N2-Acetylornithine deacetylase functions as a Cys-Gly dipeptidase in the cytosolic glutathione degradation pathway in Arabidopsis thaliana.

Glutathione (GSH) is required for various physiological processes in plants, including redox regulation and detoxification of harmful compounds. GSH also functions as a repository for assimilated sulfur and is actively catabolized in plants. In Arabidopsis, GSH is mainly degraded initially by cytosolic enzymes, γ-glutamyl cyclotransferase, and γ-glutamyl peptidase, which release cysteinylglycine (Cys-Gly). However, the subsequent enzyme responsible for catabolizing this dipeptide has not been identified to date. In the present study, we identified At4g17830 as a Cys-Gly dipeptidase, namely cysteinylglycine peptidase 1 (CGP1). CGP1 complemented the phenotype of the yeast mutant that cannot degrade Cys-Gly. The Arabidopsis cgp1 mutant had lower Cys-Gly degradation activity than the wild type and showed perturbed concentrations of thiol compounds. Recombinant CGP1 showed reasonable Cys-Gly degradation activity in vitro. Metabolomic analysis revealed that cgp1 exhibited signs of severe sulfur deficiency, such as elevated accumulation of O-acetylserine (OAS) and the decrease in sulfur-containing metabolites. Morphological changes observed in cgp1, including longer primary roots of germinating seeds, were also likely associated with sulfur starvation. Notably, At4g17830 has previously been reported to encode an N2-acetylornithine deacetylase (NAOD) that functions in the ornithine biosynthesis. The cgp1 mutant did not show a decrease in ornithine content, whereas the analysis of CGP1 structure did not rule out the possibility that CGP1 has Cys-Gly dipeptidase and NAOD activities. Therefore, we propose that CGP1 is a Cys-Gly dipeptidase that functions in the cytosolic GSH degradation pathway and may play dual roles in GSH and ornithine metabolism.

Distinct lactate metabolism between hepatocytes and myotubes revealed by live cell imaging with genetically encoded indicators.

The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

Plant metabolomics.

Plants are a treasure trove of metabolic compounds. The chemical diversity of plant cells has developed and been maintained through evolution and metabolic regulation, and plays a crucial role in plant physiology, development, and adaption to changing environmental situations. Metabolomics, when combined with genomics and proteomics, has opened up unprecedented opportunities to address the biological importance of metabolic diversity. It has also provided an avenue for metabolic engineering to produce a particular compound of interest to meet societal and economical demands, an important effort to achieve sustainable development. This Special Issue therefore focuses on current trends in plant metabolomics research, providing examples in the development of analytical technologies, the functional study of plant metabolism, and applications to synthetic and engineering biology.

An auxin signaling network translates low-sugar-state input into compensated cell enlargement in the fugu5 cotyledon.

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

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 34S- 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.

Glutathione degradation activity of γ-glutamyl peptidase 1 manifests its dual roles in primary and secondary sulfur metabolism in Arabidopsis.

Glutathione (GSH) functions as a major sulfur repository and hence occupies an important position in primary sulfur metabolism. GSH degradation results in sulfur reallocation and is believed to be carried out mainly by γ-glutamyl cyclotransferases (GGCT2;1, GGCT2;2, and GGCT2;3), which, however, do not fully explain the rapid GSH turnover. Here, we discovered that γ-glutamyl peptidase 1 (GGP1) contributes to GSH degradation through a yeast complementation assay. Recombinant proteins of GGP1, as well as GGP3, showed high degradation activity of GSH, but not of oxidized glutathione (GSSG), in vitro. Notably, the GGP1 transcripts were highly abundant in rosette leaves, in agreement with the ggp1 mutants constantly accumulating more GSH regardless of nutritional conditions. Given the lower energy requirements of the GGP- than the GGCT-mediated pathway, the GGP-mediated pathway could be a more efficient route for GSH degradation than the GGCT-mediated pathway. Therefore, we propose a model wherein cytosolic GSH is degraded chiefly by GGP1 and likely also by GGP3. Another noteworthy fact is that GGPs are known to process GSH conjugates in glucosinolate and camalexin synthesis; indeed, we confirmed that the ggp1 mutant contained higher levels of O-acetyl-l-Ser, a signaling molecule for sulfur starvation, and lower levels of glucosinolates and their degradation products. The predicted structure of GGP1 further provided a rationale for this hypothesis. In conclusion, we suggest that GGP1 and possibly GGP3 play vital roles in both primary and secondary sulfur metabolism.

The Roles of Functional Amino Acids in Plant Growth and Development.

Plants incorporate acquired carbon and nitrogen into amino acid metabolism, whereby the building blocks of proteins and the precursors of various metabolites are produced. This fundamental demand requires tight amino acid metabolism to sustain physiological homeostasis. There is increasing evidence that amino acid metabolism undergoes plastic alteration to orchestrate specific growth and developmental events. Consequently, there has been a gradual exploration of the interface at which amino acid metabolism and plant morphogenesis are mutually affected. This research progress offers an opportunity to explore amino acid metabolism, with the goal to understand how it can be modulated to serve special cellular needs and regulate specific growth and developmental pathways. Continuous improvements in the sensitivity and coverage of metabolomics technology, along with the development of chemoinformatics, have allowed the investigation of these research questions. In this review, we summarize the roles of threonine, serine, arginine and γ-aminobutyric acid as representative examples of amino acids relevant to specific developmental processes in plants ('functional amino acids'). Our objective is to expand perspectives regarding amino acid metabolism beyond the conventional view that it is merely life-supporting machinery.

Endoplasmic Reticulum Bodies in the Lateral Root Cap Are Involved in the Direct Transport of Beta-Glucosidase to Vacuoles.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the ß-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport ß-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.

Molecular Identification of UDP-Sugar-Dependent Glycosyltransferase and Acyltransferase Involved in the Phenylethanoid Glycoside Biosynthesis Induced by Methyl Jasmonate in Sesamum indicum L.

Sesame (Sesamum indicum L.) plants contain large amounts of acteoside, a typical phenylethanoid glycoside (PhG) that exhibits various pharmacological activities. Although there is increasing interest in the biosynthesis of PhGs for improved production, the pathway remains to be clarified. In this study, we established sesame-cultured cells and performed transcriptome analysis of methyl jasmonate (MeJA)-treated cultured cells to identify enzyme genes responsible for glucosylation and acylation in acteoside biosynthesis. Among the genes annotated as UDP-sugar-dependent glycosyltransferase (UGT) and acyltransferase (AT), 34 genes and one gene, respectively, were upregulated by MeJA in accordance with acteoside accumulation. Based on a phylogenetic analysis, five UGT genes (SiUGT1-5) and one AT gene (SiAT1) were selected as candidate genes involved in acteoside biosynthesis. Additionally, two AT genes (SiAT2-3) were selected based on sequence identity. Enzyme assays using recombinant SiUGT proteins revealed that SiUGT1, namely, UGT85AF10, had the highest glucosyltransferase activity among the five candidates against hydroxytyrosol to produce hydroxytyrosol 1-O-glucoside. SiUGT1 also exhibited glucosyltransferase activity against tyrosol to produce salidroside (tyrosol 1-O-glucoside). SiUGT2, namely, UGT85AF11, had similar activity against hydroxytyrosol and tyrosol. Enzyme assay using the recombinant SiATs indicated that SiAT1 and SiAT2 had activity transferring the caffeoyl group to hydroxytyrosol 1-O-glucoside and salidroside (tyrosol 1-O-glucoside) but not to decaffeoyl-acteoside. The caffeoyl group was attached mainly at the 4-position of glucose of hydroxytyrosol 1-O-glucoside, followed by attachment at the 6-position and the 3-position of glucose. Based on our results, we propose an acteoside biosynthetic pathway induced by MeJA treatment in sesame.

Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.

Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants.

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.

Diversified amino acid-mediated allosteric regulation of phosphoglycerate dehydrogenase for serine biosynthesis in land plants.

The phosphorylated pathway of serine biosynthesis is initiated with 3-phosphoglycerate dehydrogenase (PGDH). The liverwort Marchantia polymorpha possesses an amino acid-sensitive MpPGDH which is inhibited by l-serine and activated by five proteinogenic amino acids, while the eudicot Arabidopsis thaliana has amino acid-sensitive AtPGDH1 and AtPGDH3 as well as amino acid-insensitive AtPGDH2. In this study, we analyzed PGDH isozymes of the representative land plants: the monocot Oryza sativa (OsPGDH1-3), basal angiosperm Amborella trichopoda (AmtriPGDH1-2), and moss Physcomitrium (Physcomitrella) patens (PpPGDH1-4). We demonstrated that OsPGDH1, AmtriPGDH1, PpPGDH1, and PpPGDH3 were amino acid-sensitive, whereas OsPGDH2, OsPGDH3, AmtriPGDH2, PpPGDH2, and PpPGDH4 were either sensitive to only some of the six effector amino acids or insensitive to all effectors. This indicates that PGDH sensitivity to effectors has been diversified among isozymes and that the land plant species examined, except for M. polymorpha, possess different isozyme types in terms of regulation. Phylogenetic analysis suggested that the different sensitivities convergently evolved in the bryophyte and angiosperm lineages. Site-directed mutagenesis of AtPGDH1 revealed that Asp538 and Asn556 residues in the ACT domain are involved in allosteric regulation by the effectors. These findings provide insight into the evolution of PGDH isozymes, highlighting the functional diversification of allosteric regulation in land plants.

Differential regulation of fluorescent alkaloid metabolism between idioblast and lacticifer cells during leaf development in Catharanthus roseus seedlings.

Bioactive specialized (secondary) metabolites are indispensable for plant development or adjustment to their surrounding environment. In many plants, these specialized metabolites are accumulated in specifically differentiated cells. Catharanthus roseus is a well-known medicinal plant known for producing many kinds of monoterpenoid indole alkaloids (MIAs). C. roseus has two types of specifically differentiated cells accumulating MIAs, so-called idioblast cells and laticifer cells. In this study, we compared each of the cells as they changed during seedling growth, and found that the fluorescent metabolites accumulated in these cells were differentially regulated. Analysis of fluorescent compounds revealed that the fluorescence observed in these cells was emitted from the compound serpentine. Further, we found that the serpentine content of leaves increased as leaves grew. Our findings suggest that idioblast cells and laticifer cells have different biological roles in MIA biosynthesis and its regulation.

Biochemical insight into redox regulation of plastidial 3-phosphoglycerate dehydrogenase from Arabidopsis thaliana.

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants.

Overexpression of nicotinamidase 3 (NIC3) gene and the exogenous application of nicotinic acid (NA) enhance drought tolerance and increase biomass in Arabidopsis.

KEY MESSAGE: Overexpressing Nicotinamidase 3 gene, and the exogenous application of its metabolite nicotinic acid (NA), enhance drought stress tolerance and increase biomass in Arabidopsis thaliana. With progressive global climatic changes, plant productivity is threatened severely by drought stress. Deciphering the molecular mechanisms regarding genes responsible for balancing plant growth and stress amelioration could imply multiple possibilities for future sustainable goals. Nicotinamide adenine dinucleotide (NAD) biosynthesis and recycling/ distribution is a crucial feature for plant growth. The current study focuses on the functional characterization of nicotinamidase 3 (NIC3) gene, which is involved in the biochemical conversion of nicotinamide (NAM) to nicotinic acid (NA) in the salvage pathway of NAD biosynthesis. Our data show that overexpression of NIC3 gene enhances drought stress tolerance and increases plant growth. NIC3-OX plants accumulated more NA as compared to WT plants. Moreover, the upregulation of several genes related to plant growth/stress tolerance indicates that regulating the NAD salvage pathway could significantly enhance plant growth and drought stress tolerance. The exogenous application of nicotinic acid (NA) showed a similar phenotype as the effect of overexpressing NIC3 gene. In short, we contemplated the role of NIC3 gene and NA application in drought stress tolerance and plant growth. Our results would be helpful in engineering plants with enhanced drought stress tolerance and increased growth potential.

The cytochrome P450 CYP77A4 is involved in auxin-mediated patterning of the Arabidopsis thaliana embryo.

Metabolism often plays an important role in developmental control, in addition to supporting basal physiological requirements. However, our understanding of this interaction remains limited. Here, we performed quantitative phenome analysis of Arabidopsis thaliana cytochrome P450 mutants to identify a novel interaction between development and metabolism. We found that cyp77a4 mutants exhibit specific defects in cotyledon development, including asymmetric positioning and cup-shaped morphology, which could be rescued by introducing the CYP77A4 gene. Microscopy revealed that the abnormal patterning was detected at least from the 8-cell stage of the cyp77a4 embryos. We next analysed auxin distribution in mutant embryos, as the phenotypes resembled those of auxin-related mutants. We found that the auxin response pattern was severely perturbed in the cyp77a4 embryos owing to an aberrant distribution of the auxin efflux carrier PIN1. CYP77A4 intracellularly localised to the endoplasmic reticulum, which is consistent with the notion that this enzyme acts as an epoxidase of unsaturated fatty acids in the microsomal fraction. We propose that the CYP77A4-dependent metabolic pathway is an essential element for the establishment of polarity in plant embryos.

Regulation of Sugar and Storage Oil Metabolism by Phytochrome during De-etiolation.

Exposure of dark-grown (etiolated) seedlings to light induces the heterotrophic-to-photoautotrophic transition (de-etiolation) processes, including the formation of photosynthetic machinery in the chloroplast and cotyledon expansion. Phytochrome is a red (R)/far-red (FR) light photoreceptor that is involved in the various aspects of de-etiolation. However, how phytochrome regulates metabolic dynamics in response to light stimulus has remained largely unknown. In this study, to elucidate the involvement of phytochrome in the metabolic response during de-etiolation, we performed widely targeted metabolomics in Arabidopsis (Arabidopsis thaliana) wild-type and phytochrome A and B double mutant seedlings de-etiolated under R or FR light. The results revealed that phytochrome had strong impacts on the primary and secondary metabolism during the first 24 h of de-etiolation. Among those metabolites, sugar levels decreased during de-etiolation in a phytochrome-dependent manner. At the same time, phytochrome upregulated processes requiring sugars. Triacylglycerols are stored in the oil bodies as a source of sugars in Arabidopsis seedlings. Sugars are provided from triacylglycerols through fatty acid ß-oxidation and the glyoxylate cycle in glyoxysomes. We examined if and how phytochrome regulates sugar production from oil bodies. Irradiation of the etiolated seedlings with R and FR light dramatically accelerated oil body mobilization in a phytochrome-dependent manner. Glyoxylate cycle-deficient mutants not only failed to mobilize oil bodies but also failed to develop thylakoid membranes and expand cotyledon cells upon exposure to light. Hence, phytochrome plays a key role in the regulation of metabolism during de-etiolation.

Analysis of carbon flow at the metabolite level reveals that starch synthesis from hexose is a limiting factor in a high-yielding rice cultivar.

Understanding the limiting factors of grain filling is essential for the further improvement of grain yields in rice (Oryza sativa). The relatively slow grain growth of the high-yielding cultivar 'Momiroman' is not improved by increasing carbon supply, and hence low sink activity (i.e. the metabolic activity of assimilate consumption/storage in sink organs) may be a limiting factor for grain filling. However, there is no metabolic evidence to corroborate this hypothesis, partly because there is no consensus on how to define and quantify sink activity. In this study, we investigated the carbon flow at a metabolite level from photosynthesis in leaves to starch synthesis in grains of three high-yielding cultivars using the stable isotope 13C. We found that a large amount of newly fixed carbon assimilates in Momiroman was stored as hexose instead of being converted to starch. In addition, the activity of ADP-glucose pyrophosphorylase and the expression of AGPS2b, which encodes a subunit of the ADP-glucose pyrophosphorylase enzyme, were both lower in Momiroman than in the other two cultivars in grains in superior positions on panicle branches. Hence, slower starch synthesis from hexose, which is partly explained by the low expression level of AGPS2b, may be the primary metabolic reason for the lower sink activity observed in Momiroman.

Targeted permeabilization of the cell wall and extraction of charged molecules from single cells in intact plant clusters using a focused electric field.

The extraction of cellular contents from plant cells covered with cell walls remains a challenge, as it is physically hindered by the cell wall. We present a new microfluidic approach that leverages an intense pulsed electric field for permeabilizing the cell wall and a focused DC electric field for extracting the cellular contents selectively from a few targeted cells in a cluster of intact plant cells. We coupled the approach with on-chip fluorescence quantification of extracted molecules leveraging isotachophoresis as well as off-chip reverse transcription-quantitative polymerase chain reaction detecting extracted mRNA molecules. Our approach offers a workflow of about 5 min, isolating a cluster of intact plant cells, permeabilizing the cell wall, selectively extracting cytosolic molecules from a few targeted cells in the cluster, and outputting them to off-chip analyses without any enzymatic reactions. We anticipate that this approach will create a new opportunity to explore plant biology through less biased data realized by the rapid extraction of molecules from intact plant clusters.