DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana
Wednesday, 2016/09/28 | 08:32:56
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Jun Zhang, Jinshan Ella Lin, Chinchu Harris, Fernanda Campos Mastrotti Pereira, Fan Wu, Joshua J. Blakeslee, and Wendy Ann Peer SignificanceAlthough auxin oxidation has long been known to be the primary mechanism of auxin catabolism and Arabidopsis seedlings have 10–100 more 2-oxindole 3-acetic acid compared with other auxin catabolic products, the enzymes that constitutively catalyze this process remained unknown. This work fills the gap by identifying and characterizing the Arabidopsis proteins DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) and DAO2, which catalyze auxin oxidation under normal growth conditions and shows that this activity has a physiological function in planta. The protein localization and phenotypes of the loss/gain of function mutants support that DAO1 is the primary constitutive mechanism of auxin catabolism in Arabidopsis and that the temporal- and tissue-specific oxidative inactivation of auxin by DAO adjusts indole-3-acetic acid levels throughout the life of the plant to optimize growth and development. AbstractTight homeostatic regulation of the phytohormone auxin [indole-3-acetic acid (IAA)] is essential to plant growth. Auxin biosynthetic pathways and the processes that inactivate auxin by conjugation to amino acids and sugars have been thoroughly characterized. However, the enzyme that catalyzes oxidation of IAA to its primary catabolite 2-oxindole-3-acetic acid (oxIAA) remains uncharacterized. Here, we show that DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) catalyzes formation of oxIAA in vitro and in vivo and that this mechanism regulates auxin homeostasis and plant growth. Null dao1-1 mutants contain 95% less oxIAA compared with wild type, and complementation of dao1 restores wild-type oxIAA levels, indicating that DAO1 is the primary IAA oxidase in seedlings. Furthermore, dao1 loss of function plants have altered morphology, including larger cotyledons, increased lateral root density, delayed sepal opening, elongated pistils, and reduced fertility in the primary inflorescence stem. These phenotypes are tightly correlated with DAO1 spatiotemporal expression patterns as shown by DAO1pro:β-glucuronidase (GUS) activity and DAO1pro:YFP-DAO1 signals, and transformation with DAO1pro:YFP-DAO1 complemented the mutant phenotypes. The dominant dao1-2D mutant has increased oxIAA levels and decreased stature with shorter leaves and inflorescence stems, thus supporting DAO1 IAA oxidase function in vivo. A second isoform, DAO2, is very weakly expressed in seedling root apices. Together, these data confirm that IAA oxidation by DAO1 is the principal auxin catabolic process in Arabidopsis and that localized IAA oxidation plays a role in plant morphogenesis.
See: http://www.pnas.org/content/113/39/11010.abstract.html?etoc PNAS September 27 2016; vol.113; no.39: 11010–11015
Fig. S3. Heterologous expression and purification of rDAO1 and rDAO2 in E. coli. (A) SDS/PAGE analysis of rDAO1 and rDAO2 expression after IPTG induction. Lane 1, protein marker (Bio-Rad); lane 2, cell lysate of noninduced E. coli harboring rDAO1 construct; lane 3, cell lysate of IPTG-induced E. coli harboring rDAO1 construct; lane 4, purified rDAO1 from IPTG-induced E. coli; lane 5, cell lysate of noninduced E. coli harboring rDAO2 construct; lane 6, cell lysate of IPTG-induced E. coli harboring rDAO2 construct; and lane 7, purified rDAO2 from IPTG-induced E. coli. (B, Upper) Western blot analysis of noninduced (−) and purified rDAO from IPTG-induced (+) E. coli harboring either rDAO1 or rDAO2 construct as indicated. (B, Lower) Ponceau staining of the gel. |
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