Arabidopsis <i>XTH4</i> and <i>XTH9</i> Contribute to Wood Cell Expansion and Secondary Wall Formation

Abstract

Xyloglucan is the major hemicellulose of dicotyledon primary cell walls, affecting the load-bearing framework with the participation of xyloglucan <i>endo</i>-transglycosylase/hydrolases (XTHs). We used loss- and gain-of function approaches to study functions of <i>XTH4</i> and <i>XTH9</i> abundantly expressed in cambial regions during secondary growth of Arabidopsis (<i>Arabidopsis thaliana</i>). In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. They also stimulated secondary wall thickening but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition indicating an overall increase in secondary versus primary walls in mutants, indicative of higher xylem production compared with the wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers compared with the wild type was increased in <i>xth9</i> and reduced in <i>xth4</i>, whereas the double mutant <i>xth4x9</i> displayed an intermediate number of layers. These changes correlated with specific Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers, where neither <i>XTH4</i> nor <i>XTH9</i> was expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and cell wall integrity sensing, including <i>THESEUS1</i> and <i>WALL ASSOCIATED KINASE2</i>, were highly induced in the mutants, indicating that deficiency in <i>XTH4</i> and <i>XTH9</i> triggers cell wall integrity signaling, which, we propose, stimulates xylem cell production and modulates secondary wall thickening. Prominent effects of <i>XTH4</i> and <i>XTH9</i> on secondary xylem support the hypothesis that altered xyloglucan affects wood properties both directly and via cell wall integrity sensing.