| Summary: | Abstract Background Mepiquat chloride (MC) is a plant growth regulator widely used in cotton (Gossypium hirsutum L.) production to suppress excessive vegetative growth, increase root growth and avoid yield losses. To increase root growth, cotton seeds were treated with MC to increase the number of lateral root (LRs) and improve drought resistance. An increased indole-3-acetic acid (IAA) pool appeared to correlate with LR growth, and the principal source of IAA in germinating seeds is IAA conjugates. Here, the role of IAA homeostasis and signaling was investigated in cotton seedlings treated with MC. Results In the present research, MC significantly increased endogenous IAA levels in the roots, which promoted lateral root initiation (LRI) by upregulating GhARF7/19 and GhLBD18s and subsequently increasing LR quantity and elongation. The levels of IAA-amide conjugates significantly decreased in MC-treated seedlings compared with untreated control seedlings. Sixteen members of the cotton IAA amidohydrolase (IAH) gene family were identified, of which GhIAR3a, GhIAR3b, GhILR1, GhILL3 and GhILL6 were expressed during cotton seed germination. Compared with those in untreated control seedlings, the expression levels of GhIAR3a, GhIAR3b, GhILR1 and GhILL6 in the MC-treated seedlings were markedly elevated. The GhIAR3a/b and GhILR1 genes were cloned and expressed in Escherichia coli; these recombinant proteins exhibited hydrolytic activity that could cleave IAA-phenyalanine (Phe), IAA-methionine (Met), IAA-glycine (Gly) and IAA-leucine (Leu) in vitro, while only GhIAR3a hydrolyzed IAA-alanine (Ala) efficiently. The content of GhIAR3a, as detected via an established sandwich enzyme-linked immunosorbent assay (ELISA), increased in the MC-treated seedlings compared with the untreated control seedlings. In addition, the Arabidopsis iar3 mutant was less responsive to MC-induced LR growth than was wild type. Conclusions These findings suggested that MC application could mediate IAA homeostasis via increased IAA levels from IAA-amide conjugate hydrolysis by accelerating IAH gene expression, which might promote LRI and increase the LR quantity and elongation.
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