NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.
One of the most efficient plant resistance reactions to pathogen attack is the hypersensitive response, a form of programmed cell death at infection sites. The Arabidopsis transcription factor MYB30 is a positive regulator of hypersensitive cell death responses. Here we show that MIEL1 (MYB30-Interacting E3 Ligase1), an Arabidopsis RING-type E3 ubiquitin ligase that interacts with and ubiquitinates MYB30, leads to MYB30 proteasomal degradation and downregulation of its transcriptional activity. In non-infected plants, MIEL1 attenuates cell death and defence through degradation of MYB30. Following bacterial inoculation, repression of MIEL1 expression removes this negative regulation allowing sufficient MYB30 accumulation in the inoculated zone to trigger the hypersensitive response and restrict pathogen growth. Our work underlines the important role played by ubiquitination to control the hypersensitive response and highlights the sophisticated fine-tuning of plant responses to pathogen attack. Overall, this work emphasizes the importance of protein modification by ubiquitination during the regulation of transcriptional responses to stress in eukaryotic cells.
Fig. 5. AGO1 is a peripheral membrane protein. (A)Western analysis of AGO1, HMG1, and PEPC proteins in 100,000 × g supernatant (sup) and pellet (pel) fractions of cleared Col-0 inflorescence extracts prepared by hypertonic lysis. Five percent of the supernatant fraction is loaded, 20% of pellet fraction is loaded, precluding any clear estimates of relative abundance in soluble versus insoluble fractions. HMG1 is used here solely as a positive control for a transmembrane protein. (B) Western analysis of AGO1 and HMG1 proteins in pellet fractions prepared as in A. Pellets were resuspended in either microsome buffer, or microsome buffer supplemented with 1 M KCl, 0.1 M Na 2 CO 3 , or 0.5% Triton X-100, and separated into supernatant (sup) and pellet (pel) fractions again at 100,000 × g. At longer exposures, an insoluble AGO1 fraction upon Triton X-100 treatment is visible. (C) Microsome fractionation of inflorescence lysates from Col-0, ago1-25, and ago1-38. Two experiments with corresponding total (tot.) and microsome (micr.) fractions analyzed by Western blots with AGO1 antibodies are shown. (Top two panels) Western blots developed by enhanced chemiluminescence. (Bottom two panels) Western blots developed by less sensitive alkaline phosphatase staining that allows a clearer visualization of the difference in microsomal AGO1 abundance between Col-0 (WT) and ago1-38. *Unspecific cross-reacting band; RbcL, large subunit of RuBisCO; BSA, BSA added to the microsome buffer to avoid membrane association of proteins post lysis. The amount of BSA recovered in washed pellets is proportional to the amount of membrane recovered, and is used as loading control. (D) (Upper) Schematic depicting the position of several AGO1 hypomorphic mutations used in this study (black triangle) and the ago1-38 allele (red triangle) causing the G186R missense mutation. (Lower) The glycine residue mutated in Arabidopsis ago1-38 is highly conserved among various metazoan and fungal AGO proteins (highlighted in yellow). At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Sp, Schizosaccharomyces pombe. (E) Microsome fractionation of inflorescence lysates from Col-0 and hmg1-3. (Upper) Western analysis of AGO1 in total extracts. (Lower) Western analysis of AGO1 and HMG1 in microsome (pel) fraction. (F) Same analysis as in E performed with GFP171.1 and mad3 inflorescence lysates.
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