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.

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.

Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine-Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.

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