Shotgun proteomics profiling of chia seeds (Salvia hispanica L.) reveals genotypic differential responses to viability loss.

Introduction: Exposure to elevated temperatures and relative humidity expedites the seed aging process, finally leading to seed viability loss. In this context, certain proteins play a pivotal role in safeguarding the longevity of seeds. However, the seedproteomic response to loss viability in Salvia hispanica L., commonly known as chia, remains incompletely understood. Methods: This work explores the application of proteomics as a potent tool for uncovering molecular responses to viability loss caused by artificial aging in two chia genotypes, WN and MN. Results: By using a quantitative label-free proteomics analysis (LC-MS/MS), 1787 proteins wereidentified in chia seeds at a 95% confidence level, including storage proteins, heat shock proteins (HSPs), late embryogenesis abundant proteins (LEA),oleosins, reactive oxygen species (ROS)-related enzymes, and ribosomal proteins. A relatively low percentage of exclusive proteins were identified in viable and non-viable seeds. However, proteins exhibiting differential abundancebetween samples indicated variations in the genotype and physiological status. Specifically, the WN genotype showed 130 proteins with differential abundancecomparing viable and non-viable seeds, while MN displayed changes in the abundance of 174 proteins. While both showed a significant decrease in keyproteins responsible for maintaining seed functionality, longevity, and vigor withhigh-temperature and humidity conditions, such as LEA proteins or HSPs, ROS, and oleosins, distinct responses between genotypes were noted, particularly in ribosomal proteins that were accumulated in MN and diminished in WN seeds. Discussion: Overall, the results emphasize the importance of evaluating changes in proteins of viable and non-viable seeds as they offer valuable insights into the underlying biological mechanisms responsible for the maintenance of chia seed integrity throughout high-temperature and humidity exposure.

From 'Farm to Fork': Exploring the Potential of Nutrient-Rich and Stress-Resilient Emergent Crops for Sustainable and Healthy Food in the Mediterranean Region in the Face of Climate Change Challenges.

In the dynamic landscape of agriculture and food science, incorporating emergent crops appears as a pioneering solution for diversifying agriculture, unlocking possibilities for sustainable cultivation and nutritional bolstering food security, and creating economic prospects amid evolving environmental and market conditions with positive impacts on human health. This review explores the potential of utilizing emergent crops in Mediterranean environments under current climate scenarios, emphasizing the manifold benefits of agricultural and food system diversification and assessing the impact of environmental factors on their quality and consumer health. Through a deep exploration of the resilience, nutritional value, and health impacts of neglected and underutilized species (NUS) such as quinoa, amaranth, chia, moringa, buckwheat, millet, teff, hemp, or desert truffles, their capacity to thrive in the changing Mediterranean climate is highlighted, offering novel opportunities for agriculture and functional food development. By analysing how promoting agricultural diversification can enhance food system adaptability to evolving environmental conditions, fostering sustainability and resilience, we discuss recent findings that underscore the main benefits and limitations of these crops from agricultural, food science, and health perspectives, all crucial for responsible and sustainable adoption. Thus, by using a sustainable and holistic approach, this revision analyses how the integration of NUS crops into Mediterranean agrifood systems can enhance agriculture resilience and food quality addressing environmental, nutritional, biomedical, economic, and cultural dimensions, thereby mitigating the risks associated with monoculture practices and bolstering local economies and livelihoods under new climate scenarios.

Evaluating Yield, Nutritional Quality, and Environmental Impact of Quinoa Straws across Mediterranean Water Environments.

Quinoa (Chenopodium quinoa Willd.) is a promising and versatile crop due to its remarkable adaptability to diverse environments and the exceptional nutritional value of its seeds. Nevertheless, despite the recent extensive research on quinoa seeds, the straw associated with this crop has received comparatively little attention. The valorisation of this by-product provides an opportunity to improve the overall outcomes of quinoa cultivation. In this work, three quinoa varieties were evaluated for two years (2019 and 2020) under three different Mediterranean water environments (irrigation, fresh rainfed, and hard rainfed), aiming to assess the straw yield and nutritional quality and to study the changes in the crop nutritional uptake associated with different water environmental conditions. The nutritional analysis included the quantification of the ash, crude protein, crude fat, minerals (P, K, Ca, Mg), and fibre (gross fibre (GF), acid detergent fibre (ADF), neutral detergent fibre (NDF), acid detergent lignin (ADL), hemicellulose, cellulose) contents. As the results reveal, most of the parameters evaluated were susceptible to change mainly with the water environment but also with the genotype (or their interaction), including the yield, crude protein, relative feed value (RFV), and mineral content, which generally decreased under water-limiting conditions. Moreover, a comparative analysis revealed that straw Ca, Mg, and K contents were generally higher than in seeds. Overall, this study demonstrates that quinoa straw quality is genotypic and environmentally dependent, and these factors should be considered when aiming at improving straw feed value for livestock nutrition.

Assessment of the changes in seed yield and nutritional quality of quinoa grown under rainfed Mediterranean environments.

Climate change is considered a serious threat to agriculture and food security. It is linked to rising temperatures and water shortages, conditions that are expected to worsen in the coming decades. Consequently, the introduction of more drought-tolerant crops is required. Quinoa (Chenopodium quinoa Willd.) has received great attention worldwide due to the nutritional properties of its seeds and its tolerance to abiotic stress. In this work, the agronomic performance and seed nutritional quality of three quinoa varieties were studied during two consecutive years (2019-2020) under three water environmental conditions of Southwestern Europe (irrigated conditions, fresh rainfed, and hard rainfed) with the goal of determining the impact of rainfed conditions on this crop performance. High precipitations were recorded during the 2020 growing season resulting in similar grain yield under irrigation and fresh rainfed conditions. However, in 2019, significant yield differences with penalties under water-limiting conditions were found among the evaluated environmental conditions. Furthermore, nutritional and metabolomic differences were observed among seeds harvested from different water environments including the progressive accumulation of glycine betaine accompanied by an increase in saponin and a decrease in iron with water limitation. Generally, water-limiting environments were associated with increased protein contents and decreased yields preserving a high nutritional quality despite particular changes. Overall, this work contributes to gaining further knowledge about how water availability affects quinoa field performance, as it might impact both seed yield and quality. It also can help reevaluate rainfed agriculture, as water deficit can positively impact the nutritional quality of seeds.

Unveiling changes in rhizosphere-associated bacteria linked to the genotype and water stress in quinoa.

Drought is among the main abiotic factors causing agronomical losses worldwide. To minimize its impact, several strategies have been proposed, including the use of plant growth-promoting bacteria (PGPBs), as they have demonstrated roles in counteracting abiotic stress. This aspect has been little explored in emergent crops such as quinoa, which has the potential to contribute to reducing food insecurity. Thus, here we hypothesize that the genotype, water environment and the type of inoculant are determining factors in shaping quinoa rhizosphere bacterial communities, affecting plant performance. To address this, two different quinoa cultivars (with contrasting water stress tolerance), two water conditions (optimal and limiting water conditions) and different soil infusions were used to define the relevance of these factors. Different bacterial families that vary among genotypes and water conditions were identified. Certain families were enriched under water stress conditions, such as the Nocardioidaceae, highly present in the water-sensitive cultivar F15, or the Pseudomonadaceae, Burkholderiaceae and Sphingomonadaceae, more abundant in the tolerant cultivar F16, which also showed larger total polyphenol content. These changes demonstrate that the genotype and environment highly contribute to shaping the root-inhabiting bacteria in quinoa, and they suggest that this plant species is a great source of PGPBs for utilization under water-liming conditions.

Molecular, Metabolic and Physiological Responses to Boron Stress in Higher Plants.

Although the essentiality of boron (B) for plant growth has recently been questioned [...].

Shotgun proteomics of quinoa seeds reveals chitinases enrichment under rainfed conditions.

Quinoa is an Andean crop whose cultivation has been extended to many different parts of the world in the last decade. It shows a great capacity for adaptation to diverse climate conditions, including environmental stressors, and, moreover, the seeds are very nutritious in part due to their high protein content, which is rich in essential amino acids. They are gluten-free seeds and contain good amounts of other nutrients such as unsaturated fatty acids, vitamins, or minerals. Also, the use of quinoa hydrolysates and peptides has been linked to numerous health benefits. Altogether, these aspects have situated quinoa as a crop able to contribute to food security worldwide. Aiming to deepen our understanding of the protein quality and function of quinoa seeds and how they can vary when this crop is subjected to water-limiting conditions, a shotgun proteomics analysis was performed to obtain the proteomes of quinoa seeds harvested from two different water regimes in the field: rainfed and irrigated conditions. Differentially increased levels of proteins determined in seeds from each field condition were analysed, and the enrichment of chitinase-related proteins in seeds harvested from rainfed conditions was found. These proteins are described as pathogen-related proteins and can be accumulated under abiotic stress. Thus, our findings suggest that chitinase-like proteins in quinoa seeds can be potential biomarkers of drought. Also, this study points to the need for further research to unveil their role in conferring tolerance when coping with water-deficient conditions.

What Can Boron Deficiency Symptoms Tell Us about Its Function and Regulation?

On the eve of the 100th anniversary of Dr. Warington's discovery of boron (B) as a nutrient essential for higher plants, "boronists" have struggled to demonstrate a role beyond its structural function in cell walls dimerizing pectin molecules of rhamnogalacturonan II (RGII). In this regard, B deficiency has been associated with a plethora of symptoms in plants that include macroscopic symptoms like growth arrest and cell death and biochemical or molecular symptoms that include changes in cell wall pore size, apoplast acidification, or a steep ROS production that leads to an oxidative burst. Aiming to shed light on B functions in plant biology, we proposed here a unifying model integrating the current knowledge about B function(s) in plants to explain why B deficiency can cause such remarkable effects on plant growth and development, impacting crop productivity. In addition, based on recent experimental evidence that suggests the existence of different B ligands other than RGII in plant cells, namely glycolipids, and glycoproteins, we proposed an experimental pipeline to identify putative missing ligands and to determine how they would integrate into the above-mentioned model.

Changes in nutritional quality-related traits of quinoa seeds under different storage conditions.

Within the context of climate change and its impact on global food security, seed storage has become key, as it ensures long-term food and next-season seed preservation. Aiming at evaluating quality-related changes in quinoa seeds over storage time, different storage temperatures (-20, 4, 12, 25, and 37°C) and humidity conditions (use of silica gel or not) were studied and different seed nutritional parameters were evaluated at different points during a year of storage. Also, to determine if these variations could be conditioned by the genotype used, two quinoa cultivars were compared. The results proved that quinoa seed quality is highly dependent on the storage temperature but is not consistently affected by the use of silica gel if the seed moisture content (SMC) is kept between 5 and 12%. Furthermore, quality can be maintained and even improved by keeping SMC lower than 12% and storage temperatures low (4°C). Under these conditions (at 4°C in hermetic packaging with or without silica gel), and after 12 months of storage, there was an increase in amino acids like isoleucine, serine, arginine, glycine, and glutamic acid and in seed viability and germination. On the contrary, quinoa seeds stored at 37°C showed an accumulation of reactive oxygen species (ROS) which was related to a lower antioxidant capacity and a reduction in the contents of essential amino acids like isoleucine, lysine, histidine, and threonine, resulting in a delayed and reduced germination capacity, and, therefore, lower seed quality. Besides, quality-related differences appeared between cultivars highlighting differences linked to the genotype. Overall, this work demonstrates that optimal storage temperatures and SMC can preserve or even improve quinoa seed nutritional quality, which in turn can impact food safety and agriculture.

Changes in Quinoa Seed Fatty Acid Profile Under Heat Stress Field Conditions.

The nutritional quality of quinoa is often related to the high protein content of their seeds. However, and despite not being an oilseed crop, the oil composition of quinoa seeds is remarkable due to its profile, which shows a high proportion of polyunsaturated fatty acids (PUFAs), particularly in essential fatty acids such as linoleic (ω-6) and α-linolenic (ω-3). In line with this, this study aimed at evaluating the effect of elevated temperatures on the oil composition of different quinoa cultivars grown in the field in two consecutive years (i.e., 2017 and 2018). In 2017, heat stress episodes resulted in a reduced oil content and lower quality linked to decreased ratios of oleic acid:linoleic acid, larger omega-6 (ω-6) to omega-3 (ω-3) ratios, and lower monounsaturated fatty acid (MUFA) and higher PUFA contents. Furthermore, the correlations found between mineral nutrients such as phosphorous (P) and the contents of oleic and linoleic acids emphasize the possibility of optimizing oil quality by controlling fertilization. Overall, the results presented in this study show how the environmental and genetic factors and their interaction may impact oil quality in quinoa seeds.

Genotype-Dependent Variation of Nutritional Quality-Related Traits in Quinoa Seeds.

Exploiting the relationship between the nutritional properties of seeds and the genetic background constitutes an essential analysis, which contributes to broadening our knowledge regarding the control of the nutritional quality of seeds or any other edible plant structure. This is an important aspect when aiming at improving the nutritional characteristics of crops, including those of Chenopodium quinoa Willd. (quinoa), which has the potential to contribute to food security worldwide. Previous works have already described changes in the nutritional properties of quinoa seeds due to the influence of the environment, the genotype, or their interaction. However, there is an important limitation in the analyses carried out, including the outcomes that can be translated into agronomical practices and their effect on seed quality. In the present study, several seed nutritional-related parameters were analyzed in 15 quinoa cultivars grown in a particular environmental context. Important agronomical and nutritional differences were found among cultivars, such as variations in mineral or protein contents and seed viability. More importantly, our analyses revealed key correlations between seed quality-related traits in some cultivars, including those that relate yield and antioxidants or yield and the germination rate. These results highlight the importance of considering the genotypic variation in quinoa when selecting improved quinoa varieties with the best nutritional characteristics for new cultivation environments.

Heat Stress Impact on Yield and Composition of Quinoa Straw under Mediterranean Field Conditions.

Quinoa (Chenopodium quinoa Willd.) is receiving increasing attention globally due to the high nutritional value of its seeds, and the ability of this crop to cope with stress. In the current climate change scenario, valorization of crop byproducts is required to support a climate-smart agriculture. Furthermore, research works characterizing and evaluating quinoa stems and their putative uses are scarce. In this work, straw yield and composition, and the relative feed value of five quinoa varieties, were analyzed in two consecutive years (2017-2018) under field conditions in Southwestern Europe. High temperatures were recorded during the 2017 growing season resulting in significantly decreased straw yield and improved feed value, associated with compositional changes under elevated temperatures. Crude protein, ash, phosphorus, and calcium contents were higher under high temperatures, whereas fiber contents decreased. The relative feed value was also higher in 2017 and differed among varieties. Differences among varieties were also found in straw yield, and contents of phosphorus, potassium, and calcium. Overall, the results presented here support a sustainable quinoa productive system by encouraging straw valorization and shedding light on the mechanisms underlying heat-stress responses in this crop.

Studying the Impact of Different Field Environmental Conditions on Seed Quality of Quinoa: The Case of Three Different Years Changing Seed Nutritional Traits in Southern Europe.

Chenopodium quinoa Willd (quinoa) has acquired an increased agronomical and nutritional relevance due to the capacity of adaptation to different environments and the exceptional nutritional properties of their seeds. These include high mineral and protein contents, a balanced amino acid composition, an elevated antioxidant capacity related to the high phenol content, and the absence of gluten. Although it is known that these properties can be determined by the environment, limited efforts have been made to determine the exact changes occurring at a nutritional level under changing environmental conditions in this crop. To shed light on this, this study aimed at characterizing variations in nutritional-related parameters associated with the year of cultivation and different genotypes. Various nutritional and physiological traits were analyzed in seeds of different quinoa cultivars grown in the field during three consecutive years. We found differences among cultivars for most of the nutritional parameters analyzed. It was observed that the year of cultivation was a determinant factor in every parameter studied, being 2018 the year with lower yields, germination rates, and antioxidant capacity, but higher seed weights and seed protein contents. Overall, this work will greatly contribute to increase our knowledge of the impact of the environment and genotype on the nutritional properties of quinoa seeds, especially in areas that share climatic conditions to Southern Europe.

Medicago truncatula Yellow Stripe-Like7 encodes a peptide transporter participating in symbiotic nitrogen fixation.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.

Soybean Yellow Stripe-like 7 is a symbiosome membrane peptide transporter important for nitrogen fixation.

Legumes form a symbiosis with rhizobia that convert atmospheric nitrogen (N2) to ammonia and provide it to the plant in return for a carbon and nutrient supply. Nodules, developed as part of the symbiosis, harbor rhizobia that are enclosed in a plant-derived symbiosome membrane (SM) to form an organelle-like structure called the symbiosome. In mature nodules exchanges between the symbionts occur across the SM. Here we characterize Yellow Stripe-like 7 (GmYSL7), a Yellow stripe-like family member localized on the SM in soybean (Glycine max) nodules. It is expressed specifically in infected cells with expression peaking soon after nitrogenase becomes active. Unlike most YSL family members, GmYSL7 does not transport metals complexed with phytosiderophores. Rather, it transports oligopeptides of between four and 12 amino acids. Silencing GmYSL7 reduces nitrogenase activity and blocks infected cell development so that symbiosomes contain only a single bacteroid. This indicates the substrate of YSL7 is required for proper nodule development, either by promoting symbiosome development directly or by preventing inhibition of development by the plant. RNAseq of nodules where GmYSL7 was silenced suggests that the plant initiates a defense response against rhizobia with genes encoding proteins involved in amino acid export downregulated and some transcripts associated with metal homeostasis altered. These changes may result from the decrease in nitrogen fixation upon GmYSL7 silencing and suggest that the peptide(s) transported by GmYSL7 monitor the functional state of the bacteroids and regulate nodule metabolism and transport processes accordingly. Further work to identify the physiological substrate for GmYSL7 will allow clarification of this role.

MtNIP5;1, a novel Medicago truncatula boron diffusion facilitator induced under deficiency.

BACKGROUND: Legumes comprise important crops that offer major agronomic benefits, including the capacity of establishing symbiosis with rhizobia, fixing atmospheric N2. It has been proven that legumes are particularly susceptible to boron (B) stress, which leads to important yield penalties. Boron (B) deficiency or toxicity in plants causes the inhibition of growth and an altered development. Under such conditions, the participation of two distinct protein families (the major intrinsic protein family MIP and the Boron transporter family BOR) is required to minimize detrimental effects caused by B stress. However, in legumes, little is known about the transport mechanisms responsible for B uptake and distribution, especially under deficiency. RESULTS: A Medicago truncatula protein, MtNIP5;1 (Medtr1g097840) (homologous to the Arabidopsis thaliana AtNIP5;1) was identified as a novel legume B transporter involved in B uptake under deficiency. Further analyses revealed that this M. truncatula aquaporin expression was boron-regulated in roots, being induced under deficiency and repressed under toxicity. It localizes at the plasma membrane of root epidermal cells and in nodules, where B plays pivotal roles in symbiosis. Furthermore, the partial complementation of the nip5;1-1 A. thaliana mutant phenotype under B deficiency supports a functional role of MtNIP5;1 as a B transporter in this legume model plant. CONCLUSIONS: The results here presented support a functional role of MtNIP5;1 in B uptake under deficiency and provides new insights into B transport mechanisms in legume species.

The Medicago truncatula Yellow Stripe1-Like3 gene is involved in vascular delivery of transition metals to root nodules.

Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.

Altered plant organogenesis under boron deficiency is associated with changes in high-mannose N-glycan profile that also occur in animals.

Boron (B) deficiency affects the development of Pisum sativum nodules and Arabidopsis thaliana root meristems. Both organs show an alteration of cell differentiation that result in the development of tumor-like structures. The fact that B in plants is not only able to interact with components of the cell wall but also with membrane-associated glycoconjugates, led us to analyze changes in high mannose type N-glycans (HMNG). The affinoblots with concanavalin A revealed alterations in the N-glycosylation pattern during early development of nodules and roots under B deprivation. Besides, there is increasing evidence of a B role in animal physiology that brought us to investigate the impact of B deficiency on Danio rerio (zebrafish) development. When B deficiency was induced prior to early cleavage stages, embryos developed as an abnormal undifferentiated mass of cells. Additionally, when B was removed at post-hatching, larvae undergo aberrant organogenesis. Resembling the phenomenon described in plants, alteration of the N-glycosylation pattern occurred in B-deficient zebrafish larvae prior to organogenesis. Overall, these results support a common function of B in plants and animals associated with glycosylation that might be important for cell signaling and cell fate determination during development.

Nitrogen physiology of contrasting genotypes of Chenopodium quinoa Willd. (Amaranthaceae).

Quinoa has been highlighted as a promising crop to sustain food security. The selection of physiological traits that allow identification genotypes with high Nitrogen use efficiency (NUE) is a key factor to increase Quinoa cultivation. In order to unveil the underpinning mechanisms for N-stress tolerance in Quinoa, three genotypes with similar phenology, but different NUE were developed under high (HN) or low (LN) nitrogen conditions. N metabolism processes and photosynthetic performance were studied after anthesis and in correlation with productivity to identify principal traits related to NUE. We found that protein content, net photosynthesis and leaf dry-mass were determinant attributes for yield at both HN and LN conditions. Contrastingly, the enhancement of N related metabolites ([Formula: see text], proline, betacyanins) and processes related with re-assimilation of [Formula: see text], including an increment of glutamine synthetase activity and up-regulation of CqAMT1,1 transporter expression in leaves, were negatively correlated with grain yield at both N conditions. Biochemical aspects of photosynthesis and root biomass were traits exclusively associated with grain yield at LN. The impact of N supply on seed quality is discussed. These results provide new insights towards the understanding the N metabolism of Quinoa.

MtMTP2-Facilitated Zinc Transport Into Intracellular Compartments Is Essential for Nodule Development in Medicago truncatula.

Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula.