Systematic analysis of the NDR1/HIN1-like (NHL) family in Gossypium hirsutum reveals a role of GhNHL69 in responding to cold stress

NDR1/HIN1-like (NHL) proteins widely exist in the plant kingdom, and play an essential role in resistance to both biotic and abiotic stress. However, the study of NHL family in cotton is still lacking. In the present study, a total of 40, 46, 79, and 78 NHL genes were detected by genome-wide identification in G.arboreum, G.raimondii, G. barbadense, and G.hirsutum, respectively. By analyzing the duplication pattern, the whole genome duplication (WGD) and allotetraploidization events play a vital role in the expansion of GhNHLs. The analysis of gene structure, conserved protein motif, and Ka/Ks values suggest that the structure and function might remain conserved during the evolution. The cis-acting elements analysis revealed that the GhNHL genes could respond to the abiotic stress, and the further transcriptome analysis and qRT-PCR assay indicated that GhNHLs might play a role in the cold stress tolerance. The WGCNA (Weighted correlation network analysis) analysis showed that GhNHL69 co-express with many transcription factors that are related to cold stress. By performing virus-induced gene silencing (VIGS) assays, the GhNHL69 silencing plant exhibited more severe dehydration and injury compared to the control groups. Additionally, we further found the expression of many abiotic genes was inhibited in the GhNHL69 silenced plants. This study gained insight into NHL members in cotton and mined a gene related to cold stress and lays the important basis for further study on NHL genes related to cold stress tolerance.