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Abstract: We investigated the fourth subgroup of Arabidopsis aquaporin, small and basic intrinsic proteins (SIPs). When they were expressed in yeast, SIP1;1 and SIP1;2, but not SIP2;1, gave water-channel activity. The transient expression of SIPs linked with green fluorescent protein in Arabidopsis cells and the subcellular fractionation of the tissue homogenate showed their ER localization. The SIP proteins were detected in all of the tissues, except for dry seeds. Histochemical analysis of promoter-β-glucuronidase fusions revealed the cell-specific expression of SIPs. SIP1;1 and SIP1;2 may function as water channels in the ER, while SIP2;1 might act as an ER channel for other small molecules or ions. [Copyright &y& Elsevier]

By searching a Chlamydomonas expressed sequence tag database and by comparing the retrieved data with homologous sequences from a DNA database, we identified an expressed Chlamydomonas reinhardtii putative major intrinsic protein (MIP) gene. The nucleotide sequence, consisting of 1,631 bp, contains an open reading frame coding for a 300-amino-acid protein named CrMIP1. It possesses conserved NPA motifs, but is not highly homologous to known aquaporins. CrMIP1 was expressed in Saccharomyces cerevisiae and assayed for water and glycerol transport activity. By the stopped-flow spectrophotometric assay, CrMIP1 did not enhance the osmotic water permeability of membrane vesicles of the yeast transformant. However, the transformant cells showed glycerol transport activity in the in vivo assay using [14C]glycerol. This is the first report on the isolation and functional identification of a MIP member from algae. [ABSTRACT FROM AUTHOR]

Plants contain a number of aquaporin isoforms. We developed a method for determining the water channel activity of individual isoforms of aquaporin. Six plasma membrane aquaporins (RsPIPs) and two vacuolar membrane aquaporins (RsTIPs) of radish (Raphanus sativus) were expressed heterologously in Saccharomyces cerevisiae BJ5458, which is deficient in endogenous functional aquaporin. Aquaporins were detected by immunoblot analysis with corresponding antibodies. Water permeability of membranes from yeast transformants was assayed by stopped-flow spectrophotometry. The water channel activity of members of the RsPIP2 and RsTIP subfamilies was about 10 times and 5 times greater, respectively, than that of the control; however, RsPIP1s had little (RsPIP1-2 and RsPIP1-3) or no activity (RsPIP1-1). Site-directed mutation of several residues conserved in RsPIP1s or RsPIP2s markedly altered the water transport activity. Exchange of Ile244 of RsPIP1-3 with valine increased the activity to 250% of the wild type RsPIP1-3. On the other hand, exchange of Val235 of RsPIP2-2, which corresponds to RsPIP1-3 Ile244, with isoleucine caused a marked inactivation to 45% of the original RsPIP2-2. Mutation at possible phosphorylation sites at the N- and C-terminal tails also altered the activity. These results suggest that these residues in the half-helix loop E and the tails are involved in the water transport and the functional regulation of RsPIP1 and RsPIP2. [ABSTRACT FROM AUTHOR]

We investigated tissue- and cell-specific accumulation of radish aquaporin isoforms by immunocytochemical analysis. In taproots, the plasma membrane aquaporins (RsPIP1 and RsPIP2) were accumulated at high levels in the cambium, while the tonoplast aquaporin (RsTIP) was distributed in all tissues. The three isoforms were highly accumulated in the central cylinder of seedling roots and hypocotyls, and rich in the vascular tissue of the petiole of mature plants. The results suggest that RsPIP1 and RsPIP2 are abundant in the cells surrounding the sieve tube of the radish plant. The swelling rate of protoplasts in a hypotonic solution was determined individually by a newly established method to compare the osmotic water permeability of different cell types. All cells of the cortex and endodermis in seedlings showed a high water permeability of more than 300 µm s–1. There was no marked difference in the values between the root endodermis and cortex protoplasts, although the RsPIP level was lower in the cortex than in the endodermis. This inconsistency suggests two possibilities: (1) a low level of aquaporin is enough for high water permeability and (2) the water channel activity of aquaporin in the tissues is regulated individually. The uneven distribution of aquaporins in tissues is discussed along with their physiological roles. [ABSTRACT FROM PUBLISHER]

Aquaporins in the plasma and vacuolar membranes play a key role in the intercellular and intracellular water transport in plants. First, we quantitated the absolute amounts for mRNAs of eight aquaporin isoforms in hypocotyls of radish seedlings. Then, we investigated the effects of salt and water stresses (150 mM NaCl, 300 mM mannitol and 20% polyethylene glycol) and phytohormones (gibberellic acid, abscisic acid and brassinolide) on the mRNA and protein levels of aquaporins in the plasma membrane (RsPIP1-1, 1–2, 1–3, 2–1, 2–2 and 2–3) and vacuolar membrane (RsTIP1-1 and 2–1). The mRNA and protein levels of RsTIP1-1, RsTIP2-1, RsPIP1-1, RsPIP1-2 and RsPIP1-3 were comparatively constant. In contrast, mannitol treatment altered the mRNA levels of RsPIP2-1, RsPIP2-2 and RsPIP2-3 in roots. Immunoblot analysis showed that the RsPIP2-1 protein level was increased by NaCl treatment and decreased by treatment with mannitol and polyethylene glycol. Gibberellic acid and abscisic acid suppressed the levels of mRNAs of RsPIP2-1, RsPIP2-2 and RsPIP2-3 and the protein level of RsPIP2-1 in roots. On the other hand, the protein levels of RsPIP1-group members and RsTIPs were scarcely changed by these phytohormones. In the case of hypocotyls and cotyledons, the mRNA and protein levels of eight isoforms were not markedly affected by any treatment. These results indicate that aquaporins in the root, especially the RsPIP2 group, may be a stress responsive type of aquaporin at least in the protein level. [ABSTRACT FROM PUBLISHER]

A major membrane intrinsic protein (VM23) in vacuoles of radish (Raphanus) tap root was investigated. The cDNAs for two isoforms of VM23, γ- and δ-VM23, encode polypeptides of 253 and 248 amino acids, respectively, γ-and δ-VM23 correspond to the γ- and δ-TIP (tonoplast intrinsic protein) of Arabidopsis. The deduced amino acid sequences of the two VM23 isoforms were 60% identical. The ammo-terminal sequence of γ-VM23 showed agreement with the direct sequence of the purified VM23, suggesting that γ-VM23 is the most abundant molecule among the VM23 isoforms. When mRNAs of γ- and δ-VM23 were injected into Xenopus oocytes, the osmotic water permeability of oocytes increased 6-fold (60 to 200 μM s1) of the control oocytes. The transcripts of both isoforms were detected in a high level in growing hypocotyls and young leaves, but δ-VM23 was not detected in seedling roots. Light illumination enhanced the transcription of two genes of VM23 in cotyledons and roots but suppressed their expression in hypocotyls the growth of which was inhibited by light. These findings suggest that the expression of VM23 is tightly related to cell elongation [ABSTRACT FROM AUTHOR]

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 μg 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. [ABSTRACT FROM AUTHOR]

Abstract: In order to analyze the mitigation of methane (CH4) emissions and the global warming potentials (GWPs) of CH4 and nitrous oxide (N2O) emissions from paddy fields by modifying the adopted water-management technique, we conducted field experiments to measure the CH4 and N2O fluxes at nine sites across Japan. Over 2 years, we tested different water-management strategies such as prolonged midseason drainage (MD) in each site. The CH4 emission rates at each site varied considerably; the rates were dependent on the ratio of reductive and oxidative capacities of the fields. Seasonal CH4 emission was effectively reduced at most sites by prolonging MD beyond its conventional duration, especially at sites where organic matter was added to the soil before the cultivation. We attribute this result to the effective suppression of the CH4 emission peak that occurs early in the cultivation period. Despite the large variation in seasonal CH4 emissions among the sites, the rate of CH4 emission resulting from alternative water-management strategies relative to that resulting from conventional water-management strategies is highly dependent on the degree of drainage during the MD period. N2O emission at most sites, in terms of GWP-based CO2-equivalent, was much smaller than that of CH4 emission. Compared to conventional water-management strategies, the seasonal CH4 emissions and the net 100-year GWPs (CH4 +N2O) can be suppressed to 69.5±3.4 (SE)% and 72.0±3.1% while maintaining grain yields as high as 96.2±2.0% by prolonging MD on average by employing the selected alternative water-management strategies that satisfied the following conditions: the percent of CH4 emission of alternative water-management strategies was less than 90% and the grain yield was greater than 85% relative to conventional water-management strategies. [Copyright &y& Elsevier]