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 inhai1-2in 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 ofHAI1expression 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 includingShootmeristemless. These genes were also misregulated inhai1-2. 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.

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 HAI1regulated kinases including Mitogen-Activated Protein Kinases (MPKs), Sucrose-non fermenting Related Kinase 2 (SnRK2s) 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 (STM). These genes were also mis-regulated 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 also indicate that HAI1-AHL10 signaling coordinates growth with stress and defense responses.

Protein phosphorylation is a key signalling mechanism and has myriad effects on protein function. Phosphorylation by protein kinases can be reversed by protein phosphatases, thus allowing dynamic control of protein phosphorylation. Although this may suggest a straightforward kinase-phosphatase relationship, plant genomes contain five times more kinases than phosphatases. Here, we examine phospho-signalling from a protein phosphatase centred perspective and ask how relatively few phosphatases regulate many phosphorylation sites. The most abundant class of plant phosphatases, the protein phosphatase 2Cs (PP2Cs), is surrounded by a web of regulation including inhibitor and activator proteins as well as posttranslational modifications that regulate phosphatase activity, control phosphatase stability, or determine the subcellular locations where the phosphatase is present and active. These mechanisms are best established for the Clade A PP2Cs, which are key components of stress and abscisic acid signalling.We also describe other PP2C clades and illustrate how these phosphatases are highly regulated and involved in a wide range of physiological functions. Together, these examples of multiple layers of phosphatase regulation help explain the unbalanced kinase-phosphatase ratio. Continued use of phosphoproteomics to examine phosphatase targets and phosphatase-kinase relationships will be important for deeper understanding of phosphoproteome regulation.

The Highly ABA-Induced 1 (HAI1) protein phosphatase is a central component of drought-related signaling. A screen for HAI1-interacting proteins identified HAI1-Interactor 1 (HIN1), a nuclear protein of unknown function which could be dephosphorylated by HAI1 in vitro. HIN1 colocalization and interaction with serine-arginine rich (SR) splicing factors and appearance of nuclear speckle-localized HIN1 during low water potential (ψw) stress suggested a pre-mRNA splicing-related function. RNA sequencing of Arabidopsis Col-0 wild type identified more than 500 introns where moderate severity low ψw altered intron retention (IR) frequency. Surprisingly, nearly 90% of these had increased splicing efficiency (decreased IR) during stress. For one-third of these introns, ectopic HIN1 expression (35S:HIN1) in unstressed plants mimicked the increased splicing efficiency seen in stress-treated wild type. HIN1 bound to a GAA-repeat, Exonic Splicing Enhancer-like RNA motif enriched in flanking sequence around HIN1-regulated introns. Genes with stress and HIN1-affected splicing efficiency were enriched for abiotic stress and signaling-related functions. The 35S:HIN1 plants had enhanced growth maintenance during low ψw, while hin1 mutants had reduced growth, further indicating the role of HIN1 in drought response. HIN1 is annotated as an MYB/SANT domain protein but has limited homology to other MYB/SANT proteins and is not related to known yeast or metazoan RNA-binding proteins or splicing regulators. Together these data identify HIN1 as a plant-specific RNA-binding protein, show a specific effect of drought acclimation to promote splicing efficiency of IR-prone introns, and also discover HAI1–HIN1 interaction and dephosphorylation that connects stress signaling to splicing regulation.

No abstract

In response to changing environmental conditions, plants activate cellular responses to enable them to adapt to these changes. One such response is autophagy, in which cellular components, for example proteins and organelles, are delivered to the vacuole for degradation. Autophagy is activated by a wide range of conditions, and the regulatory pathways controlling this activation are now being elucidated. However, key insights into how these factors may function together to properly modulate autophagy in response to specific internal or external signals are yet to be discovered. In this review we discuss mechanisms for regulation of autophagy in response to environmental stress and disruptions in cell homeostasis. These pathways include post-translational modification of proteins required for autophagy activation and progression, control of protein stability of the autophagy machinery, and transcriptional regulation, resulting in changes in transcription of genes involved in autophagy. In particular, we highlight potential connections between the roles of key regulators and explore gaps in research, the filling of which can further our understanding of the autophagy regulatory network in plants.

Phosphorylation of AHL10, one of the AT-hook family of plant-specific DNA binding proteins, is critical for growth suppression during moderate severity drought (low water potential) stress. To understand how AHL10 phosphorylation determines drought response, we identified putative AHL10 interacting proteins and further characterized interaction with RRP6L1, a protein involved in epigenetic regulation. RRP6L1 and AHL10 mutants, as well as ahl10-1rrp6l1-2, had similar phenotype of increased growth maintenance during low water potential. Conversely, loss of AHL13, which is homologous to AHL10 and phosphorylated at similar C-terminal site, blocked the enhanced growth maintenance of ahl10-1. Chromatin precipitation demonstrated that RRP6L1 chromatin association increased during low water potential stress and was dependent upon AHL10 phosphorylation. Transcriptome analyses showed that AHL10 and RRP6L1 have concordant effects on expression of stress- and development-related genes. Stress signaling can act via AHL10 phosphorylation to control the chromatin association of the key regulatory protein RRP6L1. AHL10 and RRP6L1 interaction in meristem cells is part of a mechanism to down-regulate growth during low water potential stress. AHL10 and AHL13 are not redundant but rather have distinct roles, likely as part of AHL hetero-complexes.