Viability markers for determination of desiccation tolerance and critical stages during dehydration in Selaginella species.

While most plants die below a threshold of water content, desiccation-tolerant species display specific responses that allow them to survive extreme dehydration. Some of these responses are activated at critical stages during water loss and could represent the difference between desiccation tolerance (DT) and death. Here, we report the development of a simple and reproducible system to determine DT in Selaginella species. The system is based on exposure of excised tissue to a dehydration agent inside small containers, and subsequent evaluation for tissue viability. We evaluated several methodologies to determine viability upon desiccation including: triphenyltetrazolium chloride (TTC) staining, the quantum efficiency of PSII, antioxidant potential, and relative electrolyte leakage. Our results show that the TTC test is a simple and accurate assay to identify novel desiccation-tolerant Selaginella species, and can also indicate viability in other desiccation-tolerant models (i.e. ferns and mosses). The system we developed is particularly useful to identify critical points during the dehydration process. We found that a desiccation-sensitive Selaginella species shows a change in viability when dehydrated to 40% relative water content, indicating the onset of a critical condition at this water content. Comparative studies at critical stages could provide a better understanding of DT mechanisms and unravel insights into the key responses to survive desiccation.

Natural or light-induced pigment accumulation in grain amaranths coincides with enhanced resistance against insect herbivory.

MAIN CONCLUSION: Increased resistance to insect herbivory in grain amaranth plants is associated with increased betalain pigmentation, either naturally acquired or accumulated in response to blue-red light irradiation. Betalains are water-soluble pigments characteristic of plants of the Caryophyllales order. Their abiotic stress-induced accumulation is believed to protect against oxidative damage, while their defensive function against biotic aggressors is scarce. A previous observation of induced betalain-biosynthetic gene expression in stressed grain amaranth plants led to the proposal that these pigments play a defensive role against insect herbivory. This study provided further support for this premise. First, a comparison of "green" and "red" Amaranthus cruentus phenotypes showed that the latter suffered less insect herbivory damage. Coincidentally, growth and vitality of Manduca sexta larvae were more severely affected when fed on red-leafed A. cruentus plants or on an artificial diet supplemented with red-leaf pigment extracts. Second, the exposure of A. cruentus and A. caudatus plants, having contrasting pigmentation phenotypes, to light enriched in the blue and red wavelength spectra led to pigment accumulation throughout the plant and to increased resistance to insect herbivory. These events were accompanied by the induced expression of known betalain-biosynthetic genes, including uncharacterized DODA genes believed to participate in this biosynthetic pathway in a still undefined way. Finally, transient co-expression of different combinations of betalain-biosynthetic genes in Nicotiana benthamiana led to detectable accumulation of betalamic acid and betanidin. This outcome supported the participation of certain AhDODA and other genes in the grain amaranth betalain-biosynthetic pathway.

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.

AhDGR2, an amaranth abiotic stress-induced DUF642 protein gene, modifies cell wall structure and composition and causes salt and ABA hyper-sensibility in transgenic Arabidopsis.

MAIN CONCLUSION: An amaranth DGR gene, induced under abiotic stress, modifies cell wall structure and causes hypersensitivity to ABA and salt when overexpressed in Arabidopsis. DUF642 is a highly conserved plant-specific family of unknown cell wall-associated proteins. The AhDGR2 gene, coding for a DUF642 protein, was significantly induced in grain amaranth (Amaranthus hypochondriacus) plants subjected to water-deficit and salinity stress, thereby suggesting its participation in abiotic stress tolerance in this plant. A role in development was also inferred from the higher AhDGR2 expression rates detected in young tissues. Subsequent overexpression of AhDGR2 in transgenic Arabidopsis plants (OE-AhDGR2) supported its possible role in development processes. Thus, OE-AhDGR2 plants generated significantly longer roots when grown in normal MS medium. However, they showed a hypersensitivity to increasing concentrations of abscisic acid or NaCl in the medium, as manifested by shorter root length, smaller and slightly chlorotic rosettes, as well as highly reduced germination rates. Contrary to expectations, OE-AhDGR2 plants were intolerant to abiotic stress. Moreover, cell walls in transgenic plants were thinner, in leaves, and more disorganized, in roots, and had significantly modified pectin levels. Lower pectin methylesterase activity detected in leaves of OE-AhDGR2 plants, but not in roots, was contrary to previous reports associating DUF642 proteins and decreased pectin esterification levels in cell walls. Nonetheless, microarray data identified candidate genes whose expression levels explained the phenotypes observed in leaves of OE-AhDGR2 plants, including several involved in cell wall integrity and extension, growth and development, and resistance to abiotic stress. These results support the role of DUF642 proteins in cell wall-related processes and offer novel insights into their possible role(s) in plants.

Identification of genetic and biochemical mechanisms associated with heat shock and heat stress adaptation in grain amaranths.

Heat stress is poised to become a major factor negatively affecting plant performance worldwide. In terms of world food security, increased ambient temperatures are poised to reduce yields in cereals and other economically important crops. Grain amaranths are known to be productive under poor and/or unfavorable growing conditions that significantly affect cereals and other crops. Several physiological and biochemical attributes have been recognized to contribute to this favorable property, including a high water-use efficiency and the activation of a carbon starvation response. This study reports the behavior of the three grain amaranth species to two different stress conditions: short-term exposure to heat shock (HS) conditions using young plants kept in a conditioned growth chamber or long-term cultivation under severe heat stress in greenhouse conditions. The latter involved exposing grain amaranth plants to daylight temperatures that hovered around 50°C, or above, for at least 4 h during the day and to higher than normal nocturnal temperatures for a complete growth cycle in the summer of 2022 in central Mexico. All grain amaranth species showed a high tolerance to HS, demonstrated by a high percentage of recovery after their return to optimal growing conditions. The tolerance observed coincided with increased expression levels of unknown function genes previously shown to be induced by other (a)biotic stress conditions. Included among them were genes coding for RNA-binding and RNA-editing proteins, respectively. HS tolerance was also in accordance with favorable changes in several biochemical parameters usually induced in plants in response to abiotic stresses. Conversely, exposure to a prolonged severe heat stress seriously affected the vegetative and reproductive development of all three grain amaranth species, which yielded little or no seed. The latter data suggested that the usually stress-tolerant grain amaranths are unable to overcome severe heat stress-related damage leading to reproductive failure.

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.

Cross-kingdom effects of plant-plant signaling via volatile organic compounds emitted by tomato (Solanum lycopersicum) plants infested by the greenhouse whitefly (Trialeurodes vaporariorum).

Volatile organic compounds (VOCs) emitted from plants in response to insect infestation can function as signals for the attraction of predatory/parasitic insects and/or repulsion of herbivores. VOCs also may play a role in intra- and inter-plant communication. In this work, the kinetics and composition of VOC emissions produced by tomato (Solanum lycopersicum) plants infested with the greenhouse whitefly Trialeurodes vaporariorum was determined within a 14 days period. The VOC emission profiles varied concomitantly with the duration of whitefly infestation. A total of 36 different VOCs were detected during the experiment, 26 of which could be identified: 23 terpenoids, plus decanal, decane, and methyl salicylate (MeSA). Many VOCs were emitted exclusively by infested plants, including MeSA and 10 terpenoids. In general, individual VOC emissions increased as the infestation progressed, particularly at 7 days post-infestation (dpi). Additional tunnel experiments showed that a 3 days exposure to VOC emissions from whitefly-infested plants significantly reduced infection by a biotrophic bacterial pathogen. Infection of VOC-exposed plants induced the expression of a likely tomato homolog of a methyl salicylate esterase gene, which preceded the expression of pathogenesis-related protein genes. This expression pattern correlated with reduced susceptibility in VOC-exposed plants. The observed cross-kingdom effect of plant-plant signaling via VOCs probably represents a generalized defensive response that contributes to increased plant fitness, considering that resistance responses to whiteflies and biotrophic bacterial pathogens in tomato share many common elements.

Transcriptomic analysis of grain amaranth (Amaranthus hypochondriacus) using 454 pyrosequencing: comparison with A. tuberculatus, expression profiling in stems and in response to biotic and abiotic stress.

BACKGROUND: Amaranthus hypochondriacus, a grain amaranth, is a C4 plant noted by its ability to tolerate stressful conditions and produce highly nutritious seeds. These possess an optimal amino acid balance and constitute a rich source of health-promoting peptides. Although several recent studies, mostly involving subtractive hybridization strategies, have contributed to increase the relatively low number of grain amaranth expressed sequence tags (ESTs), transcriptomic information of this species remains limited, particularly regarding tissue-specific and biotic stress-related genes. Thus, a large scale transcriptome analysis was performed to generate stem- and (a)biotic stress-responsive gene expression profiles in grain amaranth. RESULTS: A total of 2,700,168 raw reads were obtained from six 454 pyrosequencing runs, which were assembled into 21,207 high quality sequences (20,408 isotigs + 799 contigs). The average sequence length was 1,064 bp and 930 bp for isotigs and contigs, respectively. Only 5,113 singletons were recovered after quality control. Contigs/isotigs were further incorporated into 15,667 isogroups. All unique sequences were queried against the nr, TAIR, UniRef100, UniRef50 and Amaranthaceae EST databases for annotation. Functional GO annotation was performed with all contigs/isotigs that produced significant hits with the TAIR database. Only 8,260 sequences were found to be homologous when the transcriptomes of A. tuberculatus and A. hypochondriacus were compared, most of which were associated with basic house-keeping processes. Digital expression analysis identified 1,971 differentially expressed genes in response to at least one of four stress treatments tested. These included several multiple-stress-inducible genes that could represent potential candidates for use in the engineering of stress-resistant plants. The transcriptomic data generated from pigmented stems shared similarity with findings reported in developing stems of Arabidopsis and black cottonwood (Populus trichocarpa). CONCLUSIONS: This study represents the first large-scale transcriptomic analysis of A. hypochondriacus, considered to be a highly nutritious and stress-tolerant crop. Numerous genes were found to be induced in response to (a)biotic stress, many of which could further the understanding of the mechanisms that contribute to multiple stress-resistance in plants, a trait that has potential biotechnological applications in agriculture.

Transformed tobacco (Nicotiana tabacum) plants over-expressing a peroxisome proliferator-activated receptor gene from Xenopus laevis (xPPARα) show increased susceptibility to infection by virulent Pseudomonas syringae pathogens.

Transgenic tobacco plants capable of over-expressing Xenopus PPARα (xPPARα), a transcription factor known to be required for peroxisome proliferation in animals, were recently generated. These plants (herewith referred to as PPAR-OE) were found to have increased peroxisome abundance, higher peroxisomal acyl-CoA oxidase and catalase activity and modified fatty acid metabolism. Further characterization of PPAR-OE plants revealed a higher susceptibility to virulent and a partial loss of resistance to avirulent Pseudomonas syringae pathogens, whereas the basal resistance response remained unaffected. Biochemical- and defense-related gene expression analyses showed that increased susceptibility to bacterial invasion coincided with the generalized reduction in H(2)O(2) and salicylic acid (SA) levels observed within the first 24 h of bacterial contact. Decreased H(2)O(2) levels were correlated with modified activity levels of catalase and other antioxidant enzymes. A correspondence between a rapid (within 1-24 hpi; ACCO and AOC) and sustained increase (up to 6 days pi; ACCO) in the expression levels of ethylene (ACCO) and jasmonic acid (AOC) biosynthetic genes and a higher susceptibility to virulent bacterial invasion was also observed in PPAR-OE plants. Conversely, no apparent differences in the short- and/or long-term expression levels of markers for the hypersensitive-response, oxidative burst and systemic-acquired resistance were observed between wild type and PPAR-OE plants. The results suggest that peroxisome proliferation could lead to increased susceptibility to bacterial pathogens in tobacco by altering the redox balance of the plant and the expression pattern of key defense signaling pathway genes.

Highest Defoliation Tolerance in Amaranthus cruentus Plants at Panicle Development Is Associated With Sugar Starvation Responses.

Defoliation tolerance (DT) in Amaranthus cruentus is known to reach its apex at the panicle emergence (PE) phase and to decline to minimal levels at flowering (FL). In this study, defoliation-induced changes were recorded in the content of non-structural carbohydrates and raffinose family oligosaccharides (RFOs), and in the expression and/or activity of sugar starvation response-associated genes in plants defoliated at different vegetative and reproductive stages. This strategy identified sugar-starvation-related factors that explained the opposite DT observed at these key developmental stages. Peak DT at PE was associated with increased cytosolic invertase (CI) activity in all organs and with the extensive induction of various class II trehalose-phosphate synthase (TPS) genes. Contrariwise, least DT at FL coincided with a sharp depletion of starch reserves and with sucrose (Suc) accumulation, in leaves and stems, the latter of which was consistent with very low levels of CI and vacuolar invertase activities that were not further modified by defoliation. Increased Suc suggested growth-inhibiting conditions associated with altered cytosolic Suc-to-hexose ratios in plants defoliated at FL. Augmented cell wall invertase activity in leaves and roots, probably acting in a regulatory rather than hydrolytic role, was also associated with minimal DT observed at FL. The widespread contrast in gene expression patterns in panicles also matched the opposite DT observed at PE and FL. These results reinforce the concept that a localized sugar starvation response caused by C partitioning is crucial for DT in grain amaranth.

Distinct gene expression and secondary metabolite profiles in suppressor of prosystemin-mediated responses2 (spr2) tomato mutants having impaired mycorrhizal colonization.

Arbuscular mycorrhizal fungi (AMF) colonization, sampled at 32-50 days post-inoculation (dpi), was significantly reduced in suppressor of prosystemin-mediated responses2 (spr2) mutant tomato plants impaired in the ω-3 FATTY ACID DESATURASE7 (FAD7) gene that limits the generation of linolenic acid and, consequently, the wound-responsive jasmonic acid (JA) burst. Contrary to wild-type (WT) plants, JA levels in root and leaves of spr2 mutants remained unchanged in response to AMF colonization, further supporting its regulatory role in the AM symbiosis. Decreased AMF colonization in spr2 plants was also linked to alterations associated with a disrupted FAD7 function, such as enhanced salicylic acid (SA) levels and SA-related defense gene expression and a reduction in fatty acid content in both mycorrhizal spr2 roots and leaves. Transcriptomic data revealed that lower mycorrhizal colonization efficiency in spr2 mutants coincided with the modified expression of key genes controlling gibberellin and ethylene signaling, brassinosteroid, ethylene, apocarotenoid and phenylpropanoid synthesis, and the wound response. Targeted metabolomic analysis, performed at 45 dpi, revealed augmented contents of L-threonic acid and DL-malic acid in colonized spr2 roots which suggested unfavorable conditions for AMF colonization. Additionally, time- and genotype-dependent changes in root steroid glycoalkaloid levels, including tomatine, suggested that these metabolites might positively regulate the AM symbiosis in tomato. Untargeted metabolomic analysis demonstrated that the tomato root metabolomes were distinctly affected by genotype, mycorrhizal colonization and colonization time. In conclusion, reduced AMF colonization efficiency in spr2 mutants is probably caused by multiple and interconnected JA-dependent and independent gene expression and metabolomic alterations.

Identification of Factors Linked to Higher Water-Deficit Stress Tolerance in Amaranthus hypochondriacus Compared to Other Grain Amaranths and A. hybridus, Their Shared Ancestor.

Water deficit stress (WDS)-tolerance in grain amaranths (Amaranthus hypochondriacus, A. cruentus and A. caudatus), and A. hybridus, their presumed shared ancestor, was examined. A. hypochondriacus was the most WDS-tolerant species, a trait that correlated with an enhanced osmotic adjustment (OA), a stronger expression of abscisic acid (ABA) marker genes and a more robust sugar starvation response (SSR). Superior OA was supported by higher basal hexose (Hex) levels and high Hex/sucrose (Suc) ratios in A. hypochondriacus roots, which were further increased during WDS. This coincided with increased invertase, amylase and sucrose synthase activities and a strong depletion of the starch reserves in leaves and roots. The OA was complemented by the higher accumulation of proline, raffinose, and other probable raffinose-family oligosaccharides of unknown structure in leaves and/or roots. The latter coincided with a stronger expression of Galactinol synthase 1 and Raffinose synthase in leaves. Increased SnRK1 activity and expression levels of the class II AhTPS9 and AhTPS11 trehalose phosphate synthase genes, recognized as part of the SSR network in Arabidopsis, were induced in roots of stressed A. hypochondriacus. It is concluded that these physiological modifications improved WDS in A. hypochondriacus by raising its water use efficiency.

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.

Modulation of steroidal glycoalkaloid biosynthesis in tomato (Solanum lycopersicum) by jasmonic acid.

Jasmonic acid (JA) is a phytohormone involved in plant development and defense. A major role of JA is the enhancement of secondary metabolite production, such as response to herbivory. Systemin is a bioactive plant peptide of 18 amino acids that contributes to the induction of local and systemic defense responses in tomato (Solanum lycopersicum) through JA biosynthesis. The overexpression of systemin (PS-OE) results in constitutive JA accumulation and enhances pest resistance in plants. Conversely, mutant plants affected in linolenic acid synthesis (spr2) are negatively compromised in the production of JA which favors damage and oviposition by insect herbivores. With undirected mass fingerprinting analyses, we found global metabolic differences between genotypes with modified jasmonic acid production. The spr2 mutants were enriched in di-unsaturated fatty acids and generally showed more changes. The PS-OE genotype produced an unidentified compound with a mass-to-charge ratio of 695 (MZ695). Most strikingly, the steroidal glycoalkaloid biosynthesis was negatively affected in the spr2 genotype. Complementation with jasmonic acid could restore the tomatine pathway, which strongly suggests the control of steroidal glycoalkaloid biosynthesis by jasmonic acid. spr2 plants were more susceptible to fungal infection with Fusarium oxysporum f.sp. ciceris, but not to bacterial infection with Clavibacter michiganensis subsp. michiganensis which supports the involvement of steroidal glycoalkaloids in the plant response against fungi.

Cloning of a cDNA encoding a cystatin from grain amaranth (Amaranthus hypochondriacus) showing a tissue-specific expression that is modified by germination and abiotic stress.

A cDNA, encoding a cysteine protease inhibitor (AhCPI), was isolated from an immature seed cDNA library of grain amaranth (Amaranthus hypochondriacus L.) and characterized. It encoded a polypeptide of 247 amino acids (aa), including a putative N-terminal signal peptide. Other relevant regions found in its sequence included the G and PW conserved aa motifs, the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The predicted aa sequence for AhCPI showed a significant homology to other plant cystatins. Gene expression analyses indicated that AhCPI was constitutively expressed in mature seeds, and gradually decreased during germination. In vegetative tissues, AhCPI was expressed in the radicle and hypocotyls of seedlings and in the stems and roots of young plantlets. Its expression in roots and stems increased substantially in response to water deficit, salinity-, cold- and heat-stress, whereas heat-stress induced a rapid and transient accumulation of AhCPI transcripts in leaves. The results obtained were suggestive of multiple roles for AhCPI in grain amaranth, acting as a regulator of seed germination and as a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner. The work herewith described reports a novel, and apparently, single cystatin protein in which, in agreement with other plant model systems, could have a regulatory role in germination, and further expands previous findings linking the accumulation of protease inhibitors, mostly of the serine proteinase type, with protection against (a)biotic stress in A. hypochondriacus.

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.

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.

The overexpression of an Amaranthus hypochondriacus NF-YC gene modifies growth and confers water deficit stress resistance in Arabidopsis.

Nuclear factor-Y (NF-Y), is a plant heterotrimeric transcription factor constituted by NF-YA, NF-YB and NF-YC subunits. The function of many NF-Y subunits, mostly of the A and B type, has been studied in plants, but knowledge regarding the C subunit remains fragmentary. Here, a water stress-induced NF-YC gene from Amaranthus hypochondriacus (AhNF-YC) was further characterized by its overexpression in transgenic Arabidospis thaliana plants. A role in development was inferred from modified growth rates in root, rosettes and inflorescences recorded in AhNF-YC overexpressing Arabidopsis plants, in addition to a delayed onset of flowering. Also, the overexpression of AhNF-YC caused increased seedling sensitivity to abscisic acid (ABA), and influenced the expression of several genes involved in secondary metabolism, development and ABA-related responses. An altered expression of the latter in water stressed and recovered transgenic plants, together with the observed increase in ABA sensitivity, suggested that their increased water stress resistance was partly ABA-dependent. An untargeted metabolomic analysis also revealed an altered metabolite pattern, both in normal and water stress/recovery conditions. These results suggest that AhNF-YC may play an important regulatory role in both development and stress, and represents a candidate gene for the engineering of abiotic stress resistance in commercial crops.

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.

Treatment of Amaranthus cruentus with chemical and biological inducers of resistance has contrasting effects on fitness and protection against compatible Gram positive and Gram negative bacterial pathogens.

Amaranthus cruentus (Ac) plants were treated with the synthetic systemic acquired resistance (SAR) inducer benzothiadiazole (BTH), methyl jasmonate (MeJA) and the incompatible pathogen, Pseudomonas syringae pv. syringae (Pss), under greenhouse conditions. The treatments induced a set of marker genes in the absence of pathogen infection: BTH and Pss similarly induced genes coding for pathogenesis-related and antioxidant proteins, whereas MeJA induced the arginase, LOX2 and amarandin 1 genes. BTH and Pss were effective when tested against the Gram negative pathogen Ps pv. tabaci (Pst), which was found to have a compatible interaction with grain amaranth. The resistance response appeared to be salicylic acid-independent. However, resistance against Clavibacter michiganensis subsp. michiganensis (Cmm), a Gram positive tomato pathogen also found to infect Ac, was only conferred by Pss, while BTH increased susceptibility. Conversely, MeJA was ineffective against both pathogens. Induced resistance against Pst correlated with the rapid and sustained stimulation of the above genes, including the AhPAL2 gene, which were expressed both locally and distally. The lack of protection against Cmm provided by BTH, coincided with a generalized down-regulation of defense gene expression and chitinase activity. On the other hand, Pss-treated Ac plants showed augmented expression levels of an anti-microbial peptide gene and, surprisingly, of AhACCO, an ethylene biosynthetic gene associated with susceptibility to Cmm in tomato, its main host. Pss treatment had no effect on productivity, but compromised growth, whereas MeJA reduced yield and harvest index. Conversely, BTH treatments led to smaller plants, but produced significantly increased yields. These results suggest essential differences in the mechanisms employed by biological and chemical agents to induce SAR in Ac against bacterial pathogens having different infection strategies. This may determine the outcome of a particular plant-pathogen interaction, leading to resistance or susceptibility, as in Cmm-challenged Ac plants previously induced with Pss or BTH, respectively.