Low Aquaporin Content and Low Osmotic Water Permeability of the Plasma and Vacuolar Membranes of a CAM Plant Graptopetalum paraguayense: Comparison with Radish

Abstract

Aquaporin facilitates the osmotic water transport across biomembranes and is involved in the transcellular and intracellular water flow in plants. We immunochemically quantified the aquaporin level in leaf plasma membranes (PM) and tonoplast of Graptopetalum paraguayense, a Crassulacean acid metabolism (CAM) plant. The aquaporin content in the Graptopetalum tonoplast was approximately 1% of that of radish. The content was calculated to be about 3 microg mg(-1) of tonoplast protein. The level of PM aquaporin in Graptopetalum was determined to be less than 20% of that of radish, in which an aquaporin was a major protein of the PM. The PM aquaporin was detected in the mesophyll tissue of Graptopetalum leaf by tissue print immunoblotting. The osmotic water permeability of PM and tonoplast vesicles prepared from both plants was determined with a stopped-flow spectrophotometer. The water permeability of PM was lower than that of the tonoplast in both plants. The Graptopetalum PM vesicles hardly showed water permeability, although the tonoplast showed a relatively high permeability. The water permeability changed depending on the assay temperature and was also partially inhibited by a sulfhydryl reagent. Furthermore, measurement of the rate of swelling and shrinking in different mannitol concentrations revealed that the protoplasts of Graptopetalum showed low water permeability. These results suggest that the low content of aquaporins in PM and tonoplast is one of the causes of the low water permeability of GRAPTOPETALUM: The relationship between the water-storage function of succulent leaves of CAM plants and the low aquaporin level is also discussed.

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

“…The PIP antibody was raised against N-terminal common sequence "KDYNEPPPAPLFEP-GELSSWS", among six radish PIPs including three PIP1s and three PIP2s. The antibody has broader specificity and was able to detect the PIPs in graptopetalum and radish [23]. We compared all the rice PIP proteins and found high conservation in this region.…”

“…Plasma membrane fractions were prepared by aqueous two-phase partitioning as described by Ohshima et al [23]. Protein concentration was measured according to Bradford [24].…”

“…Protein concentration was measured according to Bradford [24]. Western blots were carried out using anti-PIP antibody [23] following the standard method according to Sambrook et al [25]. The PIP antibody was raised against N-terminal common sequence "KDYNEPPPAPLFEP-GELSSWS", among six radish PIPs including three PIP1s and three PIP2s.…”

“…Standard procedures for SDS-PAGE in 12% polyacrylamide gels were used and proteins were transferred to nitrocellulose. The anti-PIP antibodies [23] were 1:500 diluted for immunoblotting. The primary antibodies were detected with goat anti-rabbit IgG-HRP (1:1000), and the signal was detected by chemiluminescence method (West Pico West Blotting, Pierce, IL, USA).…”

Water relations and an expression analysis of plasma membrane intrinsic proteins in sensitive and tolerant rice during chilling and recovery

“…Root plasma membrane fractions were prepared by the aqueous two-phase partitioning method as describe by Ohshima et al [31]. Membrane protein concentration was measured according to Bradford [32].…”