ABSTRACT
The members of early auxin response gene family, Aux/IAA, encode negative regulators of auxin signaling but play a central role in auxin-mediated plant development. Here we report the interaction of an Aux/IAA protein, AtIAA14, with Drought-induced-19 (Di19-3) protein and its possible role in auxin signaling. The Atdi19-3 mutant seedlings develop short hypocotyl, both in light and dark, and are compromised in temperature-induced hypocotyl elongation. The mutant plants accumulate more IAA and also show altered expression of NIT2, ILL5, and YUCCA genes involved in auxin biosynthesis and homeostasis, along with many auxin responsive genes like AUX1 and MYB77. Atdi19-3 seedlings show enhanced root growth inhibition when grown in the medium supplemented with auxin. Nevertheless, number of lateral roots is low in Atdi19-3 seedlings grown on the basal medium. We have shown that AtIAA14 physically interacts with AtDi19-3 in yeast two-hybrid (Y2H), bimolecular fluorescence complementation, and in vitro pull-down assays. However, the auxin-induced degradation of AtIAA14 in the Atdi19-3 seedlings was delayed. By expressing pIAA14::mIAA14-GFP in Atdi19-3 mutant background, it became apparent that both Di19-3 and AtIAA14 work in the same pathway and influence lateral root development in Arabidopsis. Gain-of-function slr-1/iaa14 (slr) mutant, like Atdi19-3, showed tolerance to abiotic stress in seed germination and cotyledon greening assays. The Atdi19-3 seedlings showed enhanced sensitivity to ethylene in triple response assay and AgNO3, an ethylene inhibitor, caused profuse lateral root formation in the mutant seedlings. These observations suggest that AtDi19-3 interacting with AtIAA14, in all probability, serves as a positive regulator of auxin signaling and also plays a role in some ethylene-mediated responses in Arabidopsis. SIGNIFICANCE STATEMENT: This study has demonstrated interaction of auxin responsive Aux/IAA with Drought-induced 19 (Di19) protein and its possible implication in abiotic stress response.
ABSTRACT
A molecular evolutionary analysis of a well conserved protein helps to determine the essential amino acids in the core catalytic region. Based on the chemical properties of amino acid residues, phylogenetic analysis of a total of 172 homologous sequences of a highly conserved enzyme, L-myo-inositol 1-phosphate synthase or MIPS from evolutionarily diverse organisms was performed. This study revealed the presence of six phylogenetically conserved blocks, out of which four embrace the catalytic core of the functional protein. Further, specific amino acid modifications targeting the lysine residues, known to be important for MIPS catalysis, were performed at the catalytic site of a MIPS from monocotyledonous model plant, Oryza sativa (OsMIPS1). Following this study, OsMIPS mutants with deletion or replacement of lysine residues in the conserved blocks were made. Based on the enzyme kinetics performed on the deletion/replacement mutants, phylogenetic and structural comparison with the already established crystal structures from non-plant sources, an evolutionarily conserved peptide stretch was identified at the active pocket which contains the two most important lysine residues essential for catalytic activity.
