Analysis of Arabidopsis AIR12 and Brassica carinata CIL1 in root development and response to abiotic stress

Abstract

The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (CIL1) from Brassica carinata and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both CIL1 and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense CIL1 B. carinata plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the CIL1 and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.