Type-B authentic response regulators regulate natural rubber biosynthesis by targeting small rubber particle proteins...

Natural rubber, predominantly derived from Hevea brasiliensis, is an indispensable industrial material whose supply faces increasing threat. Alternative rubber-producing species, including Eucommia ulmoides and Taraxacum kok-saghyz, provide promising supplementary sources. Type-B response regulators (ARR-Bs) play critical roles in plant growth and development, but their functions in natural rubber biosynthesis remain unclear. In this study, we identified 22 ARR-B genes across the three rubber-producing plants. Collinearity and phylogenetic analyses indicated that gene duplication events contributed to the expansion of the ARR-B family, while promoter analyses revealed cis-acting elements associated with hormone signaling, stress responses, growth, and light regulation. Transcriptomic and quantitative real-time PCR analyses showed nine HbARR-Bs were highly expressed in latex, with HbARR-B1 exhibiting the strongest expression. Most EuARR-Bs were preferentially expressed in peels, with EuARR-B2 being the most abundant. TksARR-B5 displayed high expression in roots, particularly in the high-yielding variety T1001. Functional studies demonstrate that HbARR-B1, EuARR-B2, and TksARR-B5 are nuclear transcription factors with transcriptional activation activity that specifically bind to the ATCT cis-acting elements. Promoter prediction revealed multiple binding sites for these regulators in key rubber biosynthesis genes, including small rubber particle proteins (SRPPs). Both in vivo and in vitro experiments confirmed that HbARR-B1 activates HbSRPP1, EuARR-B2 activates EuSRPP5, and TksARR-B5 activates TksSRPP4. Our results confirmed that HbARR-B1, EuARR-B2, and TksARR-B5 as conserved transcriptional regulators of SRPPs. This study provides new insights into ARR-Bs evolution and function and identifies promising candidate targets for molecular improvement of rubber-producing plants.