Georgina Hernández

Phosphorus Stress in Common Bean: Root Transcript and Metabolic Responses

Phosphorus (P) is an essential element for plant growth. Crop production of common bean (Phaseolus vulgaris), the most important legume for human consumption, is often limited by low P in the soil. Functional genomics were used to investigate global gene expression and metabolic responses of bean plants grown under P-deficient and P-sufficient conditions. P-deficient plants showed enhanced root to shoot ratio accompanied by reduced leaf area and net photosynthesis rates. Transcript profiling was performed through hybridization of nylon filter arrays spotted with cDNAs of 2,212 unigenes from a P deficiency root cDNA library. A total of 126 genes, representing different functional categories, showed significant differential expression in response to P: 62% of these were induced in P-deficient roots. A set of 372 bean transcription factor (TF) genes, coding for proteins with Inter-Pro domains characteristic or diagnostic for TF, were identified from The Institute of Genomic Research/Dana Farber Cancer Institute Common Bean Gene Index. Using real-time reverse transcription-polymerase chain reaction analysis, 17 TF genes were differentially expressed in P-deficient roots; four TF genes, including MYB TFs, were induced. Nonbiased metabolite profiling was used to assess the degree to which changes in gene expression in P-deficient roots affect overall metabolism. Stress-related metabolites such as polyols accumulated in P-deficient roots as well as sugars, which are known to be essential for P stress gene induction. Candidate genes have been identified that may contribute to root adaptation to P deficiency and be useful for improvement of common bean.

Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

BackgroundEscherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) are capable of coutilizing glucose and other carbon sources due to the absence of catabolite repression by glucose. In these strains, the lack of this important regulatory and transport system allows the coexistence of glycolytic and gluconeogenic pathways. Strains lacking PTS have been constructed with the goal of canalizing part of the phosphoenolpyruvate (PEP) not consumed in glucose transport to the aromatic pathway. The deletion of the ptsHIcrr operon inactivates PTS causing poor growth on this sugar; nonetheless, fast growing mutants on glucose have been isolated (PB12 strain). However, there are no reported studies concerning the growth potential of a PTS- strain in mixtures of different carbon sources to enhance the production of aromatics compounds.ResultsPB12 strain is capable of coutilizing mixtures of glucose-arabinose, glucose-gluconate and glucose-glycerol. This capacity increases its specific growth rate (μ) given that this strain metabolizes more moles of carbon source per unit time. The presence of plasmids pRW300aroGfbrand pCLtktA reduces the μ of strain PB12 in all mixtures of carbon sources, but enhances the productivity and yield of aromatic compounds, especially in the glucose-glycerol mixture, as compared to glucose or glycerol cultures. No acetate was detected in the glycerol and the glucose-glycerol batch fermentations.ConclusionDue to the lack of catabolite repression, PB12 strain carrying multicopy plasmids containing tktA and aroGfbrgenes is capable of coutilizing glucose and other carbon sources; this capacity, reduces its μ but increases the production of aromatic compounds.

Sequencing and Analysis of Common Bean ESTs. Building a Foundation for Functional Genomics

Although common bean (Phaseolus vulgaris) is the most important grain legume in the developing world for human consumption, few genomic resources exist for this species. The objectives of this research were to develop expressed sequence tag (EST) resources for common bean and assess nodule gene expression through high-density macroarrays. We sequenced a total of 21,026 ESTs derived from 5 different cDNA libraries, including nitrogen-fixing root nodules, phosphorus-deficient roots, developing pods, and leaves of the Mesoamerican genotype, Negro Jamapa 81. The fifth source of ESTs was a leaf cDNA library derived from the Andean genotype, G19833. Of the total high-quality sequences, 5,703 ESTs were classified as singletons, while 10,078 were assembled into 2,226 contigs producing a nonredundant set of 7,969 different transcripts. Sequences were grouped according to 4 main categories, metabolism (34%), cell cycle and plant development (11%), interaction with the environment (19%), and unknown function (36%), and further subdivided into 15 subcategories. Comparisons to other legume EST projects suggest that an entirely different repertoire of genes is expressed in common bean nodules. Phaseolus-specific contigs, gene families, and single nucleotide polymorphisms were also identified from the EST collection. Functional aspects of individual bean organs were reflected by the 20 contigs from each library composed of the most redundant ESTs. The abundance of transcripts corresponding to selected contigs was evaluated by RNA blots to determine whether gene expression determined by laboratory methods correlated with in silico expression. Evaluation of root nodule gene expression by macroarrays and RNA blots showed that genes related to nitrogen and carbon metabolism are integrated for ureide production. Resources developed in this project provide genetic and genomic tools for an international consortium devoted to bean improvement.

Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots.

Phosphorus (P), an essential element for plants, is one of the most limiting nutrients for plant growth. A few transcription factor (TF) genes involved in P-starvation signalling have been characterized for Arabidopsis thaliana and rice. Crop production of common bean (Phaseolus vulgaris L.), the most important legume for human consumption, is often limited by low P in the soil. Despite its agronomic importance, nothing is known about transcriptional regulation in P-deficient bean plants. We functionally characterized the P-deficiency-induced MYB TF TC3604 (Dana Farber Cancer Institute, Common Bean Gene Index v.2.0), ortholog to AtPHR1 (PvPHR1). For its study, we applied RNAi technology in bean composite plants. PvPHR1 is a positive regulator of genes implicated in P transport, remobilization and homeostasis. Although there are no reports on the regulatory roles of microRNAs (miRNA) in bean, we demonstrated that PvmiR399 is an essential component of the PvPHR1 signalling pathway. The analysis of DICER-like1 (PvDCL1) silenced bean composite plants suppressed for accumulation of PvmiR399 and other miRNAs suggested that miR399 is a negative regulator of the ubiquitin E2 conjugase: PvPHO2 expression. Our results set the basis for understanding the signalling for P-starvation responses in common bean and may contribute to crop improvement.

Improvement of Drought Tolerance and Grain Yield in Common Bean by Overexpressing Trehalose-6-Phosphate Synthase in Rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.

MicroRNA expression profile in common bean (Phaseolus vulgaris) under nutrient deficiency stresses and manganese toxicity.

*MicroRNAs (miRNAs) play a pivotal role in post-transcriptional regulation of gene expression in plants. Information on miRNAs in legumes is as yet scarce. This work investigates miRNAs in an agronomically important legume, common bean (Phaseolus vulgaris). *A hybridization approach employing miRNA macroarrays - printed with oligonucleotides complementary to 68 known miRNAs - was used to detect miRNAs in the leaves, roots and nodules of control and nutrient-stressed (phosphorus, nitrogen, or iron deficiency; acidic pH; and manganese toxicity) common bean plants. *Thirty-three miRNAs were expressed in control plants and another five were only expressed under stress conditions. The miRNA expression ratios (stress:control) were evaluated using principal component and hierarchical cluster analyses. A group of miRNAs responded to nearly all stresses in the three organs analyzed. Other miRNAs showed organ-specific responses. Most of the nodule-responsive miRNAs showed up-regulation. miRNA blot expression analysis confirmed the macroarray results. Novel miRNA target genes were proposed for common bean and the expression of selected targets was evaluated by quantitative reverse transcriptase-polymerase chain reaction. *In addition to the detection of previously reported stress-responsive miRNAs, we discovered novel common bean stress-responsive miRNAs, for manganese toxicity. Our data provide a foundation for evaluating the individual roles of miRNAs in common bean.

Global changes in the transcript and metabolic profiles during symbiotic nitrogen fixation in phosphorus-stressed common bean plants.

Phosphorus (P) deficiency is widespread in regions where the common bean (Phaseolus vulgaris), the most important legume for human consumption, is produced, and it is perhaps the factor that most limits nitrogen fixation. Global gene expression and metabolome approaches were used to investigate the responses of nodules from common bean plants inoculated with Rhizobium tropici CIAT899 grown under P-deficient and P-sufficient conditions. P-deficient inoculated plants showed drastic reduction in nodulation and nitrogenase activity as determined by acetylene reduction assay. Nodule transcript profiling was performed through hybridization of nylon filter arrays spotted with cDNAs, approximately 4,000 unigene set, from the nodule and P-deficient root library. A total of 459 genes, representing different biological processes according to updated annotation using the UniProt Knowledgebase database, showed significant differential expression in response to P: 59% of these were induced in P-deficient nodules. The expression platform for transcription factor genes based in quantitative reverse transcriptase-polymerase chain reaction revealed that 37 transcription factor genes were differentially expressed in P-deficient nodules and only one gene was repressed. Data from nontargeted metabolic profiles indicated that amino acids and other nitrogen metabolites were decreased, while organic and polyhydroxy acids were accumulated, in P-deficient nodules. Bioinformatics analyses using MapMan and PathExpress software tools, customized to common bean, were utilized for the analysis of global changes in gene expression that affected overall metabolism. Glycolysis and glycerolipid metabolism, and starch and Suc metabolism, were identified among the pathways significantly induced or repressed in P-deficient nodules, respectively.

Analysis of bacterial community during the fermentation of pulque, a traditional Mexican alcoholic beverage, using a polyphasic approach

In this study, the characterization of the bacterial community present during the fermentation of pulque, a traditional Mexican alcoholic beverage from maguey (Agave), was determined for the first time by a polyphasic approach in which both culture and non-culture dependent methods were utilized. The work included the isolation of lactic acid bacteria (LAB), aerobic mesophiles, and 16S rDNA clone libraries from total DNA extracted from the maguey sap (aguamiel) used as substrate, after inoculation with a sample of previously produced pulque and followed by 6-h fermentation. Microbiological diversity results were correlated with fermentation process parameters such as sucrose, glucose, fructose and fermentation product concentrations. In addition, medium rheological behavior analysis and scanning electron microscopy in aguamiel and during pulque fermentation were also performed. Our results showed that both culture and non-culture dependent approaches allowed the detection of several new and previously reported species within the alpha-, gamma-Proteobacteria and Firmicutes. Bacteria diversity in aguamiel was composed by the heterofermentative Leuconostoc citreum, L. mesenteroides, L. kimchi, the gamma-Proteobacteria Erwinia rhapontici, Enterobacter spp. and Acinetobacter radioresistens. Inoculation with previously fermented pulque incorporated to the system microbiota, homofermentative lactobacilli related to Lactobacillus acidophilus, several alpha-Proteobacteria such as Zymomonas mobilis and Acetobacter malorum, other gamma-Proteobacteria and an important amount of yeasts, creating a starting metabolic diversity composed by homofermentative and heterofermentative LAB, acetic and ethanol producing microorganisms. At the end of the fermentation process, the bacterial diversity was mainly composed by the homofermentative Lactobacillus acidophilus, the heterofermentative L. mesenteroides, Lactococcus lactis subsp. lactis and the alpha-Proteobacteria A. malorum. After a 6-h fermentation, 83.27% of total sugars detected after inoculation were consumed (228.4 mM hexose equivalents) and a carbon (C) recovery of 66.18% in fermentation products was estimated. They were produced 284.4 mM C as ethanol, 71.5 mM C as acetic acid and 19 mM C as lactic acid, demonstrating the presence of homo- and heterofermentative, acetic and alcoholic metabolisms in the final product. It was also found, after hydrolysis, that the exopolysaccharide produced during the fermentation was mainly composed by fructose residues, probably inulin or levan.

Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

BackgroundShikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis.ResultsBatch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (μ) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the μ decreased, whereas aromatic intermediate production reached its maximum. The double aroK-aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in μ, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).ConclusionsThe effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.

An RNA-Seq based gene expression atlas of the common bean

BackgroundCommon bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species.ResultsCombining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form.ConclusionsThese data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.