Homeostasis Of Brassinosteroids Regulated By DRL1, A Putative Acyltransferase In Arabidopsis

Brassinosteroids (BRs) play essential roles in regulating various aspects of plant growth and development and in responding to diverse environmental cues, which conclude cell elongation, cell division and differentiation, formation of the leaf vascular, stem elongation, photomorphogenesis, reproduction, senescence, and stresses. BRs have low expression levels in plant pollen, seed and young vegetable organs, so their homeostasis is a very important way in regulating their contents, and their synthesis and metabolism are both important. Up to now, the pathway of BR synthesis is known well, while the pathway of BR metabolism is still elusive.Here, we identified a dominant mutant from an activation-tagging population based on the material Col-0, dwarf and round leaf-1 (drll-D), which exhibits a weak BR-deficient or BR-insensitive mutant phenotypes, including short and round leaves, prolonged senescence and dwarfed shape. Further, through TAIL-PCR, real time qRT-PCR and the phenotype of backcross seedlings and overexpression seedlings, we confirmed that the gene DRL1 (At4g31910) was a single insertion to determine the dwarf phenotype of the mutant drll-D. Next, we studied the gene DRL1’s functions through many different aspects.First, three assays including phenotype abservations like hypocotyl lengths in different BR concentrations and root inhibition assays, molecular assays like the transcript level of marker genes responding to BR signaling, biochemical assays like the phosphorylation status of protein marker BES1 in BR signaling, suggest us that the drll-D mutant can respond to BRs, but has decreased BR signaling outputs, which indicate that the gene DRL1 may be involved in BR biosynthesis pathway or BR metabolic pathway rather than BR singal pathway.Secondly, by GC/MS methods to measure the contents of the endogenous BRs in Col-0 and the mutant drll-D, we found that the endogenous levels of several BRs, including typhasterol (TY),6-deoxotyphasterol (6-deoxoTY), and 6-deoxocastasterone (6-deoxoCS) are significantly lower in the drll-D mutant than in the wide type, while there is no accumulation of the upstream BR intermediates. This suggests that DRL1 may function in BR metabolism, not in BR biosynthesis.Thirdly, the pDRL1::GUS transgenic plant showed that DRL1 is widely expressed in leaves, roots, flowers, and siliques, and this putative acyl-transferase DRL1 may catalyze BR esterification to form BR conjugation due to the in vitro recombinant enzyme activity assays.Further, as the T-DNA insertion line DRL1-KO has no obvious phenotype compared to Col-0, and previous studies have shown that DRL1 belongs to a small gene family named BAHD family. In this branch, only overexpression of these two genes At5g17540 and At2g40230 make BR-deficient phenotypes like dwarf shape, short petioles, short and round leaves. The plants without these two genes are larger with an enhanced BR signaling. Besides these characters, the expression levels of At5g17540 and At2g40230 were also induced by eBL and inhibited by ABA like that of DRL1.At last, we overexpressed the Arabidopsis gene DRL1 into rice, and the transgenic rices have dwarf and erect phenotypes, and the BR signaling output is reduced while they bear seeds normally, which suggest that this transgenic rice may be a new cultivated variety for reasonable close planting and increasing the yields.In all, a putative acyltransferase DRL1 is involved in the BR metabolism likely by catalyzing the BR conjugation through esterification, which plays important roles in regulating the BR homeostasis and responding to abiotic stresses in Arabidopsis.