Nitrogen assimilation and photorespiration become more efficient under chloride nutrition as a beneficial macronutrient.

Chloride (Cl-) and nitrate ( NO 3 - ) are closely related anions involved in plant growth. Their similar physical and chemical properties make them to interact in cellular processes like electrical balance and osmoregulation. Since both anions share transport mechanisms, Cl- has been considered to antagonize NO 3 - uptake and accumulation in plants. However, we have recently demonstrated that Cl- provided at beneficial macronutrient levels improves nitrogen (N) use efficiency (NUE). Biochemical mechanisms by which beneficial Cl- nutrition improves NUE in plants are poorly understood. First, we determined that Cl- nutrition at beneficial macronutrient levels did not impair the NO 3 - uptake efficiency, maintaining similar NO 3 - content in the root and in the xylem sap. Second, leaf NO 3 - content was significantly reduced by the treatment of 6 mM Cl- in parallel with an increase in NO 3 - utilization and NUE. To verify whether Cl- nutrition reduces leaf NO 3 - accumulation by inducing its assimilation, we analysed the content of N forms and the activity of different enzymes and genes involved in N metabolism. Chloride supply increased transcript accumulation and activity of most enzymes involved in NO 3 - assimilation into amino acids, along with a greater accumulation of organic N (mostly proteins). A reduced glycine/serine ratio and a greater ammonium accumulation pointed to a higher activity of the photorespiration pathway in leaves of Cl--treated plants. Chloride, in turn, promoted higher transcript levels of genes encoding enzymes of the photorespiration pathway. Accordingly, microscopy observations suggested strong interactions between different cellular organelles involved in photorespiration. Therefore, in this work we demonstrate for the first time that the greater NO 3 - utilization and NUE induced by beneficial Cl- nutrition is mainly due to the stimulation of NO 3 - assimilation and photorespiration, possibly favouring the production of ammonia, reductants and intermediates that optimize C-N re-utilization and plant growth. This work demonstrates new Cl- functions and remarks on its relevance as a potential tool to manipulate NUE in plants.

Chloride nutrition improves drought resistance by enhancing water deficit avoidance and tolerance mechanisms.

Chloride (Cl-), traditionally considered harmful for agriculture, has recently been defined as a beneficial macronutrient with specific roles that result in more efficient use of water (WUE), nitrogen (NUE), and CO2 in well-watered plants. When supplied in a beneficial range of 1-5 mM, Cl- increases leaf cell size, improves leaf osmoregulation, and reduces water consumption without impairing photosynthetic efficiency, resulting in overall higher WUE. Thus, adequate management of Cl- nutrition arises as a potential strategy to increase the ability of plants to withstand water deficit. To study the relationship between Cl- nutrition and drought resistance, tobacco plants treated with 0.5-5 mM Cl- salts were subjected to sustained water deficit (WD; 60% field capacity) and water deprivation/rehydration treatments, in comparison with plants treated with equivalent concentrations of nitrate, sulfate, and phosphate salts. The results showed that Cl- application reduced stress symptoms and improved plant growth during water deficit. Drought resistance promoted by Cl- nutrition resulted from the simultaneous occurrence of water deficit avoidance and tolerance mechanisms, which improved leaf turgor, water balance, photosynthesis performance, and WUE. Thus, it is proposed that beneficial Cl- levels increase the ability of crops to withstand drought, promoting a more sustainable and resilient agriculture.

Chloride Improves Nitrate Utilization and NUE in Plants.

Chloride (Cl-) has traditionally been considered harmful to agriculture because of its toxic effects in saline soils and its antagonistic interaction with nitrate (NO3 -), which impairs NO3 - nutrition. It has been largely believed that Cl- antagonizes NO3 - uptake and accumulation in higher plants, reducing crop yield. However, we have recently uncovered that Cl- has new beneficial macronutrient, functions that improve plant growth, tissue water balance, plant water relations, photosynthetic performance, and water-use efficiency. The increased plant biomass indicates in turn that Cl- may also improve nitrogen use efficiency (NUE). Considering that N availability is a bottleneck for the plant growth, the excessive NO3 - fertilization frequently used in agriculture becomes a major environmental concern worldwide, causing excessive leaf NO3 - accumulation in crops like vegetables and, consequently, a potential risk to human health. New farming practices aimed to enhance plant NUE by reducing NO3 - fertilization should promote a healthier and more sustainable agriculture. Given the strong interaction between Cl- and NO3 - homeostasis in plants, we have verified if indeed Cl- affects NO3 - accumulation and NUE in plants. For the first time to our knowledge, we provide a direct demonstration which shows that Cl-, contrary to impairing of NO3 - nutrition, facilitates NO3 - utilization and improves NUE in plants. This is largely due to Cl- improvement of the N-NO3 - utilization efficiency (NUTE), having little or moderate effect on N-NO3 - uptake efficiency (NUPE) when NO3 - is used as the sole N source. Clear positive correlations between leaf Cl- content vs. NUE/NUTE or plant growth have been established at both intra- and interspecies levels. Optimal NO3 - vs. Cl- ratios become a useful tool to increase crop yield and quality, agricultural sustainability and reducing the negative ecological impact of NO3 - on the environment and on human health.

SILVOLIVE, a Germplasm Collection of Wild Subspecies With High Genetic Variability as a Source of Rootstocks and Resistance Genes for Olive Breeding.

Wild subspecies of Olea europaea constitute a source of genetic variability with huge potential for olive breeding to face global changes in Mediterranean-climate regions. We intend to identify wild olive genotypes with optimal adaptability to different environmental conditions to serve as a source of rootstocks and resistance genes for olive breeding. The SILVOLIVE collection includes 146 wild genotypes representative of the six O. europaea subspecies and early-generations hybrids. These genotypes came either from olive germplasm collections or from direct prospection in Spain, continental Africa and the Macaronesian archipelago. The collection was genotyped with plastid and nuclear markers, confirming the origin of the genotypes and their high genetic variability. Morphological and architectural parameters were quantified in 103 genotypes allowing the identification of three major groups of correlative traits including vigor, branching habits and the belowground-to-aboveground ratio. The occurrence of strong phenotypic variability in these traits within the germplasm collection has been shown. Furthermore, wild olive relatives are of great significance to be used as rootstocks for olive cultivation. Thus, as a proof of concept, different wild genotypes used as rootstocks were shown to regulate vigor parameters of the grafted cultivar "Picual" scion, which could improve the productivity of high-density hedgerow orchards.

Chloride as a Beneficial Macronutrient in Higher Plants: New Roles and Regulation.

Chloride (Cl-) has traditionally been considered a micronutrient largely excluded by plants due to its ubiquity and abundance in nature, its antagonism with nitrate (NO3-), and its toxicity when accumulated at high concentrations. In recent years, there has been a paradigm shift in this regard since Cl- has gone from being considered a harmful ion, accidentally absorbed through NO3- transporters, to being considered a beneficial macronutrient whose transport is finely regulated by plants. As a beneficial macronutrient, Cl- determines increased fresh and dry biomass, greater leaf expansion, increased elongation of leaf and root cells, improved water relations, higher mesophyll diffusion to CO2, and better water- and nitrogen-use efficiency. While optimal growth of plants requires the synchronic supply of both Cl- and NO3- molecules, the NO3-/Cl- plant selectivity varies between species and varieties, and in the same plant it can be modified by environmental cues such as water deficit or salinity. Recently, new genes encoding transporters mediating Cl- influx (ZmNPF6.4 and ZmNPF6.6), Cl- efflux (AtSLAH3 and AtSLAH1), and Cl- compartmentalization (AtDTX33, AtDTX35, AtALMT4, and GsCLC2) have been identified and characterized. These transporters have proven to be highly relevant for nutrition, long-distance transport and compartmentalization of Cl-, as well as for cell turgor regulation and stress tolerance in plants.

Chloride as a macronutrient increases water-use efficiency by anatomically driven reduced stomatal conductance and increased mesophyll diffusion to CO2.

Chloride (Cl- ) has been recently described as a beneficial macronutrient, playing specific roles in promoting plant growth and water-use efficiency (WUE). However, it is still unclear how Cl- could be beneficial, especially in comparison with nitrate (NO3- ), an essential source of nitrogen that shares with Cl- similar physical and osmotic properties, as well as common transport mechanisms. In tobacco plants, macronutrient levels of Cl- specifically reduce stomatal conductance (gs ) without a concomitant reduction in the net photosynthesis rate (AN ). As stomata-mediated water loss through transpiration is inherent in the need of C3 plants to capture CO2 , simultaneous increase in photosynthesis and WUE is of great relevance to achieve a sustainable increase in C3 crop productivity. Our results showed that Cl- -mediated stimulation of larger leaf cells leads to a reduction in stomatal density, which in turn reduces gs and water consumption. Conversely, Cl- improves mesophyll diffusion conductance to CO2 (gm ) and photosynthetic performance due to a higher surface area of chloroplasts exposed to the intercellular airspace of mesophyll cells, possibly as a consequence of the stimulation of chloroplast biogenesis. A key finding of this study is the simultaneous improvement of AN and WUE due to macronutrient Cl- nutrition. This work identifies relevant and specific functions in which Cl- participates as a beneficial macronutrient for higher plants, uncovering a sustainable approach to improve crop yield.

Chloride regulates leaf cell size and water relations in tobacco plants.

Chloride (Cl(-)) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl(-) when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl(-)-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5 mM Cl(-)) and no water limitation, Cl(-) specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1-5 mM range, Cl(-) played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl(-) also altered water relations at the whole-plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. In contrast to Cl(-), these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl(-) responds to adaptive functions improving water homeostasis in higher plants.