Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis

Abstract

The wall-associated kinases (WAKs) 1 are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAKdependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGs in planta. Using N 14/ N 15 isotopic in vivo metabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. The cell walls of angiosperms are composed of a complex arrangement of cellulose, hemicellulose, and pectin and are assembled through a complex, developmentally regulated coordination of synthesis, turnover, and interactions between protein and carbohydrates (1). The pectins can be selectively and locally cross-linked into a structural network that is subsequently remodeled and degraded by enzymes, and these events have dramatic effects on cell enlargement (2-6). Pathogens and mechanical disruptions also cause fragmentation and, thus, release of the pectin, leading often to a plant stress response (7-9).A number of receptor kinases such as THE1, FER, HERK, ANX, and RLP44 have been termed cell wall sensors (10 -18) and typically have extracellular domains containing leucinerich regions and a malectin carbohydrate-binding domain, although an experimentally demonstrated role for polysaccharide binding to their extracellular domains is unclear. Of the plant putative "wall sensors" only the wall-associated kinases (WAKs) are known to bind to a cell wall component, pectin, and these are distinguished also by their unique extracellular domain that lacks leucine-rich repeats and contains instead epidermal growth factor (EGF) repeats as well as a pectin-binding region (19).Pectins are synthesized in the Golgi apparatus as methyl esterified 1-4 D-galacturonic acids and are secret...

Plant mechanosensitive ion channels: an ocean of possibilities

“…Dwarfing and ectopic lesions are also observed in response to a single EMS-induced point mutation in the C-terminus of MSL10 [44], suggesting that these overexpression phenotypes reflect some aspect of the normal gene function. In addition, a recent study implicated MSL4 in pathogen-triggered immunity [45], and MSL6 phosphorylation was observed in response to oligogalacturonide treatment [46]. We propose that plasma membrane-localized MSLs serve as sensors of cellular mechanical homeostasis, or “mechanostasis”.…”

YODA MAP3K kinase regulates plant immune responses conferring broad‐spectrum disease resistance

“…Among the putative CA-YDA-associated defensive responses, there are genes, like SSP1 and SSP2, which encode putative small secreted peptides, whose mature and biologically active sequences are unknown, but they seem differ from previously described peptidic DAMPs . Also, a group of genes encoding potential PRRs were upregulated in CA-YDA, and among these there was an RLK with two wall-associated kinaselike domains that have been involved in the recognition of cellwall-derived oligogalacturonides (Kohorn et al, 2009(Kohorn et al, , 2016Brutus et al, 2010). Remarkably, mutants impaired in these genes (ssp1, ssp2, and wakl-rlk) show an enhanced susceptibility to the pathogens to which the CA-YDA plants are resistant to: PcBMM, H. arabidopsidis and P. syringae (Figs 8, S13c).…”

Early mannitol-triggered changes in the Arabidopsis leaf (phospho)proteome

“…Within the normalised phosphopeptides, two phosphosites of a remorin family member REM1.3 (AT2G45820), Thr 58 and Ser 64 , were down regulated upon mannitol treatment ( Figure 8 and Table 1 ). REM1.3 was already reported to be phosphorylated upon oligo-galacturonide treatment which elucidates a plant stress response (Kohorn et al , 2016). REM1.3 is proposed as a scaffolding protein for signalling at the plasma membrane and is thus an interesting candidate for mannitol-induced signalling (Marín et al , 2012).…”