| Mei-Hao Sun (Plant Science) Directed by Professors Wei-Ai Su and Zhang-Cheng Tang Aquaporins are proteins, which form macromolecular aqueous pores in the cellmembranes facilitating water transport across the membranes. The discovery ofaquaporins in plant gives us new insights on the water transport through cellmembranes. For example, we used to believe that water diffusion is the main way forwater transmembrane transport, now it is believed that it is aquaporins in themembranes. We investigated: 1. The roles of aquaporins in stomatal movements ofVicia faba. 2. The expression and regulation of rwc3 in rice.1.The role of aquaporins in stomatal movements of Vicia faba. One full-length cDNA named bbaq1 was isolated from a cDNA library of V. fabaepidermis. As the epidermis of leaf can easily be peeled off, we developed a simplemethod of in situ hybridization using the epidermis to substitute the sections. It hasbeen used satisfactorily to ensure the expression of broad bean aquaporin1 in guardcells. The results of RT-PCR also show that the expression of aquaporins in guardcells is much higher than other leaf cell types, which indicate the importance of AQPfor the bulk water flow across guard cell membranes and the stomatal movements. However, the function of aquaporin in stomatal movements remains largelyelusive. Here we provided some evidences that aquaporin plays a role in stomatalmovements via regulating water flow across the plasma membrane in guard cells.Aquaporin inhibitor HgCl2 decreased significantly the shrinkage rates of guard cellprotoplasts induced by PEG 6000. Consequently, HgCl2 inhibited bothfusicoccin-induced stomatal opening and ABA-induced closing. In contrast,2-mercaptoethanol, a scavenging reagent of HgCl2 suppressed partially the inhibitoryeffects of HgCl2 on both water flows across the plasma membrane of guard cells andstomatal movements. In addition, the shrinkage of guard cell protoplasts wasdecreased significantly by protein kinase inhibitor K-252a and enhanced byphosphatase inhibitor okadaic acid, which suggests that phosphorylalation ofaquaproins is involved in water transportation across the plasma membrane in guardcells. Furthermore, our result show that the aquaporin transcription level was verywell associated with the stomatal opening and closing rates. Taken together, theseresults provide important evidence that aquaporins regulate water transportation IVacross guard cell plasma membranes; and therefore play an essential role in stomatalmovements.2.The expression and regulation of rwc3 in rice Reporter gene GUS was used to investigate the expression and regulation ofwater channel protein3 (rwc3) in rice. The 5' upstream (1.2kb) of genomic clone ofrwc3 was isolated and constructed into pCAMBIA1381Z at the cloning site of SmaI(named pprwc3). The plasimid pprwc3 was validated by PCR, enzyme digestion andsequencing; and it was transformed into rice (Oryza sativa L. No.4 Shenxiangjing)by Agrobactium-mediated method. The results of PCR, Southern blot and GUShistochemical staining all indicated that GUS gene was integrated into rice genomicDNA. Histochemical analysis of GUS activity showed that the transcription of rwc3 isvery broad in rice. There are expression in root, stem, leaf, flower and seeds.Moreover, its expression levels in root hairs, root mature zone, root meristematiczone, vascular bundle of leaf and stem, pollen, ovary and immature seed coat werevery high. It distribution in these tissues indicated that RWC3 may involved in waterabsorption, water transport in leaf and stem, development of root cells and ovary andmaturation of pollen. GA affected the expression of GUS in transgenic rice leaves,calli and suspension cells. GA enhanced the GUS activity in leaves, and ancymidolthe inhibitor of endogenous GA synthesis had the opposite effect. When substitutingmannitol for sugar in N6D2 medium, there w...
|
PDF Full Text Request