Oswaldo Valdés-López

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.

Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population

Cytosine DNA methylation is one avenue for passing information through cell divisions. Here, we present epigenomic analyses of soybean recombinant inbred lines (RILs) and their parents. Identification of differentially methylated regions (DMRs) revealed that DMRs mostly cosegregated with the genotype from which they were derived, but examples of the uncoupling of genotype and epigenotype were identified. Linkage mapping of methylation states assessed from whole-genome bisulfite sequencing of 83 RILs uncovered widespread evidence for local methylQTL. This epigenomics approach provides a comprehensive study of the patterns and heritability of methylation variants in a complex genetic population over multiple generations, paving the way for understanding how methylation variants contribute to phenotypic variation.

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.

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.

An Atlas of Soybean Small RNAs Identifies Phased siRNAs from Hundreds of Coding Genes

An extensive analysis of small RNAs in soybean identified many miRNAs and phased, secondary siRNA (phasiRNA) loci; some of these miRNAs were the triggers of the phasiRNA loci. Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNA-dependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets.

miR172 Regulates Soybean Nodulation

Micro-RNAs (miRNAs) play a pivotal role in the control of gene expression and regulate plant developmental processes. miRNA 172 (miR172) is a conserved miRNA in plants reported to control the expression of genes involved in developmental phase transition, floral organ identity, and flowering time. However, the specific role of miR172 in legume nodulation is undefined. Ectopic expression of soybean miR172 resulted in an increase in nodule numbers in transgenic roots and an increase in the expression of both symbiotic leghemoglobin and nonsymbiotic hemoglobin. These nodules showed higher levels of nitrogenase activity. Further analysis revealed a complex regulatory circuit in which miR156 regulates miR172 expression and controls the level of an AP2 transcription factor. The latter, either directly or indirectly, controls the expression of nonsymbiotic hemoglobin, which is essential for regulating the levels of nodulation.

Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

MicroRNAs are recognized as important post-transcriptional regulators in plants. Information about the roles of miRNAs in common bean (Phaseolus vulgaris L.), an agronomically important legume, is yet scant. The objective of this work was to functionally characterize the conserved miRNA: miR398b and its target Cu/Zn Superoxide Dismutase 1 (CSD1) in common bean. We experimentally validated a novel miR398 target: the stress up-regulated Nodulin 19 (Nod19). Expression analysis of miR398b and target genes –CSD1 and Nod19- in bean roots, nodules and leaves, indicated their role in copper (Cu) homeostasis. In bean plants under Cu toxicity miR398b was decreased and Nod19 and CSD1, that participates in reactive oxygen species (ROS) detoxification, were up-regulated. The opposite regulation was observed in Cu deficient bean plants; lower levels of CSD1 would allow Cu delivery to essential Cu-containing proteins. Composite common bean plants with transgenic roots over-expressing miR398 showed ca. 20-fold higher mature miR398b and almost negligible target transcript levels as well as increased anthocyanin content and expression of Cu-stress responsive genes, when subjected to Cu deficiency. The down-regulation of miR398b with the consequent up-regulation of its targets was observed in common bean roots during the oxidative burst resulting from short-time exposure to high Cu. A similar response occurred at early stage of bean roots inoculated with Rhizobium tropici, where an increase in ROS was observed. In addition, the miR398b down-regulation and an increase in CSD1 and Nod19 were observed in bean leaves challenged with Sclerotinia scleortiorum fungal pathogen. Transient over-expression of miR398b in Nicotiana benthamiana leaves infected with S. sclerotiorum resulted in enhanced fungal lesions. We conclude that the miR398b-mediated up-regulation of CSD and Nod19 is relevant for common bean plants to cope with oxidative stress generated in abiotic and biotic stresses.

Identification of candidate phosphorus stress induced genes in Phaseolus vulgaris through clustering analysis across several plant species

Common bean (Phaseolus vulgaris L.) is the worlds most important grain legume for direct human consumption. However, the soils in which common bean predominate are frequently limited by the availability of phosphorus (P). Improving bean yield and quality requires an understanding of the genes controlling P acquisition and use, ultimately utilising these genes for crop improvement. Here we report an in silico approach for the identification of genes involved in adaptation of P. vulgaris and other legumes to P-deficiency. Some 22 groups of genes from four legume species and Arabidopsis thaliana, encoding diverse functions, were identified as statistically over-represented in EST contigs from P-stressed tissues. By combining bioinformatics analysis with available micro / macroarray technologies and clustering results across five species, we identified 52 P. vulgaris candidate genes belonging to 19 categories as induced by P-stress response. Transport-related, stress (defence and regulation) signal transduction genes are abundantly represented. Manipulating these genes through traditional breeding methodologies and / or biotechnology approaches may allow us to improve crop P-nutrition.

The Micro-RNA172c-APETALA2-1 Node as a Key Regulator of the Common Bean-Rhizobium etli Nitrogen Fixation Symbiosis

A common bean microRNA, that targets a trancription factor, positively controls root development and symbiotic rhizobia infection and nodulation. Micro-RNAs are recognized as important posttranscriptional regulators in plants. The relevance of micro-RNAs as regulators of the legume-rhizobia nitrogen-fixing symbiosis is emerging. The objective of this work was to functionally characterize the role of micro-RNA172 (miR172) and its conserved target APETALA2 (AP2) transcription factor in the common bean (Phaseolus vulgaris)-Rhizobium etli symbiosis. Our expression analysis revealed that mature miR172c increased upon rhizobial infection and continued increasing during nodule development, reaching its maximum in mature nodules and decaying in senescent nodules. The expression of AP2-1 target showed a negative correlation with miR172c expression. A drastic decrease in miR172c and high AP2-1 mRNA levels were observed in ineffective nodules. Phenotypic analysis of composite bean plants with transgenic roots overexpressing miR172c or a mutated AP2-1 insensitive to miR172c cleavage demonstrated the pivotal regulatory role of the miR172 node in the common bean-rhizobia symbiosis. Increased miR172 resulted in improved root growth, increased rhizobial infection, increased expression of early nodulation and autoregulation of nodulation genes, and improved nodulation and nitrogen fixation. In addition, these plants showed decreased sensitivity to nitrate inhibition of nodulation. Through transcriptome analysis, we identified 114 common bean genes that coexpressed with AP2-1 and proposed these as being targets for transcriptional activation by AP2-1. Several of these genes are related to nodule senescence, and we propose that they have to be silenced, through miR172c-induced AP2-1 cleavage, in active mature nodules. Our work sets the basis for exploring the miR172-mediated improvement of symbiotic nitrogen fixation in common bean, the most important grain legume for human consumption.