Joint research project

Response and adaptation to drought in common bean during symbiotic nitrogen fixation: exploring new mutants for better seed quality and sustainable crop production.

Project leaders
Francesca Sparvoli, Georgina Hernandez Delgado
MESSICO - CONACYT - Consejo Nacional de Ciencia y Tecnologia
CNR/CONACYT 2012-2014
Agriculture and Food
Thematic area
Biology, agriculture and food sciences
Status of the project

Research proposal

Common bean, the most important grain legume for human consumption in the world, comprises 50% of the legumes consumed worldwide and meets very well the increasing demand of consumers for a better nutritional quality and healthy properties of plant derived food. It is highly produced in Latin America and in the Mediterranean basin, however its production is limited by several biotic and abiotic stresses such as nutrient deficiency and drought.
Drought is one of the adverse conditions that more commonly affect terrestrial plants and is considered one of the most important limiting factors for plant growth and productivity. Plants adapt to drought through several types of responses at the transcriptional, post-transcriptional, translational and post-translational levels, some of which involve the maintenance of water balance. These responses are mediated by conserved signalling pathways resulting in the induction of detoxification systems that control and repair damage, modify water allocation and regulate growth.
In the developing world bean is a small farmer crop grown under rain-fed conditions thus the probability of the crop facing drought are high. The importance of abiotic stress is unquestionable, especially in low input agricultural systems. Estimates suggest that 50% of bean production suffers from phosphorus deficiency and 73% from drought. Climate change will bring even more challenges since its effects are most frequently cited in terms of risk of drought. Higher temperatures and greater evapotranspiration, combined with lower rainfall is expected to exacerbate drought in important bean producing areas.
Like other legumes, bean has the capacity to establish symbiosis with nitrogen-fixing soil bacteria (rhizobia). Rhizobial root infection elicits the formation of a specialized plant organ: the nodule, where differentiated bacteria (bacteroids) reduce atmospheric nitrogen to ammonia by the action of the nitrogenase enzyme complex (Nase). Legume-rhizobia associations confer many advantages for agricultural improvement. Symbiotic nitrogen fixation (SNF) is a key biological process with economic and ecological interests since nitrogen is limiting factor for legume production and these plants are of great importance for food and feed. This association might increase nutrient uptake from soils, and reduces the need for fertilizers preventing the accumulation of nitrates and phosphates in agricultural soils. Another important advantage of SNF is related to an increasing legume tolerance to abiotic stresses such as drought and salt or nutrient deficiencies or toxicities.
Drought is one of the most important environmental factors that drastically affect SNF and legume crop production, as a consequence of diverse physiological mechanisms that affect Nase activity. Different hypothesis have been raised to understand drought effect on nitrogen fixation. One suggestion is that it might be due to a decrease in carbon flux to the bacteroids by inhibition of the sucrose synthase causing an accumulation of sucrose in nodules and a decrease in the contribution of malate. Also, nitrogen fixation drought tolerance may be associated with levels of nitrogen-compounds, such as ureides, that are transported from the nodule to the leaves. Among the ureide producers tested, common bean showed relatively lower levels of ureides and a higher degree of nitrogen fixation drought tolerance compared to other legume crops. On the other hand, the involvement of a rapid osmotic mechanism implicated in the operation of the nodule oxygen diffusion barrier has been proposed for several years. Other evidences suggest that the cells of the inner cortex are osmocontractile and may collapse to reduce the size of intercellular spaces and/or expel water into them. Thus, oxygen availability inside the nodules decreased and bacteroid respiration cannot be maintained.
The establishment of an effective symbiotic association between rhizobia and legumes requires the expression of several bacterial and plant genes. In nitrogen-fixing bacteria, oxygen concentration regulates expression of genes related to microaerobic and symbiotic life styles. Although symbiotic nitrogen-fixing bacteria are aerobic organisms, they are exposed to an oxygen-limited environment inside the nodules, which allows Nase activity to be preserved. L. Girard group (CCG-UNAM) has reported a R. etli null mutant defective in a novel Crp/Fnr-type transcriptional regulator, named StoRd (for symbiotic terminal oxidase regulator). This mutant enhances the nodule gene expression of fixNOQP (encoding the symbiotic cbb3 terminal oxidase) and the cbb3 protein content. An outstanding feature is that the absence of the stoRd gene product improved the capacity of R. etli CFN42 for symbiotic nitrogen fixation: ca. 2-fold increase in Nase activity correlating with 30% higher shoot dry weight and 45% higher plant nitrogen content was observed in bean plants inoculated with a StoRd mutant strain. These data indicate that stoRd plays a crucial repressive role during the symbiosis of R. etli with bean plants (Granados-Baeza et al. 2007, MPMI 20: 1242-1249). Preliminary results indicate that bean plants inoculated with StoRd showed an improved drought resistance as compared to those inoculated with wild type rhizobia. This advantage may be derived from the improved respiratory capacity of the StoRd strain that is a relevant factor involved in alleviating the decreased oxygen availability found in drought stressed nodules.
The common bean "low phytic acid" (lpa1) mutant identified by the group of F. Sparvoli (IBBA-CNR) is the main subject of study in this and our past collaborative projects. Phytic acid (PA) is one of the antinutritional factors present in bean seeds that can severely impair the adsorption of nutrients. It causes losses of micronutrients since it is a strong chelator of mineral bivalent cations forming with them insoluble complexes that are indigestible for humans and monogastric animals. lpa mutants from bean and other crops have been obtained as a strategy for increasing micronutrient content, especially iron and zinc, in plant derived food by lowering seed PA content (Campion et al. 2010, TAG 118:1211-1221). From an agronomical point of view, lpa mutants are often associated with negative effects on seed and plant performance, such as compromised germination and emergence, lower stress tolerance and poor seed filling. Although there are some exceptions, like the Arabidopsis mutant Atmrp5 (bearing a mutation in the PA transporter), which is drought tolerant, or the barley lpa1-1, which showed a good agronomic performance in both irrigated and non-irrigated environments, projects aimed at obtaining lpa crops should take into account aspects regarding agronomic potential.
The bean lpa1 mutant shows several nutritionally important seed characteristics such as 90% reduction in PA, 25% reduction of raffinosaccharides and 7-fold increase of free iron cations. Moreover, unlike other strong lpa mutants (with up to 90% of PA reduction), lpa1 shows no negative pleiotropic effects on traits of agronomic relevance. The lpa1 mutation is due to a defective MRP type ABC transporter gene (Pvmrp1), orthologous to Arabidopsis AtMRP5/AtABCC5 (Panzeri et al. 2011, New Phytol 191:70-83). Biochemical and molecular investigations showed that the Pvmrp1 mutation causes a general repression of the PA pathway, indicating that transient accumulation of PA, or its degradation products, may reduce its biosynthesis through a negative feedback mechanism. Moreover, we detected increased ABA sensitivity during germination of the mutant that apparently correlates with the lower myo-inositol content observed in the in lpa1 seeds, a finding supported by data on Arabidopsis mutants with reduced myo-inositol content. These data, together with new others emerging from the literature, indicate an important role of the PA itself and/or of compounds involved in its metabolism  in plant cell signalling, development, and hormone signalling regulation.
This project aims to characterize the response and adaptation of SNF common bean to drought, exploring drastic and mild stress conditions both in wt genotypes and lpa1 mutant. For this we will take advantage of the drought tolerance improved symbionts that each of the participating groups (IBBA-CNR and CCG-UNAM) have obtained: the lpa1 bean mutant and the StoRd R. etli mutant. The preliminary characterization of the response to drought using the described biological material (bean and rhizobia mutants) has only been made separately -either for the plant or for the bacteria- by each partner that obtained the respective mutant. So the complementary analysis of the mentioned symbiotic association can only be done establishing a bilateral collaborative project such as this one, an academic association to investigate a relevant biological association.
Justification of the international cooperation
The scientists responsible for the proposed project are Drs. G. Hernández (CCG-UNAM) and F. Sparvoli (IBBA-CNR). Our groups share scientific interests and goals in plant biology (molecular biology, genetics and genomic) specifically related to common bean and have been performing collaborative research for more than four years. Our ongoing collaboration has been supported by CNR with individual formation and mobility grants to FS in 2008 and by bilateral CNR-CONACyT agreement  2009-2011 (project: "Characterization of the lpa-280-10 (low phytic acid) mutant of common bean: transcriptome and functional analyses of the response to phosphorus deficiency"). Due to the close collaborative work and the complementary expertise of both groups, sounded achievements were derived from our past project. We are performing final analyses of the obtained data and we will submitt manuscripts to high impact journals.
This new project proposal aims to extend our previous collaborative work to explore other constraints that limit bean growth taking advantage of our complementary expertise and biological materials (drought vs. SNF and lpa1 bean mutant vs. StoRd R. etli mutant). The complementary expertise, equipments and materials of the Italian and Mexican groups guarantee the success of the project and, together with young scientists training, will be reciprocally available during and after the project. Within the ongoing bilateral CNR-CONACyT project the partners developed a 90K custom microarray chip, based on Combimatrix technology, that will be used in this proposal to perform transcriptional analysis aiming to address the role of StoRd mutation in SNF in common bean nodules. The imminent setup of a genomic and bioinformatic platform at IBBA-CNR, (framed in the project INTEROMICS, CNR-MIUR) will then provide the technology and expertise needed for the RNA-seq analyses here proposed. The expertise of the Mexican partner in the study of the symbiotic interaction between bean plant and R. etli will be essential to address the understanding of the complexity of the plant-microbe interactions when plant are subjected to drought.
The advancement of our collaborative project requires a close collaboration between the two groups and the proposed bilateral visits are essential to perform experiments, train young scientists, review the progress of the project, discuss results, plan the presentation of the results in related meetings or workshops and prepare of manuscripts for the publication of the results.

Research goals

The main scope of this project is to analyze the effect of the R. etli StoRd mutant strain in SNF and in plant response to drought in the symbiotic interactions with wt and the biofortified lpa bean mutant, using physiological and functional genomics approaches.
We aim to elucidate the role of the R. etli StoRd mutant strain in the control of SNF and ammonia assimilation by the plant and probably in improving the ability of beans to respond/adapt to drought stress. In the project we will also assess if the lpa mutation could improve the ability of the plant to cope with the drought condition and to establish an efficient symbiotic interaction with R. etli wt and StoRd mutant strain. 
Specific objectives:

Set-up of the most appropriate experimental conditions for common bean growth under drastic and mild drought treatments, with and without R. etli symbiosis
Phenotypic and physiological characterization of the control vs. stressed and non-inoculated vs. SNF plants
Transcriptome analyses (chips) to address the role of StoRd mutation in common bean SNF. It is expected to identify genes that are differentially expressed in StoRd bacteroids and their host nodules, as compared to the wild type symbiosis. Based on these results, the expression of selected genes will be recorded as part of the phenotypic characterization of control vs drought stressed SNF plants.
Comprehensive transcriptome analyses (RNA-seq) to define differential gene expression in leaves and nodules from plants grown in selected conditions (previously defined in this project, objectives 1, 2) displaying the most contrasting phenotypes regarding adaptation to drought stress.

Last update: 27/11/2021