Proteomic profile of tepary bean seed storage proteins in germination with low water potential.

BACKGROUND: Tepary bean (Phaseolus acutifolius A. Gray) is one of the five species domesticated from the genus Phaseolus with genetic resistance to biotic and abiotic stress. To understand the mechanisms underlying drought responses in seed storage proteins germinated on water and polyethylene glycol (PEG-6000) at -0.49 MPa, we used a proteomics approach to identify potential molecular target proteins associated with the low water potential stress response. METHODS: Storage proteins from cotyledons of Tepary bean seeds germinated at 24, 48 and 72 h on water and PEG-6000 at -0.49 MPa were analyzed by one-dimensional electrophoresis (DE) with 2-DE analysis and shotgun mass spectrometry. Using computational database searching and bioinformatics analyses, we performed Gene Ontology (GO) and protein interactome (functional protein association network) String analyses. RESULTS: Comparative analysis showed that the effect of PEG-6000 on root growth was parallel to that on germination. Based on the SDS‒PAGE protein banding patterns and 2-DE analysis, ten differentially abundant seed storage proteins showed changes in storage proteins, principally in the phaseolin and lectin fractions. We found many proteins that are recognized as drought stress-responsive proteins, and several of them are predicted to be intrinsically related to abiotic stress. The shotgun analysis searched against UniProt's legume database, and Gene Ontology (GO) analysis indicated that most of the seed proteins were cytosolic, with catalytic activity and associated with carbohydrate metabolism. The protein‒protein interaction networks from functional enrichment analysis showed that phytohemagglutinin interacts with proteins associated with the degradation of storage proteins in the cotyledons of common bean during germination. CONCLUSION: These findings suggest that Tepary bean seed proteins provide valuable information with the potential to be used in genetic improvement and are part of the drought stress response, making our approach a potentially useful strategy for discovering novel drought-responsive proteins in other plant models.

High-Throughput Screening to Examine the Dynamic of Stay-Green by an Imaging System.

The development of RGB (red, green, blue) sensors has opened the way for plant phenotyping. This is relevant because plant phenotyping allows us to visualize the product of the interaction between the plant ontogeny, anatomy, physiology, and biochemistry. Better yet, this can be achieved at any stage of plant development, i.e., from seedling to maturity. Here, we describe the use of phenotyping, based on the stay-green trait, of common bean (Phaseolus vulgaris L.) plant, as a model, stressed by water deficit, to elucidate the result of that interaction. Description is based on interpretation of RGB digital images acquired using a phenomic platform and a specific software. These images allow us to obtain a data group related to the color parameters that quantify the changes and alterations in each plant growth and development.

Phenotypic, Anatomical, and Diel Variation in Sugar Concentration Linked to Cell Wall Invertases in Common Bean Pod Racemes under Water Restriction.

The common bean (Phaseolus vulgaris L.) pod wall is essential for seed formation and to protect seeds. To address the effect of water restriction on sugar metabolism in fruits differing in sink strength under light-dark cycles, we used plants of cv. OTI at 100% field capacity (FC) and at 50% FC over 10 days at the beginning of pod filling. Water restriction intensified the symptoms of leaf senescence. However, pods maintained a green color for several days longer than leaves did. In addition, the functionality of pods of the same raceme was anatomically demonstrated, and no differences were observed between water regimes. The glucose and starch concentrations were lower than those of sucrose, independent of pod wall size. Remarkably, the fructose concentration decreased only under water restriction. The cell wall invertase activity was twofold higher in the walls of small pods than in those of large ones in both water regimes; similar differences were not evident for cytosolic or vacuolar invertase. Using bioinformatics tools, six sequences of invertase genes were identified in the P. vulgaris genome. The PvINVCW4 protein sequence contains substitutions for conserved residues in the sucrose-binding site, while qPCR showed that transcript levels were induced in the walls of small pods under stress. The findings support a promising strategy for addressing sink strength under water restriction.

Starch accumulation in bean fruit pericarp is mediated by the differentiation of chloroplasts into amyloplasts.

The sucrose supply to bean fruits remains almost constant during seed development, and the early stages of this process are characterized by a significant amount of starch and soluble sugars (glucose, fructose and sucrose) accumulated in the pericarp. Bean fruits are photosynthetically active; however, our results indicated that starch synthesis in the pericarp was largely dependent on the photosynthetic activity of the leaves. The photosynthetic activity and the amount of the Rubisco large subunit were gradually reduced in the fruit pericarp, and a large increase in the amount of the ADP-glucose pyrophosphorylase small subunit (AGPase SS) was observed. These changes suggested differentiation of chloroplasts into amyloplasts. Pericarp chloroplasts imported glucose 1-P to support starch synthesis, and their differentiation into amyloplasts allowed the surplus sucrose to be used in the synthesis of starch, which was later degraded to meet the needs of fast-growing seeds. Starch stored in the bean fruit pericarp was not degraded in response to drought stress, but it was rapidly used under severe nutrient restriction. Together, this work indicated that starch accumulation in the pericarp of bean fruits is important to adjust the needs of developing seeds to the amount of sucrose that is provided to fruits.

Physcomitrium patens Infection by Colletotrichum gloeosporioides: Understanding the Fungal-Bryophyte Interaction by Microscopy, Phenomics and RNA Sequencing.

Anthracnose caused by the hemibiotroph fungus Colletotrichum gloeosporioides is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of C. gloeosporioides has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss Physcomitrium patens Hedw. B&S (former Physcomitrella patens) is sensitive to known bacterial and fungal phytopathogens, including C. gloeosporioides, which cause infection and cell death. P. patens responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of P. patens and C. gloeosporioides have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of P. patens to C. gloeosporioides. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 P. patens genes were differentially expressed in response to Colletotrichum infection. The analysis of differentially expressed gene function showed that the C. gloeosporioides infection led to a transcription reprogramming in P. patens that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, P. patens changes its transcription from primary metabolism to defend itself from the pathogen.

Interaction among species, time-of-day, and soil water potential on biochemical and physiological characteristics of cladodes of Opuntia.

The physiology and biochemistry of young Opuntia spp. cladodes relate with their Crassulacean acid metabolism, which extends over the day-night cycle in four phases, is species-dependent and is affected by water availability. This study aimed to assess the interaction among species, time-of-day, and the soil water potential (ΨW) on biochemical and physiological characteristics of cladodes of Opuntia species. Three-week-old cladodes were harvested at 7:00 a.m. and 3:00 p.m. from plants with or without irrigation for 30 d (-0.17 and -5.72 MPa soil ΨW), from O. albicarpa, O. ficus-indica, O. hyptiacantha, O. megacantha, and O. streptacantha. The experimental design was a factorial 5 x 2 x 2 (species, sampling time and soil ΨW). The experimental unit was one cladode per plant, and six repetitions were evaluated. Total acids, glucose, fructose, sucrose, starch, total phenolics, free amino acids, and soluble proteins concentrations were evaluated, as well as acid invertase and neutral invertase activities. The interaction among species x soil ΨW and species x time of the day was significant (P ≤ 0.05) in all variables evaluated. An exception was the species x soil ΨW on starch concentration (P = 0.1827). The biochemical and physiological characteristics of Opuntia cladodes were modified by the time of the day and soil ΨW interaction, but most of the characteristics were positively or inversely affected depending on the species, frequently displaying a descending trend following O. streptacantha, O. hyptiacantha, O. megacantha, O. albicarpa and O. ficus-indica. The total acids, glucose, fructose, starch, soluble proteins, and free amino acids concentrations revealed that domestication significantly modifies C and N metabolism in Opuntia.

Isolation and characterization of psychrophilic and psychrotolerant plant-growth promoting microorganisms from a high-altitude volcano crater in Mexico.

Extreme ecosystems are a possible source of new interesting microorganisms, in this study the isolation of psychrophilic and psychrotolerant plant growth promoting microorganisms was pursued in a cold habitat, with the aim of finding novel microbes that can protect crops from cold. Eight yeast and four bacterial strains were isolated from rhizospheric soil collected from the Xinantécatl volcano in Mexico, and characterized for plant growth promoting properties. Most of the yeasts produced indole acetic acid and hydrolytic enzymes (cellulases, xilanases and chitinases), but none of them produced siderophores, in contrast to their bacterial counterparts. Inorganic phosphate solubilization was detected for all the bacterial strains and for two yeast strains. Yeast and bacterial strains may inhibit growth of various pathogenic fungi, propounding a role in biological control. Microorganisms were identified up to genera level, by applying ribotyping techniques and phylogenetic analysis. Bacterial strains belonged to the genus Pseudomonas, whereas yeast strains consisted of Rhodotorula sp. (4), Mrakia sp. (3) and Naganishia sp. (1). New species belonging to the aforementioned genera seem to have been isolated from both bacteria and yeasts. Germination promoting activity on Solanum lycopersicum seeds was detected for all strains compared to a control, whereas tomato plantlets, grown at 15 °C in the presence of some of the strains, performed better than the non-inoculated plantlets. This study offers the possibility of using these strains as an additive to improve culture conditions of S. lycopersicum in a more environmentally compatible way. This is the first study to propose psychrophilic/psychrotolerant yeasts, as plant growth promoting microbes.

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression.

Subcellular compartmentation of sugar signaling: links among carbon cellular status, route of sucrolysis, sink-source allocation, and metabolic partitioning.

Recent findings suggest that both subcellular compartmentation and route of sucrolysis are important for plant development, growth, and yield. Signaling effects are dependent on the tissue, cell type, and stage of development. Downstream effects also depend on the amount and localization of hexoses and disaccharides. All enzymes of sucrose metabolism (e.g., invertase, hexokinase, fructokinase, sucrose synthase, and sucrose 6-phosphate synthase) are not produced from single genes, but from paralog families in plant genomes. Each paralog has unique expression across plant organs and developmental stages. Multiple isoforms can be targeted to different cellular compartments (e.g., plastids, mitochondria, nuclei, and cytosol). Many of the key enzymes are regulated by post-transcriptional modifications and associate in multimeric protein complexes. Some isoforms have regulatory functions, either in addition to or in replacement of their catalytic activity. This explains why some isozymes are not redundant, but also complicates elucidation of their specific involvement in sugar signaling. The subcellular compartmentation of sucrose metabolism forces refinement of some of the paradigms of sugar signaling during physiological processes. For example, the catalytic and signaling functions of diverse paralogs needs to be more carefully analyzed in the context of post-genomic biology. It is important to note that it is the differential localization of both the sugars themselves as well as the sugar-metabolizing enzymes that ultimately led to sugar signaling. We conclude that a combination of subcellular complexity and gene duplication/subfunctionalization gave rise to sugar signaling as a regulatory mechanism in plant cells.

Heterologous expression of yeast Hxt2 in Arabidopsis thaliana alters sugar uptake, carbon metabolism and gene expression leading to glucose tolerance of germinating seedlings.

The hexose transporter 2 gene (Hxt2) from Saccharomyces cerevisiae was expressed in Arabidopsis thaliana under control of the 35S promoter. Several independent transgenic lines were selected after confirming single gene insertion by southern blot analysis in the T4 generation. Northern blots revealed the presence of heterologous transcript. Radiolabeling experiments revealed an increased rate of incorporation of the non-metabolizable analog 3-O-methyl-[U-14C]-glucose. This confirmed that the yeast Hxt2 transporter was functional in Arabidopsis. No phenotypic changes at the vegetative and reproductive stages could be detected in the transgenic lines when compared to wild type plants. Shortly after germination some differences in development and glucose signaling were observed. Transgenic seedlings cultivated in liquid medium or on solid agar plates were able to grow with 3% glucose (producing bigger plants and longer roots), while development of wild type plants was delayed under those conditions. Metabolite analysis revealed that the Hxt2 transgenic lines had higher rates of sugar utilization. Transcriptional profiling showed that particular genes were significantly up- or down-regulated. Some transcription factors like At1g27000 were repressed, while others, such as At3g58780, were induced. The mRNA from classical sugar signaling genes such as STP1, Hxk1, and ApL3 behaved similarly in transgenic lines and wild type lines. Results suggest that the Hxt2 transgene altered some developmental processes related to the perception of high carbon availability after the germination stage. We conclude that the developmental arrest of wild type plants at 3% glucose not only depends on Hxk1 as the only sugar sensor but might also be influenced by the route of hexose transport across the plasma membrane.