Roles of endogenous alpha-expansins and xyloglucan in cell wall growth and mechanical properties in Arabiodpsis thaliana.

摘要

Expansins which consists of four families named EXPA, EXPB, EXLA, and EXLB were first discovered as unique cell wall loosening proteins under acidic-pH conditions using isolated cell walls. Expansins are hypothesized to disrupt non-covalent bonds between cellulose microfibrilis and hemicelluloses or other wall matrix polysaccharides in a non-enzymatic fashion. Land plants typically show acid-induced growth responses mediated by expansins. Auxin activates a proton pump that lowers the apoplast pH of plant cells. Therefore, pH-dependent wall loosening by expansins has been implicated as being important for auxin growth responses of land plants.;Etiolated Arabidopsis hypocotyls are an excellent system for studying the fundamental processes of cell growth because etiolated hypocotyls are mainly composed of growing primary cell walls. For this reason, I studied endogenous expansin functions particularly with respect to auxin and acid growth responses using etiolated Arabidopsis hypocotyls.;To understand the endogenous functions of expansins in-vivo as well as in-vitro, I identified six alpha-expansins (EXPA1, EXPA2, EXPA8, EXPA9, EXPA11, and EXPA12) expressed in etiolated Arabidopsis hypocotyls and examined their functional roles for auxin- and acid-growth responses in-vitro and in-vivo. I employed Salk T-DNA insertion mutants of EXPA9 (expa9-1 and expa9-2) and established transgenic plants expressing silencing constructs for the other five alpha-expansins through RNA interference (RNAi) or artificial microRNA (amiRNA). Plants with knock-out mutation or silencing of alpha- expansins expressed in etiolated hypocotyls showed significantly reduced hypocotyl length in dark-grown seedlings. Isolated cell walls of the transgenic plants with reduced alpha-expansin expression showed slower cell wall elongation and stress relaxation rates, when compared to wild type. Based on these results, I conclude that endogenous alpha- expansins are required for the normal molecular rearrangements or reorientations of cell wall polymers underlying hypocotyl elongation. Additionally, auxin- and FC-induced growth studies with the transgenic etiolated hypocotyl segments provide the evidence for endogenous alpha-expansins stimulating for the in-vivo 'acid growth' responses.;In addition, I made two amiRNA constructs to silence most of the Arabidopsis alpha- expansins. The constructs were theoretically predicted to suppress 17 alpha-expansins. The silencing plants containing the precursor of two pre-amiRNAs showed impaired leaf inclination compared to control plants. This suggests another functional role of alpha- expansins, required for normal shading avoidance.;Moreover, I studied Arabidopsis double mutant (xxt1xxt2), which is defective in xyloglucan (XyG) synthesis, with respect to cell wall mechanical properties. 28-day-old rosette petioles of xxt1xxt2 showed decreased wall elongation in response to acidic buffer and exogenous alpha-expansins, compared to wild type. These results suggest that alpha- expansins are required for normal cell wall loosening and elongation through disrupting the cellulose-XyG network. Also, results of wall extension assays using seven different wall loosening enzymes or proteins support the conclusion that when cell walls lack XyG, cellulose-xylan and cellulose-pectin networks can take over the main load bearing role, replacing cellulose-XyG.;In my thesis, I drew three major conclusions concerning in-vivo functions of alpha- expansins in wall growth; (1) endogenous alpha-expansins are required for normal wall growth responses in response to acidic wall pH and auxin in living cells, (2) alpha-expansins are necessary for normal shading avoidance, and (3) the cellulose-XyG network, a main substrate of alpha-expansins, plays a major role for the extensibility of primary cell walls in Arabidopsis. The main load-bearing structure, however, can be substituted with cellulose-xylan and cellulose-pectin networks, when cell walls have insufficient XyG content.