Amaranth calcium oxalate crystals are associated with chloroplast structures and proteins.

Calcium oxalate (CaOx) crystals in plants are formed in crystal idioblasts cells and have specific geometric shapes. Their proposed functions include calcium homeostasis and carbon source, among others. Amaranth is a plant that presents high tolerance to abiotic stresses and accumulates considerable amounts of CaOx crystals; however, few studies have focused on characterizing the crystals ultrastructure and none is related to identifying proteins bound to them. This information is of great interest to understand the mechanisms related to CaOx crystal formation and to support their proposed functions. Thus, this work aimed to characterize CaOx crystals in amaranth leaves. Crystals were purified and the proteins bound to them were isolated and identified by nLC-MS/MS. Leaf sections were analyzed by light and electron microscopy. The identified proteins were related to the chloroplast such as ATPb synthase, RuBisCO large subunit, and cell wall-related proteins, which were validated by immunohistochemistry and immunogold labeling. In addition, it was observed that CaOx crystal idioblasts were formed from parenchyma cells associated with mesophyll and veins, in which the thylakoid membranes of degraded chloroplasts turned into crystal chambers. These results significantly advance our understanding of the mechanisms of CaOx crystal formation and the potential function as an alternative carbon source in leaves.

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.

Biomass, chlorophyll fluorescence, and osmoregulation traits let differentiation of wild and cultivated Amaranthus under water stress.

Amaranths are recognized by their high nutritive value and their natural tolerance to environmental stresses. In this study, physiological differences in response to water stress were compared between A. hybridus, a wild species considered as weed, and A. hypochondriacus, the most cultivated species for grain production, under the hypothesis that wild species have better adaptation to stress. In both species, photosynthetic parameters, pigments, and gene expression of selected genes were assessed. Biomass, effective quantum efficiency (ΦPSII), photochemical quenching (qP), and electron transport rate (ETR) values were reduced only in A. hybridus due to water deficit. Drought stress promoted proline accumulation by twice in A. hybridus but until three times in A. hypochondriacus. In both species, drought stress reduced net assimilation rate (A), transpiration rate (E), stomatal conductance (gs), and the expression of phosphoenol pyruvate carboxylase (PEPC). While, maximum quantum efficiency (Fv/Fm), chlorophyll, betacyanins, and the expression of ribulose1-5, bisphosphate carboxylase/oxygenase large subunit (LSU) did not change when plants were subjected to water stress. Likewise, both species accumulated total phenolic compounds and Oxalyl-CoA gene was up-regulated in response to drought. Our results have shown that A. hypochondriacus, the cultivated species, exhibited better tolerance to drought than A. hybridus, the wild species, probably due to an unconsciously selected trait during the domestication process.

Molecular characterization and in vitro interaction analysis of Op14-3-3 µ protein from Opuntia ficus-indica: identification of a new client protein from shikimate pathway.

In plants, 14-3-3 proteins are important modulators of protein-protein interactions in response to environmental stresses. The aim of the present work was to characterize one Opuntia ficus-indica 14-3-3 and get information about its client proteins. To achieve this goal, O. ficus-indica 14-3-3 cDNA, named as Op14-3-3 µ, was amplified by 3'-RACE methodology. Op14-3-3 µ contains an Open Reading Frame of 786 bp encoding a 261 amino acids protein. Op14-3-3 µ cDNA was cloned into a bacterial expression system and recombinant protein was purified. Differential Scanning Fluorimetry, Dynamic Light Scattering, and Ion Mobility-Mass Spectrometry were used for Op14-3-3 µ protein characterization, and Affinity-Purification-Mass Spectrometry analysis approach was used to obtain information about their potential client proteins. Pyrophosphate-fructose 6-phosphate 1-phosphotransferase, ribulose bisphosphate carboxylase large subunit, and vacuolar-type H+-ATPase were identified. Interestingly chorismate mutase p-prephenate dehydratase was also identified. Op14-3-3 µ down-regulation was observed in Opuntia calluses when they were induced with Jasmonic Acid, while increased accumulation of Op14-3-3 µ protein was observed. The putative interaction of 14-3-3 µ with chorismate mutase, which have not been reported before, suggest that Op14-3-3 µ could be an important regulator of metabolites biosynthesis and responses to stress in Opuntia spp. SIGNIFICANCE: Opuntia species are important crops in arid and semiarid areas worldwide, but despite its relevance, little information about their tolerance mechanism to cope with harsh environmental conditions is reported. 14-3-3 proteins have gained attention due to its participation as protein-protein regulators and have been linked with primary metabolism and hormones responses. Here we present the characterization of the first Opuntia ficus-indica 14-3-3 (Op14-3-3) protein using affinity purification-mass spectrometry (AP-MS) strategy. Op14-3-3 has high homology with other 14-3-3 from Caryophyllales. A novel Op14-3-3 client protein has been identified; the chorismate mutase p-prephenate dehydratase, key enzyme that links the primary with secondary metabolism. The present results open new questions about the Opuntia spp. pathways mechanisms in response to environmental stress and the importance of 14-3-3 proteins in betalains biosynthesis.

Tecia solanivora infestation increases tuber starch accumulation in Pastusa Suprema potatoes.

In response to infestation with larvae of the Guatemalan tuber moth (Tecia solanivora), some Solanum tuberosum (potato) varieties exhibit an overcompensation response, whereby the total dry mass of uninfested tubers is increased. Here, we describe early responses, within the first few days, of T. solanivora feeding, in the Colombian potato variety Pastusa Suprema. Non-targeted metabolite profiling showed significant secondary metabolism changes in T. solanivora-infested tubers, but not in uninfested systemic tubers. In contrast, changes in primary metabolism were greater in uninfested systemic tubers than in the infested tubers, with a notable 80% decline in systemic tuber sucrose levels within 1 d of T. solanivora infestation. This suggested either decreased sucrose transport from the leaves or increased sink strength, i.e., more rapid sucrose to starch conversion in the tubers. Increased sucrose synthesis was indicated by higher rubisco activase and lower starch synthase gene expression in the leaves of infested plants. Elevated sink strength was demonstrated by 45% more total starch deposition in systemic tubers of T. solanivora-infested plants compared to uninfested control plants. Thus, rather than investing in increased defense of uninfested tubers, Pastusa Suprema promotes deposition of photoassimilates in the form of starch as a response to T. solanivora infestation.

Genetic mapping identifies loci that influence tomato resistance against Colorado potato beetles.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), the most economically important insect pest on potato (Solanum tuberosum L.), also feeds on other Solanaceae, including cultivated tomato (Solanum lycopersicum L.). We used tomato genetic mapping populations to investigate natural variation in CPB resistance. CPB bioassays with 74 tomato lines carrying introgressions of Solanum pennellii in S. lycopersicum cv. M82 identified introgressions from S. pennellii on chromosomes 1 and 6 conferring CPB susceptibility, whereas introgressions on chromosomes 1, 8 and 10 conferred higher resistance. Mapping of CPB resistance using 113 recombinant inbred lines derived from a cross between S. lycopersicum cv UC-204B and Solanum galapagense identified significant quantitative trait loci on chromosomes 6 and 8. In each case, the S. galapagense alleles were associated with lower leaf damage and reduced larval growth. Results of both genetic mapping approaches converged on the same region of chromosome 6, which may have important functions in tomato defense against CPB herbivory. Although genetic mapping identified quantitative trait loci encompassing known genes for tomato acyl sugar and glycoalkaloid biosynthesis, experiments with acyl sugar near-isogenic lines and transgenic GAME9 glycoalkaloid-deficient and overproducing lines showed no significant effect of these otherwise insect-defensive metabolites on CPB performance.

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.

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.

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.