Shoji Segami

Keep an Eye on PPi: The Vacuolar-Type H+-Pyrophosphatase Regulates Postgerminative Development in Arabidopsis

Oilseed germination requires gluconeogenesis, the conversion of storage lipids into carbohydrates, to sustain seedling heterotrophic growth. Pyrophosphate (PPi), a byproduct of ATP hydrolysis, is released by active metabolism in imbibed seeds. We report that the removal of PPi by plant vacuolar H+-pyrophosphatase is central for successful gluconeogenesis and resumption of postembryonic growth. Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H+-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1Pro:IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.

AtHMA1 contributes to the detoxification of excess Zn(II) in Arabidopsis

AtHMA1 is a member of the heavy metal-transporting ATPase family. It exhibits amino acid sequence similarity to two other Zn(II) transporters, AtHMA2 and AtHMA4, and contains poly-His motifs that are commonly found in Zn(II)-binding proteins, but lacks some amino acids that are typical for this class of transporters. AtHMA1 localizes to the chloroplast envelope. In comparison with wild-type plants, we observed a more pronounced sensitivity in the presence of high Zn(II) concentrations, and increased accumulation of Zn in the chloroplast of T-DNA insertional mutants in AtHMA1. The Zn(II)-sensitive phenotype of AtHMA1 knock-out plants was complemented by the expression of AtHMA1 under the control of its own promoter. The Zn(II)-transporting activity of AtHMA1 was confirmed in a heterologous expression system, Saccharomyces cerevisiae. The sensitivity of yeast to high concentrations of Zn(II) was altered by the expression of AtHMA1 lacking its N-terminal chloroplast-targeting signal. Taken together, these results suggest that under conditions of excess Zn(II), AtHMA1 contributes to Zn(II) detoxification by reducing the Zn content of Arabidopsis thaliana plastids.

Rapid Structural Changes and Acidification of Guard Cell Vacuoles during Stomatal Closure Require Phosphatidylinositol 3,5-Bisphosphate

Stomatal closure is critical for water conservation in plants. The biochemical basis of vacuolar dynamics seen in guard cells during stomatal closing remains unclear. This work shows that guard cell vacuoles acidify upon abscisic acid treatment; this vacuolar acidification is required for normal stomatal closure and requires phosphatidylinositol 3,5-bisphosphate. Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)–induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H+-ATPase vha-a2 vha-a3 and vacuolar H+-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution.

Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein in Arabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures

In this work, expressing monomeric green fluorescent protein (GFP)-linked vacuolar H+-pyrophosphatase (H+-PPase) under the control of its own promoter revealed dynamic morphological changes in vacuoles and tonoplasts and tissue-specific expression patterns of H+-PPase. However, weak homodimerization of highly expressed GFP-tagged protein triggered the generation of artifactual bulbs in plant cells. We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H+-pyrophosphatase (H+-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H+-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H+-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H+-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H+-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.

Quantification, Organ-Specific Accumulation and Intracellular Localization of Type II H+-Pyrophosphatase in Arabidopsis thaliana

Most plants have two types of H(+)-translocating inorganic pyrophosphatases (H(+)-PPases), I and II, which differ in primary sequence and K(+) dependence of enzyme function. Arabidopsis thaliana has three genes for H(+)-PPases: one for type I and two for type II. The type I H(+)-PPase requires K(+) for maximal enzyme activity and functions together with H(+)-ATPase in vacuolar membranes. The physiological role of the type II enzyme, which does not require K(+), is not clear. We focused on the type II enzymes (AtVHP2;1 and AtVHP2;2) of A. thaliana. Total amounts of AtVHP2s were quantified immunochemically using a specific antibody and determined to be 22 and 12 ng mg(-1) of total protein in the microsomal fractions of suspension-cultured cells and young roots, respectively, and the values are approximately 0.1 and 0.2%, respectively, of the vacuolar H(+)-PPase. In plants, AtVHP2s were detected immunochemically in all tissues except mature leaves, and were abundant in roots and flowers. The intracellular localization of AtVHP2s in suspension cells was determined by sucrose density gradient centrifugation and immunoblotting. Comparison with a number of marker proteins revealed localization in the Golgi apparatus and the trans-Golgi network. These results suggest that the type II H(+)-PPase functions as a proton pump in the Golgi and related vesicles in young tissues, although its content is very low compared with the type I enzyme.

Immunochemical Analysis of Aquaporin Isoforms in Arabidopsis Suspension-Cultured Cells

Aquaporins mediate the movement of water across biomembranes. Arabidopsis thaliana contains 35 aquaporins that belong to four subfamilies (PIP, TIP, SIP, and NIP). We investigated their expression profiles immunochemically in suspension-cultured Arabidopsis thaliana cells during growth and in response to salt and osmotic stresses. Protein amounts of all aquaporins were much lower in cultured cells than in the plant tissues. This is consistent with the low water permeability of protoplasts from cultured cells. After treatment with NaCl, the protein amounts of PIP2;1, PIP2;2, and PIP2;3 in the cells increased several-fold, and those of TIP1;1 and TIP1;2, 15- and 3-fold respectively. PIP1 did not change under the stress. Cell death began after 19 d in culture, accompanied by marked accumulation of PIPs and TIPs and a gradual decrease in SIPs. Our results suggest the followings: (i) Accumulation of aquaporin isoforms was individually regulated at low levels in single cells. (ii) At least PIP2;2, PIP2;3, TIP1;1, and TIP1;2 are stress-responsive aquaporins in suspension cells. (iii) A sudden increment of several members of PIP2 and TIP1 subfamilies might be related to cell death.

Vacuolar Proton Pumps and Aquaporins Involved in Rapid Internode Elongation of Deepwater Rice

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.

Contribution of PPi-Hydrolyzing Function of Vacuolar H+-Pyrophosphatase in Vegetative Growth of Arabidopsis: Evidenced by Expression of Uncoupling Mutated Enzymes

The vacuolar-type H+-pyrophosphatase (H+-PPase) catalyzes a coupled reaction of pyrophosphate (PPi) hydrolysis and active proton translocation across the tonoplast. Overexpression of H+-PPase improves growth in various plant species, and loss-of-function mutants (fugu5s) of H+-PPase in Arabidopsis thaliana have post-germinative developmental defects. Here, to further clarify the physiological significance of this important enzyme, we newly generated three varieties of H+-PPase overexpressing lines with different levels of activity that we analyzed together with the loss-of-function mutant fugu5-3. The H+-PPase overexpressors exhibited enhanced activity of H+-PPase during vegetative growth, but no change in the activity of vacuolar H+-ATPase. Overexpressors with high enzymatic activity grew more vigorously with fresh weight increased by more than 24 and 44%, compared to the wild type and fugu5-3, respectively. Consistently, the overexpressors had larger rosette leaves and nearly 30% more cells in leaves than the wild type. When uncoupling mutated variants of H+-PPase, that could hydrolyze PPi but could not translocate protons, were introduced into the fugu5-3 mutant background, shoot growth defects recovered to the same levels as when a normal H+-PPase was introduced. Taken together, our findings clearly demonstrate that additional expression of H+-PPase improves plant growth by increasing cell number, predominantly as a consequence of the PPi-hydrolyzing activity of the enzyme.

Regulation of pyrophosphate levels by H+-PPase is central for proper resumption of early plant development.

The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H+PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H+PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H+PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H+PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.

Regulation of PPi Levels Through the Vacuolar Membrane H+-Pyrophosphatase

Inorganic pyrophosphate (PPi) is a high-energy compound, although the free energy change of its hydrolysis is approximately 60 % that of ATP. PPi is generated as a by-product of macromolecule biosyntheses in plants, especially in proliferating cells. In living cells, the accumulation of PPi causes the suppression of these metabolic processes and the formation of insoluble Ca–PPi complexes. To avoid these negative effects, the vacuolar H+-pyrophosphatase (H+-PPase) hydrolyzes PPi and pumps H+ across the vacuolar membrane to maintain their acidic state. Importantly, recent studies on fugu5, the H+-PPase loss-of-function mutants, have clearly demonstrated that their phenotype is rescued by the expression of the yeast cytosolic PPase IPP1, which hydrolyzes cytosolic PPi but has no effect on vacuolar acidification, thus strongly suggesting that the role of the H+-PPase lies in the consumption of the inhibitory PPi rather than vacuolar acidification. In this chapter we describe the chemical properties and metabolic role of PPi, in addition to the physiological functions of H+-PPase and soluble PPase revealed by using several mutant lines.