| 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. |
