Cytochemical localization and synthesis mechanism of the glucomannan in pseudobulbs of Bletilla striata Reichb. f.

Abstract

The dried pseudobulbs of Bletilla striata, an important traditional Chinese medicine named BaiJi, have an extraordinary polysaccharide content and excellent prospects for medicinal effects. However, the distribution and molecular mechanism underlying biosynthesis are poorly understood. In this study, chemical and immunologic analyses were performed in representative tissues of B. striata, and the results showed that what are conventionally termed Bletilla striata polysaccharides (BSPs) are water-soluble polysaccharides deposited only in pseudobulbs. The structural component of BSPs is glucomannan, with a mannose:glucose mass ratio of ~3:2. BSPs are present in the parenchyma of the pseudobulbs in cells known as glucomannan idioblasts and distributed in the cytoplasm within cellular membranes, but are not contained in the vacuole. Comparative transcriptomics and bioinformatics analyses mapped the pathway from sucrose to BSP and identified BsGPI, BsmanA, and BsCSLAs as the key genes of BSP biosynthesis, suggesting that the functional differentiation of the cellulose synthase-like family A (CSLA) may be critical for the flow of glucomannan to the BSP or cell wall. Subsequently, virus-mediated gene silencing showed that silencing of two CSLAs (Bs03G11846 and Bs03G11849) led to a decrease in BSP content, and yeast two-hybrid and luciferase complementation experiments confirmed that four CSLAs (Bs03G11846, Bs03G11847, Bs03G11848, and Bs03G11849) can form homo- or heterodimers, suggesting that multiple CSLAs may form a large complex that functions in BSP synthesis. Our results provide cytological evidence of BSP and describe the isolation and characterization of candidate genes involved in BSP synthesis, laying a solid foundation for further research on its regulation mechanisms and the genetic engineering breeding of B. striata.