Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook–Like10 phosphorylation required for stress growth regulation

Abstract

Significance Plant growth is highly plastic, and coordination of environmental signals with intrinsic developmental pathways has implications for plant productivity and stress resistance. However, signaling mechanisms involved in such coordination remain unclear. Phosphoproteomics found a range of phosphorylation sites affected by Highly ABA-Induced1 (HAI1), a protein phosphatase involved in abiotic stress and abscisic acid signaling. Of these, HAI1 could directly dephosphorylate the DNA-binding protein AT-Hook–Like 10 (AHL10). AHL10 phosphorylation was critical for growth regulation and expression of development and hormone-related genes during water limitation stress. By identifying HAI1-affected phosphoproteins and the functionally important AHL10 phosphorylation site, these results elucidate HAI1 and AHL10 function and demonstrate a mechanism plants use to balance maximal growth versus robust response to environmental stress. The clade A protein phosphatase 2C Highly ABA-Induced 1 (HAI1) plays an important role in stress signaling, yet little information is available on HAI1-regulated phosphoproteins. Quantitative phosphoproteomics identified phosphopeptides of increased abundance in hai1-2 in unstressed plants and in plants exposed to low-water potential (drought) stress. The identity and localization of the phosphoproteins as well as enrichment of specific phosphorylation motifs indicated that these phosphorylation sites may be regulated directly by HAI1 or by HAI1-regulated kinases including mitogen-activated protein kinases, sucrose non-fermenting–related kinase 2, or casein kinases. One of the phosphosites putatively regulated by HAI1 was S313/S314 of AT-Hook–Like10 (AHL10), a DNA-binding protein of unclear function. HAI1 could directly dephosphorylate AHL10 in vitro, and the level of HAI1 expression affected the abundance of phosphorylated AHL10 in vivo. AHL10 S314 phosphorylation was critical for restriction of plant growth under low-water potential stress and for regulation of jasmonic acid and auxin-related gene expression as well as expression of developmental regulators including Shootmeristemless. These genes were also misregulated in hai1-2. AHL10 S314 phosphorylation was required for AHL10 complexes to form foci within the nucleoplasm, suggesting that S314 phosphorylation may control AHL10 association with the nuclear matrix or with other transcriptional regulators. These data identify a set of HAI1-affected phosphorylation sites, show that HAI1-regulated phosphorylation of AHL10 S314 controls AHL10 function and localization, and indicate that HAI1-AHL10 signaling coordinates growth with stress and defense responses.