John Paul Délano-Frier

Jasmonic acid influences mycorrhizal colonization in tomato plants by modifying the expression of genes involved in carbohydrate partitioning

The role of jasmonic acid (JA) on mycorrhizal colonization by Glomus fasciculatum in tomato plants was examined using mutant plants overexpressing prosystemin (PS) or affected in the synthesis of JA (suppressor of prosystemin-mediated responses 2, spr2). The degree of mycorrhizal colonization was determined by measuring frequency (F%) and intensity (M%) of colonization and arbuscule abundance (A%). Gene expression and biochemical analyses were also performed in roots to detect changes in carbon (C) partitioning. Colonization was similar in mycorrhizal PS and wild-type roots, except for a higher A% in the former. Conversely, colonization was severely reduced in roots of spr2 mutants. No association was found between levels of expression of genes coding for systemic wound responsive proteins (or SWRPs) and other defense-related proteins in roots and mycorrhization levels in these plants. On the other hand, the degree of mycorrhizal colonization correlated with changes in the transcriptional regulation of a number of genes involved in sucrose hydrolysis and transport, cell wall invertase activity and mycorrhizal-specific fatty acid content in roots. The results obtained suggest that one of the mechanisms by which JA might operate to modulate the mycorrhization process could be through its influence on the regulation of C partitioning in the plant. The significant colonization increase observed in mycorrhizal spr2 plants supplied with exogenous methyl jasmonate supports its role as a positive regulator of the symbiosis.

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.

BackgroundAmaranthus 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.ResultsA 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).ConclusionsThis 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.

The effect of exogenous jasmonic acid on induced resistance and productivity in amaranth (Amaranthus hypochondriacus) is influenced by environmental conditions.

Amaranthus hypochondriacus is a C4 pseudocereal crop capable of producing reasonable grain yields in adverse environmental conditions that limit cereal performance. It accumulates trypsin inhibitors and α-amylase inhibitors in seeds and leaves that are considered to act as insect feeding deterrents. Foliar trypsin and α-amylase inhibitors also accumulate by treatment with exogenous jasmonic acid (JA) in controlled laboratory conditions. Three field experiments were performed in successive years to test if two nonphytotoxic dosages of JA were capable of inducing inhibitor activity in A. hypochondriacus in agronomical settings, and if this induced response reduced insect herbivory and insect abundance in foliage and seed heads. The performance of JA-treated plants was compared to insecticide-treated plants and untreated controls. The effect of exogenous JA on the foliar levels of six additional putatively defence proteins was also evaluated. Possible adverse effects of JA induction on productivity were evaluated by measuring grain yield, seed protein content, and germination efficiency. The results present a complex pattern and were not consistent from year to year. To some extent, the yearly variability observed could have been consequence of growth under drought versus nondrought conditions. In a drought year, JA-treated plants had lower levels of insect herbivory-derived damage in apical leaves and panicle than control plants, whereas in nondrought years, there was an inconsistent effect on aphids, with no effect on lepidopteran larvae. JA treatments reduced the size of the insect community in seed heads. The effect varied with year. Exogenous JA did not adversely affect productivity, and in the absence of drought stress, the higher dosage enhanced grain yield. Induction of defensive proteins by JA, although sporadic, was more effective in nondrought conditions. The patterns of foliar protein accumulation observed suggest that they may be part of a constitutive, rather than inducible, chemical defense mechanism that is developmentally regulated and critically dependent on the environment. The results emphasize the difficulties that are often encountered when evaluating the performance of chemical elicitors of induced resistance in field settings.

Differential gene expression in whitefly Bemisia tabaci-infested tomato (Solanum lycopersicum) plants at progressing developmental stages of the insect's life cycle.

A suppression-subtractive-hybridization (SSH) strategy was used to identify genes whose expression was modified in response to virus-free whitefly Bemisia tabaci (Bt, biotype A) infestation in tomato (Solanum lycopersicum) plants. Thus, forward and reverse SSH gene libraries were generated at four points in the whiteflys life cycle, namely at (1) 2 days (adult feeding and oviposition: phase I); (2) 7 days (mobile crawler stage: phase II); (3) 12 days (second to third instar nymphal transition: phase III) and (4) 18 days (fourth instar nymphal stage: phase IV). The 169 genes with altered expression (up and downregulated) that were identified in the eight generated SSH libraries, together with 75 additional genes that were selected on the basis of their involvement in resistance responses against phytofagous insects and pathogens, were printed on a Nexterion(®) Slide MPX 16 to monitor their pattern of expression at the above phases. The results indicated that Bt infestation in tomato led to distinctive phase-specific expression/repression patterns of several genes associated predominantly with photosynthesis, senescence, secondary metabolism and (a)biotic stress. Most of the gene expression modifications were detected in phase III, coinciding with intense larval feeding, whereas fewer changes were detected in phases I and IV. These results complement previously reported gene expression profiles in Bt-infested tomato and Arabidopisis, and support and expand the opinion that Bt infestation leads to the downregulation of specific defense responses in addition to those controlled by jasmonic acid.

Exogenous systemin has a contrasting effect on disease resistance in mycorrhizal tomato ( Solanum lycopersicum ) plants infected with necrotrophic or hemibiotrophic pathogens

A study was performed to determine the effect of the systemin polypeptide on the bio-protective effect of arbuscular mycorrhizal fungi (AMF) in tomato plants infected with Alternaria solani, Phytophthora infestans or P. parasitica. Before infection, tomato plants were colonized with two different AMF, Glomus fasciculatum or G. clarum. In addition, a group of inoculated plants was treated with systemin, just after emergence. The exogenous application of systemin marginally suppressed the resistance against A. solani leaf blight observed in G. fasciculatum mycorrhizal plants but significantly enhanced it in plants colonized with G. clarum. Systemin induced resistance to P. parasitica in leaves of G. fasciculatum mycorrhizal plants, in which AMF colonization alone was shown to have no protective effect. Conversely, none of the treatments led to resistance to root or stem rots caused by P. infestans or P. parasitica. The above effects did not correlate with changes in the activity levels of β-1,3-glucanase (BG), chitinase (CHI), peroxidase (PRX), and phenylalanine ammonium lyase (PAL) in leaves of infected plants. However, they corroborated previous reports showing that colonization by AMF can lead to a systemic resistance response against A. solani. Systemic resistance to A. solani was similarly observed in non-mycorrhizal systemin-treated plants, which, in contrast, showed increased susceptibility to P. infestans and P. parasitica. The results indicated that the pattern of systemic disease resistance conferred by mycorrhizal colonization was dependent on the AMF employed and could be altered by the exogenous application of systemin, by means of a still undefined mechanism.

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

BackgroundAmaranthus 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.ResultsDefoliation 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.ConclusionsIt 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.

Expression of the 1-SST and 1-FFT genes and consequent fructan accumulation in Agave tequilana and A. inaequidens is differentially induced by diverse (a)biotic-stress related elicitors.

The expression of genes coding for sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100), both fructan biosynthesizing enzymes, characterization by TLC and HPAEC-PAD, as well as the quantification of the fructo-oligosaccharides (FOS) accumulating in response to the exogenous application of sucrose, kinetin (cytokinin) or other plant hormones associated with (a)biotic stress responses were determined in two Agave species grown in vitro, domesticated Agave tequilana var. azul and wild A. inaequidens. It was found that elicitors such as salicylic acid (SA), and jasmonic acid methyl ester (MeJA) had the strongest effect on fructo-oligosaccharide (FOS) accumulation. The exogenous application of 1mM SA induced a 36-fold accumulation of FOS of various degrees of polymerization (DP) in stems of A. tequilana. Other treatments, such as 50mM abscisic acid (ABA), 8% Sucrose (Suc), and 1.0 mg L(-1) kinetin (KIN) also led to a significant accumulation of low and high DP FOS in this species. Conversely, treatment with 200 μM MeJA, which was toxic to A. tequilana, induced an 85-fold accumulation of FOS in the stems of A. inaequidens. Significant FOS accumulation in this species also occurred in response to treatments with 1mM SA, 8% Suc, and 10% polyethylene glycol (PEG). Maximum yields of 13.6 and 8.9 mg FOS per g FW were obtained in stems of A. tequilana and A. inaequidens, respectively. FOS accumulation in the above treatments was tightly associated with increased expression levels of either the 1-FFT or the 1-SST gene in tissues of both Agave species.

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

An analysis of key genes and enzymes of the betacyanin biosynthetic pathway in Amaranthus hypochondriacus (Ah) was performed. Complete cDNA sequence of Ah genes coding for cyclo-DOPA 5-O glucosyltransferase (AhcDOPA5-GT), two 4, 5-DOPA-extradiol-dioxygenase isoforms (AhDODA-1 and AhDODA-2, respectively), and a betanidin 5-O-glucosyltransferase (AhB5-GT), plus the partial sequence of an orthologue of the cytochrome P-450 R gene (CYP76AD1) were obtained. With the exception AhDODA-2, which had a closer phylogenetic relationship to DODA-like 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 Ah genotypes having contrasting pigmentation levels (green to red-purple) was determined. Additional analyses were performed in Ah 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 AhDODA-1 and AhB5-GT, 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 Ah 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 Ah. 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.

Burkholderia ambifaria and B. caribensis promote growth and increase yield in grain amaranth (Amaranthus cruentus and A. hypochondriacus) by improving plant nitrogen uptake.

Grain amaranth is an emerging crop that produces seeds having high quality protein with balanced amino-acid content. However, production is restricted by agronomic limitations that result in yields that are lower than those normally produced by cereals. In this work, the use of five different rhizobacteria were explored as a strategy to promote growth and yields in Amaranthus hypochondriacus cv. Nutrisol and A. cruentus cv. Candil, two commercially important grain amaranth cultivars. The plants were grown in a rich substrate, high in organic matter, nitrogen (N), and phosphorus (P) and under greenhouse conditions. Burkholderia ambifaria Mex-5 and B. caribensis XV proved to be the most efficient strains and significantly promoted growth in both grain amaranth species tested. Increased grain yield and harvest index occurred in combination with chemical fertilization when tested in A. cruentus. Growth-promotion and improved yields correlated with increased N content in all tissues examined. Positive effects on growth also occurred in A. cruentus plants grown in a poor soil, even after N and P fertilization. No correlation between non-structural carbohydrate levels in roots of inoculated plants and growth promotion was observed. Conversely, gene expression assays performed at 3-, 5- and 7-weeks after seed inoculation in plants inoculated with B. caribensis XV identified a tissue-specific induction of several genes involved in photosynthesis, sugar- and N- metabolism and transport. It is concluded that strains of Burkholderia effectively promote growth and increase seed yields in grain amaranth. Growth promotion was particularly noticeable in plants grown in an infertile soil but also occurred in a well fertilized rich substrate. The positive effects observed may be attributed to a bio-fertilization effect that led to increased N levels in roots and shoots. The latter effect correlated with the differential induction of several genes involved in carbon and N metabolism and transport.

Grain Amaranths Are Defoliation Tolerant Crop Species Capable of Utilizing Stem and Root Carbohydrate Reserves to Sustain Vegetative and Reproductive Growth after Leaf Loss

Tolerance to defoliation can be defined as the degree to which productivity is affected by photosynthetic area reduction. This trait was studied in grain amaranth (Amaranthus cruentus and A. hypochondriacus), which are considered to be a highly defoliation-tolerant species. The physiological and biochemical responses to increasing levels of mechanical leaf removal up to total defoliation were quantified. Tolerance appeared to be dependent on various factors: ( i) amount of lost tissue; (ii) mechanics of leaf tissue removal; (iii) environment, and (iv) species tested. Thus, grain amaranth was found to be a highly tolerant species under green-house conditions when leaf tissue loss was performed by gradual perforation. However, tolerance was compromised under similar conditions when defoliation was done by gradual cutting of the leaf. Also tolerance in completely defoliated plants tended to decrease under field conditions, where differences between A. cruentus and A. hypochondriacus were observed. All non-structural carbohydrate (NSC) levels were reduced in stems and roots of totally defoliated amaranths one day after treatment. Such depletion probably provided the carbon (C) resources needed to sustain the early recovery process in the absence of photosynthetic capacity. This was corroborated by shading of intact plants, which produced the same rapid and drastic reduction of NSC levels in these tissues. These results emphasize the role of stored NSCs, particularly starch, in buffering the impact of severe defoliation in amaranth. The fall in sucrose synthase and cell wall invertase activity observed in stems and roots soon after defoliation was consistent with their predicted shift from sink to source tissues. It is concluded that mobilization of C stores in stems and roots, is a physiologically important trait underlying tolerance to defoliation in grain amaranth.