Transcriptional activation of auxin biosynthesis drives developmental reprogramming of differentiated cells.

Plant cells exhibit remarkable plasticity of their differentiation states, enabling regeneration of whole plants from differentiated somatic cells. How they revert cell fate and express pluripotency, however, remains unclear. In this study, we demonstrate that transcriptional activation of auxin biosynthesis is crucial for reprogramming differentiated Arabidopsis (Arabidopsis thaliana) leaf cells. Our data show that interfering with the activity of histone acetyltransferases dramatically reduces callus formation from leaf mesophyll protoplasts. Histone acetylation permits transcriptional activation of PLETHORAs, leading to the induction of their downstream YUCCA1 gene encoding an enzyme for auxin biosynthesis. Auxin biosynthesis is in turn required to accomplish initial cell division through the activation of G2/M phase genes mediated by MYB DOMAIN PROTEIN 3-RELATED (MYB3Rs). We further show that the AUXIN RESPONSE FACTOR 7 (ARF7)/ARF19 and INDOLE-3-ACETIC ACID INDUCIBLE 3 (IAA3)/IAA18-mediated auxin signaling pathway is responsible for cell cycle reactivation by transcriptionally upregulating MYB3R4. These findings provide a mechanistic model of how differentiated plant cells revert their fate and reinitiate the cell cycle to become pluripotent.

Polar vacuolar distribution is essential for accurate asymmetric division of Arabidopsis zygotes.

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.

The plasma membrane-associated Ca2+ -binding protein, PCaP1, is required for oligogalacturonide and flagellin-induced priming and immunity.

Early signalling events in response to elicitation include reversible protein phosphorylation and re-localization of plasma membrane (PM) proteins. Oligogalacturonides (OGs) are a class of damage-associated molecular patterns (DAMPs) that act as endogenous signals to activate the plant immune response. Previous data on early phosphoproteome changes in Arabidopsis thaliana upon OG perception uncovered the immune-related phospho-regulation of several membrane proteins, among which PCaP1, a PM-anchored protein with actin filament-severing activity, was chosen for its potential involvement in OG- and flagellin-triggered responses. Here, we demonstrate that PCaP1 is required for late, but not early, responses induced by OGs and flagellin. Moreover, pcap1 mutants, unlike the wild type, are impaired in the recovery of full responsiveness to a second treatment with OGs performed 24 h after the first one. Localization studies on PCaP1 upon OG treatment in plants expressing a functional PCaP1-GFP fusion under the control of PCaP1 promoter revealed fluorescence on the PM, organized in densely packed punctate structures, previously reported as microdomains. Fluorescence was found to be associated also with endocytic vesicles, the number of which rapidly increased after OG treatment, suggesting both an endocytic turnover of PCaP1 for maintaining its homeostasis at the PM and an OG-induced endocytosis.

Vacuolar H+-Pyrophosphatase and Cytosolic Soluble Pyrophosphatases Cooperatively Regulate Pyrophosphate Levels in Arabidopsis thaliana.

Inorganic pyrophosphate (PPi) is a phosphate donor and energy source. Many metabolic reactions that generate PPi are suppressed by high levels of PPi. Here, we investigated how proper levels of cytosolic PPi are maintained, focusing on soluble pyrophosphatases (AtPPa1 to AtPPa5; hereafter PPa1 to PPa5) and vacuolar H+-pyrophosphatase (H+-PPase, AtVHP1/FUGU5) in Arabidopsis thaliana In planta, five PPa isozymes tagged with GFP were detected in the cytosol and nuclei. Immunochemical analyses revealed a high abundance of PPa1 and the absence of PPa3 in vegetative tissue. In addition, the heterologous expression of each PPa restored growth in a soluble PPase-defective yeast strain. Although the quadruple knockout mutant plant ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes, H+-PPase and PPa1 double mutants (fugu5 ppa1) exhibited significant phenotypes, including dwarfism, high PPi concentrations, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Altered cell arrangements and weakened cell walls in the root tip were particularly evident in fugu5 ppa1 and were more severe than in fugu5 Our results indicate that H+-PPase is essential for maintaining adequate PPi levels and that the cytosolic PPa isozymes, particularly PPa1, prevent increases in PPi concentrations to toxic levels. We discuss fugu5 ppa1 phenotypes in relation to metabolic reactions and PPi homeostasis.

Arabidopsis PCaP2 modulates the phosphatidylinositol 4,5-bisphosphate signal on the plasma membrane and attenuates root hair elongation.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] serves as a subcellular signal on the plasma membrane, mediating various cell-polarized phenomena including polar cell growth. Here, we investigated the involvement of Arabidopsis thaliana PCaP2, a plant-unique plasma membrane protein with phosphoinositide-binding activity, in PtdIns(4,5)P2 signaling for root hair tip growth. The long-root-hair phenotype of the pcap2 knockdown mutant was found to stem from its higher average root hair elongation rate compared with the wild type and to counteract the low average rate caused by a defect in the PtdIns(4,5)P2 -producing enzyme gene PIP5K3. On the plasma membrane of elongating root hairs, the PCaP2 promoter-driven PCaP2-green fluorescent protein (GFP), which complemented the pcap2 mutant phenotype, overlapped with the PtdIns(4,5)P2 marker 2xCHERRY-2xPHPLC in the subapical region, but not at the apex, suggesting that PCaP2 attenuates root hair elongation via PtdIns(4,5)P2 signaling on the subapical plasma membrane. Consistent with this, a GFP fusion with the PCaP2 phosphoinositide-binding domain PCaP2N23 , root hair-specific overexpression of which caused a low average root hair elongation rate, localized more intense to the subapical plasma membrane than to the apical plasma membrane similar to PCaP2-GFP. Inducibly overexpressed PCaP2-GFP, but not its derivative lacking the PCaP2N23 domain, replaced 2xCHERRY-2xPHPLC on the plasma membrane in root meristematic epidermal cells, and suppressed FM4-64 internalization in elongating root hairs. Moreover, inducibly overexpressed PCaP2 arrested an endocytic process of PIN2-GFP recycling. Based on these results, we conclude that PCaP2 functions as a negative modulator of PtdIns(4,5)P2 signaling on the subapical plasma membrane probably through competitive binding to PtdIns(4,5)P2 and attenuates root hair elongation.

Plasma Membrane Aquaporin Members PIPs Act in Concert to Regulate Cold Acclimation and Freezing Tolerance Responses in Arabidopsis thaliana.

Aquaporins play a major role in plant water uptake at both optimal and environmentally stressed conditions. However, the functional specificity of aquaporins under cold remains obscure. To get a better insight to the role of aquaporins in cold acclimation and freezing tolerance, we took an integrated approach of physiology, transcript profiling and cell biology in Arabidopsis thaliana. Cold acclimation resulted in specific upregulation of PIP1;4 and PIP2;5 aquaporin (plasma membrane intrinsic proteins) expression, and immunoblotting analysis confirmed the increase in amount of PIP2;5 protein and total amount of PIPs during cold acclimation, suggesting that PIP2;5 plays a major role in tackling the cold milieu. Although single mutants of pip1;4 and pip2;5 or their double mutant showed no phenotypic changes in freezing tolerance, they were more sensitive in root elongation and cell survival response under freezing stress conditions compared with the wild type. Consistently, a single mutation in either PIP1;4 or PIP2;5 altered the expression of a number of aquaporins both at the transcriptional and translational levels. Collectively, our results suggest that aquaporin members including PIP1;4 and PIP2;5 function in concert to regulate cold acclimation and freezing tolerance responses.

Non-intrinsic ATP-binding cassette proteins ABCI19, ABCI20 and ABCI21 modulate cytokinin response at the endoplasmic reticulum in Arabidopsis thaliana.

KEY MESSAGE: The non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.

The ER-localized aquaporin SIP2;1 is involved in pollen germination and pollen tube elongation in Arabidopsis thaliana.

KEY MESSAGE: The ER membrane localized aquaporin SIP2;1 is involved in adaptation to ER stresses during pollen tube elongation. Aquaporins play multifaceted roles through selective transport of water and small neutral substrates. Here, we focused on the physiological roles of Arabidopsis thaliana aquaporins, namely SIP1;1, SIP1;2 and SIP2;1, which are localized to the endoplasmic reticulum (ER). While their loss-of-function mutants displayed normal vegetative growth. We identified defects in pollen of sip2;1. Whereas the germination rate of sip2;1 pollen was ~ 60% that of the wild type (WT), in vitro germinated sip2;1 pollen tube length was reduced up to 82% compared to the WT. Importantly, most pollen tubes on pistils from sip2;1 stopped elongation in the mid-region of pistils, and the bottom region of sip2;1 siliques lacked seeds. Consistently, silique of sip2;1 were short, whereby the average seed number per silique was nearly the half of the WT. The above phenotypes recovered in SIP2;1 complementation lines. We detected mRNA of SIP2;1 and protein in pollen, and further revealed that the GFP-linked SIP2;1 localization in the ER of growing pollen tubes. The basal mRNA level of BINDING PROTEIN 3 (BiP3), a key gene induced by ER stress, in pollen was markedly higher than that in roots, suggesting that the pollen underwent ER stress under normal growth conditions. BiP3 mRNA was dramatically increased in sip2;1 pollen. Altogether, our findings suggest that the aquaporin SIP2;1 is probably involved in the alleviation of ER stress and that the lack of SIP2;1 reduces both pollen germination and pollen tube elongation.

Plasma Membrane-Associated Ca2+-Binding Protein PCaP1 is Involved in Root Hydrotropism of Arabidopsis thaliana.

Root hydrotropism is an essential growth response to water potential gradients in plants. To understand the mechanism, fundamental elements such as MIZU-KUSSEI 1 (MIZ1) have been investigated extensively. We investigated the physiological role of a plasma membrane-associated cation-binding protein (PCaP1) and examined the effect of PCaP1 loss-of-function mutations on root hydrotropism. pcap1 knockout mutants showed a defect in root bending as a hydrotropic response, although gravitropism was normal in pcap1 mutants. When pcap1 seedlings were treated with abscisic acid, a negative regulator of gravitropism, the seedlings showed normal gravitropism. The hydrotropism defect in pcap1 mutants was clearly rescued by introducing the genomic sequence of PCaP1 with an endodermis-specific promoter. Analysis of PCaP1-greenfluorescent protein-expressing roots by confocal laser scanning microscopy revealed that PCaP1 was stably associated with the plasma membrane in most cells, but in the cytoplasm of endodermal cells at the bending region. Furthermore, we prepared a transgenic line overexpressing MIZ1 on the pcap1 background and found that the pcap1 hydrotropism defect was rescued. Our results indicate that PCaP1 in the endodermal cells of the root elongation zone is involved in the hydrotropic response. We suggest that PCaP1 contributes to hydrotropism through a MIZ1-independent pathway or as one of the upstream components that transduce water potential signals to MIZ1.

Excess Pyrophosphate within Guard Cells Delays Stomatal Closure.

A variety of cellular metabolic reactions generate inorganic pyrophosphate (PPi) as an ATP hydrolysis byproduct. The vacuolar H+-translocating pyrophosphatase (H+-PPase) loss-of-function fugu5 mutant is susceptible to drought and displays pleotropic postgerminative growth defects due to excess PPi. It was recently reported that stomatal closure after abscisic acid (ABA) treatment is delayed in vhp1-1, a fugu5 allele. In contrast, we found that specific removal of PPi rescued all of the above fugu5 developmental and growth defects. Hence, we speculated that excess PPi itself, rather than vacuolar acidification, might delay stomatal closure. To test this hypothesis, we constructed transgenic plants expressing the yeast IPP1 gene (encoding a cytosolic pyrophosphatase) driven by a guard cell-specific promoter (pGC1::IPP1) in the fugu5 background. Our measurements confirmed stomatal closure defects in fugu5, further supporting a role for H+-PPase in stomatal functioning. Importantly, while pGC1::IPP1 transgenics morphologically mimicked fugu5, stomatal closure was restored in response to ABA and darkness. Quantification of water loss revealed that fugu5 stomata were almost completely insensitive to ABA. In addition, growth of pGC1::IPP1 plants was promoted compared to fugu5 throughout their life; however, it did not reach the wild type level. fugu5 also displayed an increased stomatal index, in violation of the one-cell-spacing rule, and phenotypes recovered upon removal of PPi by pAVP1::IPP1 (FUGU5, VHP1 and AVP1 are the same gene encoding H+-PPase), but not in the pGC1::IPP1 line. Taken together, these results clearly support our hypothesis that dysfunction in stomata is triggered by excess PPi within guard cells, probably via perturbed guard cell metabolism.

A cell-wall protein SRPP provides physiological integrity to the Arabidopsis seed.

Seed and root hair protective protein (SRPP) is expressed in seeds and root hairs, localized in the cell wall, and involved in cell wall integrity. We analyzed a loss-of-function mutant of SRPP, focusing on siliques and seeds. The srpp-1 plants generated dark brown shrunken seeds at a high rate. The germination rate of these defect seeds of srpp-1 was less than 6%, although apparently normal srpp-1 seeds germinated at a rate of 83%. The production ratio of severe phenotypic seeds was dependent on the growth conditions. When the srpp-1 plants were cultivated at low humidity, the defect ratio was 73%, which was significantly higher than that at normal humidity. Defects of the silique and seeds could be detected on day 7 after pollination and the apical region of the siliques displayed a severe phenotype at a high frequency. Complementation with an SRPP gene under the control of promoters specific to the embryo, seed coat, or valve (carpel) partially rescued the phenotype, and complementation using the SRPP promoter fully rescued the phenotype. Furthermore, overexpression of SRPP enhanced the thermotolerance. After the treatment of seeds at 50 °C for 2 h, the germination rate of the seeds from overexpression with the 35S promoter increased to levels twice that of the wild-type seeds. Under the same conditions, no srpp-1 seeds germinated. These results indicate that SRPP is essential for the production of normal viable seeds in siliques under stress conditions. It is possible that modification of the SRPP gene improves seed integrity.

Biochemical, Structural and Physiological Characteristics of Vacuolar H+-Pyrophosphatase.

Proton-translocating inorganic pyrophosphatase (H+-PPase) actively translocates protons across membranes coupled with the hydrolysis of inorganic pyrophosphate (PPi). H+-PPase, which is composed of a single protein and uses a simple compound as a substrate, has been recognized as a new type of ion pump in addition to the P-, F- and V-type ion-translocating ATPases. H+- and Na+-PPases are distributed in various organisms including plants, parasitic protozoa, Archaebacteria and bacteria, but are not present in animals or yeast. Vacuolar H+-PPase has dual functions in plant cells: hydrolysis of cytosolic PPi to maintain the levels of PPi, and translocation of protons into vacuoles to maintain the acidity of the vacuolar lumen. Acidification performed with the vacuolar-type H+-ATPase and H+-PPase is essential to maintain acidic conditions, which are necessary for vacuolar hydrolytic enzymes and for supplying energy to secondary active transporters. Recent studies using loss-of-function mutant lines of H+-PPase and complementation lines with soluble PPases have emphasized the physiological importance of the scavenging role of PPi. An overview of the main features of H+-PPases present in the vacuolar membrane is provided in terms of tissue distribution in plants, intracellular localization, structure-function relationship, biochemical potential as a proton pump and functional stability.

SRPP, a Cell Wall Protein is Involved in Development and Protection of Seeds and Root Hairs in Arabidopsis thaliana.

Enhancement of root hair development in response to phosphate (Pi) deficit has been reported extensively. Root hairs are involved in major root functions such as the absorption of water, acquisition of nutrients and secretion of organic acids and enzymes. Individual root hair cells maintain these functions and appropriate structure under various physiological conditions. We carried out a study to identify protein(s) which maintain the structure and function of root hairs, and identified a protein (SEED AND ROOT HAIR PROTECTIVE PROTEIN, SRPP) that was induced in root hairs under Pi-deficient conditions. Promoter assay and mRNA quantification revealed that SRPP was expressed in root hairs and seeds. A knockout mutant, srpp-1, consistently displayed defects in root hairs and seeds. Root hairs in srpp-1 were short and the phenotypes observed under Pi-deficient conditions were also detected in ethylene-treated srpp-1 plants. Propidium iodide stained most root hairs of srpp-1 grown under Pi-deficient conditions, suggesting cell death. In addition to root hairs, most srpp-1 seeds were withered and their embryos were dead. SRPP tagged with green fluorescent protein was detected in the cell wall. Electron microscopy showed abnormal morphology of the cell wall. Wild-type phenotypes were restored when the SRPP gene was expressed in srpp-1. These data strongly suggest that SRPP contributes to the construction of robust cell walls, whereby it plays a key role in the development of root hairs and seeds.

Compensated Cell Enlargement in fugu5 is Specifically Triggered by Lowered Sucrose Production from Seed Storage Lipids.

To reveal the logic of size regulation in multicellular organisms, we have used Arabidopsis thaliana as a model organism and its leaves as a model organ. We discovered the existence of a compensatory system, whereby a decrease in leaf cell number often triggers unusual cell enlargement. However, despite the large number of compensation-exhibiting mutants analyzed to date, we have only a limited understanding of the detailed molecular mechanisms triggering the decrease in cell number and subsequent compensated cell enlargement (CCE). CCE in fugu5, the vacuolar type H+-pyrophosphatase loss-of-function mutant, is specific to cotyledons and completely suppressed when sucrose (Suc) is supplied or cytosolic pyrophosphate (PPi) is specifically removed. In addition, several lines of evidence suggest that excess cytosolic PPi in fugu5 impairs gluconeogenesis from triacylglycerol (TAG) to Suc. Here, detailed cellular phenotyping revealed that the loss-of-function mutants icl-2, mls-2 and pck1-2 triggered CCE in cotyledons. All double mutant combinations between fugu5-1 and the above three mutants exhibited compensation, but did not display a further increase in cell size. Importantly, similar phenotypes were observed in icl-2 mls-2, icl-2 pck1-2 and mls-2 pck1-2. Quantification of TAG breakdown and Suc contents further supported our findings. Taken together, we demonstrate that de novo Suc synthesis from TAG is fundamentally important for proper resumption of post-germinative cotyledon development. Moreover, provided that icl-2, mls-2 and pck1-2 are only compromised in Suc biosynthesis de novo from TAG, our findings clearly indicate that lowered Suc production in fugu5, rather than excess cytosolic PPi, is the direct trigger of CCE.

Dynamics of vacuoles and H+-pyrophosphatase visualized by monomeric green fluorescent protein in Arabidopsis: artifactual bulbs and native intravacuolar spherical structures.

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.

Arabidopsis TWISTED DWARF1 functionally interacts with auxin exporter ABCB1 on the root plasma membrane.

Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological assays, we demonstrate a predominant lateral, mainly outward-facing, plasma membrane location for TWD1 in the root epidermis characterized by the lateral marker ABC transporter G36/PLEIOTROPIC DRUG-RESISTANCE8/PENETRATION3. At these epidermal plasma membrane domains, TWD1 colocalizes with nonpolar ABCB1. In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)-TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein-protein interaction at the plasma membrane, minimizing reflux from the root apoplast into the cytoplasm.

Rapid structural changes and acidification of guard cell vacuoles during stomatal closure require 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.

A novel-type phosphatidylinositol phosphate-interactive, Ca-binding protein PCaP1 in Arabidopsis thaliana: stable association with plasma membrane and partial involvement in stomata closure.

The Ca(2+)-binding protein-1 (PCaP1) of Arabidopsis thaliana is a new type protein that binds to phosphatidylinositol phosphates and Ca(2+)-calmodulin complex as well as free Ca(2+). Although biochemical properties, such as binding to ligands and N-myristoylation, have been revealed, the intracellular localization, tissue and cell specificity, integrity of membrane association and physiological roles of PCaP1 are unknown. We investigated the tissue and intracellular distribution of PCaP1 by using transgenic lines expressing PCaP1 linked with a green fluorescence protein (GFP) at the carboxyl terminus of PCaP1. GFP fluorescence was obviously detected in most tissues including root, stem, leaf and flower. In these tissues, PCaP1-GFP signal was observed predominantly in the plasma membrane even under physiological stress conditions but not in other organelles. The fluorescence was detected in the cytosol when the 25-residue N-terminal sequence was deleted from PCaP1 indicating essential contribution of N-myristoylation to the plasma membrane anchoring. Fluorescence intensity of PCaP1-GFP in roots was slightly decreased in seedlings grown in medium supplemented with high concentrations of iron for 1 week and increased in those grown with copper. In stomatal guard cells, PCaP1-GFP was strictly, specifically localized to the plasma membrane at the epidermal-cell side but not at the pore side. A T-DNA insertion mutant line of PCaP1 did not show marked phenotype in a life cycle except for well growth under high CO2 conditions. However, stomata of the mutant line did not close entirely even in high osmolarity, which usually induces stomata closure. These results suggest that PCaP1 is involved in the stomatal movement, especially closure process, in leaves and response to excessive copper in root and leaf as a mineral nutrient as a physiological role.

AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum.

Fatty acids, the building blocks of biological lipids, are synthesized in plastids and then transported to the endoplasmic reticulum (ER) for assimilation into specific lipid classes. The mechanism of fatty acid transport from plastids to the ER has not been identified. Here we report that AtABCA9, an ABC transporter in Arabidopsis thaliana, mediates this transport. AtABCA9 was localized to the ER, and atabca9 null mutations reduced seed triacylglycerol (TAG) content by 35% compared with WT. Developing atabca9 seeds incorporated 35% less (14)C-oleoyl-CoA into TAG compared with WT seeds. Furthermore, overexpression of AtABCA9 enhanced TAG deposition by up to 40%. These data strongly support a role for AtABCA9 as a supplier of fatty acid substrates for TAG biosynthesis at the ER during the seed-filling stage. AtABCA9 may be a powerful tool for increasing lipid production in oilseeds.