ABSTRACT
Zinc (Zn) is an essential element for plants. Numerous proteins in different cellular compartments require Zn for their structure and function. Zn can be toxic when it accumulates in high levels in the cytoplasm. Therefore, Zn homeostasis at tissue, cell, and organelle levels is vital for plant growth. A part of the metal tolerance protein (MTP) / Cation Diffusion Facilitator (CDF) transporters functions as Zn transporters, exporting Zn from the cytosol to various membrane compartments. In Arabidopsis thaliana, MTP1, MTP2, MTP3, MTP4, MTP5, and MTP12 are classified as Zn transporters (Zn-CDF). In this study, we systematically analyzed the localization of GFP-fused Zn-CDFs in the leaf epidermal cells of Nicotiana benthamiana. As previously reported, MTP1 and MTP3 were localized to tonoplast, MTP2 to endoplasmic reticulum, and MTP5 to Golgi. In addition, we identified the localization of MTP4 to trans-Golgi Network (TGN). Since MTP4 is specifically expressed in pollen, we analyzed the localization of MTP4-GFP in the Arabidopsis pollen tubes and confirmed that it is in the TGN. We also showed the Zn transport capability of MTP4 in yeast cells. We then analyzed the phenotype of an mtp4 T-DNA insertion mutant under both limited and excess Zn conditions. We found that their growth and fertility were not largely different from the wild-type. Our study has paved the way for investigating the possible roles of MTP4 in metallating proteins in the secretory pathway or in exporting excess Zn through exocytosis. In addition, our system of GFP-fused MTPs will help study the mechanisms for targeting transporters to specific membrane compartments.
ABSTRACT
We investigated the effect of cold plasma application on the yield and grain quality of rice (Oryza sativa L.), focusing on the brewer's rice cultivar, Yamadanishiki. Two treatment methods were examined in a paddy; direct plasma irradiation of seedlings and indirect treatment with plasma-activated Ringer's lactate solution (PAL) during the vegetative growth phase. Periodic direct irradiation for 30 s increased whole plant weight and grain yield. Treatment with PAL promoted some growth of panicles relatively and partially suppressed the growth of culms and leaves. Both treatments affected the grain quality; an increase of the ratio of white-core grains to total number of grains, which is suited for producing Japanese sake rice, as well as a decrease of the ratio of immature grains. The results showed that the effective production of rice grains for sake production can be improved by the application of cold plasma treatment of rice seedlings in a paddy.HighlightRice plants of brewer's rice cultivar in a paddy were treated with cold plasma, by the direct irradiation of plants and the immersed of plants in plasma-activated Ringer's lactate (PAL).Direct plasma irradiation promoted plant weight, grain ripening, and increased yield.PAL treatment affected the growth of main stem and promoted the growth of panicles relatively.Both treatments improved the producing white-core grains, in addition to promotion of grain ripening.Cold plasma treatment can be applied to produce stable and high-quality food in various agriculture and food industries, which can achieve the sustainable developmental goals (SDGs).
Subject(s)
Oryza , Plasma Gases , Plasma Gases/pharmacology , Alcoholic Beverages , Ringer's Lactate/pharmacology , Fermentation , Edible GrainABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant/genetics , Transcriptional Activation , Plant Roots/metabolism , Transcription Factors/metabolism , Indoleacetic Acids/metabolism , Arabidopsis/metabolismABSTRACT
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.
Subject(s)
Alarmins/metabolism , Arabidopsis Proteins/physiology , Arabidopsis/immunology , Calcium-Binding Proteins/physiology , Cell Membrane/metabolism , Flagellin/metabolism , Polynucleotides/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Botrytis , Calcium-Binding Proteins/metabolism , Gene Expression Regulation, Plant , Glucans/metabolism , Microscopy, Confocal , Phosphoproteins/metabolism , Plant Diseases/immunology , Plant Diseases/microbiology , Reactive Oxygen Species/metabolism , TranscriptomeABSTRACT
The cytosolic level of inorganic pyrophosphate (PPi) is finely regulated, with PPi hydrolyzed primarily by the vacuolar H+-pyrophosphatase (H+-PPase, VHP1/FUGU5/AVP1) and secondarily by five cytosolic soluble pyrophosphatases (sPPases; PPa1-PPa5) in Arabidopsis thaliana. Loss-of-function mutants of H+-PPase (fugu5s) have been reported to show atrophic phenotypes in their rosette leaves when nitrate is the sole nitrogen source in the culture medium. For this phenotype, two questions remain unanswered: why does atrophy depend on physical contact between shoots and the medium, and how does ammonium prevent such atrophy. To understand the mechanism driving this phenotype, we analyzed the growth and phenotypes of mutants on ammonium-free medium in detail. fugu5-1 showed cuticle defects, cell swelling, reduced ß-glucan levels, and vein malformation in the leaves, suggesting cell wall weakening and cell lethality. Based on the observation in the double mutants fugu5-1 ppa1 and fugu5-1 ppa4 of more severe atrophy compared to fugu5-1, the nitrogen-dependent phenotype might be linked to PPi metabolism. To elucidate the role of ammonium in this process, we examined the fluctuations of sPPase mRNA levels and the possibility of alternative PPi-removing factors, such as other types of pyrophosphatase. First, we found that both the protein and mRNA levels of sPPases were unaffected by the nitrogen source. Second, to assess the influence of other PPi-removing factors, we examined the phenotypes of triple knockout mutants of H+-PPase and two sPPases on ammonium-containing medium. Both fugu5 ppa1 ppa2 and fugu5 ppa1 ppa4 had nearly lethal embryonic phenotypes, with the survivors showing striking dwarfism and abnormal morphology. Moreover, fugu5 ppa1+/- ppa4 showed severe atrophy at the leaf margins. The other triple mutants, fugu5 ppa1 ppa5 and fugu5 ppa2 ppa4, exhibited death of root hairs and were nearly sterile due to deformed pistils, respectively, even when grown on standard medium. Together, these results suggest that H+-PPase and sPPases act in concert to maintain PPi homeostasis, that the existence of other PPi removers is unlikely, and that ammonium may suppress the production of PPi during nitrogen metabolism rather than stimulating PPi hydrolysis.
ABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Cytokinins/metabolism , Endoplasmic Reticulum/metabolism , ATP-Binding Cassette Transporters/genetics , Amino Acid Motifs , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Cytokinins/genetics , Endoplasmic Reticulum/radiation effects , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/radiation effects , Gene Knockout Techniques , Light , Phylogeny , Plants, Genetically Modified , Promoter Regions, Genetic , Protein Binding , Seedlings/genetics , Seedlings/growth & development , Seedlings/metabolism , Seedlings/radiation effectsABSTRACT
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.
Subject(s)
Acclimatization/genetics , Aquaporins/genetics , Arabidopsis/genetics , Cell Membrane/genetics , Cold-Shock Response , Aquaporins/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Membrane/metabolism , Chlorophyll/metabolism , Freezing , Gene Expression Regulation, Plant , Mercuric Chloride/metabolism , Optical Imaging , RNA, Plant/genetics , RNA, Plant/isolation & purification , Sequence Analysis, RNAABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
ABSTRACT
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.
Subject(s)
Aquaporins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Endoplasmic Reticulum/metabolism , Germination , Pollen Tube/metabolism , Pollen/metabolism , Aquaporins/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cell Survival , Fertility , Flowers/metabolism , Gene Expression Regulation, Plant , Molecular Chaperones , Mutation , Phenotype , Plant Proteins/metabolism , Plant Roots , Pollen/growth & development , Pollen Tube/growth & development , RNA, MessengerABSTRACT
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.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/growth & development , Calcium-Binding Proteins/physiology , Plant Roots/growth & development , Tropism , Abscisic Acid/metabolism , Arabidopsis/metabolism , Cell Membrane/metabolism , Gene Knockdown Techniques , Gravitropism , Plant Growth Regulators/metabolism , Plant Roots/metabolism , WaterABSTRACT
Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H+-pumping pyrophosphatases (H+-PPase) convert the substantial energy present in PPi into an electrochemical gradient. We show here, that both cold- and heat stress sensitivity of fugu5 mutants lacking the major H+-PPase isoform AVP1 is correlated with reduced SUMOylation. In addition, we show that increased PPi concentrations interfere with SUMOylation in yeast and we provide evidence that SUMO activating E1-enzymes are inhibited by micromolar concentrations of PPi in a non-competitive manner. Taken together, our results do not only provide a mechanistic explanation for the beneficial effects of AVP1 overexpression in plants but they also highlight PPi as an important integrator of metabolism and stress tolerance.
Subject(s)
Arabidopsis/physiology , Diphosphates/metabolism , Stress, Physiological , Sumoylation , Acclimatization , Arabidopsis/enzymology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cold Temperature , Hot Temperature , Inorganic Pyrophosphatase/metabolism , Isoenzymes/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cell Membrane/genetics , Cell Membrane/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Phosphatidylinositol 4,5-Diphosphate/genetics , Phosphatidylinositol 4,5-Diphosphate/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Signal Transduction/physiologyABSTRACT
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.
Subject(s)
Diphosphates/metabolism , Plant Stomata/metabolism , Abscisic Acid/pharmacology , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/metabolism , Inorganic Pyrophosphatase/genetics , Inorganic Pyrophosphatase/metabolism , Mutation/genetics , Plant Stomata/drug effects , Plant Stomata/physiologyABSTRACT
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.
Subject(s)
Arabidopsis/physiology , Asymmetric Cell Division , Cell Polarity , Vacuoles/metabolism , Zygote/cytology , Zygote/metabolism , Actin Cytoskeleton/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , Chloroplast Proteins/genetics , Chloroplast Proteins/metabolism , Fluorescent Antibody Technique , MutationABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Seeds/physiology , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Cell Wall/metabolism , Germination , Mutation , Phenotype , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Seeds/genetics , Seeds/growth & developmentABSTRACT
Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H+-PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1pro:IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H+-PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.
Subject(s)
Arabidopsis/metabolism , Diphosphates/metabolism , Gluconeogenesis , Uridine Diphosphate Glucose/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Carbon-Carbon Double Bond Isomerases/genetics , Carbon-Carbon Double Bond Isomerases/metabolism , Hemiterpenes , Inorganic Pyrophosphatase/genetics , Inorganic Pyrophosphatase/metabolism , Metabolomics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Seedlings/metabolismABSTRACT
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.
Subject(s)
Arabidopsis/metabolism , Cytosol/enzymology , Diphosphates/metabolism , Inorganic Pyrophosphatase/metabolism , Pyrophosphatases/metabolism , Vacuoles/enzymology , Vacuoles/metabolismABSTRACT
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.