The F-box E3 ubiquitin ligase BAF1 mediates the degradation of the brassinosteroid-activated transcription factor BES1 through selective autophagy in Arabidopsis

Brassinosteroids (BRs) regulate plant growth, development, and stress responses by activating the core transcription factor BRI1-EMS-SUPPRESSOR1 (BES1), whose degradation occurs through the proteasome and autophagy pathways. The E3 ubiquitin ligase(s) that modify BES1 for autophagy-mediated degradation remain to be fully defined. Here, we identified an F-box family E3 ubiquitin ligase named BES1-ASSOCIATED F-BOX1 (BAF1) in Arabidopsis thaliana. BAF1 interacts with BES1 and mediates its ubiquitination and degradation. Our genetic data demonstrated that BAF1 inhibits BR signaling in a BES1-dependent manner. Moreover, BAF1 targets BES1 for autophagic degradation in a selective manner. BAF1-triggered selective autophagy of BES1 depends on the ubiquitin binding receptor DOMINANT SUPPRESSOR OF KAR2 (DSK2). Sucrose starvation-induced selective autophagy of BES1, but not bulk autophagy, was significantly compromised in baf1 mutant and BAF1-ΔF (BAF1 F-box decoy) overexpression plants, but clearly increased by BAF1 overexpression. The baf1 and BAF1-ΔF overexpression plants had increased BR-regulated growth but were sensitive to long-term sucrose starvation, while BAF1 overexpression plants had decreased BR-regulated growth but were highly tolerant of sucrose starvation. Our results not only established BAF1 as an E3 ubiquitin ligase that targets BES1 for degradation through selective autophagy pathway, but also revealed a mechanism for plants to reduce growth during sucrose starvation.

Influence of sucrose starvation, osmotic and salt stresses on expression profiles of genes involved in the development of autophagy by means of microtubules

Aim. The aim of this work was to investigate changes in expression profiles of key genes involved in the development of autophagy by means of microtubules under the influence of sucrose starvation, osmotic and salt stresses. Methods. Arabidopsis thaliana seeds were sown aseptically on Murashige and Skoog solid medium. Salt and osmotic stresses were simulated by seed germination and seedlings cultivation on the media containing 150 mM NaCl and 10 mM mannitol, respectively. For investigation of starvation-induced autophagy plants were germinated and grown on sucrose-free medium. Results. Changes in expression of α-tubulin and atg8 genes had clearly defined stressdependent nature. Overexpression of tua1 and atg8e under starvation; tua3 and atg8f under osmotic stress; tua3 and atg8f, atg8e during salt stress indirectly testifies interaction between the structural units of autophagosomes and microtubules. It was shown that influence of investigated abiotic stimuli results in overexpression of elp3 and hda6 genes. Small increase in expression levels of hexokinase 2 and 3 was demonstrated. Conclusions. Transcriptome analysis of key genes involved in realization of autophagy induced by sucrose starvation, osmotic and salt stresses in Arabidopsis thaliana cells was conducted. Received data indirectly testifies interaction between the structural units of autophagosomes and microtubules and enables to point α-tubulin and atg8 genes, which are specific for the realization of autophagy induced by a certain abiotic stimuli. Expression profiles of elp3/deacetylases as well as hexokinases indicate the critical role of α-tubulin acetylation for autophagic response, that is involved in the development of programmed cell death.Keywords: autophagy, sucrose starvation, osmotic stress, salt stress, transcriptome analysis, α-tubulin, atg8.

Induction of a bZIP Type Transcription Factor and Amino Acid Catabolism-Related Genes in Soybean Seedling in Response to Starvation Stress

To address roles of bZIP transcription factors on regulation of amino acid catabolism under autophagy-induced plant cells, we examined the effect of nutrient starvation on the expression of low energy stress-related transcription factor homologs, GmbZIP53A and GmbZIP53B, and amino acid catabolism-related genes in soybean (Glycine max (L.) Merr.). Sucrose starvation treatment significantly enhanced the expressions of GmbZIP53A, but not GmbZIP53B asparagine synthase (GmASN1), proline dehydrogenase1 (GmProDH), and branched chain amino acid transaminase 3 (GmBCAT3). GmbZIP53-related immunoreactive signals were upregulated under severe starvation with sucrose starvation and protease inhibitors, while 3% sucrose and sucrose starvation had no or marginal effects on the signal. Profiles of induction of GmASN1, GmProDH and GmBCAT3 under various nutrient conditions were consistent with the profiles of GmbZIP53 protein levels but not with those of GmbZIP mRNA levels. These results indicate that GmbZIP53 proteins levels are regulated by posttranslational mechanism in response to severe starvation stress and that the increased protein of GmbZIP53 under severe starvation accelerates transcriptional induction of GmASN1, GmProDH, and GmBCAT3. Furthermore, it is conceivable that decrease of branched chain amino acid level by the BCAT-mediated degradation eventually enhances autophagy under severe starvation.