Mechanism Of SKU5 And SKS1 Proteins Coordinating Cell Wall Formation In Arabidopsis

The primary cell wall is a fundamental plant constituent for plant cell,which is a compact network constituted by polysaccharides.This special structure gives the cell wall plasticity and an extremely high mechanical strength for normal growth and morphogenesis of plant cells.The formation of cell wall is influenced by several factors,among which reactive oxygen species(ROS)act as an important messenger to change the structure of cell wall and affect the normal growth of cells,but the regulatory mechanism of ROS on the maintenance of cell wall homeostasis needs to be studied more deeply.Pervious study had found that Arabidopsis multi-copper oxidases Skewed 5(SKU5)-similar proteins(SKSs)may participate in primary cell wall formation by regulating apoplastic ROS homeostasis,but the mechanism still unclear.Therefore,we used the multicopper oxidase SKU5 and its homolog SKS1 as the basis to investigate the regulatory role of ROS on cell wall formation.First,we obtained the SKU5 and SKS1 protein loss-of-function mutant sku5 sks1,whose primary root growth was severely inhibited.Observation of cell morphology of sku5 sks1 mutant roots showed that the longitudinal cell wall exhibited abnormal protrusions in epidermal and cortical cell layers,which coincided with the location of SKU5 and SKS1 expression.Further observation of the structure of longitudinal cell walls of sku5 sksl mutant revealed a severe disruption of the compact network formed by cellulose microfibers.It indicated that SKU5 and SKS1 are involved in the synthesis of cellulose and the regulation of cell wall structure.To investigate the cause of defective cell wall development,we observed the production and distribution of ROS in sku5 sksl mutant roots.We found that ROS,especially in the form of Superoxide(O2·-),were overproduced in apoplast in sku5 sksl mutant roots.The transcriptional level of NADPH oxidase,a synthetic protein of apoplastic O2·-,was also upregulated in sku5 sks1 mutant roots.Scavenging of O2·-or inhibition of NADPH oxidase activity by biochemical and genetic approaches partially restored the abnormal phenotype of sku5 sksl mutant,suggesting that SKU5 and SKS1 are involved in cell wall formation by regulating NADPH oxidase mediated ROS production.Further studies on SKU5 and SKS1 protein functions revealed that both of them were activated by iron treatment,and iron over-accumulated in the longitudinal cell walls of sku5 sks1 mutant.Reducing iron content in sku5 sks1 mutant roots inhibited ROS overproduction and restored the phenotype of abnormal cell wall.It implies that SKU5 and SKS1 regulate cell wall formation by regulating iron-mediated ROS production in apoplast.In summary,we identified SKU5 and SKS1 as novel regulators for apoplastic ROS homeostasis,further regulating cell wall formation and cell morphogenesis.