Genome-wide identification of major latex protein gene family and functional analysis of EuMLP22.1 in Eucommia ulmoides Oliver.

Eucommia ulmoides produces natural rubber, known as Eucommia rubber, which is widely used in the pharmaceutical and industrial fields. Major latex proteins (MLPs) play crucial roles in plant stress responses. In this study, we identified 437 binding peptides from Eucommia rubber particles by LC-MS/MS. Furthermore, EuMLP22, which harbors the peptide "SDGDVFHELFR", was retrieved from the whole-genome annotation database of E. ulmoides. Genome-wide analysis further identified 28 EuMLP family members containing the conserved Bet v 1 domain, which are distributed across five chromosomes. Seven EuMLP genes (EuMLP17, EuMLP18, EuMLP20, EuMLP21, EuMLP22.1, EuMLP22.2, and EuMLP26) were cloned and characterized. Most exhibited tissue-specific expression with predominant expression in stem bark, whereas EuMLP26 was highly expressed in samaras. Focusing on EuMLP22.1, which contains the rubber particle-associated peptide, we demonstrated that it is localized to both the cell membrane and the nucleus. Promoter-GUS assays revealed its upregulation under methyl jasmonate (MeJA), low temperature, and shading treatments. In E. ulmoides seedlings, EuMLP22.1 gene expression was induced by high temperature and shading treatments. Functional analysis of transgenic tobacco overexpressing EuMLP22.1 demonstrated that its overexpression enhanced cold tolerance. Conversely, gene silencing of EuMLP22.1 downregulated two key rubber biosynthesis genes (EuSRPP3 and EuREF1), suggesting its regulatory role in rubber biosynthesis. These findings provide new insights into the molecular mechanisms underlying Eucommia rubber biosynthesis and stress responses and identify EuMLP22.1 as a promising target for the genetic improvement of E. ulmoides.