Cucumber malate decarboxylase,CsNADP-ME2,functions in the balance of carbon and amino acid metabolism in fruit

Central metabolism produces carbohydrates and amino acids that are tightly correlated to plant growth and thereby crop productivity.Malate is reported to link mitochondrial respiratory metabolism with cytosolic biosynthetic pathways.Although the function of malate metabolism-related enzymes in providing carbon has been characterized in some plants,evidence for this role in the fleshy fruit of cucumber is lacking.Here,radiolabeled bicarbonate fed into the xylem stream from the cucumber roots was incorporated into amino acids,soluble sugars,and organic acids in the exocarp and vasculature of fruits.The activities of decarboxylases,especially decarboxylation from NADP-dependent malic enzyme(NADP-ME),were higher in cucumber fruit than in the leaf lamina.Histochemical localization revealed that CsNADP-ME2 was mainly located in the exocarp and vascular bundle system of fruit.Radiotracer and gas-exchange analysis indicated that overexpression of CsNADP-ME2 could promote carbon flux into soluble sugars and starch in fruits.Further studies combined with metabolic profiling revealed that the downregulation of CsNADP-ME2 in RNA interference(RNAi)lines caused the accumulation of its substrate,malate,in the exocarp.In addition to inhibition of glycolysis-related gene expression and reduction of the activities of the corresponding enzymes,increased amino acid synthesis and decreased sugar abundance were also observed in these lines.The opposite effect was found in CsNADP-ME2-overexpressing lines,suggesting that there may be a continuous bottom-up feedback regulation of glycolysis in cucumber fruits.Overall,our studies indicate that CsNADP-ME2 may play potential roles in both central carbon reactions and amino acid metabolism in cucumber fruits.

Modulating effects of acyl-CoA synthetase 5-derived mitochondrial Wnt2B palmitoylation on intestinal Wnt activity

AIM:To investigate the role of acyl-CoA synthetase 5(ACSL5)activity in Wnt signaling in intestinal surface epithelia.METHODS:Several cell lines were used to investigate the ACSL5-dependent expression and synthesis of Wnt2B,a mitochondrially expressed protein of the Wnt signaling family.Wnt activity was functionally assessed with a luciferase reporter assay.ACSL5-related biochemical Wnt2B modifications were investigatedwith a modified acyl-exchange assay.The findings from the cell culture models were verified using an Apcmin/+mouse model as well as normal and neoplastic diseased human intestinal tissues.RESULTS:In the presence of ACSL5,Wnt2B was unable to translocate into the nucleus and was enriched in mitochondria,which was paralleled by a significant decrease in Wnt activity.ACSL5-dependent S-palmitoylation of Wnt2B was identified as a molecular reason for mitochondrial Wnt2B accumulation.In cell culture systems,a strong relation of ACSL5 expression,Wnt2B palmitoylation,and degree of malignancy were found.Using normal mucosa,the association of ACSL5 and Wnt2B was seen,but in intestinal neoplasias the mechanism was only rudimentarily observed.CONCLUSION:ACSL5 mediates antiproliferative activities via Wnt2B palmitoylation with diminished Wnt activity.The molecular pathway is probably relevant for intestinal homeostasis,overwhelmed by other pathways in carcinogenesis.

Targeted approaches to improve tomato fruit taste

Tomato(Solanum lycopersicum)is the most valuable fruit and horticultural crop species worldwide.Compared with the fruits of their progenitors,those of modern tomato cultivars are,however,often described as having unsatisfactory taste or lacking f lavor.The f lavor of a tomato fruit arises from a complex mix of tastes and volatile metabolites,including sugars,acids,amino acids,and various volatiles.However,considerable differences in fruit f lavor occur among tomato varieties,resulting in mixed consumer experiences.While tomato breeding has traditionally been driven by the desire for continual increases in yield and the introduction of traits that provide a long shelf-life,consumers are prepared to pay a reasonable premium for taste.Therefore,it is necessary to characterize preferences of tomato f lavor and to define its underlying genetic basis.Here,we review recent conceptual and technological advances that have rendered this more feasible,including multi-omics-based QTL and association analyses,along with the use of trained testing panels,and machine learning approaches.This review proposes how the comprehensive datasets compiled to date could allow a precise rational design of tomato germplasm resources with improved organoleptic quality for the future.

Plant gene co-expression defines the biosynthetic pathway of neuroactive alkaloids

Transcriptomes comprise gene co-expression clustersor modules,showcasing distinct patterns of coordinated expression among groups of genes across independent biological samples(Serin et al.,2016).The"guilt-by-association"hypothesis suggests the functional importance of these clusters;for example,co-expressed genes associated with metabolism typically contain genes encoding several enzymes associated with the same pathways.

Revealing the specific regulations of nitric oxide on the postharvest ripening and senescence of bitter melon fruit

Bitter melon fruit is susceptible to yellowing,softening,and rotting under room-temperature storage conditions,resulting in reduced commercial value.Nitric oxide(NO)is an important signaling molecule and plays a crucial role in regulating the fruit postharvest quality.In this study,we investigated the effects of NO treatment on changes in sensory and firmness of bitter melon fruit during postharvest storage.Moreover,transcriptomic,metabolomic,and proteomic analyses were performed to elucidate the regulatory mechanisms through which No treatment delays the ripening and senescence of bitter melon fruit.Our results show that differentially expressed genes(DEGs)were involved in fruit texture(CSLE,β-Gal,and PME),plant hormone signal transduction(ACS,JAR4,and AUX28),and fruit flavor and aroma(SUS2,LOX,and GDH2).In addition,proteins differentially abundant were associated with fruit texture(PLY,PME,and PGA)and plant hormone signal transduction(PBL15,JAR1,and PYL9).Moreover,No significantly increased the abundance of key enzymes involved in the phenylpropanoid biosynthetic pathway,thus enhancing the disease resistance and alleviating softening of bitter melon fruit.Finally,differential metabolites mainly included phenolic acids,terpenoids,and flavonoids.These results provide a theoretical basis for further studies on the physiological changes associated with postharvest ripening and senescence of bitter melon fruit.

Non-aqueous fractionation revealed changing subcellular metabolite distribution during apple fruit development

In developing apple fruit,metabolic compartmentation is poorly understood due to the lack of experimental data.Distinguishing subcellular compartments in fruit using non-aqueous fractionation has been technically difficult due to the excess amount of sugars present in the different subcellular compartments limiting the resolution of the technique.The work described in this study represents the first attempt to apply non-aqueous fractionation to developing apple fruit,covering the major events occurring during fruit development(cell division,cell expansion,and maturation).Here we describe the non-aqueous fractionation method to study the subcellular compartmentation of metabolites during apple fruit development considering three main cellular compartments(cytosol,plastids,and vacuole).Evidence is presented that most of the sugars and organic acids were predominantly located in the vacuole,whereas some of the amino acids were distributed between the cytosol and the vacuole.The results showed a shift in the plastid marker from the lightest fractions in the early growth stage to the dense fractions in the later fruit growth stages.This implies that the accumulation of starch content with progressing fruit development substantially influenced the distribution of plastidial fragments within the non-aqueous density gradient applied.Results from this study provide substantial baseline information on assessing the subcellular compartmentation of metabolites in apple fruit in general and during fruit growth in particular.

Metabolomics-centered mining of plant metabolic diversity and function:Past decade and future perspectives

Plants are natural experts in organic synthesis,being able to generate large numbers of specific metabolites with widely varying structures that help them adapt to variable survival challenges.Metabolomics is a research discipline that integrates the capabilities of several types of research including analytical chemistry,statistics,and biochemistry.Its ongoing development provides strategies for gaining a systematic understanding of quantitative changes in the levels of metabolites.Metabolomics is usually performed by targeting either a specific cell,a specific tissue,or the entire organism.Considerable advances in science and technology over the last three decades have propelled us into the era of multi-omics,in which metabolomics,despite at an earlier developmental stage than genomics,transcriptomics,and proteomics,offers the distinct advantage of studying the cellular entities that have the greatest influence on end phenotype.Here,we summarize the state of the art of metabolite detection and identification,and illustrate these techniques with four case study applications:(i)comparing metabolite composition within and between species,(ii)assessing spatio-temporal metabolic changes during plant development,(iii)mining characteristic metabolites of plants in different ecological environments and upon exposure to various stresses,and(iv)assessing the performance of metabolomics as a means of functional gene identification,metabolic pathway elucidation,and metabolomics-assisted breeding through analyzing plant populations with diverse genetic variations.In addition,we highlight the prominent contributions of joint analyses of plant metabolomics and other omics datasets,including those from genomics,transcriptomics,proteomics,epigenomics,phenomics,microbiomes,and ion-omics studies.Finally,we discuss future directions and challenges exploiting metabolomics-centered approaches in understanding plant metabolic diversity.

Disease resistance conferred by components of essential chrysanthemum oil and the epigenetic regulation of OsTPS1

The sesquiterpene alpha-bisabolol is the predominant active ingredient in essential oils that are highly valued in the cosmetics industry due to its wound healing,anti-inflammatory,and skin-soothing properties.Alpha-bisabolol was thought to be restricted to Compositae plants.Here we reveal that alpha-bisabolol is also synthesized in rice,a non-Compositae plant,where it acts as a novel sesquiterpene phytoalexin.Overexpressing the gene responsible for the biosynthesis of alpha-bisabolol,Os TPS1,conferred bacterial blight resistance in rice.Phylogenomic analyses revealed that alpha-bisabolol-synthesizing enzymes in rice and Compositae evolved independently.Further experiments demonstrated that the natural variation in the disease resistance level was associated with differential transcription of Os TPS1 due to polymorphisms in its promoter.We demonstrated that Os TPS1 was regulated at the epigenetic level by JMJ705 through the methyl jasmonate pathway.These data reveal the cross-family accumulation and regulatory mechanisms of alpha-bisabolol production.

Integrating multiomics data accelerates elucidation of plant primary and secondary metabolic pathways

Plants are the most important sources of food for humans,as well as supplying many ingredients that are of great importance for human health.Developing an understanding of the functional components of plant metabolism has attracted considerable attention.The rapid development of liquid chromatography and gas chromatography,coupled with mass spectrometry,has allowed the detection and characterization of many thousands of metabolites of plant origin.Nowadays,elucidating the detailed biosynthesis and degradation pathways of these metabolites represents a major bottleneck in our understanding.Recently,the decreased cost of genome and transcriptome sequencing rendered it possible to identify the genes involving in metabolic pathways.Here,we review the recent research which integrates metabolomic with different omics methods,to comprehensively identify structural and regulatory genes of the primary and secondary metabolic pathways.Finally,we discuss other novel methods that can accelerate the process of identification of metabolic pathways and,ultimately,identify metabolite function(s).

Elucidation of the melitidin biosynthesis pathway in pummelo

Specialized plant metabolism is a rich resource of compounds for drug discovery.The acylated flavonoid glycoside melitidin is being developed as an anti-cholesterol statin drug candidate,but its biosynthetic route in plants has not yet been fully characterized.Here,we describe the gene discovery and functional characterization of a new flavonoid gene cluster(UDP-glucuronosyltransferases(Cg UGTs),1,2rhamnosyltransferase(Cg1,2Rha T),acyltransferases(Cg ATs))that is responsible for melitidin biosynthesis in pummelo(Citrus grandis(L.)Osbeck).Population variation analysis indicated that the tailoring of acyltransferases,specific for bitter substrates,mainly determine the natural abundance of melitidin.Moreover,3-hydroxy-3-methylglutaryl-Co A reductase enzyme inhibition assays showed that the product from this metabolic gene cluster,melitidin,may be an effective anti-cholesterol statin drug candidate.Co-expression of these clustered genes in Nicotiana benthamiana resulted in the formation of melitidin,demonstrating the potential for metabolic engineering of melitidin in a heterologous plant system.This study establishes a biosynthetic pathway for melitidin,which provides genetic resources for the breeding and genetic improvement of pummelo aimed at fortifying the content of biologically active metabolites.

Diel dynamics of multi-omics in elkhorn fern provide new insights into weak CAM photosynthesis

Crassulacean acid metabolism(CAM)has high water-use efficiency(WUE)and is widely recognized to have evolved from C3 photosynthesis.Different plant lineages have convergently evolved CAM,but the molecular mechanism that underlies C3-to-CAM evolution remains to be clarified.Platycerium bifurcatum(elkhorn fern)provides an opportunity to study the molecular changes underlying the transition from C3 to CAM photosynthesis because both modes of photosynthesis occur in this species,with sporotrophophyll leaves(SLs)and cover leaves(CLs)performing C3 and weak CAM photosynthesis,respectively.Here,we report that the physiological and biochemical attributes of CAM in weak CAM-performing CLs differed from those in strong CAM species.We investigated the diel dynamics of the metabolome,proteome,and transcriptome in these dimorphic leaves within the same genetic background and under identical environmental conditions.We found that multi-omic diel dynamics in P.bifurcatum exhibit both tissue and diel effects.Our analysis revealed temporal rewiring of biochemistry relevant to the energy-producing pathway(TCA cycle),CAM pathway,and stomatal movement in CLs compared with SLs.We also confirmed that PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE(PPCK)exhibits convergence in gene expression among highly divergent CAM lineages.Gene regulatory network analysis identified candidate transcription factors regulating the CAM pathway and stomatal movement.Taken together,our results provide new insights into weak CAM photosynthesis and new avenues for CAM bioengineering.

Natural variations of OsAUX5,a target gene of OsWRKY78,control the neutral essential amino acid content in rice grains

Grain essential amino acid(EAA)levels contribute to rice nutritional quality.However,the molecular mechanisms underlying EAA accumulation and natural variation in rice grains remain unclear.Here we report the identification of a previously unrecognized auxin influx carrier subfamily gene,OsAUX5,which encodes an amino acid transporter that functions in uptake of multiple amino acids.We identified an elite haplotype of Pro::OsAUX5^(Hap2) that enhances grain EAA accumulation without an apparent negative effect on agronomic traits.Natural variations of OsAUX5 occur in the cis elements of its promoter,which are differentially activated because of the different binding affinity between OsWRKY78 and the W-box,contributing to grain EAA variation among rice varieties.The two distinct haplotypes were shown to have originated from different Oryza rufipogon progenitors,which contributed to the divergence between japonica and indica.Introduction of the indica-type Pro::OsAUX5^(Hap2) genotype into japonica could significantly increase EAA levels,indicating that indica-type Pro::OsAUX5^(Hap2) can be utilized to increase grain EAAs of japonica varieties.Collectively,our study uncovers an WRKY78–OsAUX5-based regulatory mechanism controlling grain EAA accumulation and provides a potential target for breeding EAA-rich rice.

Promotion of liquid-to-solid phase transition of cGAS by Baicalein suppresses lung tumorigenesis

Dear Editor,Kras and p53 mutation are among the most common gene mutations in lung cancer,which has both the highest incidence and mortality rate among cancers.1 Kras/p53 mutation also causes mitochondrial dysfunction,which has been implicated to promote the inflammation-to-cancer transition.2 We established a lung adenocarcinoma model by using conditional alleles of KrasLSLG12D/p53flox/flox in mice3 to evaluate the effect of Baicalein(5,6,7-trihydroxyflavone),a principal component of Scutellaria baicalensis in traditional Chinese medicine,4 on the initiation and progression of lung cancer.Cre-mediated expression of KrasG12D and deletion of p53 caused obvious tumor lesions in the lung,which were strongly inhibited by the administration of Baicalein(Fig.1a,b and Supplementary Fig.1a,b),indicating that Baicalein is highly potent in inhibiting the progression of primary lung cancer.

Contemporary synthesis of bioactive cyclobutane natural products

Many cyclobutane natural products have intriguing biological properties that arise from their fascinating chemical structures.Cyclobutane natural products feature a cyclobutane scaffold as the core or as a part of the spirocyclic or fused cyclic core.However,the highly functionalized nature and the inherent stereochemistry of these cyclobutane natural products,which are associated with their biological activities,pose tremendous challenges to their preparation.In this perspective,we present contemporary advancements in synthetic methods and/or strategies en route to the bioactive cyclobutane natural products.We begin by describing the representative bioactive cyclobutane natural products and then focus on illustrative examples of their syntheses reported from 2018 to 2021.These advances will enable efficient syntheses of cyclobutanes of structural and biological importance.

The light and hypoxia induced gene ZmPORB1 determines tocopherol content in the maize kernel

Tocopherol is an important lipid-soluble antioxidant beneficial for both human health and plant growth. Here, we fine mapped a major QTLqVE1 affecting γ-tocopherol content in maize kernel, positionally cloned and confirmed the underlying gene ZmPORB1(por1), as a protochlorophyllide oxidoreductase. A 13.7 kb insertion reduced the tocopherol and chlorophyll content, and the photosynthetic activity by repressing ZmPORB1 expression in embryos of NIL-K22, but did not affect the levels of the tocopherol precursors HGA(homogentisic acid)and PMP(phytyl monophosphate). Furthermore, ZmPORB1 is inducible by low oxygen and light, thereby involved in the hypoxia response in developing embryos. Concurrent with natural hypoxia in embryos, the redox state has been changed with NO increasing and H_(2)O_(2) decreasing, which lowered γ-tocopherol content via scavenging reactive nitrogen species. In conclusion, we proposed that the lower lightharvesting chlorophyll content weakened embryo photosynthesis, leading to fewer oxygen supplies and consequently diverse hypoxic responses including an elevated γ-tocopherol consumption. Our findings shed light on the mechanism for fine-tuning endogenous oxygen concentration in the maize embryo through a novel feedback pathway involving the light and low oxygen regulation of ZmPORB1 expression and chlorophyll content.

The Structure and Function of Major Plant Metabolite Modifications

Plants produce a myriad of structurally and functionally diverse metabolites that play many different roles in plant growth and development and in plant response to continually changing environmental conditions as well as abiotic and biotic stresses. This metabolic diversity is, to a large extent, due to chemical modification of the basic skeletons of metabolites. Here, we review the major known plant metabolite modifications and summarize the progress that has been achieved and the challenges we are facing in the field. We focus on discussing both technical and functional aspects in studying the influences that various modifications have on biosynthesis, degradation, transport, and storage of metabolites, as well as their bioactivity and toxicity. Finally, we discuss some emerging insights into the evolution of metabolic pathways and metabolite functionality.

Arabidopsis Coordinates the Diurnal Regulation of Carbon Allocation and Growth across a Wide Range of Photoperiods

In short photoperiods, plants accumulate starch more rapidly in the light and degrade it more slowly at night, ensuring that their starch reserves last until dawn. To investigate the accompanying changes in the timing of growth, Arabidopsis was grown in a range of photoperiods and analyzed for rosette biomass, photosynthesis, respiration, ribosome abundance, polysome loading, starch, and over 40 metabolites at dawn and dusk. The data set was used to model growth rates in the daytime and night, and to identify metabolites that correlate with growth. Modeled growth rates and polysome loading were high in the daytime and at night in long photoperiods, but decreased at night in short photoperiods. Ribosome abundance was similar in all photoperiods. It is discussed how the amount of starch accumulated in the light period, the length of the night, and maintenance costs interact to constrain growth at night in short photoperiods, and alter the strategy for optimizing ribosome use. Significant correlations were found in the day- time and the night between growth rates and the levels of the sugar-signal trehalose 6-phosphate and the amino acid biosynthesis intermediate shikimate, identifying these metabolites as hubs in a network that coordinates growth with diurnal changes in the carbon supply.

Carbon Supply and the Regulation of Cell Wa Synthesis

All plant cells are surrounded by a cell wall that determines the directionality of cell growth and protects the cell against its environment. Plant cell walls are comprised primarily of polysaccharides and represent the largest sink for photosynthetically fixed carbon, both for individual plants and in the terrestrial biosphere as a whole. Cell wall synthesis is a highly sophisticated process, involving multiple enzymes and metabolic intermediates, intracellular trafficking of proteins and cell wall precursors, assembly of cell wall polymers into the extracellular matrix, remodeling of polymers and their interactions, and recycling of cell wall sugars. In this review we discuss how newly fixed carbon, in the form of UDP-glucose and other nucleotide sugars, contributes to the synthesis of cell wall polysaccharides, and how cell wall synthesis is influenced by the carbon status of the plant, with a focus on the model species Arabidopsis （Arabidopsis thaliana）.

Sarm1-mediated neurodegeneration within the enteric nervous system protects against local inflammation of the colon

Axonal degeneration is one of the key features of neu-rodegenerative disorders.In the canonical view,axonal degeneration destructs neural connections and promotes detrimental disease defects.Here,we assessed the enteric nervous system(ENS)of the mouse,nonhuman primate,and human by advanced 3D imaging.We observed the profound neurodegeneration of catecholaminergic axons in human colons with ulcerative colitis,and similarly,in mouse colons during acute dextran sulfate sodium-induced colitis.However,we unexpectedly revealed that blockage of such axonal degeneration by the Sarml deletion in mice exacerbated the colitis condition.In contrast,pharmacologic ablation or chemogenetic inhibition of catecholaminergic axons suppressed the colon inflammation.We further showed that the catecholaminergic neurotransmitter norepinephrine exerted a pro-inflammatory function by enhancing the expression of IL-17 cytokines.Together,this study demonstrated that Sarm1-mediated neurodegeneration within the ENS mitigated local inflammation of the colon,uncovering a previously-unrecognized beneficial role of axonal degeneration in this disease context.

Combined Transcriptomics and Metabolomics of Arabidopsis thaliana Seedlings Exposed to Exogenous GABA Suggest Its Role in Plants Is Predominantly Metabolic

Dear Editor, y-Aminobutyric acid （GABA） is a non-protein amino acid （AA） metabolized via the GABA shunt; a three-enzyme pathway that includes glutamate decarboxylase （GAD）, GABA transaminases （GABA-T）, and succinic semialdehyde dehydrogenase （SSADH） （Bown and Shelp, 1997; Bouch~ and Fromm, 2004）. The majority of work on GABA, to date, has focused on its role as an inhibitory neurotransmitter in mammals. In plants, however, due to its intermediate posi- tion between carbon （C） and nitrogen （N）, the metabolism of GABA has been suggested as a modulator of C-N balance （Fair et al., 2008）. In addition, the numerous observations of a rapid accumulation of GABA in response to （a）biotic stresses has prompted different hypotheses to be postu- lated on its role including a regulator of cytosolic pH and a herbivore deterrent （Bouche and Fromm, 2004; Roberts, 2007）. Additionally, a signaling role has been hypothesized in plants, though evidence remains elusive and the debate on the dual function of GABA as a signaling molecule and as a metabolite in plants remains ongoing. In the present work, we report changes in the metabolite and transcript profiles in Arabidopsis seedlings exposed to exogenous GABA. Liquid-grown seedlings were cultured under C and N limitation to reveal a possible role of GABA as either C or N substrate and to ascertain its influence on transcriptional programs.