leupeptins and methyl-jasmonate

The final expression level of a transgene-derived protein in transgenic plants depends on transcriptional and post-transcriptional processes. Here, we focus on methods to improve protein stability without comprising biological function. We found that the four isoforms of the Arabidopsis RAD23 protein family are relatively stable. The UBA2 domain derived from RAD23a can be used as a portable stabilizing signal to prolong the half-life of two unstable transcription factors (TFs), HFR1 and PIF3. The increased stability of the TF-UBA2 fusion proteins results in an enhanced phenotype in transgenic plants compared to expression of the TF alone. Similar results were obtained for the RAD23a UBA1 domain. In addition to UBA1/2 of RAD23a, the UBA domain from the Arabidopsis DDI1 protein also increased the half-life of the unstable protein JAZ10.1, which is involved in jasmonate signaling. Taken together, our results suggest that UBA fusions can be used to increase the stability of unstable proteins for basic plant biology research as well as crop improvement.

Topics: Acetates; Arabidopsis; Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; Cycloheximide; Cyclopentanes; DNA-Binding Proteins; Gene Expression Regulation, Plant; Half-Life; Leupeptins; Nuclear Proteins; Oxylipins; Phenotype; Plant Growth Regulators; Plants, Genetically Modified; Protein Isoforms; Protein Stability; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Seedlings; Transcription Factors

Biosynthesis of many plant alkaloids is enhanced by endogenous accumulation and exogenous application of jasmonates, but the general and specific signaling components are not well understood. In Arabidopsis, jasmonate-induced ZIM-domain-containing (JAZ) proteins have recently been found to be critical transcriptional repressors linking CORONATINE INSENSTIVE1 (COI1)-mediated jasmonate perception and jasmonate-regulated transcriptional regulation. Insect herbivory on tobacco leaves activates the jasmonate signaling pathway, leading to up-regulation of nicotine biosynthesis genes in roots. We show here that roots of COI1-silenced tobacco plants are insensitive to growth inhibition by methyl jasmonate, and do not activate nicotine biosynthesis genes after jasmonate treatment or wounding of leaves. Tobacco JAZ proteins appeared to be rapidly degraded after jasmonate treatment, whereas a C-terminally truncated form lacking the conserved Jas motif did not. When the non-degradable JAZ forms were expressed in tobacco hairy roots, jasmonate induction of nicotine biosynthesis was strongly inhibited. Formation of tobacco alkaloids in jasmonate-elicited tobacco BY-2 cells was also effectively suppressed by the COI1 RNAi (RNA interference) construct and by the dominant-negative truncated JAZ constructs. In addition, jasmonate-mediated induction of nicotine biosynthesis genes was diminished by treatment with a proteasome inhibitor MG132. These results indicate that jasmonate-triggered, COI1-mediated degradation of JAZ repressors activates transcriptional regulation of nicotine biosynthesis genes in tobacco roots.

Topics: Acetates; Cyclopentanes; Gene Expression Regulation, Plant; Genes, Dominant; Genes, Plant; Leupeptins; Molecular Sequence Data; Nicotiana; Nicotine; Oxylipins; Plant Proteins; Plant Roots; Proteasome Inhibitors; Protein Processing, Post-Translational; RNA Interference; Suppression, Genetic