Neutral/alkaline invertases (N/AINVs) are sucrose hydrolases with important roles in plants. In this study, 15, 15, 15, 29, and 30 N/AINVs were identified in the Gossypium species, G. raimondii, G. herbaceum, G. arboreum, G. hirsutum, and G. barbadense, respectively. Along with two previously discovered branches, α and β, a new clade γ was first discovered in our study. Investigation of gene collinearity showed that whole-genome duplication (WGD) and polyploidization were responsible for the expansion of the N/AINV gene family in allopolyploid Gossypium. Moreover, expression patterns revealed that GhN/AINV3/13/17/23/24/28 from the β clade is highly expressed during the period of fiber initiation. The invertase activity of GhN/AINV13 and GhN/AINV23 were confirmed by restoring defects of invertase-deficient yeast mutant SEY2102. Treatments of abiotic stress showed that most GhN/AINVs were induced in response to polyethylene glycol (PEG) or salt stress. A virus-induced gene-silencing (VIGS) experiment and yeast two-hybrid assay demonstrated that GhN/AINV13 may interact with their positive regulators Gh14-3-3 proteins and participate in the fiber initiation or stress tolerance of cotton. Our results provided fundamental information regarding N/AINVs and highlight their potential functions in cotton stress tolerance.
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