Identification and Functional Analysis of Key Autophosphorylation Residues of Arabidopsis Senescence Associated Receptor-like Kinase

Reversible protein phosphorylation mediated by protein kinases and phosphatases plays important roles in the regulation of leaf senescence. We previously reported that the senescence-associated leucine-rich repeat receptor-like kinase AtSARK autophosphorylates on both serine/threonine and tyrosine residues and functions as a positive regulator of Arabidopsis leaf senescence; the senescence-suppressed protein phosphatase SSPP interacts with and dephosphorylates the cytoplasmic domain of AtSARK, thereby negatively regulating leaf senescence. Here, 27 autophosphorylation residues of AtSARK were revealed by mass spectrometry analysis, and six of them, including two Ser, two Thr, and two Tyr residues, were further found to be important for the biological functions of AtSARK. All site-directed mutations of these six residues that resulted in decreased autophosphorylation level of AtSARK could significantly inhibit AtSARK-induced leaf senescence. In addition, mutations mimicking the dephosphorylation form of Ser384 (S384A) or the phosphorylation form of Tyr413 (Y413E) substantially reduced the interaction between AtSARK and SSPP. All results suggest that autophosphorylation of AtSARK is essential for its functions in promoting leaf senescence. The possible roles of S384 and Y413 residues in fine-tuning the interaction between AtSARK and SSPP are discussed herein.     

The Xyloglucan Endotransglucosylase/Hydrolase Gene XTH22/TCH4 Regulates Plant Growth by Disrupting the Cell Wall Homeostasis in Arabidopsis under Boron Deficiency

TCH4 is a xyloglucan endotransglucosylase/hydrolase (XTH) family member. Extensive studies have shown that XTHs are very important in cell wall homeostasis for plant growth and development. Boron (B), as an essential micronutrient for plants, plays an essential role in the cross-linking of cell wall pectin. However, the effect of B on cell wall organization is unclear. This study aimed to explore the mechanism of plant adaption to B stress by investigating the role of TCH4 in cell wall homeostasis. We conducted both plate and hydroponic cultures of wild-type Col-0 and overexpression and gene knockout lines of XTH22/TCH4 to analyze the phenotype, components, and characteristics of the cell wall using immunofluorescence, atomic force microscopy (AFM), and transmission electron microscopy (TEM). B deficiency induces the expression of TCH4. The overexpression lines of TCH4 presented more sensitivity to B deficiency than the wild-type Col-0, while the knockout lines of TCH4 were more resistant to low B stress. Up-regulation of TCH4 influenced the ratio of chelator-soluble pectin to alkali-soluble pectin and decreased the degree of methylesterification of pectin under B-deficient conditions. Moreover, we found that B deficiency disturbed the arrangement of cellulose, enlarged the gap between cellulose microfibrils, and decreased the mechanical strength of the cell wall, leading to the formation of a thickened and deformed triangular region of the cell wall. These symptoms were more profound in the TCH4 overexpression lines. Consistently, compared with Col-0, the O₂⁻ and MDA contents in the TCH4 overexpression lines increased under B-deficient conditions. This study identified the B-deficiency-induced TCH4 gene, which regulates cell wall homeostasis to influence plant growth under B-deficient conditions.