Asparagine-linked glycosylation (ALG, N-glycosylation) is one of the most prevalent protein modifications in eukaryotes and regulates protein folding, trafficking and function. Recently, we reported that the mutation of N154Q significantly led to the ER retention of brassinosteroids insensitive 1 (BRI1), the receptor of brassinosteroids (BRs). However, the mechanism of how the N154 site affects BRI1 structure is still not completely clear. In current study, we found that the removal of N154-glycan with S156A replacement significantly enhanced the ability of bri1 to complement bri1-301 mutant and plasma membrane localization compared with N154Q. In addition, the various mutations on N154 site resulted in bri1 retention in the ER, except for N154D. The 3D modeling suggested that there existed polar contacts around N154 site and the mutations not only destroyed the addition of N-glycan on the site, but also led to the disorder of hydrogen bonds formation. The sequence analysis showed that the N275 shared more similarity with N154 site and the removal of N275-glycan further enhanced the retention of bri1 carrying S156A mutation in the ER. Our results showed that N154 was special and essential for maintaining BRI1 structure and explored the role of those residues and key N-glycans lying in the LRR inner surface on protein conformation.

Brassinosteroids (BRs) play important roles in many aspects of plant growth and development, including regulation of vascular cambium activities and cell elongation. BR-induced BEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response. Here, we identified a poplar (Populus alba × Populus glandulosa) BEE3-like gene, PagBEE3L, encoding a putative basic helix-loop-helix (bHLH)-type transcription factor. Expression of PagBEE3L was induced by brassinolide (BL). Transcripts of PagBEE3L were mainly detected in stems, with the internode having a low level of transcription and the node having a relatively higher level. The function of the PagBEE3L gene was investigated through phenotypic analyses with PagBEE3L-overexpressing (ox) transgenic lines. This work particularly focused on a potential role of PagBEE3L in stem growth and development of polar. The PagBEE3L-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3L-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3L-ox plants and PagBEE3L plays a role in stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems.

Rice leaf angle is an important agronomic trait determining plant architecture and crop yield. Brassinosteroids (BRs) play crucial roles in controlling rice leaf angle, thus an increasing number of researches were focused on the BR signaling pathway in rice. However, the orthologs of some important components in Arabidopsis BR signaling have not yet been characterized in rice. In this study, we identified a rice bHLH transcription factor named OsBIM1, as the closest rice homolog of AtBIM1 (BES1-Interacting MYC-like Protein1). Overexpression of OsBIM1 significantly increases rice leaf angles, whereas the T-DNA knock-out mutant osbim1 and wide type (WT) showed similar leaf inclination. OsBIM1 overexpression enhances the sensitivity and response to BR treatment in rice. Gene expression analysis showed that the overexpression of OsBIM1 significantly increased the transcripts of INCREASED LEAF INCLINATION1 (OsILI1) that functions as a key transcription factor promoting BR signaling and response. Meanwhile, OsBIM1 inhibited the expression of DWARF2 (OsD2, a key enzyme in BR biosynthesis pathway). OsBIM1 can bind with OsILI1 promoter and enhance OsILI1 expression in response to BR treatment. The promoting effect of OsBIM1 overexpression on leaf angle can still be observed at harvest stage, but overexpression of OsBIM1 resulted in smaller grain size and reduced yield. These results indicate that OsBIM1 functions as a positive regulator in BR signaling, and its overexpression increases rice lamina inclination by promoting BR sensitivity and response.