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Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH., Competing Interests: YK, HK, SO, KS, VD, MS, GZ, Cd, HI, CK, MH, YT, WS No competing interests declared, (© 2023, Kato, Kuroda, Ozawa et al.)

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls., (© 2023. The Author(s).)

Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34 S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana , especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4' coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8' coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds., (© The Author(s) 2020. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report the identification and characterization of Arabidopsis ( Arabidopsis thaliana ) genes encoding three enzymes shared between the mitochondria- and plastid-localized type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β-ketoacyl-acyl carrier protein (ACP) reductase and enoyl-ACP reductase, catalyze two of the reactions that constitute the core four-reaction cycle of the FAS system, which iteratively elongates the acyl chain by two carbon atoms per cycle. The third enzyme, malonyl-coenzyme A:ACP transacylase, catalyzes the reaction that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl cofactor of ACP. GFP fusion experiments revealed that the these enzymes localize to both chloroplasts and mitochondria. This localization was validated by characterization of mutant alleles, which were rescued by transgenes expressing enzyme variants that were retargeted only to plastids or only to mitochondria. The singular retargeting of these proteins to plastids rescued the embryo lethality associated with disruption of the essential ptFAS system, but these rescued plants displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, and reduced growth. However, these latter traits were reversible in an elevated-CO 2 atmosphere, which suppresses mtFAS-associated photorespiration-dependent chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these nonredundant fatty acid biosynthetic machineries., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Plants strictly regulate the levels of sterol in their cells, as high sterol levels are toxic. However, how plants achieve sterol homeostasis is not fully understood. We isolated an Arabidopsis thaliana mutant that abundantly accumulated sterol esters in structures of about 1 µm in diameter in leaf cells. We designated the mutant high sterol ester 1 (hise1) and called the structures sterol ester bodies. Here, we show that HISE1, the gene product that is altered in this mutant, functions as a key factor in plant sterol homeostasis on the endoplasmic reticulum (ER) and participates in a fail-safe regulatory system comprising two processes. First, HISE1 downregulates the protein levels of the β-hydroxy β-methylglutaryl-CoA reductases HMGR1 and HMGR2, which are rate-limiting enzymes in the sterol synthesis pathway, resulting in suppression of sterol overproduction. Second, if the first process is not successful, excess sterols are converted to sterol esters by phospholipid sterol acyltransferase1 (PSAT1) on ER microdomains and then segregated in SE bodies.

Lysine decarboxylase converts l-lysine to cadaverine as a branching point for the biosynthesis of plant Lys-derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys-derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) and identified cadaverine-derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys-derived alkaloids in the transgenic lines. Moreover, we found several cadaverine-derived metabolites specifically detected in the transgenic lines compared with the non-transformed control. Among these, three specific metabolites were identified and confirmed as 5-aminopentanal, 5-aminopentanoate and δ-valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope-labeled [α- 15 N]- or [ε- 15 N]-l-lysine. Our results show similar 15 N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys-derived alkaloid biosynthesis., (© 2019 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Environmental stresses cause membrane damage in terrestrial plants. Studies on the lipids obtained from these plants are required to understand their adaptation to climate change. Recently, a number of plant leaf lipidomic studies converged on the topic of chloroplastic glycerolipid remodeling and triacylglycerol production. In this review, we show that among various abiotic stresses, plant leaves under heat stress specifically increase the levels of galactolipids containing linoleate (18:2) in chloroplasts; phospholipids containing palmitate (16:0), stearate (18:0), and oleate (18:1) in the endoplasmic reticulum and plasma membrane; and triacylglycerol containing α-linolenate (18:3) and hexadecatrienoic acid (16:3) as lipid droplets in the leaves of Arabidopsis thaliana. Recent studies have proposed responsible genes for the lipid remodeling under heat stress, highlighting the importance of the catabolic process of chloroplastic monogalactosyldiacylglycerol. This review comprehensively describes glycerolipid compositional changes in plant leaves under heat stress detected by lipidomic analyses and compares them with those under other abiotic stresses. We will discuss the physiological significance underlying the observed lipid metabolism under heat stress. Detailed knowledge about plant lipid remodeling can aid in the development of solutions to deal with the consequences of climate change, including global warming., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 AVP1 pro :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.

Under heat stress, polyunsaturated acyl groups, such as α-linolenate (18:3) and hexadecatrienoate (16:3), are removed from chloroplastic glycerolipids in various plant species. Here, we showed that a lipase designated HEAT INDUCIBLE LIPASE1 (HIL1) induces the catabolism of monogalactosyldiacylglycerol (MGDG) under heat stress in Arabidopsis thaliana leaves. Using thermotolerance tests, a T-DNA insertion mutant with disrupted HIL1 was shown to have a heat stress-sensitive phenotype. Lipidomic analysis indicated that the decrease of 34:6-MGDG under heat stress was partially impaired in the hil1 mutant. Concomitantly, the heat-induced increment of 54:9-triacylglycerol in the hil1 mutant was 18% lower than that in the wild-type plants. Recombinant HIL1 protein digested MGDG to produce 18:3-free fatty acid (18:3-FFA), but not 18:0- and 16:0-FFAs. A transient assay using fluorescent fusion proteins confirmed chloroplastic localization of HIL1. Transcriptome coexpression network analysis using public databases demonstrated that the HIL1 homolog expression levels in various terrestrial plants are tightly associated with chloroplastic heat stress responses. Thus, HIL1 encodes a chloroplastic MGDG lipase that releases 18:3-FFA in the first committed step of 34:6 (18:3/16:3)-containing galactolipid turnover, suggesting that HIL1 has an important role in the lipid remodeling process induced by heat stress in plants., (© 2018 American Society of Plant Biologists. All rights reserved.)

We analyzed the metabolites and proteins contained in pure intact vacuoles isolated from Arabidopsis suspension-cultured cells using capillary electrophoresis-mass spectrometry (CE-MS), Fourier transform-ion cyclotron resonance (FT-ICR)-MS and liquid chromatography (LC)-MS. We identified 21 amino acids and five organic acids as major primary metabolites in the vacuoles with CE-MS. Further, we identified small amounts of 27 substances including well-known vacuolar molecules, but also some unexpected substances (e.g. organic phosphate compounds). Non-target analysis of the vacuolar sample with FT-ICR-MS suggested that there are 1,106 m/z peaks that could predict the 5,090 molecular formulae, and we have annotated 34 compounds in these peaks using the KNapSAck database. By conducting proteomic analysis of vacuolar sap, we found 186 proteins in the same vacuole samples. Since the vacuole is known as a major degradative compartment, many of these were hydrolases, but we also found various oxidoreductases and transferases. The relationships between the proteins and metabolites in the vacuole are discussed.

The regulatory nucleotides, guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp), were originally identified in Escherichia coli, and control a large set of gene expression and enzyme activities. The (p)ppGpp-dependent control of cell activities is referred to as the stringent response. A growing number of (p)ppGpp synthase/hydrolase homologs have been identified in plants, which are localized in plastids in Arabidopsis thaliana. We recently reported that the Arabidopsis mutant overproducing ppGpp in plastids showed dwarf chloroplasts, and transcript levels in the mutant plastids were significantly suppressed. Furthermore, the mutant showed more robust growth than the wild type (WT), especially under nutrient-deficient conditions, although the mechanisms are unclear. To better understand the impact of the ppGpp accumulation on plant responses to nutrient deficiency, photosynthetic activities and metabolic changes in the ppGpp-overproducing mutant were characterized here. Upon transition to the nitrogen-deficient conditions, the mutant showed reduction of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) contents, and effective and maximum quantum yield of photosystem II compared with WT. The mutant also showed more obvious changes in key metabolite levels including some amino acid contents than WT; similar metabolic change is known to be critical for plants to maintain carbon-nitrogen balance in their cells. These results suggest that artificially overproducing ppGpp modulates the organelle functions that play an important role in controlling photosynthetic performance and metabolite balance during nitrogen starvation.

Lineage-specific gene duplications contribute to a large variation in specialized metabolites among different plant species. There is also considerable variability in the specialized metabolites within a single plant species. However, it is unclear whether copy number variations (CNVs) derived from gene duplication events contribute to the diversity of specialized metabolites within species. We identified metabolome quantitative trait genes (mQTGs) associated with quantitative metabolite variations and examined the relationship between mQTGs and CNVs. We obtained 1,335 specialized metabolite signals from 53 worldwide A. thaliana accessions using liquid chromatography-quadrupole time-of-flight mass spectrometry. In this study, genes associated with specialized metabolites were inferred by either a generally authorized genome-wide association study (GWAS) approach or a novel analysis of the association between gene expression and metabolite accumulation. Genes qualified by both analyses are defined to be mQTGs. The integrated method enabled us to detect mQTGs with a low false positive rate (=5.71 × 10-4). We also identified 5,654 genes associated with 1,335 specialized metabolites. Of these genes, 4.4% were affected by CNVs, which was more than expected (χ2 test: P < 0.01). This result suggests that CNVs contribute to variations in specialized metabolites within a species. To assess the contribution of CNVs to adaptive evolution in A. thaliana, we examined the selective sweeps around the mQTGs. We observed that the mQTGs with CNVs tended to undergo selective sweeps. These observations imply that variations in specialized metabolites caused by CNVs contribute to the adaptive evolution of A. thaliana., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non-transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Growth of eukaryotic cells is regulated by the target of rapamycin (TOR). The strongest activator of TOR in metazoa is amino acid availability. The established transducers of amino acid sensing to TOR in metazoa are absent in plants. Hence, a fundamental question is how amino acid sensing is achieved in photo-autotrophic organisms. Here we demonstrate that the plant Arabidopsis does not sense the sulfur-containing amino acid cysteine itself, but its biosynthetic precursors. We identify the kinase GCN2 as a sensor of the carbon/nitrogen precursor availability, whereas limitation of the sulfur precursor is transduced to TOR by downregulation of glucose metabolism. The downregulated TOR activity caused decreased translation, lowered meristematic activity, and elevated autophagy. Our results uncover a plant-specific adaptation of TOR function. In concert with GCN2, TOR allows photo-autotrophic eukaryotes to coordinate the fluxes of carbon, nitrogen, and sulfur for efficient cysteine biosynthesis under varying external nutrient supply.

Water deficit caused by global climate changes seriously endangers the survival of organisms and crop productivity, and increases environmental deterioration 1,2 . Plants' resistance to drought involves global reprogramming of transcription, cellular metabolism, hormone signalling and chromatin modification 3-8 . However, how these regulatory responses are coordinated via the various pathways, and the underlying mechanisms, are largely unknown. Herein, we report an essential drought-responsive network in which plants trigger a dynamic metabolic flux conversion from glycolysis into acetate synthesis to stimulate the jasmonate (JA) signalling pathway to confer drought tolerance. In Arabidopsis, the ON/OFF switching of this whole network is directly dependent on histone deacetylase HDA6. In addition, exogenous acetic acid promotes de novo JA synthesis and enrichment of histone H4 acetylation, which influences the priming of the JA signalling pathway for plant drought tolerance. This novel acetate function is evolutionarily conserved as a survival strategy against environmental changes in plants. Furthermore, the external application of acetic acid successfully enhanced the drought tolerance in Arabidopsis, rapeseed, maize, rice and wheat plants. Our findings highlight a radically new survival strategy that exploits an epigenetic switch of metabolic flux conversion and hormone signalling by which plants adapt to drought.

We report the characterization of the Arabidopsis ( Arabidopsis thaliana ) 3-hydroxyacyl-acyl carrier protein dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein transgenesis experiment and by in vivo complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO 2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO 2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA sequencing analysis revealed that mthd-rnai and mtkas mutants are nearly equivalent to each other in altering the transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system but independent of photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Glutamine synthetase (GS) is an important enzyme for nitrogen assimilation, and GS2, encoded by GLN2, is the only plastid-type GS in Arabidopsis thaliana. A co-expression analysis suggested that the expression level of the gene encoding a uridylyltransferase-like protein, ACR11, is strongly correlated with GLN2 expression levels. Here we showed that the recombinant ACR11 protein increased GS2 activity in vitro by reducing the Km values of its substrate glutamine. A T-DNA insertion mutant of ACR11 exhibited a reduced GS activity under low nitrate conditions and reduced glutamine levels. Biochemical analyses revealed that ACR11 and GS2 interacted both in vitro and in vivo. These data demonstrate that ACR11 is an activator of GS2, giving it a mechanistic role in the nitrogen assimilation of A. thaliana., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Phytoaccumulation is a technique to extract metals from soil utilising ability of plants. Cesium is a valuable metal while radioactive isotopes of cesium can be hazardous. In order to establish a more efficient phytoaccumulation system, small molecules which promote plants to accumulate cesium were investigated. Through chemical library screening, 14 chemicals were isolated as 'cesium accumulators' in Arabidopsis thaliana. Of those, methyl cysteinate, a derivative of cysteine, was found to function within the plant to accumulate externally supplemented cesium. Moreover, metabolite profiling demonstrated that cesium treatment increased cysteine levels in Arabidopsis. The cesium accumulation effect was not observed for other cysteine derivatives or amino acids on the cysteine metabolic pathway tested. Our results suggest that methyl cysteinate, potentially metabolised from cysteine, binds with cesium on the surface of the roots or inside plant cells and improve phytoaccumulation.

Glucosinolates (GSLs) in the plant order of the Brassicales are sulfur-rich secondary metabolites that harbor antipathogenic and antiherbivory plant-protective functions and have medicinal properties, such as carcinopreventive and antibiotic activities. Plants repress GSL biosynthesis upon sulfur deficiency (-S); hence, field performance and medicinal quality are impaired by inadequate sulfate supply. The molecular mechanism that links -S to GSL biosynthesis has remained understudied. We report here the identification of the -S marker genes sulfur deficiency induced 1 ( SDI1 ) and SDI2 acting as major repressors controlling GSL biosynthesis in Arabidopsis under -S condition. SDI1 and SDI2 expression negatively correlated with GSL biosynthesis in both transcript and metabolite levels. Principal components analysis of transcriptome data indicated that SDI1 regulates aliphatic GSL biosynthesis as part of -S response. SDI1 was localized to the nucleus and interacted with MYB28, a major transcription factor that promotes aliphatic GSL biosynthesis, in both yeast and plant cells. SDI1 inhibited the transcription of aliphatic GSL biosynthetic genes by maintaining the DNA binding composition in the form of an SDI1-MYB28 complex, leading to down-regulation of GSL biosynthesis and prioritization of sulfate usage for primary metabolites under sulfur-deprived conditions.

Plants rapidly perceive tissue damage, such as that inflicted by insects, and activate several key defense responses. The importance of the fatty acid-derived hormone jasmonates (JA) in dictating these wound responses has been recognized for many years. However, important features pertaining to the regulation of the JA pathway are still not well understood. One key unknown is the inactivation mechanism of the JA pathway and its relationship with plant response to wounding. Arabidopsis cytochrome P450 enzymes in the CYP94 clade metabolize jasmonoyl-L-isoleucine (JA-Ile), a major metabolite of JA responsible for many biological effects attributed to the JA signaling pathway; thus, CYP94s are expected to contribute to the attenuation of JA-Ile-dependent wound responses. To directly test this, we created the double and triple knock-out mutants of three CYP94 genes, CYP94B1, CYP94B3, and CYP94C1. The mutations blocked the oxidation steps and caused JA-Ile to accumulate 3-4-fold the WT levels in the wounded leaves. Surprisingly, over accumulation of JA-Ile did not lead to a stronger wound response. On the contrary, the mutants displayed a series of symptoms reminiscent of JA-Ile deficiency, including resistance to wound-induced growth inhibition, decreased anthocyanin and trichomes, and increased susceptibility to insects. The mutants, however, responded normally to exogenous JA treatments, indicating that JA perception or signaling pathways were intact. Untargeted metabolite analyses revealed >40% reduction in wound-inducible metabolites in the mutants. These observations raise questions about the current JA signaling model and point toward a more complex model perhaps involving JA derivatives and/or feedback mechanisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Incidence of natural light stress renders it important to enhance our understanding of the mechanisms by which plants protect themselves from harmful effects of UV-B irradiation, as this is critical for fitness of land plant species. Here we describe natural variation of a class of phenylacylated-flavonols (saiginols), which accumulate to high levels in floral tissues of Arabidopsis. They were identified in a subset of accessions, especially those deriving from latitudes between 16° and 43° North. Investigation of introgression line populations using metabolic and transcript profiling, combined with genomic sequence analysis, allowed the identification of flavonol-phenylacyltransferase 2 (FPT2) that is responsible for the production of saiginols and conferring greater UV light tolerance in planta. Furthermore, analysis of polymorphism within the FPT duplicated region provides an evolutionary framework of the natural history of this locus in the Brassicaceae., Competing Interests: T.T., R.W., H.I., R.S., M.S. and A.R.F. have filed patent applications on saiginol derivatives and FPT gene function. The remaining authors declare no competing financial interests.

Adaptation to environmental stress requires genome-wide changes in gene expression. Histone modifications are involved in gene regulation, but the role of histone modifications under environmental stress is not well understood. To reveal the relationship between histone modification and environmental stress, we assessed the effects of inhibitors of histone modification enzymes during salinity stress. Treatment with Ky-2, a histone deacetylase inhibitor, enhanced high-salinity stress tolerance in Arabidopsis. We confirmed that Ky-2 possessed inhibition activity towards histone deacetylases by immunoblot analysis. To investigate how Ky-2 improved salt stress tolerance, we performed transcriptome and metabolome analysis. These data showed that the expression of salt-responsive genes and salt stress-related metabolites were increased by Ky-2 treatment under salinity stress. A mutant deficient in AtSOS1(Arabidopis thaliana SALT OVERLY SENSITIVE 1), which encodes an Na(+)/H(+)antiporter and was among the up-regulated genes, lost the salinity stress tolerance conferred by Ky-2. We confirmed that acetylation of histone H4 at AtSOS1 was increased by Ky-2 treatment. Moreover, Ky-2 treatment decreased the intracellular Na(+)accumulation under salinity stress, suggesting that enhancement of SOS1-dependent Na(+)efflux contributes to increased high-salinity stress tolerance caused by Ky-2 treatment., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Key Message: A candidate gene for phenylcoumaran benzylic ether reductase in Arabidopsis thaliana encodes a peptide with predicted functional activity and plays a crucial role in secondary metabolism. Phenylcoumaran benzylic ether reductase (PCBER) is thought to be an enzyme crucial in the biosynthesis of 8-5'-linked neolignans. Genes of the enzyme have been isolated and characterized in several plant species. In this study, we cloned cDNA and the 5'-untranslated region of one PCBER candidate gene (At4g39230, designated AtPCBER1) from Arabidopsis thaliana. At the amino acid level, AtPCBER1 shows high sequence identity (64-71 %) with PCBERs identified from other plant species. Expression analyses of AtPCBER1 by reverse transcriptase-polymerase chain reaction and histochemical analysis of transgenic plants harboring the 5'-untranslated region of AtPCBER1 linked with gus coding sequence indicate that expression is induced by wounding and is expressed in most tissues, including flower, stem, leaf, and root. Catalytic analysis of recombinant AtPCBER1 with neolignan and lignans in the presence of NADPH suggests that the protein can reduce not only the 8-5'-linked neolignan, dehydrodiconiferyl alcohol, but also 8-8' linked lignans, pinoresinol, and lariciresinol, with lower activities. To investigate further, we performed metabolomic analyses of transgenic plants in which the target gene was up- or down-regulated. Our results indicate no significant effects of AtPCBER1 gene regulation on plant growth and development; however, levels of some secondary metabolites, including lignans, flavonoids, and glucosinolates, differ between wild-type and transgenic plants. Taken together, our findings indicate that AtPCBER1 encodes a polypeptide with PCBER activity and has a critical role in the biosynthesis of secondary metabolites in A. thaliana.

Environmental stress causes membrane damage in plants. Lipid studies are required to understand the adaptation of plants to climate change. Here, LC-MS-based lipidomic and microarray transcriptome analyses were carried out to elucidate the effect of short-term heat stress on the Arabidopsis thaliana leaf membrane. Vegetative plants were subjected to high temperatures for one day, and then grown under normal conditions. Sixty-six detected glycerolipid species were classified according to patterns of compositional change by Spearman's correlation coefficient. Triacylglycerols, 36:4- and 36:5-monogalactosyldiacylglycerol, 34:2- and 36:2-digalactosyldiacylglycerol, 34:1-, 36:1- and 36:6-phosphatidylcholine, and 34:1-phosphatidylethanolamine increased by the stress and immediately decreased during recovery. The relative amount of one triacylglycerol species (54:9) containing α-linolenic acid (18:3) increased under heat stress. These results suggest that heat stress in Arabidopsis leaves induces an increase in triacylglycerol levels, which functions as an intermediate of lipid turnover, and results in a decrease in membrane polyunsaturated fatty acids. Microarray data revealed candidate genes responsible for the observed metabolic changes.

Under sulfur deficiency (-S), plants induce expression of the sulfate transport systems in roots to increase uptake and root-to-shoot transport of sulfate. The low-affinity sulfate transporter SULTR2;1 is predominantly expressed in xylem parenchyma and pericycle cells in Arabidopsis thaliana roots under -S. The mechanisms underlying -S-inducible expression of SULTR2;1 in roots have remained unclear, despite the possible significance of SULTR2;1 for acclimation to low-sulfur conditions. In this investigation, examination of deletions and base substitutions in the 3'-intergenic region of SULTR2;1 revealed novel sulfur-responsive elements, SURE21A (5'-CAATGTATC-3') and SURE21B (5'-CTAGTAC-3'), located downstream of the SULTR2;1 3'-untranslated region. SURE21A and SULTR21B effectively induced reporter gene expression from fusion constructs under -S in combination with minimal promoters or promoters not inducible by -S, suggesting their versatility in controlling transcription. T-DNA insertions near SURE21A and SULTR21B abolished -S-inducible expression of SULTR2;1 in roots and reduced the uptake and root-to-shoot transport of sulfate. In addition, these mutations partially suppressed SULTR2;1 expression in shoots, without changing its -S-responsive expression. These findings indicate that SULTR2;1 contributes to the increase in uptake and internal translocation of sulfate driven by gene expression induced under the control of sulfur-responsive elements in the 3'-nontranscribed intergenic region of SULTR2;1., (© 2015 American Society of Plant Biologists. All rights reserved.)

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4'-feruloyl ethers. In-depth quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.

Flavonol 3-O-diglucosides with a 1→2 inter-glycosidic linkage are representative pollen-specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild-type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild-type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP-glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen-specific flavonol structure. Kaempferol and quercetin 3-O-glucosyl-(1→2)-glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild-type plants. Recombinant UGT79B6 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucosyl-(1→2)-glucoside. UGT79B6 recognized 3-O-glucosylated/galactosylated anthocyanins/flavonols but not 3,5- or 3,7-diglycosylated flavonoids, and prefers UDP-glucose, indicating that UGT79B6 encodes flavonoid 3-O-glucoside:2″-O-glucosyltransferase. A UGT79B6-GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers., (© 2014 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.

Surface functionalization of nanoparticles has become an important tool for in vivo delivery of bioactive agents to their target sites. Here we describe the reverse strategy, nanoharvesting, in which nanoparticles are used as a tool to isolate bioactive compounds from living cells. Anatase TiO₂ nanoparticles smaller than 20 nm form strong bonds with molecules bearing enediol and especially catechol groups. We show that these nanoparticles enter plant cells, conjugate enediol and catechol group-rich flavonoids in situ, and exit plant cells as flavonoid-nanoparticle conjugates. The source plant tissues remain viable after treatment. As predicted by the surface chemistry of anatase TiO₂ nanoparticles, quercetin-based flavonoids were enriched amongst the nanoharvested flavonoid species. Nanoharvesting eliminates the use of organic solvents, allows spectral identification of the isolated compounds, and opens new avenues for use of nanomaterials for coupled isolation and testing of bioactive properties of plant-synthesized compounds., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

The notion that plants use specialized metabolism to protect against environmental stresses needs to be experimentally proven by addressing the question of whether stress tolerance by specialized metabolism is directly due to metabolites such as flavonoids. We report that flavonoids with radical scavenging activity mitigate against oxidative and drought stress in Arabidopsis thaliana. Metabolome and transcriptome profiling and experiments with oxidative and drought stress in wild-type, single overexpressors of MYB12/PFG1 (PRODUCTION OF FLAVONOL GLYCOSIDES1) or MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), double overexpressors of MYB12 and PAP1, transparent testa4 (tt4) as a flavonoid-deficient mutant, and flavonoid-deficient MYB12 or PAP1 overexpressing lines (obtained by crossing tt4 and the individual MYB overexpressor) demonstrated that flavonoid overaccumulation was key to enhanced tolerance to such stresses. Antioxidative activity assays using 2,2-diphenyl-1-picrylhydrazyl, methyl viologen, and 3,3'-diaminobenzidine clearly showed that anthocyanin overaccumulation with strong in vitro antioxidative activity mitigated the accumulation of reactive oxygen species in vivo under oxidative and drought stress. These data confirm the usefulness of flavonoids for enhancing both biotic and abiotic stress tolerance in crops., (© 2013 The Authors. The Plant Journal © 2013 John Wiley & Sons Ltd.)

Oil bodies are intracellular structures present in the seed and leaf cells of many land plants. Seed oil bodies are known to function as storage compartments for lipids. However, the physiological function of leaf oil bodies is unknown. Here, we show that leaf oil bodies function as subcellular factories for the production of a stable phytoalexin in response to fungal infection and senescence. Proteomic analysis of oil bodies prepared from Arabidopsis (Arabidopsis thaliana) leaves identified caleosin (CLO3) and α-dioxygenase (α-DOX1). Both CLO3 and α-DOX1 were localized on the surface of oil bodies. Infection with the pathogenic fungus Colletotrichum higginsianum promoted the formation of CLO3- and α-DOX1-positive oil bodies in perilesional areas surrounding the site of infection. α-DOX1 catalyzes the reaction from α-linolenic acid (a major fatty acid component of oil bodies) to an unstable compound, 2-hydroperoxy-octadecatrienoic acid (2-HPOT). Intriguingly, a combination of α-DOX1 and CLO3 produced a stable compound, 2-hydroxy-octadecatrienoic acid (2-HOT), from α-linolenic acid. This suggests that the colocalization of α-DOX1 and CLO3 on oil bodies might prevent the degradation of unstable 2-HPOT by efficiently converting 2-HPOT into the stable compound 2-HOT. We found that 2-HOT had antifungal activity against members of the genus Colletotrichum and that infection with C. higginsianum induced 2-HOT production. These results defined 2-HOT as an Arabidopsis phytoalexin. This study provides, to our knowledge, the first evidence that leaf oil bodies produce a phytoalexin under a pathological condition, which suggests a new mechanism of plant defense.

Plants have developed mechanisms to protect themselves against both biotic and abiotic environmental stress. Specialized/secondary metabolism is one of the stress response mechanisms. Recently, we reported that flavonoids, a class of specialized metabolites, including flavonols and anthocyanins with strong radical scavenging activity contributed to the mitigation of oxidative and drought stress in Arabidopsis thaliana (Arabidopsis). However, the behavior of flavonoids during drought stress is still not well-documented. Herein we investigated the time-series alternation of flavonoids in the aerial part of Arabidopsis (wild type, Col-0) during drought stress by LC-QTOF-MS. The drastic alternation of 5 flavonols and 5 anthocyanins was revealed together with changes in marker metabolites of drought stress, e.g., proline, raffinose, and galactinol. These findings indicate that flavonols and anthocyanins can mitigate drought stress.

Flavonoids are representative plant secondary products. In the model plant Arabidopsis thaliana, at least 54 flavonoid molecules (35 flavonols, 11 anthocyanins and 8 proanthocyanidins) are found. Scaffold structures of flavonoids in Arabidopsis are relatively simple. These include kaempferol, quercetin and isorhamnetin for flavonols, cyanidin for anthocyanins and epicatechin for proanthocyanidins. The chemical diversity of flavonoids increases enormously by tailoring reactions which modify these scaffolds, including glycosylation, methylation and acylation. Genes responsible for the formation of flavonoid aglycone structures and their subsequent modification reactions have been extensively characterized by functional genomic efforts - mostly the integration of transcriptomics and metabolic profiling followed by reverse genetic experimentation. This review describes the state-of-art of flavonoid biosynthetic pathway in Arabidopsis regarding both structural and genetic diversity, focusing on the genes encoding enzymes for the biosynthetic reactions and vacuole translocation., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

In this article we discuss current trends in the techniques available for plant metabolomics. Chemical assignment of unknown metabolites leads to understanding of biosynthetic mechanisms at the gene level for genome-sequenced plants. Metabolomics using mass spectrometry has achieved innovative results in phytochemical genomics for primary and secondary metabolism in the model plant Arabidopsis thaliana by using publicly and commercially available information and standard compounds. However, finding a consolidated analytical technique for elucidation of structural information (e.g., elemental composition and structure) remains challenging. Recently, hyphenated analytical techniques and computer-assisted structural analysis with high-throughput and high-accuracy have been developing. Metabolite-driven approaches using such technology will be of central importance in phytochemical genomics.

Phytochemical genomics is a recently emerging field, which investigates the genomic basis of the synthesis and function of phytochemicals (plant metabolites), particularly based on advanced metabolomics. The chemical diversity of the model plant Arabidopsis thaliana is larger than previously expected, and the gene-to-metabolite correlations have been elucidated mostly by an integrated analysis of transcriptomes and metabolomes. For example, most genes involved in the biosynthesis of flavonoids in Arabidopsis have been characterized by this method. A similar approach has been applied to the functional genomics for production of phytochemicals in crops and medicinal plants. Great promise is seen in metabolic quantitative loci analysis in major crops such as rice and tomato, and identification of novel genes involved in the biosynthesis of bioactive specialized metabolites in medicinal plants., (Copyright © 2013 The Author. Published by Elsevier Ltd.. All rights reserved.)

Transcriptome coexpression analysis is an excellent tool for predicting the physiological functions of genes. It is based on the "guilt-by-association" principle. Generally, genes involved in certain metabolic processes are coordinately regulated. In other words, coexpressed genes tend to be involved in common or closely related biological processes. Genes of which the metabolic functions have been identified are preselected as "guide" genes and are used to check the transcriptome coexpression fidelity to the pathway and to determine the threshold value of correlation coefficients to be used for subsequent analysis. The coexpression analysis provides a network of the relationships between "guide" and candidate genes that serves to create the criteria by which gene functions can be predicted. Here we describe a procedure to narrow down the number of candidate genes by means of the publicly available database, designated Arabidopsis thaliana trans-factor and cis-element prediction database (ATTED-II).

Phosphorus supply is a major factor responsible for reduced crop yields. As a result, plants utilize various adaptive mechanisms against phosphorus depletion, including lipid remodelling. Here we report the involvement of a novel plant lipid, glucuronosyldiacylglycerol, against phosphorus depletion. Lipidomic analysis of Arabidopsis plants cultured in phosphorus-depleted conditions revealed inducible accumulation of glucuronosyldiacylglycerol. Investigation using a series of sulfolipid sulfoquinovosyldiacylglycerol synthesis-deficient mutants of Arabidopsis determined that the biosynthesis of glucuronosyldiacylglycerol shares the pathway of sulfoquinovosyldiacylglycerol synthesis in chloroplasts. Under phosphorus-depleted conditions, the Arabidopsis sqd2 mutant, which does not accumulate either sulfoquinovosyldiacylglycerol or glucuronosyldiacylglycerol, was the most severely damaged of three sulfoquinovosyldiacylglycerol-deficient mutants. As glucuronosyldiacylglycerol is still present in the other two mutants, this result indicates that glucuronosyldiacylglycerol has a role in the protection of plants against phosphorus limitation stress. Glucuronosyldiacylglycerol was also found in rice, and its concentration increased significantly following phosphorus limitation, suggesting a shared physiological significance of this novel lipid against phosphorus depletion in plants.

To identify candidate genes involved in Arabidopsis flavonoid biosynthesis, we applied transcriptome coexpression analysis and independent component analyses with 1388 microarray data from publicly available databases. Two glycosyltransferases, UGT79B1 and UGT84A2 were found to cluster with anthocyanin biosynthetic genes. Anthocyanin was drastically reduced in ugt79b1 knockout mutants. Recombinant UGT79B1 protein converted cyanidin 3-O-glucoside to cyanidin 3-O-xylosyl(1→2)glucoside. UGT79B1 recognized 3-O-glucosylated anthocyanidins/flavonols and uridine diphosphate (UDP)-xylose, but not 3,5-O-diglucosylated anthocyanidins, indicating that UGT79B1 encodes anthocyanin 3-O-glucoside: 2''-O-xylosyltransferase. UGT84A2 is known to encode sinapic acid: UDP-glucosyltransferase. In ugt84a2 knockout mutants, a major sinapoylated anthocyanin was drastically reduced. A comparison of anthocyanin profiles in ugt84a knockout mutants indicated that UGT84A2 plays a major role in sinapoylation of anthocyanin, and that other UGT84As contribute the production of 1-O-sinapoylglucose to a lesser extent. These data suggest major routes from cyanidin 3-O-glucoside to the most highly modified cyanidin in the potential intricate anthocyanin modification pathways in Arabidopsis., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Seed germination is a result of the competition of embryonic growth potential and mechanical constraint by surrounding tissues such as the endosperm. To understand the processes occurring in the endosperm during germination, we analyzed tiling array expression data on dissected endosperm and embryo from 6 and 24 h-imbibed Arabidopsis seeds. The genes preferentially expressed in the endosperm of both 6 and 24 h-imbibed seeds were enriched for those related to cell wall biosynthesis/modifications, flavonol biosynthesis, defense responses and cellular transport. Loss of function of AtXTH31/XTR8, an endosperm-specific gene for a putative xyloglucan endotransglycosylase/hydrolase, led to faster germination. This suggests that AtXTH31/XTR8 is involved in the reinforcement of the cell wall of the endosperm during germination. In vivo flavonol staining by diphenyl boric acid aminoethyl ester (DPBA) showed flavonols accumulated in the endosperm of both dormant and non-dormant seeds, suggesting that this event is independent of germination. Notably, DPBA fluorescence was also intense in the embryo, but the fluorescent region was diminished around the radicle and lower half of the hypocotyl during germination. DPBA fluorescence was localized in the vacuoles during germination. Vacuolation was not seen in imbibed dormant seeds, suggesting that vacuolation is associated with germination. A gene for δVPE (vacuolar processing enzyme), a caspase-1-like cysteine proteinase involved in cell death, is expressed specifically in endosperms of 24 h-imbibed seeds. The δvpe mutant showed retardation of vacuolation, but this mutation did not affect the kinetics of germination. This suggests that vacuolation is a consequence, and not a trigger, of germination.

In order to adapt to environmental changes of light species and intensity, higher plants furnish complicate signaling systems such as the UVR/COP/HY5 cascade which links several diverse classes of photoreceptors. In addition UV-B light provokes accelerated production of UV-B protectants such as flavonoids and vitamins. Following intensive research efforts, genes in the UV-B signaling cascade have been characterized via forward genetics approaches following mutant screens relying on sensitivity to UV-B irradiation. However detailed processes of the linkage between light signaling and the upregulation of metabolite accumulation remain unclear. Here we review both the light signal cascades and metabolite pathways responding to UV-B exposure. Finally we generate co-expression network analysis using published data in order to find novel candidate genes which link light signaling and transcriptional regulation to metabolic biosynthesis in attempt to describe how these processes are interlinked., (© 2011 Landes Bioscience)

Background: 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets., Results: In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA) cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds., Conclusion: Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway.

Polarity along the adaxial-abaxial axis of the leaf is essential for leaf development and morphogenesis. One of the genes that encodes a putative transcription factor regulating adaxial-abaxial polarity, FILAMENTOUS FLOWER (FIL), is expressed in the abaxial region of the leaf primordia. However, the molecular mechanisms controlling the polarized expression of FIL remain unclear. Here, we analyzed an enlarged fil expression domain1 (enf1) mutant of Arabidopsis, which forms both abaxialized leaves and adaxialized leaves. The ENF1 gene encodes SUCCINIC SEMIALDEHYDE DEHYDROGENASE (SSADH), which catalyzes the conversion of succinic semialdehyde (SSA) to succinate. The enf1 phenotype was suppressed by an additional mutation in GAMMA-AMINOBUTYRIC ACID AMINOTRANSFERASE1 (GABAT1), which encodes an SSA-producing enzyme, suggesting that SSA or its derivatives is the metabolite responsible for the defect in the adaxial-abaxial axis-dependent gene expression of enf1. In the shoot apical meristem, GABAT1 was expressed in the outermost layer but SSADH was not. Exogenous application of SSA induced adaxial characters on the abaxial side of the newly developed leaves. We suggest that a GABA shunt metabolite, SSA or its close derivatives, is involved in the robust leaf patterning and structure along the adaxial-abaxial axis.

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate-derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen-challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long-chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate-derived isothiocyanates was dependent on side chain elongation and modification, with 8-methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short-chained aliphatic glucosinolates., (© 2011 RIKEN (Growth Regulation Research Group). The Plant Journal © 2011 Blackwell Publishing Ltd.)

Because of ever-increasing environmental deterioration it is likely that the influx of UV-B radiation (280-320 nm) will increase as a result of the depletion of stratospheric ozone. Given this fact it is essential that we better understand both the rapid and the adaptive responses of plants to UV-B stress. Here, we compare the metabolic responses of wild-type Arabidopsis with that of mutants impaired in flavonoid (transparent testa 4, tt4; transparent testa 5, tt5) or sinapoyl-malate (sinapoylglucose accumulator 1, sng1) biosynthesis, exposed to a short 24-h or a longer 96-h exposure to this photo-oxidative stress. In control experiments we subjected the genotypes to long-day conditions as well as to 24- and 96-h treatments of continuous light. Following these treatments we evaluated the dynamic response of metabolites including flavonoids, sinapoyl-malate precursors and ascorbate, which are well known to play a role in cellular protection from UV-B stress, as well as a broader range of primary metabolites, in an attempt to more fully comprehend the metabolic shift following the cellular perception of this stress. Our data reveals that short-term responses occur only at the level of primary metabolites, suggesting that these effectively prime the cell to facilitate the later production of UV-B-absorbing secondary metabolites. The combined results of these studies together with transcript profiles using samples irradiated by 24-h UV-B light are discussed in the context of current models concerning the metabolic response of plants to the stress imposed by excessive UV-B irradiation., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

MicroRNAs play a key role in the control of plant development and response to adverse environmental conditions. For example, microRNA395 (miR395), which targets three out of four isoforms of ATP sulfurylase, the first enzyme of sulfate assimilation, as well as a low-affinity sulfate transporter, SULTR2;1, is strongly induced by sulfate deficiency. However, other components of sulfate assimilation are induced by sulfate starvation, so that the role of miR395 is counterintuitive. Here, we describe the regulation of miR395 and its targets by sulfate starvation. We show that miR395 is important for the increased translocation of sulfate to the shoots during sulfate starvation. MiR395 together with the SULFUR LIMITATION 1 transcription factor maintain optimal levels of ATP sulfurylase transcripts to enable increased flux through the sulfate assimilation pathway in sulfate-deficient plants. Reduced expression of ATP sulfurylase (ATPS) alone affects both sulfate translocation and flux, but SULTR2;1 is important for the full rate of sulfate translocation to the shoots. Thus, miR395 is an integral part of the regulatory circuit controlling plant sulfate assimilation with a complex mechanism of action., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Despite its relevance for agricultural production, environmental stress-induced growth inhibition, which is responsible for significant yield reductions, is only poorly understood. Here, we investigated the molecular mechanisms underlying cell cycle inhibition in young proliferating leaves of the model plant Arabidopsis thaliana when subjected to mild osmotic stress. A detailed cellular analysis demonstrated that as soon as osmotic stress is sensed, cell cycle progression rapidly arrests, but cells are kept in a latent ambivalent state allowing a quick recovery (pause). Remarkably, cell cycle arrest coincides with an increase in 1-aminocyclopropane-1-carboxylate levels and the activation of ethylene signaling. Our work showed that ethylene acts on cell cycle progression via inhibition of cyclin-dependent kinase A activity independently of EIN3 transcriptional control. When the stress persists, cells exit the mitotic cell cycle and initiate the differentiation process (stop). This stop is reflected by early endoreduplication onset, in a process independent of ethylene. Nonetheless, the potential to partially recover the decreased cell numbers remains due to the activity of meristemoids. Together, these data present a conceptual framework to understand how environmental stress reduces plant growth.

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.