Regulatory Potential of bHLH-Type Transcription Factors on the Road to Rubber Biosynthesis in Hevea brasiliensis.

Natural rubber is the main component of latex obtained from laticifer cells of Hevea brasiliensis. For improving rubber yield, it is essential to understand the genetic molecular mechanisms responsible for laticifer differentiation and rubber biosynthesis. Jasmonate enhances both secondary laticifer differentiation and rubber biosynthesis. Here, we carried out time-course RNA-seq analysis in suspension-cultured cells treated with methyljasmonic acid (MeJA) to characterize the gene expression profile. Gene Ontology (GO) analysis showed that the term "cell differentiation" was enriched in upregulated genes at 24 hours after treatment, but inversely, the term was enriched in downregulated genes at 5 days, indicating that MeJA could induce cell differentiation at an early stage of the response. Jasmonate signaling is activated by MYC2, a basic helix-loop-helix (bHLH)-type transcription factor (TF). The aim of this work was to find any links between transcriptomic changes after MeJA application and regulation by TFs. Using an in vitro binding assay, we traced candidate genes throughout the whole genome that were targeted by four bHLH TFs: Hb_MYC2-1, Hb_MYC2-2, Hb_bHLH1, and Hb_bHLH2. The latter two are highly expressed in laticifer cells. Their physical binding sites were found in the promoter regions of a variety of other TF genes, which are differentially expressed upon MeJA exposure, and rubber biogenesis-related genes including SRPP1 and REF3. These studies suggest the possibilities that Hb_MYC2-1 and Hb_MYC2-2 regulate cell differentiation and that Hb_bHLH1 and Hb_bHLH2 promote rubber biosynthesis. We expect that our findings will help to increase natural rubber yield through genetic control in the future.