Differential Induction of Two Glucoamylases in Candida pelliculosa

Rapid growth of the submerged shoots of deepwater rice is essential for survival during the rainy season. We investigated changes in the expression of vacuolar H⁺-ATPase (V-ATPase), H⁺-pyrophosphatase (V-PPase), and aquaporins under submerged conditions. The amounts of vacuolar proton pumps, which support the active transport of ions into the vacuoles, were maintained on a membrane protein basis in the developing vacuoles. Among the six isogenes of V-PPase, OsVHP1;3 was markedly enhanced by submersion. The gene expression of efficient water channels, OsTIP1;1, OsTIP2;2, OsPIP1;1, OsPIP2;1, and OsPIP2;2, was markedly enhanced by submersion. The increase in aquaporin expression might support quick elongation of internodes. The mRNA levels of OsNIP2;2 and OsNIP3;1, which transport silicic and boric acids respectively, clearly decreased. The present study indicates that internodes of deepwater rice upregulate vacuolar proton pumps and water channel aquaporins and downregulate aquaporins that allow permeation of the substrates that suppress internode growth.     

Tissue and cell-specific localization of rice aquaporins and their water transport activities

Water transport in plants is greatly dependent on the expression and activity of water transport channels, called aquaporins. Here, we have clarified the tissue- and cell-specific localization of aquaporins in rice plants by immunoblotting and immunocytochemistry using seven isoform-specific aquaporin antibodies. We also examined water transport activities of typical aquaporin family members using a yeast expression system in combination with a stopped-flow spectrophotometry assay. OsPIP1 members, OsPIP2;1, OsTIP1;1 and OsTIP2;2 were expressed in both leaf blades and roots, while OsPIP2;3, OsPIP2;5 and OsTIP2;1 were expressed only in roots. In roots, large amounts of aquaporins accumulated in the region adjacent to the root tip (around 1.5-4 mm from the root tip). In this region, cell-specific localization of the various aquaporin members was observed. OsPIP1 members and OsTIP2;2 accumulated predominantly in the endodermis and the central cylinder, respectively. OsTIP1;1 showed specific localization in the rhizodermis and exodermis. OsPIP2;1, OsPIP2;3 and OsPIP2;5 accumulated in all root cells, but they showed higher levels of accumulation in endodermis than other cells. In the region at 35 mm from the root tip, where aerenchyma develops, aquaporins accumulated at low levels. In leaf blades, OsPIP1 members and OsPIP2;1 were localized mainly in mesophyll cells. OsPIP2;1, OsPIP2;3, OsPIP2;5 and OsTIP2;2 expressed in yeast showed high water transport activities. These results suggest that rice aquaporins with various water transport activities may play distinct roles in facilitating water flux and maintaining the water potential in different tissues and cells.     

Aquaporins in developing rice grains

During rice grain filling, grain moisture content and weight show dynamic changes. We focused on the expression of all 33 rice aquaporins in developing grains. Only two aquaporin genes, OsPIP2;1 and OsTIP3;1, were highly expressed in the period 10–25 days after heading (DAH). High-temperature treatment from 7 to 21 DAH abolished the dynamic up-regulation of OsPIP2;1 in the period 15–20 DAH, whereas OsTIP3;1 expression was not affected. Immunohistochemical analysis revealed that OsPIP2;1 was present in the starchy endosperm, nucellar projection, nucellar epidermis, and dorsal vascular bundles, but not in the aleurone layer. OsTIP3;1 was present in the aleurone layer and starchy endosperm. Water transport activity of recombinant OsTIP3;1 was low, in contrast to the high activity of recombinant OsPIP2;1 we reported previously. Our data suggest that OsPIP2;1 and OsTIP3;1 have distinct roles in developing grains.     

Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice

The vacuole, a multifunctional organelle of most plant cells, has very important roles in space filling, osmotic adjustment, storage and digestion. Previous researches suggested that aquaporins in the tonoplast were involved in vacuolar functions. The rice genome contains 33 aquaporin genes, 10 of which encode tonoplast intrinsic proteins (TIPs). However, the function of each individual TIP isoform and the integrated function of TIPs under various physiological conditions remain elusive. Here, five rice TIP members were characterized with water and/or glycerol transport activities using the Xenopus oocyte expression system. OsTIP1;2, OsTIP2;2, OsTIP4;1 and OsTIP5;1 possessed water transport activity. OsTIP1;2, OsTIP3;2 and OsTIP4;1 were demonstrated with glycerol transport activity. Rice TIP expression patterns under various abiotic stress conditions including dehydration, high salinity, abscisic acid (ABA) and during seed germination were investigated by real-time PCR. OsTIP1s (OsTIP1;1 and OsTIP1;2) were highly expressed during seed germination, whereas OsTIP3s (OsTIP3;1 and OsTIP3;2) were specifically expressed in mature seeds with a decrease in expression levels upon germination. The results of this research provided a functional and expression profiles of rice TIPs.     

Regulation and reversibility of vacuolar H(+)-ATPase

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.     

Chilling-induced changes of vacuolar proton pumps in hypocotyls of <Emphasis Type="Italic">Vigna unguiculata</Emphasis>

Vigna unguiculata (cowpea) is a legume adapted to high temperatures and is sensitive to low temperatures. Temperature is one of the limiting factors of growth and yield for many crops but its effect on cowpea metabolism is not known. We investigated the effect of chilling on activity of vacuolar proton pumps (V-ATPase and V-PPase) and their protein content in tonoplast vesicles of cowpea hypocotyls. Seedlings grown for 7 days at 10 or 4°C were used for experiments. Chilling treatment at 10 or 4°C markedly suppressed growth of cowpea seedlings. Following chilling at 10 and 4°C, activity of both proton pumps and the relative amount of V-PPase and subunit A of V-ATPase were significantly increased. Both substrate hydrolysis and H⁺ transport activities of V-PPase remained at relatively high levels during chilling treatment. For V-ATPase, treatment at 10°C for 6 days increased the ATP hydrolysis activity. However, the H⁺ transport activity of the enzyme was increased when treated for 4 days but was markedly decreased when treated for 6 days. Our results provide evidence for different regulation for these vacuolar proton pumps, indicating that V-PPase is the more stable proton pump throughout chilling stress.     

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

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

The transmembrane domain 6 of vacuolar H(+)-pyrophosphatase mediates protein targeting and proton transport

Vacuolar H(+)-pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role in the maintenance of the pH in cytoplasm and vacuoles via proton translocation from the cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. The topology of V-PPase as predicted by TopPred II suggests that the catalytic site is putatively located in loop e and exposed to the cytosol. The adjacent transmembrane domain 6 (TM6) is highly conserved and believed to participate in the catalytic function and conformational stability of V-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean V-PPase was carried out to identify its structural and functional role. Mutants Y299A, A306S and L317A exhibited gross impairment in both PP(i) hydrolysis and proton translocation. Meanwhile, mutations at L307 and N318 completely abolished the targeting of the enzyme, causing broad cytosolic localization and implicating a possible role of these residues in protein translocation. The location of these amino acid residues was on the same side of the helix wheel, suggesting their involvement in maintaining the stability of enzyme conformation. G297A, E301A and A305S mutants showed declines in proton translocation but not in PP(i) hydrolysis, consequently resulting in decreases in the coupling efficiency. These amino acid residues cluster at one face of the helix wheel, indicating their direct/indirect participation in proton translocation. Taken together, these data indicate that TM6 is crucial to vacuolar H(+)-pyrophosphatase, probably mediating protein targeting, proton transport, and the maintenance of enzyme structure.     

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+-pumping pyrophosphatase in pepper plants

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes.     

Transport processes of solutes across the vacuolar membrane of higher plants

The central vacuole is the largest compartment of a mature plant cell and may occupy more than 80% of the total cell volume. However, recent results indicate that beside the large central vacuole, several small vacuoles may exist in a plant cell. These vacuoles often belong to different classes and can be distinguished either by their contents in soluble proteins or by different types of a major vacuolar membrane protein, the aquaporins. Two vacuolar proton pumps, an ATPase and a PPase energize vacuolar uptake of most solutes. The electrochemical gradient generated by these pumps can be utilized to accumulate cations by a proton antiport mechanism or anions due to the membrane potential difference. Uptake can be catalyzed by channels or by transporters. Growing evidence shows that for most ions more than one transporter/channel exist at the vacuolar membrane. Furthermore, plant secondary products may be accumulated by proton antiport mechanisms. The transport of some solutes such as sucrose is energized in some plants but occurs by facilitated diffusion in others. A new class of transporters has been discovered recently: the ABC type transporters are directly energized by MgATP and do not depend on the electrochemical force. Their substrates are organic anions formed by conjugation, e.g. to glutathione. In this review we discuss the different transport processes occurring at the vacuolar membrane and focus on some new results obtained in this field.     

Differential localization of tonoplast intrinsic proteins on the membrane of protein body Type II and aleurone grain in rice seeds

Tonoplast intrinsic proteins (TIPs) belong to an aquaporin family of proteins that function as water-transport channels. In this study, we isolated and characterized three novel rice cDNAs for OsTIP1, OsTIP2, and OsTIP3 that are homologous to rice gamma-TIP cDNA. Northern blot hybridization analyses revealed that rice gamma-TIP was expressed in all plant organs. OsTIP1 was expressed in mature seed embryos and during early seed germination. OsTIP2 was expressed exclusively in roots. OsTIP3 was specifically expressed in seeds. These results suggest that the OsTIP1, OsTIP2, and OsTIP3 genes encode discrete, functionally specialized TIPs. Immunocytochemical analysis in rice endosperm cells revealed that rice gamma-TIP was localized only on the protein body type II (PB-II) membranes, whereas OsTIP3 was localized on the PB-II and the aleurone grain membranes. Although both the PB-II and the aleurone grain are derived from vacuoles, these results suggest that they may be derived from different types of vacuoles.     

Endomembrane proton pumps: connecting membrane and vesicle transport

pH-homeostasis in the endomembrane system requires the activity of proton-pumps. In animals, the progressive acidification of compartments along the endocytic and secretory pathways is critical for protein sorting and vesicle trafficking, and is achieved by the activity of the vacuolar H(+)-ATPase (V-ATPase). Plants have an additional endomembrane pump, the vacuolar H(+)-pyrophosphatase (V-PPase), and previous research was largely focused on the respective functions of the two pumps in secondary active transport across the tonoplast. Recent approaches, including reverse genetics, have not only provided evidence that both enzymes play unique and essential roles but have also highlighted the important functions of the two proton pumps in endocytic and secretory trafficking.     

Functional modulation of an aquaporin to intensify photosynthesis and abrogate bacterial virulence in rice

Plant aquaporins are a recently noted biological resource with a great potential to improve crop growth and defense traits. Here, we report the functional modulation of the rice (Oryza sativa) aquaporin OsPIP1;3 to enhance rice photosynthesis and grain production and to control bacterial blight and leaf streak, the most devastating worldwide bacterial diseases in the crop. We characterize OsPIP1;3 as a physiologically relevant CO₂-transporting facilitator, which supports 30% of rice photosynthesis on average. This role is nullified by interaction of OsPIP1;3 with the bacterial protein Hpa1, an essential component of the Type III translocon that supports translocation of the bacterial Type III effectors PthXo1 and TALi into rice cells to induce leaf blight and streak, respectively. Hpa1 binding shifts OsPIP1;3 from CO₂ transport to effector translocation, aggravates bacterial virulence, and blocks rice photosynthesis. On the contrary, the external application of isolated Hpa1 to rice plants effectively prevents OsPIP1;3 from interaction with Hpa1 secreted by the bacteria that are infecting the plants. Blockage of the OsPIP1;3–Hpa1 interaction reverts OsPIP1;3 from effector translocation to CO₂ transport, abrogates bacterial virulence, and meanwhile induces defense responses in rice. These beneficial effects can combine to enhance photosynthesis by 29–30%, reduce bacterial disease by 58–75%, and increase grain yield by 11–34% in different rice varieties investigated in small-scale field trials conducted during the past years. Our results suggest that crop productivity and immunity can be coordinated by modulating the physiological and pathological functions of a single aquaporin to break the growth–defense tradeoff barrier.     

The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue

The petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red to blue during flower opening. This color change is caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals, we focused on vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) and an isoform of Na+/H+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in bud and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower opening is due to active transport of Na+ and/or K+ from the cytosol into vacuoles through a sodium- or potassium-driven Na+(K+)/H+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.