Identification and functional analysis of Wall-Associated Kinase (WAK) genes in Nicotiana tabacum

Ling Li ¹ Linggai Cao ² Jintao Li ¹ Zhiqiang Zhang ³ Jie Liu ² Zhongying Ren ³ Jie Zhang ² Rengang Wang ² Yangfan Miao ¹ Shizhou Yu ^2* Wei Li ^3*

• ¹ Zhengzhou University, Zhengzhou, Henan Province, China
• ² Guizhou Academy of Tobacco Science, Guiyang, China
• ³ Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, China

Wall-associated kinases (WAKs) are pivotal in linking plant cell walls to intracellular signaling networks, thereby playing essential roles in plant growth, development, and stress responses. In this study, we conducted a comprehensive genome-wide identification and analysis of the WAK gene family in tobacco (Nicotiana tabacum). A total of 44 WAK genes were identified in the tobacco genome, which were further classified into three distinct groups. Phylogenetic analysis comparing tobacco WAKs (NtWAKs) with Arabidopsis WAKs (AtWAKs) revealed speciesspecific expansion of these genes. The WAK proteins within each group displayed similar gene structures and conserved motif distributions. Promoter region analysis indicated that cis-elements of NtWAK genes are primarily involved in regulating plant growth and development, phytohormone signaling, and stress responses. Expression profiling under NaCl, PEG, and ABA treatments suggested that certain NtWAK genes may play key roles in modulating responses to abiotic stress. Three-dimensional structural predictions and subcellular localization analysis showed that NtWAK proteins from the three subgroups exhibit high cytoplasmic similarity and are primarily located to the plasma membrane. Kinase activity assay confirmed that they possess phosphorylation activity.This study represents the first genome-wide analysis of the WAK gene family in N. tabacum, laying the groundwork for future functional investigations.