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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.

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.

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., This work was supported by the Ministerio de Ciencia e Innovación (MICINN, Spain) (PID2019-105748RA-I00), the Madrid Government (Comunidad de Madrid-Spain) under the Multiannual Agreement with Universidad Autónoma de Madrid in the line of action encouraging youth research doctors, in the context of the V PRICIT (Regional Programme of Research and Technological Innovation) (SI1/PJI/2019-00124), the CYTED (ValSe-Food 119RT0567), the FPI UAM Fellowship Programme 2019 (to SG-R), and the Ramón y Cajal Programme 2019 (to MR).

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.

13 Pags.- 5 Figs. © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology., 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., This research was funded by a European Research Council Starting Grant (ERC-2013-StG-335284) and the Spanish State Research Agency (AEI) grant (AGL2015-65866-P) to MGG, and a AEI grant (AGL2016-75226-R) to JA and AA-F co-financed by the European Regional Development Fund (FEDER). RC-R and FJJ-P were supported by a Formación del Personal Investigador fellowship (BES2013-062674 and BES-2017-082913, respectively). IA was the recipient of a Juan de la Cierva- Formación postdoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FJCI-2017- 33222). VE was partially funded by the Severo Ochoa Programme for Centres of Excellence in R&D from the AEI (grant SEV-2016- 0672) received by the Centro de Biotecnología y Genómica de Plantas (UPM-INIA). Development of the M. truncatula Tnt1 mutant population was, in part, funded by the National Science Foundation, USA (DBI-0703285) to KSM. AM and HK and the µXRF measurements were supported by the Ministry of Education, Youth and Sports of the Czech Republic with co-financing from the European Union (grant ‘KOROLID’, CZ.02.1.01/0.0/0.0/15_003/0000336) and the Czech Academy of Sciences (RVO: 60077344).

Yellow Stripe-Like (YSL) proteins are a family of plant transporters typically involved in transition metal homeostasis. The substrate of three of the four YSL clades (clades I, II, and IV) are metal complexes with non-proteinogenic amino acid nicotianamine or its derivatives. No such transport capabilities have been shown for any member of the remaining clade (clade III), which is able to translocate short peptides across the membranes instead. The connection between clade III YSL members and metal homeostasis might have been masked by the functional redundancy characteristic of this family. This might have been circumvented in legumes through neofunctionalization of YSLs to ensure a steady supply of transition metals for symbiotic nitrogen fixation in root nodules. To test this possibility,Medicago truncatulaclade III transporter MtYSL7 has been studied both when the plant was fertilized with ammonium nitrate or when nitrogen had to be provided by endosymbiotic rhizobia within the root nodules. MtYSL7 is a plasma membrane protein expressed in the vasculature and in the nodule cortex. This protein is able to transport short peptides into the cytosol, although none with known metal homeostasis roles. ReducingMtYSL7expression levels resulted in diminished nitrogen fixation rates. In addition, nodules of mutant lines lackingYSL7accumulated more copper and iron, the later the likely result of increased expression in roots of iron uptake and delivery genes. The available data is indicative of a role of MtYSL7, and likely other clade III YSLs, in transition metal homeostasis.ONE SENTENCE SUMMARYMedicago truncatulaYSL7 is a peptide transporter required for symbiotic nitrogen fixation in legume nodules, likely controlling transition metal allocation to these organs.

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 with the nodule cells, a process in which vascular transport is essential. As occurs in root-to-shoot transport, members of the Yellow Stripe-Like (YSL) family of metal transporters should also be required for root-to-nodule metal delivery. The genome of the model legumeMedicago truncatulaencodes for eight YSL proteins, four of them with a high degree of similarity toArabidopsis thalianaYSLs 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 expression levels 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 theysl3mutant. These data suggest a role of MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.HighlightMedicago truncatulaYSL3 transporter is required for optimal nitrogen fixation in root nodules, mediating iron and zinc distribution in these organs.

Significant advances have been made in the last years trying to identify regulatory pathways that control plant responses to boron (B) deficiency. Still, there is a lack of a deep understanding of how they act regulating growth and development under B limiting conditions. Here, we analyzed the impact of B deficit on cell division leading to root apical meristem (RAM) disorganization. Our results reveal that inhibition of cell proliferation under the regulatory control of cytokinins (CKs) is an early event contributing to root growth arrest under B deficiency. An early recovery of QC46:GUS expression after transferring B-deficient seedlings to control conditions revealed a role of B in the maintenance of QC identity whose loss under deficiency occurred at later stages of the stress. Additionally, the D-type cyclin CYCD3 overexpressor and triple mutant cycd3;1-3 were used to evaluate the effect on mitosis inhibition at the G1-S boundary. Overall, this study supports the hypothesis that meristem activity is inhibited by B deficiency at early stages of the stress as it does cell elongation. Likewise, distinct regulatory mechanisms seem to take place depending on the severity of the stress. The results presented here are key to better understand early signaling responses under B deficiency, This work was supported by the MINECO BIO2012-32796 Spanish grant, the Juan de la Cierva Fellowship Program (JCI-2012-14172) (MINECO, Spain) (to M.R), the Postgraduate Studies Scholarship Programme (UAM, Spain) (to L.P.V.) and the FPU Fellowship Program (AP2010-4786) (MECD, Spain) (to I.A)

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.

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.

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 ($${{\rm{NH}}}_{4}^{+}$$ NH 4 + , proline, betacyanins) and processes related with re-assimilation of $${{\rm{NH}}}_{4}^{+}$$ NH 4 + , 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.

The present study was aimed at investigating the role of intracellular free calcium, [Ca2+]c, in the early cellular response of the green alga Chlamydomonas reinhardtii to the emergent pollutant Triclosan (13.8μM; 24h of exposure). There is a growing concern about the persistence and toxicity of this antimicrobial in aquatic environments, where non-target organisms such as C. reinhardtii, a primary producer of ecological relevance, might be severely impacted. A mechanistic study was undertaken which combined flow cytometry protocols, physiological as well as gene expression analysis. As an early response, Triclosan strongly altered [Ca2+]c homeostasis which could be prevented by prechelation with the intracellular calcium chelator BAPTA-AM. Triclosan induced ROS overproduction which ultimately leads to oxidative stress with loss of membrane integrity, membrane depolarization, photosynthesis inhibition and mitochondrial membrane depolarization; within this context, Triclosan also induced an increase in caspase 3/7 activity and altered the expression of metacaspase genes which are indicative of apoptosis. All these adverse outcomes were dependent on [Ca2+]c. Interestingly, an interconnection between [Ca2+]c alterations and increased ROS formation by Triclosan was found. Taken altogether these results shed light on the mechanisms behind Triclosan toxicity in the green alga Chlamydomonas reinhardtii and demonstrate the role of [Ca2+]c in mediating the observed toxicity.

Aiming to counteract B deficiency impacts, plants have developed different strategies in order to reach an optimal growth in soils with limited B availability. These include B transport mechanisms that involves a facilitated transport, via channel proteins, and a high-affinity active transport driven by borate transporters. The AtNIP5;1 channel protein is a member of Major Intrinsic Protein family which facilitates B influx into the roots under low B supply. In order to explore the phytohormone-dependent regulation of AtNIP5;1, the effects of abscisic acid (ABA), ethylene, auxins and cytokinins on the activity of AtNIP5;1 promoter were evaluated using the reporter line pNIP5;1-GUS. The results show that ABA treatment increased pAtNIP5;1 activity. Besides, a larger B uptake was found following ABA treatment under B deficiency suggesting a role of ABA inducing B uptake. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) caused an induction of AtNIP5;1 expression although did not correlate with higher B concentrations nor with an improvement in root growth. On the contrary, auxins and cytokinins caused slight changes in pAtNIP5;1 induction. Altogether, these results show a regulatory role of phytohormones in AtNIP5;1 promoter what may affect B transport. The herein provided information may contribute to better understand the regulation of B transport in plants towards minimizing B deficiency impacts on agriculture.

Cells depend on the homeostatic maintenance of pH within specific cellular compartments to ensure optimal conditions for metabolic and enzymatic processes as well as protein structure and function. In the animal secretory pathway, cells maintain distinct luminal pHs within various compartments (Paroutis et al., 2004). Among the many molecular players that contribute to pH and ion homeostasis in plants, Na+(K+)/H+ exchangers (also known as NHX-type cation/H+ antiporters) appear to be particularly important for the regulation of a wide variety of physiological processes, including cell expansion, cell volume regulation, osmotic adjustment, pH regulation, membrane trafficking, protein processing, and cellular stress responses (Pardo et al., 2006; Rodriguez-Rosales et al., 2009; Bassil et al., 2012).

Quinoa cultivation has been expanded around the world in the last decade and is considered an exceptional crop with the potential of contributing to food security worldwide. The exceptional nutritional value of quinoa seeds relies on their high protein content, their amino acid profile that includes a good balance of essential amino acids, the mineral composition and the presence of antioxidants and other important nutrients such as fiber or vitamins. Although several studies have pointed to the influence of different environmental stresses in certain nutritional components little attention has been paid to the effect of the agroecological context on the nutritional properties of the seeds what may strongly impact on the consumer food's quality. Thus, aiming to evaluate the effect of the agroecological conditions on the nutritional profile of quinoa seeds we analyzed three quinoa cultivars (Salcedo-INIA, Titicaca and Regalona) at different locations (Spain, Peru and Chile). The results revealed that several nutritional parameters such as the amino acid profile, the protein content, the mineral composition and the phytate amount in the seeds depend on the location and cultivar while other parameters such as saponin or fiber were more stable across locations. Our results support the notion that nutritional characteristics of seeds may be determined by seed's origin and further analysis are needed to define the exact mechanisms that control the changes in the seeds nutritional properties., This work was supported by the CEAL-AL/2015-27 Banco Santander-UAM grant (Spain), the PROMETEO/2017/189 grant from the Generalitat Valenciana (Spain) and the Juan de la Cierva Fellowship Program (JCI-2012-14172) (MINECO, Spain) (to María Reguera).

Water deficit stress followed by re-watering during grain filling resulted in the induction of the ornithine pathway and in changes in Quinoa grain quality. The genetic diversity of Chenopodium quinoa Willd. (Quinoa) is accompanied by an outstanding environmental adaptability and high nutritional properties of the grains. However, little is known about the biochemical and physiological mechanisms associated with the abiotic stress tolerance of Quinoa. Here, we characterized carbon and nitrogen metabolic changes in Quinoa leaves and grains in response to water deficit stress analyzing their impact on the grain quality of two lowland ecotypes (Faro and BO78). Differences in the stress recovery response were found between genotypes including changes in the activity of nitrogen assimilation-associated enzymes that resulted in differences in grain quality. Both genotypes showed a common strategy to overcome water stress including the stress-induced synthesis of reactive oxygen species scavengers and osmolytes. Particularly, water deficit stress induced the stimulation of the ornithine and raffinose pathways. Our results would suggest that the regulation of C- and N partitioning in Quinoa during grain filling could be used for the improvement of the grain quality without altering grain yields.

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH −1.5) and Arabidopsis thaliana root cells (ΔpH −2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H+ ATPase–dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.