Abstract:

【Objective】The sheaths of Bambusa multiplex, containing numerous chloroplasts capable of photosynthesis, serve as a significant carbon source during the fast growth stage of B. multiplex. The sheath senescence profoundly impacts the growth of the culm elongation. While the WRKY gene family is known for its pivotal role in regulating various plant developmental processes, limited information is available regarding its role in B. multiplex. Therefore, this work aimed to identify and analyze the senescence-related WRKY genes in the sheath of B. multiplex. 【Method】Based on the full-length transcripts of the B. multiplex sheath, HMMER software was adopted to the primary search of WRKY transcription factors. Then, the candidate WRKYs in the sheath transcripts were obtained by conservation domain analysis and with redundancy removal. To predict the candidate BmWRKYs involved in regulating the sheath senescence, the phylogenetic analysis between BmWRKYs and the senescence-related AtWRKYs (Arabidopsis) genes was determined. Then, based on second-generation transcriptome data obtained from different developmental stages of the sheaths of B. multiplex, the expression patterns of BmWRKYs at different developmental stages of sheaths were analyzed for functional prediction. In addition, the developmental stages of sheaths that attached to the third internode with different lengths were identified by detecting chlorophyll content and membrane ion leakage. The expression profiles of BmWRKYs in the sheath with different developmental stages were also detected by qRT-PCR to further determine the roles of BmWRKYs during the sheath senescence. 【Result】A total of 83 BmWRKYs were identified, with protein lengths ranging from 119 to 1 751. Based on the protein structure and phylogenetic analysis, these 83 BmWRKYs were classified into three subgroups: Ⅰ, Ⅱ a-e, and Ⅲ. Specifically, 39 BmWRKYs belonged to subgroup I, while 43 BmWRKYs belonged to subgroup Ⅱ a-e. Within the subgroup Ⅱ a-e, 2, 16, 11, 12, and 2 BmWRKYs were categorized into subgroups Ⅱ a, Ⅱ b, Ⅱ c, Ⅱ d, and Ⅱ e, respectively. Only one BmWRKY was classified into subgroup Ⅲ. Additionally, 31 BmWRKYs likely involved in regulating sheath senescence were identified through homologous analysis with senescence-related AtWRKYs. These 31 BmWRKYs were further categorized into four types (a, b, c and d) based on their expression patterns during different stages of the sheath senescence. BmWRKY59, BmWRKY76, BmWRKY6, BmWRKY4, BmWRKY41, BmWRKY51, BmWRKY26, BmWRKY48, BmWRKY53, BmWRKY36, BmWRKY42, BmWRKY18 and BmWRKY10 were a-type. BmWRKY2, BmWRKY74, BmWRKY38, BmWRKY76, BmWRKY30, BmWRKY43, BmWRKY69 and BmWRKY72 were b-type. BmWRKY15, BmWRKY68, BmWRKY28, BmWRKY12 and BmWRKY7 were c-type. BmWRKY3, BmWRKY21, BmWRKY1, BmWRKY25 and BmWRKY5 were d-type. Sheaths that attached to internodes with different length had different developmental stages. When the length of the third internode was between 20-25 cm, chlorophyll began to degrade. When the internode length was about 30 cm, the sheath had significant senescence. Based on these observations, few BmWRKYs were further selected to measure their expression profiles in the sheath at different developmental stages. The results suggested that BmWRKY4, BmWRKY48 and BmWRKY53 (a-type) appeared to be positive regulators of sheath senescence, while BmWRKY21 (d-type) was a potential negative regulator, acting during the early stages of sheath senescence. 【Conclusion】The sheaths play a crucial role as carbon sources during the rapid growth stage of B. multiplex internodes, and their senescence involves nutrient mobilization. Investigating sheath senescence regulation serves as a key entry point for understanding the mechanism of rapid internode elongation. This study identified multiple BmWRKYs with significant potential roles in the sheath senescence process. These results lay the foundation for further analysis of the molecular mechanism of WRKY transcription factors regulating the sheath senescence in the future.

Key words: WRKY transcription factor, Bambusa multiplex, sheath, senescence