Brassinosteroids (BRs) are a class of steroid hormones regulating multiple aspects of plant growth, development, and adaptation. Compared with extensive studies in Arabidopsis (Arabidopsis thaliana), the mechanism of BR signaling in rice (Oryza sativa) is less understood. Here, we identified OsWRKY53, a transcription factor involved in defense responses, as an important regulator of rice BR signaling. Phenotypic analyses showed that OsWRKY53 overexpression led to enlarged leaf angles and increased grain size, in contrast to the erect leaves and smaller seeds in oswrky53 mutant. In addition, the oswrky53 exhibited decreased BR sensitivity, whereas OsWRKY53 overexpression plants were hypersensitive to BR, suggesting that OsWRKY53 positively regulates rice BR signaling. Moreover, we show that OsWRKY53 can interact with and be phosphorylated by the OsMAPKK4-OsMAPK6 cascade, and the phosphorylation is required for the biological function of OsWRKY53 in regulating BR responses. Furthermore, we found that BR promotes OsWRKY53 protein accumulation but represses OsWRKY53 transcript level. Taken together, this study revealed the novel role of OsWRKY53 as a regulator of rice BR signaling and also suggested a potential role of OsWRKY53 in mediating the cross talk between the hormone and other signaling pathways.

Cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzyme-encoding genes and transcription factors involved in wax biosynthesis have been identified, but the underlying posttranslational regulatory mechanisms are poorly understood. Here, we demonstrate that DROUGHT HYPERSENSITIVE (DHS), encoding a Really Interesting New Gene (RING)-type protein, is a critical regulator of wax biosynthesis in rice (Oryza sativa). The cuticular wax contents were significantly reduced in DHS overexpression plants but increased in dhs mutants compared with the wild type, which resulted in a response opposite that of drought stress. DHS exhibited E3 ubiquitin ligase activity and interacted with the homeodomain-leucine zipper IV protein ROC4. Analysis of ROC4 overexpression plants and roc4 mutants indicated that ROC4 positively regulates cuticular wax biosynthesis and the drought stress response. ROC4 is ubiquitinated in vivo and subjected to ubiquitin/26S proteasome-mediated degradation. ROC4 degradation was promoted by DHS but delayed in dhs mutants. ROC4 acts downstream of DHS, and Os-BDG is a direct downstream target of the DHS-ROC4 cascade. These results suggest a mechanism whereby DHS negatively regulates wax biosynthesis by promoting the degradation of ROC4, and they suggest that DHS and ROC4 are valuable targets for the engineering of drought-tolerant rice cultivars.

Lipid droplets (LDs) stored during seed development are mobilized and provide essential energy and lipids to support seedling growth upon germination. Triacylglycerols (TAGs) are the main neutral lipids stored in LDs. The lipase SUGAR DEPENDENT 1 (SDP1), which hydrolyzes TAGs in Arabidopsis thaliana, is localized on peroxisomes and traffics to the LD surface through peroxisomal extension, but the underlying mechanism remains elusive. Here, we report a previously unknown function of a plant-unique endosomal sorting complex required for transport (ESCRT) component FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) in regulating peroxisome/SDP1-mediated LD turnover in Arabidopsis. We showed that LD degradation was impaired in germinating free1 mutant; moreover, the tubulation of SDP1- or PEROXIN 11e (PEX11e)-marked peroxisomes and the migration of SDP1-positive peroxisomes to the LD surface were altered in the free1 mutant. Electron tomography analysis showed that peroxisomes failed to form tubules to engulf LDs in free1, unlike in the wild-type. FREE1 interacted directly with both PEX11e and SDP1, suggesting that these interactions may regulate peroxisomal extension and trafficking of the lipase SDP1 to LDs. Taken together, our results demonstrate a pivotal role for FREE1 in LD degradation in germinating seedlings via regulating peroxisomal tubulation and SDP1 targeting.

Recent studies on membrane imaging in the plant endomembrane system by 2-D/3-D CLSM and TEM provide future perspectives of whole-cell ET and cryo-FIB-aided cryo-ET analysis.