Functional Redundancy Of CESA6 With CESA2 And CESA5 In Arabidopsis Under Light/Dark Treatments

At least 10 CESA sequences (AtCesA1～AtCesA10) have been identified in Arabid-opsis to involve in cellulose synthesis. AtCESA1, AtCESA3 and AtCESA6 are characterized for primary cell wall synthesis, whereas AtCESA4, AtCESA7 and AtCESA8 are for secondary cell wall. It has been reported that AtCESA2, AtCESA5 and AtCESA9 might be partial functional redundancy with AtCESA6. However, it is not clear about the redundancy mechanism for cellulose biosynthesis in plant.In this study, we amplified cDNA fragments of AtCesAs variable region by RT-PCR, isolated GST-AtCESAs fusion proteins, and prepared their antibodies. We observed gene expression, detected proteins and analyze CESA activities in vitro using AtCesA6 mutant (prc1-1, cesa6).Major results were described below:1. Preparation and detection of AtCesAs AntibodiesWe prepared their (AtCesA1～AtCesA8) antibodies. Western-blotting analysis indicat-ed that the antibodies (Anti-AtCESA1, Anti-AtCESA3, Anti-AtCESA6, Anti-AtCESA2, Anti-AtCESA5, Anti-AtCESA8) showed specific bands against proteins extracted from the plasma membrane fraction in Arabidopsis. But, I found cross-reaction bands in Anti-AtCESA4 and Anti-AtCESA7.2. Expression observation of AtCesAs in hypocotyls under light/dark treatmentsGUS staining and the Real-time PCR showed that AtCesA1, AtCesA3 and AtCesA6 expression levels were significantly increased under dark treatment. AtCesA2 showed a decreased expression under dark and was remarkable increased under ligh. AtCesA5 was little changed under both treatments.3. Expression analysis of AtCesAs in mutantsCompared with wild type, AtCesAs in mutants were decreased remarkably under 9-day-old light/dark conditions. Under dark treatment, AtCesA1, AtCesA3 and AtCesA5 in mutants were expressed much higher than ones under light.4. Test of AtCESA proteins and analysis of AtCESA activity in vitroAtCESA6 protein was reduced whereas AtCESA5 was raised in the plasma membrane fractions of mutants (prc1-1, cesa6) under light/dark treatments, compared with wild type. AtCESA2 showed no difference under light, but was reduced under dark. It suggested that AtCESA5 may partially replace AtCESA6 for the survival of mutants under both light and dark treatments, while AtCESA2 can only play a partial substite role under light condition. However, amounts of the complex composed of AtCESAl, AtCESA3, and AtCESA6/AtCESA2/AtCESA5 in mutants were much less than ones in wild type.Cellulose synthesis in vitro indicated thatβ-1,4-glucan product was much higher thanβ-1,3-glucan in mutants and wild type under light condition, but bothβ-1,4-glucan andβ-1,3-glucan were remarably decreased under dark condition, especially for prc1-1 mutant.5. Determination of cell wall compositionUnder light treatment, mutants(prc1-1 & cesa6) were determined with much less crystalline cellulose and more non-crystalline cellulose than wild type, and mutant (cesa6) showed additionally increased hemicellulose, pectin and soluble sugar. As a contrast, mutant prc1-1 under dark was decreased in crystalline cellulose, but increased in non-crystalline cellulose, hemicelluloses, pectins and soluble sugar. Mutant cesa6 showed a decrease in crystalline cellulose and an increase in hemicellulose, pectins and soluble sugars.6. The effect of secondary cell wall caused by the mutation of AtCesA6Compared with wild type, prc1-1 and cesa6 were determined with much less hemi-cellulose, non-crystalline cellulose, crystalline cellulose and more pectin, but showed no difference in lignin. The mutants(prc1-1 & cesa6) were increased in side chains of hemi-cellulose, H unit of lignin, but decreased in G and S units of lignin. However, DP and CrI of cellulose were much unchanged in mutants' straw. Under the treatment of cellulase, the relased sugars in cesa6 and prc1-1 can be increased by 8.83±0.87% and 5.68±0.49%, respectively, and IRX-3 can be increased by 36.63±2.67%.