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.

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.

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.

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.

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.

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.

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.

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.

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.

Down-regulation of PvTRX1h increases nodule number and affects auxin, starch, and metabolic fingerprints in the common bean (Phaseolus vulgaris L.).

The legume-rhizobium symbiotic relationship has been widely studied and characterized. However, little information is available about the role of histone lysine methyltransferases in the legume-rhizobium interaction and in the formation of nitrogen-fixing nodules in the common bean. Thus, this study aimed to gain a better understanding of the epigenetic control of nodulation in the common bean. Specifically, we studied the role of PvTRX1h, a histone lysine methyltransferase coding gene, in nodule development and auxin biosynthesis. Through a reverse genetics approach, we generated common bean composite plants to knock-down PvTRX1h expression. Here we found that the down-regulation of PvTRX1h increased the number of nodules per plant, but reduced the number of colony-forming units recovered from nodules. Genes coding for enzymes involved in the synthesis of the indole-3-acetic acid were up-regulated, as was the concentration of this hormone. In addition, PvTRX1h down-regulation altered starch accumulation as determined by the number of amyloplasts per nodule. Metabolic fingerprinting by direct liquid introduction-electrospray ionization-mass spectrometry (DLI-ESI-MS) revealed that the root nodules were globally affected by PvTRX1h down-regulation. Therefore, PvTRX1h likely acts through chromatin histone modifications that alter the auxin signaling network to determine bacterial colonization, nodule number, starch accumulation, hormone levels, and cell proliferation.

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.

Seasonal variation in non-structural carbohydrates, sucrolytic activity and secondary metabolites in deciduous and perennial Diospyros species sampled in Western Mexico.

This study was performed to test the working hypothesis that the primary determinants influencing seasonal driven modifications in carbon mobilization and other key biochemical parameters in leaves of poorly known Diospyros digyna (Ddg; semi-domesticated; perennial) and D. rekoi (Dre; undomesticated; deciduous) trees are determined by environmental growing conditions, agronomic management and physiological plasticity. Thus, biochemical changes in leaves of both trees were recorded seasonally during two successive fruiting years. Trees were randomly sampled in Western Mexico habitats with differing soil quality, climatic conditions, luminosity, and cultivation practices. Leaves of Ddg had consistently higher total chlorophyll contents (CT) that, unexpectedly, peaked in the winter of 2015. In Dre, the highest leaf CT values recorded in the summer of 2015 inversely correlated with low average luminosity and high Chl a/ Chlb ratios. The seasonal CT variations in Dre were congruent with varying luminosity, whereas those in Ddg were probably affected by other factors, such as fluctuating leaf protein contents and the funneling of light energy to foliar non-structural carbohydrates (NSCs) accumulation, which were consistently higher than those detected in Dre leaves. Seasonal foliar NSC fluctuations in both species were in agreement with the carbon (C) demands of flowering, fruiting and/ or leaf regrowth. Seasonal changes in foliar hexose to sucrose (Hex/ Suc) ratios coincided with cell wall invertase activity in both species. In Dre, high Hex/ Suc ratios in spring leaves possibly allowed an accumulation of phenolic acids, not observed in Ddg. The above results supported the hypothesis proposed by showing that leaf responses to changing environmental conditions differ in perennial and deciduous Diospyros trees, including a dynamic adjustment of NSCs to supply the C demands imposed by reproduction, leaf regrowth and, possibly, stress.

Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins.

Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa), average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt)]. Streptophyta have on average only ∼5.7 exons of medium size (∼230nt). Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400nt). Among subcellular compartments, membrane proteins are the largest (∼520aa), whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240aa). Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes.

Overexpression of Grain Amaranth (Amaranthus hypochondriacus) AhERF or AhDOF Transcription Factors in Arabidopsis thaliana Increases Water Deficit- and Salt-Stress Tolerance, Respectively, via Contrasting Stress-Amelioration Mechanisms.

Two grain amaranth transcription factor (TF) genes were overexpressed in Arabidopsis plants. The first, coding for a group VII ethylene response factor TF (i.e., AhERF-VII) conferred tolerance to water-deficit stress (WS) in transgenic Arabidopsis without affecting vegetative or reproductive growth. A significantly lower water-loss rate in detached leaves coupled to a reduced stomatal opening in leaves of plants subjected to WS was associated with this trait. WS tolerance was also associated with an increased antioxidant enzyme activity and the accumulation of putative stress-related secondary metabolites. However, microarray and GO data did not indicate an obvious correlation between WS tolerance, stomatal closure, and abscisic acid (ABA)-related signaling. This scenario suggested that stomatal closure during WS in these plants involved ABA-independent mechanisms, possibly involving reactive oxygen species (ROS). WS tolerance may have also involved other protective processes, such as those employed for methyl glyoxal detoxification. The second, coding for a class A and cluster I DNA binding with one finger TF (i.e., AhDof-AI) provided salt-stress (SS) tolerance with no evident fitness penalties. The lack of an obvious development-related phenotype contrasted with microarray and GO data showing an enrichment of categories and genes related to developmental processes, particularly flowering. SS tolerance also correlated with increased superoxide dismutase activity but not with augmented stomatal closure. Additionally, microarray and GO data indicated that, contrary to AhERF-VII, SS tolerance conferred by AhDof-AI in Arabidopsis involved ABA-dependent and ABA-independent stress amelioration mechanisms.

Non-severe thermochemical hydrolysis of stover from white corn and sequential enzymatic saccharification and fermentation to ethanol.

A parametric study, with an initial load of 15%w/w of dry stover from white corn, was conducted to evaluate the sequential thermochemical hydrolysis (TH), enzymatic saccharification (ES) and fermentation of the whole slurry with ethanologenic Escherichia coli. The TH was designed to release the maximum amount of xylose with a concomitant formation of minimal amounts of furans. It was found that 29.0% or 93.2% of the xylan was recovered as free xylose at 130°C after 8 min in the presence of 1% or 2%w/w H2SO4 and produced only 0.06 or 0.44 g/L of total furans, respectively. After 24h of ES, 76.14-77.18 g/L of monosaccharides (pentoses and hexoses) were obtained. These slurries, which contained 0.03-0.26 g/L of total furans and 5.14-5.91 g/L of acetate, were fermented with 3.7 g/L of ethanologenic E. coli to produce 24.5-23.5 g/L of ethanol.

The novel and taxonomically restricted Ah24 gene from grain amaranth (Amaranthus hypochondriacus) has a dual role in development and defense.

Grain amaranths tolerate stress and produce highly nutritious seeds. We have identified several (a)biotic stress-responsive genes of unknown function in Amaranthus hypochondriacus, including the so-called Ah24 gene. Ah24 was expressed in young or developing tissues; it was also strongly induced by mechanical damage, insect herbivory and methyl jasmonate and in meristems and newly emerging leaves of severely defoliated plants. Interestingly, an in silico analysis of its 1304 bp promoter region showed a predominance of regulatory boxes involved in development, but not in defense. The Ah24 cDNA encodes a predicted cytosolic protein of 164 amino acids, the localization of which was confirmed by confocal microscopy. Additional in silico analysis identified several other Ah24 homologs, present almost exclusively in plants belonging to the Caryophyllales. The possible function of this gene in planta was examined in transgenic Ah24 overexpressing Arabidopsis thaliana and Nicotiana tabacum plants. Transformed Arabidopsis showed enhanced vegetative growth and increased leaf number with no penalty in one fitness component, such as seed yield, in experimental conditions. Transgenic tobacco plants, which grew and reproduced normally, had increased insect herbivory resistance. Modified vegetative growth in transgenic Arabidopsis coincided with significant changes in the expression of genes controlling phytohormone synthesis or signaling, whereas increased resistance to insect herbivory in transgenic tobacco coincided with higher jasmonic acid and proteinase inhibitor activity levels, plus the accumulation of nicotine and several other putative defense-related metabolites. It is proposed that the primary role of the Ah24 gene in A. hypochondriacus is to contribute to a rapid recovery post-wounding or defoliation, although its participation in defense against insect herbivory is also plausible.

The tolerance of grain amaranth (Amaranthus cruentus L.) to defoliation during vegetative growth is compromised during flowering.

The biochemical processes underlying variations of tolerance are often accompanied by source-sink transitions affecting carbon (C) metabolism. We investigated the tolerance of Amaranthus cruentus L. to total mechanical defoliation through development and in different growing seasons. Defoliated A. cruentus recovered ∼80% of their above-ground biomass and ∼100% of grain yield compared to intact plants if defoliation occurred early during ontogeny, but could not compensate when defoliation occurred during flowering. Tolerance index was higher in the summer season (-0.3) than in the winter season (-0.7). Overall, defoliation tolerance was closely related to phosphoenolpyruvate carboxylase (PEPC) activity in leaves and the subsequent accumulation of starch (∼500 µmol/gDW) and sucrose (∼140 µmol/gDW) in stems and roots. Thus, A. cruentus accumulated sufficient C in roots and stem to allow branching and shoot re-growth after defoliation, but it only possessed sufficient C reserves to maintain <19% seed yield in the absence of new vegetative tissue. Seed size was larger during the warm season but it was not affected by foliar damage. Seed chemical composition was altered by defoliation at flowering. We conclude that A. cruentus defoliation tolerance depends on both, the re-allocation of starch from stem and roots, and the activation of dormant meristems before flowering to generate new photosynthetic capacity to sustain seed filling.