPO gene by both MeJA and ethylene was via dephosphorylated proteins. The presence of a calcium ion chelator or channel blockers also inhibited the expression of the IPO gene after wounding. However, investigation by confocal scanning microscopy further pointed out that mechanical wounding rather than the application of MeJA induced the accumulation of the calcium ion. These results may indicate that the calcium ion is also involved in the activation of IPO mRNA. In addition, wounding signals the accumulation of calcium ion first and then stimulates the biosynthesis of MeJA in sweet potato. Hence, the reaction sequence of signal transducers, including the calcium ion, MeJA and protein kinase/phosphatase, in the wounding signalling pathway of sweet potato is suggested in this report.