Endo-β-1,4-glucanases in Arabidopsis thaliana: expression and putative roles in sexual reproduction

Plant endoglucanases of glycosyl hydrolase family 9 (GH9), which cleave 1,4-β-glucosidic bonds in glycan chains, have been shown to play pivotal roles in cellulose degradation, as well as in the relaxation or construction of cell walls during diverse plant growth and developmental processes. However, their specific functions in plant sexual reproduction remain unclear. In this study, we systematically analyzed expression profiles of the GH9 gene family using in vivo observations of GFP-fusion proteins. Our results revealed that numerous GH9 genes were expressed across both vegetative and reproductive tissues: 5 in stomata, 12 in roots, 11 in mature pollen and 15 in mature ovules or seeds. Further analysis of reproductive tissues uncovered distinct expression specificities: GH9A4 as pollen-specific, GH9B6 as sperm cell-specific, GH9B12 as central cell- and endosperm cell-specific, GH9B11 as enriched in pollen and chalazal endosperm, and GH9B8 as vascular tissue-preferential. Additionally, GFP signals of up to 7 GH9 members were detected in the filiform apparatus, while 12 GH9 members showed signals in integuments and seed coats—hinting at diverse GH9 functions in reproduction. Furthermore, we conducted gene editing and phenotypic analysis on two subsets of GH9 genes highly expressed in pollen. While in vivo pollen germination and growth were unaffected, in vitro germination rates decreased significantly when GH9B5, GH9B7, and GH9A4 functionally deficient. Taken together, this study highlights the potential roles of the GH9 family in pollen germination and establishes a foundation for future functional analyses aimed at elucidating the putative roles of the GH9 family in plant reproduction.