Backcrossing Modulates the Metabolic Profiles of Anthocyanin-Pigmented 'Vitamaize' Lines Derived from Elite Maize Lines.

Vitamaize lines (VMLs) were created by backcrossing the pigmented aleurone trait into Centro Internacional de Mejoramiento de Maíz y Trigo (CIMMYT) maize lines (CMLs). This study evaluates metabolic differences between the VMLs and their original CMLs. Direct infusion mass spectrometry (DIMS) analyses, carotenoid profiling, total anthocyanins content (TAC) determination, and biochemical evaluation of the quality protein maize (QPM) endosperm trait allowed a comprehensive chemical characterization of the maize lines. DIMS data indicate higher hexoses and trigonelline content for most VMLs; the carotenoid profile revealed a decrease in ß-cryptoxanthin to less than half of the original parent content for two VMLs but an augmentation for one VML. The pigmented aleurone VMLs did not inherit the complex QPM endosperm trait of the QPM CMLs. Except for anthocyanin accumulation, no other metabolites were consistently modified across all the backcross-generated maize lines with a pigmented aleurone trait. These findings suggest using genetic or metabolic markers rather than morphological or visual traits for future breeding programs.

Maize Flavonoid Biosynthesis, Regulation, and Human Health Relevance: A Review.

Maize is one of the most important crops for human and animal consumption and contains a chemical arsenal essential for survival: flavonoids. Moreover, flavonoids are well known for their beneficial effects on human health. In this review, we decided to organize the information about maize flavonoids into three sections. In the first section, we include updated information about the enzymatic pathway of maize flavonoids. We describe a total of twenty-one genes for the flavonoid pathway of maize. The first three genes participate in the general phenylpropanoid pathway. Four genes are common biosynthetic early genes for flavonoids, and fourteen are specific genes for the flavonoid subgroups, the anthocyanins, and flavone C-glycosides. The second section explains the tissue accumulation and regulation of flavonoids by environmental factors affecting the expression of the MYB-bHLH-WD40 (MBW) transcriptional complex. The study of transcription factors of the MBW complex is fundamental for understanding how the flavonoid profiles generate a palette of colors in the plant tissues. Finally, we also include an update of the biological activities of C3G, the major maize anthocyanin, including anticancer, antidiabetic, and antioxidant effects, among others. This review intends to disclose and integrate the existing knowledge regarding maize flavonoid pigmentation and its relevance in the human health sector.

ADP-Glucose Pyrophosphorylase Is Located in the Plastid and Cytosol in the Pulp of Tropical Banana Fruit (Musa acuminata).

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch synthesis in seeds, tubers and fruits. UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of sucrose metabolism in the cytosol while alkaline phosphatase (ALP) is a marker enzyme of the amyloplast that keeps the production of ADPG by removing PPi. Unripe banana accumulates starch in the pulp during development, while ripe fruits are characterized by the accumulation of soluble sugars. The aim of the study was to compare starch granule structure, carbohydrate levels, subcellular location and activities of three enzymes: AGPase, UGPase and ALP. Protein extracts from the cytosolic and amyloplastidial fractions were obtained from the pulp of banana fruit at three developmental stages (11, 16 and 21 weeks after flowering) and analyzed by electrophoresis and immunodetection. Protein profiles were similar during ripening, showing a main electrophoretic band at 50-55 kDa. Higher protein content was found in the cytosolic than in the amyloplastidial fraction. Starch granules and ALP activity were enriched in the amyloplast, whereas AGPase showed a subcellular distribution similar to UGPase. Immunoblot analysis also confirmed the presence of AGPase in both cytosol and amyloplast. AGPase activity was higher in the cytosol than in the amyloplast. Both AGPase activity and western blot band intensity were highest at 16 weeks. UGPase activity was highest at 21 weeks. We conclude that cytosolic production of ADP-glucose is not an exclusive feature of cereal endosperms due to plant breeding, but it also occurs in fruits of non-domesticated plants such as tropical banana (Musa acuminata). This work increases our understanding about pyrophosphorylase activities in the pulp of banana fruit.

Fruits of wild and semi-domesticated Diospyros tree species have contrasting phenological, metabolic, and antioxidant activity profiles.

BACKGROUND: In contrast to commercial Diospyros species, Mesoamerican fruit-producing species are scarcely known, particularly wild species that might harbor desirable traits suitable for breeding. Thus, metabolomic, chemical, and antioxidant profiles of fruits harvested from cultivated Diospyros digyna and wild Diospyros rekoi trees during consecutive winter seasons were obtained. Fruits were harvested in habitats having marked differences in soil quality, climate, and luminosity. RESULTS: D. digyna fruits were larger and less acid than D. rekoi fruits, whereas antioxidant activity tended to be higher in D. rekoi fruits. Phenolic, flavonoid, and sugar contents also varied significantly between species. Metabolomic analysis allowed the pre-identification of 519 and 1665 metabolites in negative and positive electrospray ionization (ESI) modes, respectively. Principal component analysis of the positive ESI data explained 51.8% of the variance and indicated clear metabolomic differences between D. rekoi and D. digyna fruits that were confirmed by direct-injection ESI mass spectrometry profiles. Twenty-one discriminating metabolites were detected in fruits of both species; D. digyna fruits differentially accumulated lysophospholipids, whereas discriminating metabolites in D. rekoi fruits were chemically more diverse than those in D. digyna fruits. CONCLUSION: Domesticated D. digyna fruits have improved physicochemical fruit traits compared with wild D. rekoi fruits, including larger size and lower acidity. The metabolomic and chemical composition of their respective fruits were also significantly different, which in D. rekoi was manifested as a notable season-dependent increase in antioxidant capacity. Therefore, wild D. rekoi can be considered as an important genetic resource for the improvement of commercial Diospyros fruit quality. © 2019 Society of Chemical Industry.

Mass Fingerprints of Tomatoes Fertilized with Different Nitrogen Sources Reveal Potential Biomarkers of Organic Farming.

Direct-injection electron spray ionization mass spectrometry (DIESI-MS) can be used to quantify the whole set of positive and negative ions in complex biological samples. A cherry tomato cultivar was grown inside a greenhouse in soil pots supplemented with different nitrogen sources. Organic cultivation increased fruit dry matter while conventional chemical fertilizers increased yield due to higher water content. While soluble sugars were unaltered, secondary metabolism of tomato fruit was highly sensitive to compost soil supplied to the roots. From a total of ~1647 DIESI-MS signals, 725 revealed significant differences between treatments. Heatmap biclustering showed that ionomic differences were robustly maintained in independent experiments carried out during three consecutive years. The ionomic fingerprints allowed reproducible sample classification, reflecting the effect of organic farming on tomato fruit quality. Specific biomarker ions could be identified for various nitrogen sources. We propose DIESI-MS as an up-front strategy for plant food characterization aiming to identify the ions with the most significant differences across genotypes or agronomic conditions.

Genome-wide analysis of the invertase gene family from maize.

KEY MESSAGE: The recent release of the maize genome (AGPv4) contains annotation errors of invertase genes and therefore the enzymes are bestly curated manually at the protein level in a comprehensible fashion The synthesis, transport and degradation of sucrose are determining factors for biomass allocation and yield of crop plants. Invertase (INV) is a key enzyme of carbon metabolism in both source and sink tissues. Current releases of the maize genome correctly annotates only two vacuolar invertases (ivr1 and ivr2) and four cell wall invertases (incw1, incw2 (mn1), incw3, and incw4). Our comprehensive survey identified 21 INV isogenes for which we propose a standard nomenclature grouped phylogenetically by amino acid similarity: three vacuolar (INVVR), eight cell wall (INVCW), and ten alkaline/neutral (INVAN) isogenes which form separate dendogram branches due to distinct molecular features. The acidic enzymes were curated for the presence of the DPN tripeptide which is coded by one of the smallest exons reported in plants. Particular attention was placed on the molecular role of INV in vascular tissues such as the nodes, internodes, leaf sheath, husk leaves and roots. We report the expression profile of most members of the maize INV family in nine tissues in two developmental stages, R1 and R3. INVCW7, INVVR2, INVAN8, INVAN9, INVAN10, and INVAN3 displayed the highest absolute expressions in most tissues. INVVR3, INVCW5, INVCW8, and INVAN1 showed low mRNA levels. Expressions of most INVs were repressed from stage R1 to R3, except for INVCW7 which increased significantly in all tissues after flowering. The mRNA levels of INVCW7 in the vegetative stem correlated with a higher transport rate of assimilates from leaves to the cob which led to starch accumulation and growth of the female reproductive organs.

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris).

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S-13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.

Regulation of AtSUS2 and AtSUS3 by glucose and the transcription factor LEC2 in different tissues and at different stages of Arabidopsis seed development.

Sucrose synthase (SUS) is a key enzyme of carbon metabolism in heterotrophic tissues of plants. The Arabidopsis genome contains six SUS genes. Two members of this family, namely AtSUS2 (At5g49190) and AtSUS3 (At4g02280) are strongly and differentially expressed in Arabidopsis seed. Expression analysis was carried out using SUS:promoter-GUS fusion lines in a wild-type genetic background or in a mutant carrying a lesion in the transcription factor LEAFY COTYLEDON 2 (LEC2; At1g28300). The accumulation patterns of mRNA, protein, and SUS activity were altered in the lec2 mutant during seed development 9-18 days after flowering. This indicates that LEC2 acts epistatically on the expression of AtSUS2 and AtSUS3. It appears that LEC2 is required for cotyledon-specific expression of both SUS genes but it is not responsible for expression in the radicle tip during embryo development. The AtSUS2 promoter was induced in planta by feeding of glucose but less so by sucrose and trehalose. Non-phosphorylable glucose analogs such as 3-O-methyl-glucose and 2-deoxyglucose also caused an induction, suggesting that sugar signaling proceeds by a hexokinase-independent pathway, possibly involving hexose sensing. Analysis of transgenic lines carrying of truncated versions of the AtSUS2:promoter fused to Beta-glucuronidase activity revealed an internal 421 bp region that was responsible for expression in seeds. Bioinformatic sequence analysis revealed regulatory cis-elements putatively responsible for the spatio-temporal pattern of AtSUS2 expression such as the SEF3 (aaccca) and W-box (ttgact) motifs. These findings are discussed in relation to the roles played by AtSUS2, AtSUS3 and LEC2 in the biosynthesis of seed storage products in Arabidopsis.

Metabolic and enzymatic changes associated with carbon mobilization, utilization and replenishment triggered in grain amaranth (Amaranthus cruentus) in response to partial defoliation by mechanical injury or insect herbivory.

BACKGROUND: Amaranthus cruentus and A. hypochondriacus are crop plants grown for grain production in subtropical countries. Recently, the generation of large-scale transcriptomic data opened the possibility to study representative genes of primary metabolism to gain a better understanding of the biochemical mechanisms underlying tolerance to defoliation in these species. A multi-level approach was followed involving gene expression analysis, enzyme activity and metabolite measurements. RESULTS: Defoliation by insect herbivory (HD) or mechanical damage (MD) led to a rapid and transient reduction of non-structural carbohydrates (NSC) in all tissues examined. This correlated with a short-term induction of foliar sucrolytic activity, differential gene expression of a vacuolar invertase and its inhibitor, and induction of a sucrose transporter gene. Leaf starch in defoliated plants correlated negatively with amylolytic activity and expression of a ß-amylase-1 gene and positively with a soluble starch synthase gene. Fatty-acid accumulation in roots coincided with a high expression of a phosphoenolpyruvate/phosphate transporter gene. In all tissues there was a long-term replenishment of most metabolite pools, which allowed damaged plants to maintain unaltered growth and grain yield. Promoter analysis of ADP-glucose pyrophosphorylase and vacuolar invertase genes indicated the presence of cis-regulatory elements that supported their responsiveness to defoliation. HD and MD had differential effects on transcripts, enzyme activities and metabolites. However, the correlation between transcript abundance and enzymatic activities was very limited. A better correlation was found between enzymes, metabolite levels and growth and reproductive parameters. CONCLUSIONS: It is concluded that a rapid reduction of NSC reserves in leaves, stems and roots followed by their long-term recovery underlies tolerance to defoliation in grain amaranth. This requires the coordinate action of genes/enzymes that are differentially affected by the way leaf damage is performed. Defoliation tolerance in grain is a complex process that can't be fully explained at the transcriptomic level only.

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method.

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.

Analysis of Global Gene Expression in Maize (Zea mays) Vegetative and Reproductive Tissues That Differ in Accumulation of Starch and Sucrose.

Carbon allocation between vegetative and reproductive tissues impacts cereal grain production. Despite great agricultural importance, sink-source relationships have not been fully characterized at the early reproductive stages in maize. Here, we quantify the accumulation of non-structural carbohydrates and patterns of gene expression in the top internode of the stem and the female inflorescence of maize at the onset of grain filling (reproductive stage R1). Top internode stem and female inflorescence tissues of the Puma maize inbred line were collected at reproductive stage R1 (without pollination) and non-structural carbohydrates were quantified by spectrophotometry. The female inflorescence accumulated starch at higher levels than the top internode of the stem. Global mRNA transcript levels were then evaluated in both tissues by RNA sequencing. Gene expression analysis identified 491 genes differentially expressed between the female inflorescence and the top stem internode. Gene ontology classification of differentially expressed genes showed enrichment for sucrose synthesis, the light-dependent reactions of photosynthesis, and transmembrane transporters. Our results suggest that sugar transporters play a key role in sugar partitioning in the maize stem and reveal previously uncharacterized differences between the female inflorescence and the top internode of the stem at early reproductive stages.

Genome-wide analysis of the beta-glucosidase gene family in maize (Zea mays L. var B73).

The hydrolysis of beta-D: -glucosidic bonds which is required for the liberation of many physiologically important compounds is catalyzed by the enzyme beta-glucosidase (BGLU, EC 3.2.1.21). BGLUs are implicated in several processes in plants, such as the timely response to biotic and abiotic stresses through activation of phytohormones and defense compounds. We identified 26 BGLU isozymes in the genome of the maize inbred B73 and propose a standardized nomenclature for all Zea mays BGLU paralogs (Zmbglu1-Zmbglu26). We characterized their intron-exon structure, protein features, phylogenetic relationships, and measured their expression and activity in various tissues under different environmental conditions. Sequence alignments revealed some characteristic motifs (conserved amino acids) and specific differences among different isozymes. Analysis of putative signal peptides suggested that some BGLUs are plastidic, whereas others are mitochondrial, cytosolic, vacuolar or secreted. Microarray and RT-PCR analysis showed that each member of the Zmbglu family had a characteristic expression pattern with regard to tissue specificity and response to different abiotic conditions. The source of variance for gene expression was highest for the type of organ analyzed (tissue variance) than for the growth conditions (environmental variance) or genotype (genetic variance). Analysis of promoter sequences revealed that each Zmbglu paralog possesses a distinct set of cis elements and transcription factor binding sites. Since there are no two Zmbglu paralogs that have identical molecular properties, we conclude that gene subfunctionalization in maize occurs much more rapidly than gene duplication.

Heterologous expression of yeast Hxt2 in Arabidopsis thaliana alters sugar uptake, carbon metabolism and gene expression leading to glucose tolerance of germinating seedlings.

The hexose transporter 2 gene (Hxt2) from Saccharomyces cerevisiae was expressed in Arabidopsis thaliana under control of the 35S promoter. Several independent transgenic lines were selected after confirming single gene insertion by southern blot analysis in the T4 generation. Northern blots revealed the presence of heterologous transcript. Radiolabeling experiments revealed an increased rate of incorporation of the non-metabolizable analog 3-O-methyl-[U-14C]-glucose. This confirmed that the yeast Hxt2 transporter was functional in Arabidopsis. No phenotypic changes at the vegetative and reproductive stages could be detected in the transgenic lines when compared to wild type plants. Shortly after germination some differences in development and glucose signaling were observed. Transgenic seedlings cultivated in liquid medium or on solid agar plates were able to grow with 3% glucose (producing bigger plants and longer roots), while development of wild type plants was delayed under those conditions. Metabolite analysis revealed that the Hxt2 transgenic lines had higher rates of sugar utilization. Transcriptional profiling showed that particular genes were significantly up- or down-regulated. Some transcription factors like At1g27000 were repressed, while others, such as At3g58780, were induced. The mRNA from classical sugar signaling genes such as STP1, Hxk1, and ApL3 behaved similarly in transgenic lines and wild type lines. Results suggest that the Hxt2 transgene altered some developmental processes related to the perception of high carbon availability after the germination stage. We conclude that the developmental arrest of wild type plants at 3% glucose not only depends on Hxk1 as the only sugar sensor but might also be influenced by the route of hexose transport across the plasma membrane.

Arabidopsis sucrose synthase 2 and 3 modulate metabolic homeostasis and direct carbon towards starch synthesis in developing seeds.

Two genes encoding sucrose synthase (SUS), namely SUS2 (At5g49190) and SUS3 (At4g02280), are strongly and differentially expressed in Arabidopsis seed. Detailed biochemical analysis was carried out in developing seeds 9-21 days after flowering (DAF) of wild type and two knockouts. SUS2 and SUS3 are not redundant genes since single knockouts show a phenotype in developing seeds. The mutants had 30-50% less SUS activity and therefore accumulated 40% more sucrose and 50% less fructose at 15 DAF. This did not affect the hexose-P pool, but led to 30-70% less starch in embryo and seed coat. Lipids were 55% higher in both mutants at 9-15 DAF. It seems that sucrolysis via SUS is not required for oil or protein synthesis but rather for channeling carbon toward ADP-glucose and starch in seeds. Metabolite profiling with GC-TOF revealed specific downstream changes in primary metabolism as a consequence of signaling or regulatory fine-tuning. While sucrose increased, hexoses and specific amino acids decreased reciprocally. There was a developmental shift regarding an earlier timing of dry weight accumulation, germinative maturity, oil deposition, sugar levels, transient starch buildup, and protein storage. Nevertheless, final seed size and composition were unaltered due to an earlier cessation of growth, thus giving rise to an apparent silent phenotype of mature mutant seeds. We conclude that SUS is important for metabolite homeostasis and timing of seed development, and propose that an altered sucrose/hexose ratio can modify carbon partitioning and the pattern of storage compounds in Arabidopsis.

Anthocyanin Profiling of Maize Grains Using DIESI-MSQD Reveals That Cyanidin-Based Derivatives Predominate in Purple Corn, whereas Pelargonidin-Based Molecules Occur in Red-Pink Varieties from Mexico.

Corn seeds contain natural pigments and antioxidants, such as the molecular variants of flavonoids and carotenoids. The aleurone and pericarp tissues from pigmented genotypes were extracted for metabolic fingerprinting and evaluated using UV-vis and mass spectrometry (MS). MS ionomic fingerprints classified samples according to genetic background and kernel color. The MS/MS fragmentation pattern (Daughter and Neutral Loss methods) allowed the tentative identification of 18 anthocyanins with glycosyl, malonyl, and succinyl moieties, including 535 m/z for cyanidin-3-O-(6″-malonyl-glucoside) and 621 m/z for cyanidin-3-O-(3″,6″-dimalonyl-glucoside). We also detected 663 m/z for pelargonidin-3-O-(disuccinyl-glucoside) and 633 m/z for peonidin-3-O-(disuccinyl-glucoside). Cyanidin-based anthocyanins were the most abundant in dark purple colored kernels, while pelargonidins predominated in the red-pink kernels of the "Elote occidental" landrace. Grains of "Conico negro" had a simultaneous pigmentation of aleurone and pericarp, while Vitamaize had purple pigmentation only in the aleurone layer. Most landraces had a white endosperm, while Vitamaize had a yellow endosperm and a dark seed coat. We conclude that Vitamaize grains contain both carotenes and anthocyanins, and therefore it is proposed as a nontransgenic agronomically improved variety of tropical purple maize, a good source for organic superfoods.

The Phosphoglycerate Kinase (PGK) Gene Family of Maize (Zea mays var. B73).

Phosphoglycerate kinase (PGK, E.C. 2.7.2.3) interconverts ADP + 1,3-bisphospho-glycerate (1,3-bPGA) to ATP + 3-phosphoglycerate (3PGA). While most bacteria have a single pgk gene and mammals possess two copies, plant genomes contain three or more PGK genes. In this study, we identified five Pgk genes in the Zea mays var. B73 genome, predicted to encode proteins targeted to different subcellular compartments: ZmPgk1, ZmPgk2, and ZmPgk4 (chloroplast), ZmPgk3 (cytosol), and ZmPgk5 (nucleus). The expression of ZmPgk3 was highest in non-photosynthetic tissues (roots and cobs), where PGK activity was also greatest, consistent with a function in glycolysis. Green tissues (leaf blade and husk leaf) showed intermediate levels of PGK activity, and predominantly expressed ZmPgk1 and ZmPgk2, suggesting involvement in photosynthetic metabolism. ZmPgk5 was weakly expressed and ZmPgk4 was not detected in any tissue. Phylogenetic analysis showed that the photosynthetic and glycolytic isozymes of plants clustered together, but were distinct from PGKs of animals, fungi, protozoa, and bacteria, indicating that photosynthetic and glycolytic isozymes of plants diversified after the divergence of the plant lineage from other groups. These results show the distinct role of each PGK in maize and provide the basis for future studies into the regulation and function of this key enzyme.

Exploring regulatory networks in plants: transcription factors of starch metabolism.

Biological networks are complex (non-linear), redundant (cyclic) and compartmentalized at the subcellular level. Rational manipulation of plant metabolism may have failed due to inherent difficulties of a comprehensive understanding of regulatory loops. We first need to identify key factors controlling the regulatory loops of primary metabolism. The paradigms of plant networks are revised in order to highlight the differences between metabolic and transcriptional networks. Comparison between animal and plant transcription factors (TFs) reveal some important differences. Plant transcriptional networks function at a lower hierarchy compared to animal regulatory networks. Plant genomes contain more TFs than animal genomes, but plant proteins are smaller and have less domains as animal proteins which are often multifunctional. We briefly summarize mutant analysis and co-expression results pinpointing some TFs regulating starch enzymes in plants. Detailed information is provided about biochemical reactions, TFs and cis regulatory motifs involved in sucrose-starch metabolism, in both source and sink tissues. Examples about coordinated responses to hormones and environmental cues in different tissues and species are listed. Further advancements require combined data from single-cell transcriptomic and metabolomic approaches. Cell fractionation and subcellular inspection may provide valuable insights. We propose that shuffling of promoter elements might be a promising strategy to improve in the near future starch content, crop yield or food quality.

Seasonal Changes in the Metabolic Profiles and Biological Activity in Leaves of Diospyros digyna and D. rekoi "Zapote" Trees.

Leaves of semi-domesticated Diospyros digyna and wild D. rekoi trees, sampled seasonally in Mexico in 2014, were analyzed. Metabolic fingerprints revealed higher metabolite diversity in D. rekoi leaves. The TLC bands characteristic of glycosylated flavonoids, predominant in this species, matched the detection of quercetin and quercetin 3-O-glucuronides by liquid chromatography (UPLC-MS) of spring leaf extracts (LEs). Further gas chromatography (GC-MS) analysis revealed abundant fatty acids, organic acids, and secondary metabolites including trigonelline, p-coumaric, and ferulic and nicotinic acids. Phenolic-like compounds prevailed in D. digyna LEs, while unidentified triterpenoids and dihydroxylated coumarins were detected by UPLC-MS and GC-MS. A paucity of leaf metabolites in leaves of this species, compared to D. rekoi, was evident. Higher antioxidant capacity (AOC) was detected in D. digyna LEs. The AOC was season-independent in D. digyna but not in D. rekoi. The AOC in both species was concentrated in distinct TLC single bands, although seasonal variation in band intensity was observed among trees sampled. The AOC in D. digyna LEs could be ascribed to the coumarin esculetin. The LEs moderately inhibited phytopathogenic bacteria but not fungi. Leaf chemistry differences in these Mesoamerican Diospyros species substantiated previous variability reported in tree physiology and fruit physical chemistry, postulated to result from domestication and seasonality.

Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase.

Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial adenylate kinase in growing tubers leads to increased rates of respiratory oxygen consumption and increased carbon fluxes into starch. Increased rates of starch synthesis were accompanied by post-translational redox-activation of ADP-glucose pyrophosphorylase (AGPase), catalysing the key regulatory step of starch synthesis in the plastid, while there were no substantial changes in metabolic intermediates or sugar levels. A similar increase in post-translational redox-activation of AGPase was found after supplying adenine to wild-type potato tuber discs to increase adenine nucleotide levels. Results provide first evidence for a link between redox-activation of AGPase and adenine nucleotide levels in plants.

The fluorescent blue glow of banana fruits is not due to symplasmic plastidial catabolism but arises from insoluble phenols estherified to the cell wall.

Banana fruits are firstly green due to chlorophyll, then yellow due to carotenoids and finally turn black due to polyphenols. However, bananas glow blue when observed under UV light. It has been reported that chlorophylls fade to give rise to fluorescent chlorophyll catabolites (FCCs) in senescent banana leaves and in ripening banana peels. FCCs are short lived catabolic intermediates that ultimately lead to non-fluorescent chlorophyll catabolites (NCCs). FCCs are abundant in bananas due to hypermodification; therefore, it was concluded that FCC caused yellow bananas to glow blue. Experiments were performed in order to shed new light into the autofluorescence phenomenon. Microscopy performed on living plant samples contradict the interpretation that the fluorescent blue glow is mainly caused by FCC inside the cell. Blue fluorescence in banana emerges from the cell wall, not from the symplasm. It is not primarily caused by soluble chlorophyll catabolites in the vacuoles or senescing plastids. Insoluble phenolics from the apoplast make bananas shine strongly blue under black light. Chlorophyll is a light trap that generates black holes of blue fluorescence, and therefore cells with chloroplasts glow less blue. The white pulp of banana fruits shine more strongly than the outer peel. In both tissues autofluorescence arises from insoluble phenols that are estherified to the cell wall. In monocot species (banana, maize, sugarcanne), blue fluorescense was strongest in the cell wall, whereas in dicots (e.g. arabidopsis, spearmint, hibiscus), blue fluorescence may be dominant from cytosolic, vacuolar or plastidial compartments.