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 β-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

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

Expresión de genes relacionados con defensa en plantas de tomate durante la fase presimbiótica de la simbiosis micorrízica arbuscular

La mayoría de las plantas angioespermas forman una asociación simbiótica con los hongos micorrízicos arbusculares (HMA). Se ha observado que en esta simbiosis ocurre una supresión o una regulación de las respuestas de defensa para permitir la colonización micorrízica, relacionada con la degradación de los fragmentos de quitooligosacáridos por las quitinasas micorriza-específica. En este trabajo se describe el efecto del uso exógeno de la sistemina sobre el proceso de micorrización. Se observó un efecto diferencial de los HMA (G. cubense, F. mosseae y R. intraradices) sobre la actividad de las b-glucanasas y las quitinasas a las 12 horas de inducción, en las raíces del tomate, potenciada por la aplicación de la sistemina. La hormona polipeptídica indujo la expresión rápida de una batería de genes relacionados con los procesos de señalización y respuestas de defensa en tomate; sin embargo, su acción conjunta con los HMA moduló, de forma diferenciada, la expresión génica. Se observó la inducción de los genes RbohD, LoxD y PLA2 en las primeras dos horas después de la aplicación del HMA y de la sistemina. Estos resultados sugieren un papel de las oxylipinas y la especie reactiva del oxígeno en el proceso inicial del reconocimiento. Por otra parte, se demuestra que la sistemina, aplicada de forma exógena, está siendo percibida en las raíces donde modula las respuestas de defensa local en el proceso de micorrización

Betacyanin Biosynthetic Genes and Enzymes Are Differentially Induced by (a)biotic Stress in <i>Amaranthus hypochondriacus</i>

<div><p>An analysis of key genes and enzymes of the betacyanin biosynthetic pathway in <i>Amaranthus hypochondriacus</i> (<i>Ah</i>) was performed. Complete cDNA sequence of <i>Ah</i> genes coding for cyclo-DOPA 5-O glucosyltransferase (<i>AhcDOPA5-GT</i>), two 4, 5-DOPA-extradiol-dioxygenase isoforms (<i>AhDODA-1</i> and <i>AhDODA-2</i>, respectively), and a betanidin 5-<i>O</i>-glucosyltransferase (<i>AhB5-GT</i>), plus the partial sequence of an orthologue of the cytochrome P-450 <i>R</i> gene (<i>CYP76AD1</i>) were obtained. With the exception <i>AhDODA-2</i>, which had a closer phylogenetic relationship to <i>DODA-like</i> genes in anthocyanin-synthesizing plants, all genes analyzed closely resembled those reported in related Caryophyllales species. The measurement of basal gene expression levels, in addition to the DOPA oxidase tyrosinase (DOT) activity, in different tissues of three <i>Ah</i> genotypes having contrasting pigmentation levels (green to red-purple) was determined. Additional analyses were performed in <i>Ah</i> plants subjected to salt and drought stress and to two different insect herbivory regimes. Basal pigmentation accumulation in leaves, stems and roots of betacyanic plants correlated with higher expression levels of <i>AhDODA-1</i> and <i>AhB5-GT</i>, whereas DOT activity levels coincided with pigment accumulation in stems and roots and with the acyanic nature of green plants, respectively, but not with pigmentation in leaves. Although the abiotic stress treatments tested produced changes in pigment levels in different tissues, pigment accumulation was the highest in leaves and stems of drought stressed betacyanic plants, respectively. However, tissue pigment accumulation in stressed <i>Ah</i> plants did not always correlate with betacyanin biosynthetic gene expression levels and/or DOT activity. This effect was tissue- and genotype-dependent, and further suggested that other unexamined factors were influencing pigment content in stressed <i>Ah</i>. The results obtained from the insect herbivory assays, particularly in acyanic plants, also support the proposal that these genes could have functions other than betacyanin biosynthesis.</p></div

Changes in pigment levels and tyrosinase activity in amaranth plants subjected to <i>discontinuous</i> insect herbivory.

<p>Amaranthine (<b>A</b>) and DOPA oxidase tyrosinase (DOT) activity (<b>B</b>) levels measured in leaves, stems and roots of plants of <i>A. hypochondriacus</i> genotypes (<i>AhNut</i> [N], <i>AhIR</i> [R] and <i>AhIG</i> [G]) having different patterns of pigmentation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone-0099012-g001" target="_blank">Figure 1</a>, and subjected to <i>discontinuous</i> (samples were taken on removing the larvae) insect herbivory for 3, 9 and 15 h. Mean values ± SE in control (C: empty bars) and treated (colored bars) plants are presented (n = 6). Asterisks over the bars represent statistically different values at <i>P</i>≤0.05 (Dunnetts test). Experiments were performed twice, and representative results are shown. FW  =  fresh weight.</p

Changes in pigment levels and tyrosinase activity in amaranth plants subjected to <i>continuous</i> insect herbivory.

<p>Amaranthine (<b>A</b>) and DOPA oxidase tyrosinase (DOT) activity (<b>B</b>) levels measured in leaves, stems and roots of plants of <i>A. hypochondriacus</i> genotypes (<i>AhNut</i> [N], <i>AhIR</i> [R] and <i>AhIG</i> [G]) having different patterns of pigmentation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone-0099012-g001" target="_blank">Figure 1</a>, and subjected to <i>continuous</i> insect herbivory for 18 h and then sampled at 6, 24 and 72 h after larval removal. Mean values ± SE in control (C: empty bars) and treated (T: colored bars) plants are presented (n = 6). Asterisks over the bars represent statistically different values at <i>P</i>≤0.05 (Dunnetts test). Experiments were performed twice, and representative results are shown. FW  =  fresh weight.</p

Development and yield traits indicate that the constitutive wound response phenotype of prosystemin overexpressing tomato plants entails no fitness penalty.

Systemin is a peptide hormone that regulates the wound response in tomato plants. Consequently, the overexpression of its prosystemin (ProSys) precursor protein leads to a resource-demanding constitutive activation of tomato’s wound-response. According to the growth vs. defense resource allocation premise, ProSys overexpression should negatively affect the physiological fitness of tomato plants. The present study was performed to explore why the opposite effect was steadily observed, instead. It was based on the premise that a better understanding of this unexpected outcome could help establish improved wound and related defense responses without negatively affecting crop productivity. To this effect, an experimental strategy was deployed to measure various physiological, biochemical and molecular parameters associated with either development, productivity, defense or in combination in untransformed (WT) and ProSys overexpressing (ProSys-OE) tomato plants. Thus, the chlorophyll fluorescence data obtained from plants grown under greenhouse experiments indicated that photosynthetic performance was not affected in ProSys-OE plants which also grew 7–14% taller than WT plants. Moreover, they showed accelerated flowering and yielded fruits of increased size (7–16% taller and wider) and weight (16–58% heavier), with modified fruit quality in terms of firmness (28% higher), titratable acidity (27–32% higher) and chemical composition. These findings suggest two complementary possibilities: (i) systemin is able to modulate both the wound response and plant development through the activation of jasmonic acid biosynthesis and signaling, and (ii) ProSys, an intrinsically disordered protein, acts as a signaling hub to regulate development and defense programs. These results shed light on the understanding of this plant regulatory mechanism and further suggest that systemin/ProSys-based regulation is central to control the defense-development balance in tomato. This knowledge could eventually lead to improved and more environmentally sound agricultural production practices

Expression of betacyanin biosynthetic genes in response to <i>discontinuous</i> insect herbivory.

<p>Relative expression levels³ were determined in stems of <i>A. hypochondriacus</i> plants, with contrasting pigmentation patterns, subjected to insect herbivory. Induced or repressed levels of expression (i.e. relative expression ≥1.5 or ≤0.5) are shown in bold text and italics, respectively.</p>1<p>h =  time, in hours, spent on the plant by the feeding larvae before they were removed and the tissues sampled.</p>2<p>The genotypes examined in this study were <i>Ah</i> cv. Nutrisol (<i>AhNut</i>; with predominantly betacyanic leaves), <i>Ah</i> India Red (<i>AhIR</i>; with predominantly betacyanic stems) and <i>Ah</i> India Green (<i>AhIG</i>; with all tissues acyanic).</p>3<p>The fold change in the expression of the target genes was calculated using the 2^−ΔΔCt method according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone.0099012-Livak1" target="_blank">[76]</a>.</p

Changes in pigment levels and tyrosinase activity in water-stressed amaranth plants with contrasting pigmentation patterns.

<p>Amaranthine <b>(A)</b> and DOPA oxidase tyrosinase (DOT) activity <b>(B)</b> levels measured in leaves, stems and roots of water-stressed plants of <i>A. hypochondriacus</i> genotypes (<i>AhNut</i> [N], <i>AhIR</i> [R] and <i>AhIG</i> [G]) having different patterns of pigmentation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099012#pone-0099012-g001" target="_blank">Figure 1</a>. Mean values ± SE in control (C; empty bars) and treated (colored bars) are presented (n = 6). Asterisks over the bars represent statistically different values at <i>P</i>≤0.05 (Tukey Kramer test). Experiments were performed twice, and representative results are shown. FW  =  fresh weight.</p