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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  GhLBD18 s 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.    Electronic supplementary material  The online version of this article (10.1186/s12870-018-1599-4) contains supplementary material, which is available to authorized users.

The effects of mepiquat chloride on the lateral root initiation of cotton seedlings are associated with auxin and auxin-conjugate homeostasis