Raising root zone temperature improves plant productivity and metabolites in hydroponic lettuce production.

While it is commonly understood that air temperature can greatly affect the process of photosynthesis and the growth of higher plants, the impact of root zone temperature (RZT) on plant growth, metabolism, essential elements, as well as key metabolites like chlorophyll and carotenoids, remains an area that necessitates extensive research. Therefore, this study aimed to investigate the impact of raising the RZT on the growth, metabolites, elements, and proteins of red leaf lettuce. Lettuce was hydroponically grown in a plant factory with artificial light at four different air temperatures (17, 22, 27, and 30°C) and two treatments with different RZTs. The RZT was raised 3°C above the air temperature in one group, while it was not in the other group. Increasing the RZT 3°C above the air temperature improved plant growth and metabolites, including carotenoids, ascorbic acids, and chlorophyll, in all four air temperature treatments. Moreover, raising the RZT increased Mg, K, Fe, Cu, Se, Rb, amino acids, and total soluble proteins in the leaf tissue at all four air temperatures. These results showed that raising the RZT by 3°C improved plant productivity and the metabolites of the hydroponic lettuce by enhancing nutrient uptake and activating the metabolism in the roots at all four air temperatures. Overall, this research demonstrates that plant growth and metabolites can be improved simultaneously with an increased RZT relative to air temperature. This study serves as a foundation for future research on optimizing RZT in relation to air temperature. Further recommended studies include investigating the differential effects of multiple RZT variations relative to air temperature for increased optimization, examining the effects of RZT during nighttime versus daytime, and exploring the impact of stem heating. This research has the potential to make a valuable contribution to the ongoing growth and progress of the plant factory industry and fundamental advancements in root zone physiology. Overall, this research demonstrates that plant growth and metabolites can be improved simultaneously with an increased RZT relative to air temperature. This study serves as a foundation for future research on optimizing RZT in relation to air temperature. Further recommended studies include investigating the differential effects of multiple RZT variations relative to air temperature for increased optimization, examining the effects of RZT during nighttime versus daytime, and exploring the impact of stem heating. This research has the potential to make a valuable contribution to the ongoing growth and progress of the plant factory industry and fundamental advancements in root zone physiology.

Application of ethanol alleviates heat damage to leaf growth and yield in tomato.

Chemical priming has emerged as a promising area in agricultural research. Our previous studies have demonstrated that pretreatment with a low concentration of ethanol enhances abiotic stress tolerance in Arabidopsis and cassava. Here, we show that ethanol treatment induces heat stress tolerance in tomato (Solanum lycopersicon L.) plants. Seedlings of the tomato cultivar 'Micro-Tom' were pretreated with ethanol solution and then subjected to heat stress. The survival rates of the ethanol-pretreated plants were significantly higher than those of the water-treated control plants. Similarly, the fruit numbers of the ethanol-pretreated plants were greater than those of the water-treated ones. Transcriptome analysis identified sets of genes that were differentially expressed in shoots and roots of seedlings and in mature green fruits of ethanol-pretreated plants compared with those in water-treated plants. Gene ontology analysis using these genes showed that stress-related gene ontology terms were found in the set of ethanol-induced genes. Metabolome analysis revealed that the contents of a wide range of metabolites differed between water- and ethanol-treated samples. They included sugars such as trehalose, sucrose, glucose, and fructose. From our results, we speculate that ethanol-induced heat stress tolerance in tomato is mainly the result of increased expression of stress-related genes encoding late embryogenesis abundant (LEA) proteins, reactive oxygen species (ROS) elimination enzymes, and activated gluconeogenesis. Our results will be useful for establishing ethanol-based chemical priming technology to reduce heat stress damage in crops, especially in Solanaceae.

Induced production of specialized steroids by transcriptional reprogramming in Petunia hybrida.

Plants produce specialized metabolites with defensive properties that are often synthesized through the coordinated regulation of metabolic genes by transcription factors in various biological contexts. In this study, we investigated the regulatory function of the transcription factor PhERF1 from petunia (Petunia hybrida), which belongs to a small group of ETHYLENE RESPONSE FACTOR (ERF) family members that regulate the biosynthesis of bioactive alkaloids and terpenoids in various plant lineages. We examined the effects of transiently overexpressing PhERF1 in petunia leaves on the transcriptome and metabolome, demonstrating the production of a class of specialized steroids, petuniolides, and petuniasterones in these leaves. We also observed the activation of many metabolic genes, including those involved in sterol biosynthesis, as well as clustered genes that encode new metabolic enzymes, such as cytochrome P450 oxidoreductases, 2-oxoglutarate-dependent dioxygenases, and BAHD acyltransferases. Furthermore, we determined that PhERF1 transcriptionally induces downstream metabolic genes by recognizing specific cis-regulatory elements in their promoters. This study highlights the potential of evolutionarily conserved transcriptional regulators to induce the production of specialized products through transcriptional reprogramming.

Controlling root zone temperature improves plant growth and pigments in hydroponic lettuce.

BACKGROUND AND AIMS: Air and root zone temperatures are important environmental factors affecting plant growth and yield. Numerous studies have demonstrated that air temperature strongly affects plant growth and development. Despite the extensive literature on air temperature, comprehensive studies on the effects of root zone temperature (RZT) on plant growth, elemental composition, and pigments are limited. In this study, we carefully observed the effects of RZT in red leaf lettuce to understand its effect on lettuce growth and pigment content. METHODS: Lettuce (Lactuca sativa, red leaf cultivar 'Red Fire') was grown hydroponically in a plant factory with artificial light under three RZT treatments (15, 25, or 35 °C) for 13 days. We investigated the comprehensive effects of RZT on the production of red leaf lettuce by metabolome and ionome analyses. KEY RESULTS: The 25 °C RZT treatment achieved maximum shoot and root dry weight. The 35 °C RZT decreased plant growth but significantly increased pigment contents (e.g. anthocyanins, carotenoids). In addition, a RZT heating treatment during plant cultivation that changed from 25 to 35 °C RZT for 8 days before harvest significantly increased shoot dry weight compared with the 35 °C RZT and significantly increased pigments compared with the 25 °C RZT. The 15 °C RZT resulted in significantly less pigment content relative to the 35 °C RZT. The 15 °C RZT also resulted in shoot and root dry weights greater than the 35 °C RZT but less than the 25 °C RZT. CONCLUSIONS: This study demonstrated that plant growth and pigments can be enhanced by adjusting RZT during different stages of plant growth to attain enhanced pigment contents while minimizing yield loss. This suggests that controlling RZT could be a viable method to improve lettuce quality via enhancement of pigment content quality while maintaining acceptable yields.

Multivariate Profiling of Metabolites and Volatile Organic Compounds in Citrus depressa Hayata Fruits from Kagoshima, Okinawa, and Taiwan.

Citrus depressa Hayata is a small-fruit citrus species; it is indigenous to Kagoshima, Okinawa, and Taiwan. The metabolites and volatile organic compounds (VOCs) that affect the flavor of its fruits have not been investigated based on geographical origin. In the present study, we investigated the metabolite and VOC profiles of 18 C. depressa cultivation lines from these regions. Multivariate analysis revealed differences in the metabolites of C. depressa based on its cultivation origins; variations in sugar, sugar alcohol, and amino acid contents were also observed. Fruits from Kagoshima and Okinawa had higher galactinol, trehalose, xylose, glucose, and sucrose intensities than fruits from Taiwan (log2-fold change; 2.65-3.44, 1.68-2.13, 1.37-2.01, 1.33-1.57, and 1.07-1.43, respectively), whereas the Taiwanese lines contained higher leucine, isoleucine, serine, and alanine. In contrast to the Taiwanese Nantou line, other cultivation lines had comparable total VOC contents, and the VOCs of all lines were dominated by limonene, γ-terpinene, and p-cymene. Accordingly, the highest VOC intensities were recorded in the Nantou line, which was followed by Shikunin sweet (Kagoshima) and Taoyuan (Taiwan) (log10 normalize concentration; 5.11, 3.08, and 3.01, respectively). Moreover, multivariate analysis plots elucidated the difference in the VOCs of Ishikunibu (Okinawa), Shikunin sweet, and Taoyuan and between those of most Kagoshima and Okinawa cultivation lines. These results suggest that both the cultivation line and origin influence the metabolites and VOCs of C. depressa, thus possibly affecting its flavor quality; the data provide a valuable insight for utilizing C. depressa of different cultivation lines and origins to produce foods and beverages.

Morphological and metabolomics profiling of intraspecific Arabidopsis hybrids in relation to biomass heterosis.

Heterosis contributes greatly to the worldwide agricultural yield. However, the molecular mechanism underlying heterosis remains unclear. This study took advantage of Arabidopsis intraspecific hybrids to identify heterosis-related metabolites. Forty-six intraspecific hybrids were used to examine parental effects on seed area and germination time. The degree of heterosis was evaluated based on biomass: combinations showing high heterosis of F1 hybrids exhibited a biomass increase from 6.1 to 44% over the better parent value (BPV), whereas that of the low- and no-heterosis hybrids ranged from - 19.8 to 9.8% over the BPV. Metabolomics analyses of F1 hybrids with high heterosis and those with low one suggested that changes in TCA cycle intermediates are key factors that control growth. Notably, higher fumarate/malate ratios were observed in the high heterosis F1 hybrids, suggesting they provide metabolic support associated with the increased biomass. These hybrids may produce more energy-intensive biomass by speeding up the efficiency of TCA fluxes. However, the expression levels of TCA-process-related genes in F1 hybrids were not associated with the intensity of heterosis, suggesting that the post-transcriptional or post-translational regulation of these genes may affect the productivity of the intermediates in the TCA cycle.

Exploring Volatile Organic Compounds in Rhizomes and Leaves of Kaempferia parviflora Wall. Ex Baker Using HS-SPME and GC-TOF/MS Combined with Multivariate Analysis.

Volatile organic compounds (VOCs) play an important role in the biological activities of the medicinal Zingiberaceae species. In commercial preparations of VOCs from Kaempferia parviflora rhizomes, its leaves are wasted as by-products. The foliage could be an alternative source to rhizome, but its VOCs composition has not been explored previously. In this study, the VOCs in the leaves and rhizomes of K. parviflora plants grown in a growth room and in the field were analyzed using the headspace solid-phase microextraction (HS-SPME) method coupled with gas chromatography and time-of-flight mass spectrometry (GC-TOF-MS). The results showed a total of 75 and 78 VOCs identified from the leaves and rhizomes, respectively, of plants grown in the growth room. In the field samples, 96 VOCs were detected from the leaves and 98 from the rhizomes. These numbers are higher compared to the previous reports, which can be attributed to the analytical techniques used. It was also observed that monoterpenes were dominant in leaves, whereas sesquiterpenes were more abundant in rhizomes. Principal component analysis (PCA) revealed significantly higher abundance and diversity of VOCs in plants grown in the field than in the growth room. A high level of similarity of identified VOCs between the two tissues was also observed, as they shared 68 and 94 VOCs in the growth room and field samples, respectively. The difference lies in the relative abundance of VOCs, as most of them are abundant in rhizomes. Overall, the current study showed that the leaves of K. parviflora, grown in any growth conditions, can be further utilized as an alternative source of VOCs for rhizomes.

Using the organelle glue technique to engineer the plant cell metabolome.

KEY MESSAGE: By using the organelle glue technique, we artificially manipulated organelle interactions and controlled the plant metabolome at the pathway level. Plant cell metabolic activity changes with fluctuating environmental conditions, in part via adjustments in the arrangement and interaction of organelles. This hints at the potential for designing plants with desirable metabolic activities for food and pharmaceutical industries by artificially controlling the interaction of organelles through genetic modification. We previously developed a method called the organelle glue technique, in which chloroplast-chloroplast adhesion is induced in plant cells using the multimerization properties of split fluorescent proteins. Here, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants in which chloroplasts adhere to each other and performed metabolome analysis to examine the metabolic changes in these lines. In plant cells expressing a construct encoding the red fluorescent protein mCherry targeted to the chloroplast outer envelope by fusion with a signal sequence (cTP-mCherry), chloroplasts adhered to each other and formed chloroplast aggregations. Mitochondria and peroxisomes were embedded in the aggregates, suggesting that normal interactions between chloroplasts and these organelles were also affected. Metabolome analysis of the cTP-mCherry-expressing Arabidopsis shoots revealed significantly higher levels of glycine, serine, and glycerate compared to control plants. Notably, these are photorespiratory metabolites that are normally transported between chloroplasts, mitochondria, and peroxisomes. Together, our data indicate that chloroplast-chloroplast adhesion alters organellar interactions with mitochondria and peroxisomes and disrupts photorespiratory metabolite transport. These results highlight the possibility of controlling plant metabolism at the pathway level by manipulating organelle interactions.

A developed system to extract specific responses of increment length in rice shoots under gradient changes in nitrogen concentration regimes.

Nitrogen (N) fertilization is one of the most crucial factors that contribute to increasing food production requiring the generation of rice cultivars with improved N use efficiency (NUE) to maintain yield during low N fertilizer application. To assay NUE extent, we developed a screening system to evaluate shoot growth of each rice cultivar under gradient changes in N concentrations. This system comprises a gradient hydroponic culture and growth visualization systems. The former allows gradient changes in ammonium concentrations, while the latter records the increment in shoot length of individual rice seedlings at given time periods using a fixed-point camera. We chose 69 cultivars including two controls (Oryza sativa L. cv. Nipponbare [WRC01] and Kasalath [WRC02]) from the World Rice Core Collection to investigate shoot growth responses under ammonium-sufficient, ammonium-limited, and low ammonium concentration gradients without transplanting stress. We observed three growth patterns in response to different ammonium concentrations. Subsequently, we selected three representative cultivars (Kasalath, WRC03, and WRC05) for the characteristic responses under the different ammonium environments. Distinct expression patterns of glutamine synthetase 1;2 (OsGS1;2) but OsGS1;1 were observed in response to varying ammonium concentration regimes, indicating that the expression patterns of OsGS1;2 may be a growth marker in terms of shoot growth when transitioning from ammonium-limited to low ammonium concentrations. This system with the level of OsGS1;2 allows us to screen for candidate cultivars that return high NUE in low N environments.

Increased ACS Enzyme Dosage Causes Initiation of Climacteric Ethylene Production in Tomato.

Fruits of wild tomato species show different ethylene-dependent ripening characteristics, such as variations in fruit color and whether they exhibit a climacteric or nonclimacteric ripening transition. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) are key enzymes in the ethylene biosynthetic pathway encoded by multigene families. Gene duplication is a primary driver of plant diversification and angiosperm evolution. Here, interspecific variations in the molecular regulation of ethylene biosynthesis and perception during fruit ripening in domesticated and wild tomatoes were investigated. Results showed that the activated ACS genes were increased in number in red-ripe tomato fruits than in green-ripe tomato fruits; therefore, elevated dosage of ACS enzyme promoted ripening ethylene production. Results showed that the expression of three ACS isogenes ACS1A, ACS2, and ACS4, which are involved in autocatalytic ethylene production, was higher in red-ripe tomato fruits than in green-ripe tomato fruits. Elevated ACS enzyme dosage promoted ethylene production, which corresponded to the climacteric response of red-ripe tomato fruits. The data suggest that autoinhibitory ethylene production is common to all tomato species, while autocatalytic ethylene production is specific to red-ripe species. The essential regulators Non-ripening (NOR) and Ripening-Inhibitor (RIN) have experienced gene activation and overlapped with increasing ACS enzyme dosage. These complex levels of transcript regulation link higher ethylene production with spatiotemporal modulation of gene expression in red-ripe tomato species. Taken together, this study shows that bursts in ethylene production that accompany fruit color changes in red-ripe tomatoes are likely to be an evolutionary adaptation for seed dispersal.

Effects of light quality, photoperiod, CO2 concentration, and air temperature on chlorogenic acid and rutin accumulation in young lettuce plants.

Environmental stimuli modulate plant metabolite accumulation, facilitating adaptation to stressful conditions. In this study, the effects of blue and red light, photoperiod, CO2 concentration, and air temperature on the chlorogenic acid (CGA) and rutin contents of lettuce (Lactuca sativa L.) were evaluated. Under continuous blue light and a high CO2 concentration (1000 ppm), the CGA level increased. The increased expression of phenylalanine ammonia-lyase (PAL) and activity of its product were correlated with high expression of cinnamate 4-hydroxylase (C4H) and coumarate 3-hydroxylase (C3H). Furthermore, changes in PAL activity altered the CGA content in lettuce exposed to the three environmental factors, blue light, continuous lighting and high CO2 concentration. In addition, the expression levels of genes related to flavonoid biosynthesis increased in accordance with the promotion of CGA accumulation by the environmental factors. Under continuous blue light, 400 ppm CO2 promoted rutin accumulation to a greater degree compared to 1000 ppm CO2, by downregulating DFR expression. Low air temperature induced CGA accumulation in lettuce grown under continuous blue light and 1000 ppm CO2. Therefore, light quality, photoperiod, CO2 concentration, and air temperature exert synergistic effects on the CGA and rutin contents of lettuce by modulating activity in the corresponding biosynthesis pathways.

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.

Differential metabolic rearrangements in the roots and leaves of Cicer arietinum caused by single or double nitrate and/or phosphate deficiencies.

Nitrate (NO3 - ) and phosphate (Pi) deficiencies are the major constraints for chickpea productivity, significantly impacting global food security. However, excessive fertilization is expensive and can also lead to environmental pollution. Therefore, there is an urgent need to develop chickpea cultivars that are able to grow on soils deficient in both NO3 - and Pi. This study focused on the identification of key NO3 - and/or Pi starvation-responsive metabolic pathways in the leaves and roots of chickpea grown under single and double nutrient deficiencies of NO3 - and Pi, in comparison with nutrient-sufficient conditions. A global metabolite analysis revealed organ-specific differences in the metabolic adaptation to nutrient deficiencies. Moreover, we found stronger adaptive responses in the roots and leaves to any single than combined nutrient-deficient stresses. For example, chickpea enhanced the allocation of carbon among nitrogen-rich amino acids (AAs) and increased the production of organic acids in roots under NO3 - deficiency, whereas this adaptive response was not found under double nutrient deficiency. Nitrogen remobilization through the transport of AAs from leaves to roots was greater under NO3 - deficiency than double nutrient deficiency conditions. Glucose-6-phosphate and fructose-6-phosphate accumulated in the roots under single nutrient deficiencies, but not under double nutrient deficiency, and higher glycolytic pathway activities were observed in both roots and leaves under single nutrient deficiency than double nutrient deficiency. Hence, the simultaneous deficiency generated a unique profile of metabolic changes that could not be simply described as the result of the combined deficiencies of the two nutrients.

Ethanol induces heat tolerance in plants by stimulating unfolded protein response.

KEY MESSAGE: Ethanol priming induces heat stress tolerance by the stimulation of unfolded protein response. Global warming increases the risk of heat stress-related yield losses in agricultural crops. Chemical priming, using safe agents, that can flexibly activate adaptive regulatory responses to adverse conditions, is a complementary approach to genetic improvement for stress adaptation. In the present study, we demonstrated that pretreatment of Arabidopsis with a low concentration of ethanol enhances heat tolerance without suppressing plant growth. We also demonstrated that ethanol pretreatment improved leaf growth in lettuce (Lactuca sativa L.) plants grown in the field conditions under high temperatures. Transcriptome analysis revealed a set of genes that were up-regulated in ethanol-pretreated plants, relative to water-pretreated controls. Binding Protein 3 (BIP3), an endoplasmic reticulum (ER)-stress marker chaperone gene, was among the identified up-regulated genes. The expression levels of BIP3 were confirmed by RT-qPCR. Root-uptake of ethanol was metabolized to organic acids, nucleic acids, amines and other molecules, followed by an increase in putrescine content, which substantially promoted unfolded protein response (UPR) signaling and high-temperature acclimation. We also showed that inhibition of polyamine production and UPR signaling negated the heat stress tolerance induced by ethanol pretreatment. These findings collectively indicate that ethanol priming activates UPR signaling via putrescine accumulation, leading to enhanced heat stress tolerance. The information gained from this study will be useful for establishing ethanol-mediated chemical priming strategies that can be used to help maintain crop production under heat stress conditions.

Transcriptomic, Hormonomic and Metabolomic Analyses Highlighted the Common Modules Related to Photosynthesis, Sugar Metabolism and Cell Division in Parthenocarpic Tomato Fruits during Early Fruit Set.

Parthenocarpy, the pollination-independent fruit set, can raise the productivity of the fruit set even under adverse factors during the reproductive phase. The application of plant hormones stimulates parthenocarpy, but artificial hormones incur extra financial and labour costs to farmers and can induce the formation of deformed fruit. This study examines the performance of parthenocarpic mutants having no transcription factors of SlIAA9 and SlTAP3 and sldella that do not have the protein-coding gene, SlDELLA, in tomato (cv. Micro-Tom). At 0 day after the flowering (DAF) stage and DAFs after pollination, the sliaa9 mutant demonstrated increased pistil development compared to the other two mutants and wild type (WT). In contrast to WT and the other mutants, the sliaa9 mutant with pollination efficiently stimulated the build-up of auxin and GAs after flowering. Alterations in both transcript and metabolite profiles existed for WT with and without pollination, while the three mutants without pollination demonstrated the comparable metabolomic status of pollinated WT. Network analysis showed key modules linked to photosynthesis, sugar metabolism and cell proliferation. Equivalent modules were noticed in the famous parthenocarpic cultivars 'Severianin', particularly for emasculated samples. Our discovery indicates that controlling the genes and metabolites proffers future breeding policies for tomatoes.

Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava.

KEY MESSAGE: Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava. Tuberous root formation is a complex process consisting of phase changes as well as cell division and elongation for radial growth. We performed an integrated analysis to clarify the relationships among metabolites, phytohormones, and gene transcription during tuberous root formation in cassava (Manihot esculenta Crantz). We also confirmed the effects of the auxin (AUX), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), gibberellin (GA), brassinosteroid (BR), salicylic acid, and indole-3-acetic acid conjugated with aspartic acid on tuberous root development. An integrated analysis of metabolites and gene expression indicated the expression levels of several genes encoding enzymes involved in starch biosynthesis and sucrose metabolism are up-regulated during tuberous root development, which is consistent with the accumulation of starch, sugar phosphates, and nucleotides. An integrated analysis of phytohormones and gene transcripts revealed a relationship among AUX signaling, CK signaling, and BR signaling, with AUX, CK, and BR inducing tuberous root development. In contrast, ABA and JA inhibited tuberous root development. These phenomena might represent the differences between stem tubers (e.g., potato) and root tubers (e.g., cassava). On the basis of these results, a phytohormonal regulatory model for tuberous root development was constructed. This model may be useful for future phytohormonal studies involving cassava.

Development of RIKEN Plant Metabolome MetaDatabase.

The advancement of metabolomics in terms of techniques for measuring small molecules has enabled the rapid detection and quantification of numerous cellular metabolites. Metabolomic data provide new opportunities to gain a deeper understanding of plant metabolism that can improve the health of both plants and humans that consume them. Although major public repositories for general metabolomic data have been established, the community still has shortcomings related to data sharing, especially in terms of data reanalysis, reusability and reproducibility. To address these issues, we developed the RIKEN Plant Metabolome MetaDatabase (RIKEN PMM, http://metabobank.riken.jp/pmm/db/plantMetabolomics), which stores mass spectrometry-based (e.g. gas chromatography-MS-based) metabolite profiling data of plants together with their detailed, structured experimental metadata, including sampling and experimental procedures. Our metadata are described as Linked Open Data based on the Resource Description Framework using standardized and controlled vocabularies, such as the Metabolomics Standards Initiative Ontology, which are to be integrated with various life and biomedical science data using the World Wide Web. RIKEN PMM implements intuitive and interactive operations for plant metabolome data, including raw data (netCDF format), mass spectra (NIST MSP format) and metabolite annotations. The feature is suitable not only for biologists who are interested in metabolomic phenotypes, but also for researchers who would like to investigate life science in general through plant metabolomic approaches.

Functional disruption of cell wall invertase inhibitor by genome editing increases sugar content of tomato fruit without decrease fruit weight.

Sugar content is one of the most important quality traits of tomato. Cell wall invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source leaves and fruits, while invertase inhibitor (INVINH) regulates this process. In this study, knock-out of cell wall INVINH in tomato (SlINVINH1) was performed by genome editing using, CRISPR/Cas9 and Target-AID technologies. Most of the genome-edited lines set higher soluble solid content (SSC) fruit than the original cultivar 'Suzukoma', while fruit weight was different among the genome-edited lines. From these genome-edited lines, three lines (193-3, 199-2, and 247-2), whose SSC was significantly higher than 'Suzukoma' and fruit weight were almost the same as the original cultivar, were selected. The fruit weight and overall plant growth of the two lines were comparable to those of the original cultivar. In contrast, the fructose and glucose contents in the mature fruits of the two lines were significantly higher than those of the original cultivar. The mature fruits of genome edited line 193-3 showed the highest sugar content, and the fructose and glucose contents were 29% and 36% higher than that of the original cultivar, respectively. Whole genome sequence data showed no off-target mutations in the genome-edited lines. Non-target metabolome analysis of mature fruits revealed that fructose was the highest loading factor in principal component analysis (PCA) between the genome-edited line and the original cultivar, and no unexpected metabolites appeared in the genome-edited line. In this study, we succeeded in producing tomato lines with high sugar content without a decrease in fruit weight and deterioration of plant growth by knock-out of SlINVINH1 using genome editing technology. This study showed that functional disruption of SlINVINH1 is an effective approach to produce tomato cultivars with high sugar content.

Defective cytokinin signaling reprograms lipid and flavonoid gene-to-metabolite networks to mitigate high salinity in Arabidopsis.

Cytokinin (CK) in plants regulates both developmental processes and adaptation to environmental stresses. Arabidopsis histidine phosphotransfer ahp2,3,5 and type-B Arabidopsis response regulator arr1,10,12 triple mutants are almost completely defective in CK signaling, and the ahp2,3,5 mutant was reported to be salt tolerant. Here, we demonstrate that the arr1,10,12 mutant is also more tolerant to salt stress than wild-type (WT) plants. A comprehensive metabolite profiling coupled with transcriptome analysis of the ahp2,3,5 and arr1,10,12 mutants was conducted to elucidate the salt tolerance mechanisms mediated by CK signaling. Numerous primary (e.g., sugars, amino acids, and lipids) and secondary (e.g., flavonoids and sterols) metabolites accumulated in these mutants under nonsaline and saline conditions, suggesting that both prestress and poststress accumulations of stress-related metabolites contribute to improved salt tolerance in CK-signaling mutants. Specifically, the levels of sugars (e.g., trehalose and galactinol), amino acids (e.g., branched-chain amino acids and γ-aminobutyric acid), anthocyanins, sterols, and unsaturated triacylglycerols were higher in the mutant plants than in WT plants. Notably, the reprograming of flavonoid and lipid pools was highly coordinated and concomitant with the changes in transcriptional levels, indicating that these metabolic pathways are transcriptionally regulated by CK signaling. The discovery of the regulatory role of CK signaling on membrane lipid reprogramming provides a greater understanding of CK-mediated salt tolerance in plants. This knowledge will contribute to the development of salt-tolerant crops with the ability to withstand salinity as a key driver to ensure global food security in the era of climate crisis.

High-Throughput Chlorophyll and Carotenoid Profiling Reveals Positive Associations with Sugar and Apocarotenoid Volatile Content in Fruits of Tomato Varieties in Modern and Wild Accessions.

Flavor and nutritional quality has been negatively impacted during the course of domestication and improvement of the cultivated tomato (Solanum lycopersicum). Recent emphasis on consumers has emphasized breeding strategies that focus on flavor-associated chemicals, including sugars, acids, and aroma compounds. Carotenoids indirectly affect flavor as precursors of aroma compounds, while chlorophylls contribute to sugar production through photosynthesis. However, the relationships between these pigments and flavor content are still unclear. In this study, we developed a simple and high-throughput method to quantify chlorophylls and carotenoids. This method was applied to over one hundred tomato varieties, including S. lycopersicum and its wild relatives (S. l. var. cerasiforme and S. pimpinellifolium), for quantification of these pigments in fruits. The results obtained by integrating data of the pigments, soluble solids, sugars, and aroma compounds indicate that (i) chlorophyll-abundant varieties have relatively higher sugar accumulations and (ii) prolycopene is associated with an abundance of linear carotenoid-derived aroma compounds in one of the orange-fruited varieties, "Dixie Golden Giant". Our results suggest the importance of these pigments not only as components of fruit color but also as factors influencing flavor traits, such as sugars and aroma.