Chloride reduces plant nitrate requirement and alleviates low nitrogen stress symptoms.

Chloride (Cl-) is traditionally categorized as an antagonist of nitrate (NO3-) because Cl- hinders plant NO3- transport and accumulation. However, we have recently defined Cl- as a beneficial macronutrient for higher plants, due to specific functions that lead to more efficient use of water, nitrogen (N) and CO2 under optimal N and water supply. When accumulated in leaves at macronutrient levels, Cl- promotes growth through osmotic, physiological, metabolic, anatomical and cellular changes that improve plant performance under optimal NO3- nutrition. Nitrate over-fertilization in agriculture can adversely affect crop yield and nature, while its deficiency limits plant growth. To study the relationship between Cl- nutrition and NO3- availability, we have characterized different physiological responses such as growth and yield, N-use efficiency, water status, photosynthesis, leaf anatomy, pigments and antioxidants in tomato plants treated with or without 5 mM Cl- salts and increasing NO3- treatments (3-15 mM). First, we have demonstrated that 5 mM Cl- application can reduce the use of NO3- in the nutrient solution by up to half without detriment to plant growth and yield in tomato and other horticultural plants. Second, Cl- application reduced stress symptoms and improved plant growth under low-NO3- conditions. The Cl--dependent resistance to low-N stress resulted from: more efficient use of the available NO3-; improved plant osmotic and water status regulation; improved stomatal conductance and photosynthetic rate; and better antioxidant response. We proposed that beneficial Cl- levels increase the crop ability to grow better with lower NO3- requirements and withstand N deficiency, promoting a more sustainable and resilient agriculture.

Avoidant/resistant rather than tolerant olive rootstocks are more effective in controlling Verticillium wilt.

The identification of rootstocks of low susceptibility to Verticillium dahliae can become a valuable procedure to achieve effective control of Verticillium wilt in the olive grove. This not only involves the identification of suitable genotypes, but also the study of the interaction between the rootstock and the grafted scion. Thus, a rootstock that prevents or minimizes V. dahliae proliferation (avoidance/resistance strategy) can have very different effects on a susceptible scion compared to a rootstock that shows few or no symptoms despite being infected (tolerance strategy). Both resistance and tolerance mechanisms have been recently identified in wild olive genotypes with low susceptibility to V. dahliae. When used as rootstocks of the highly susceptible variety 'Picual', we found that resistant genotypes, including the cultivar 'Frantoio', were more effective than tolerant genotypes in controlling Verticillium wilt. Furthermore, tolerant genotypes were as ineffective as susceptible or extremely susceptible genotypes in controlling Verticillium wilt. We also identified rootstock-scion combinations with behaviours that were not expected according to the degree of susceptibility previously observed in the non-grafted rootstock. Although the rootstocks were able to control Verticillium wilt according to its degree of susceptibility to V. dahliae, the ability to control the infection was not adequately transferred to the grafted scion. Our results confirmed that: the degree of susceptibility to Verticillium wilt of an olive variety does not predict its performance as a rootstock; to use a very low susceptible genotype as rootstock of a susceptible scion increases the susceptibility of the genotype used as rootstock; in any case, avoidant/resistant rootstocks are more effective than tolerant rootstocks in reducing the susceptibility of the grafted plant to V. dahliae.

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.

From physiology to salt marsh management challenges with sea level rise: the case of native Spartina foliosa, invasive S. densiflora and their hybrid.

Sea level rise (SLR) imposes increasing salinity and inundation stresses in salt marshes which simultaneously face invasions by exotic plant species. We aimed to improve and apply knowledge on the ecophysiological responses of halophytes to SLR to conservation management of salt marshes. In a mesocosm experiment, we measured and compared phosphoenolpyruvate carboxylase (PEPC) activity and related functional traits of the California-native Spartina foliosa, invasive S. densiflora and their hybrid S. densiflora × foliosa in response to increasing levels of salinity and inundation. S. foliosa was moderately sensitive to salinity, showing a 57% reduction in PEPC specific activity from freshwater to hypersalinity. This native species compensated for the reduction of PEPC activity with increased salinity through 80% higher enzyme activation by phosphorylation. PEPC functional trait responses of S. foliosa were mostly independent of inundation depth. In view of these results, managers should conserve undeveloped lands for accommodation space above current high tide lines to facilitate colonization of stress-tolerant S. foliosa. Our results on functional responses of PEPC traits recorded high sensitivity to salinity for S. densiflora. This was reflected by 65% lower PEPC specific activity together with increasing accumulation of free proline (+96%) and total proteins (+23%) with elevated salinity. These results suggest prioritized eradication of S. densiflora populations in brackish habitats. Measured PEPC responses support the high stress tolerance of the S. densiflora × foliosa hybrid. PEPC traits for the hybrid were mostly independent of salinity and inundation. The hybrid showed higher PEPC-specific activity than S. foliosa (+70%) and S. densiflora (+15%) in freshwater under intermediate inundation. Results suggest that eradication of the hybrid should be the highest management priority. Our study shows that the responses of key functional physiological traits to environmental stresses serve as biological indicators that can guide ecosystem management practices in a scenario of climate change.

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.

Changes to the functional traits of phosphoenolpyruvate carboxylase following hybridization in C-4 halophytes.

Hybridization is a relevant evolutionary mechanism linked to the invasiveness of plant species, but little is known about its effect on enzymatic activities in response to stress. We analyzed the effects of salinity on key mechanistic traits of phosphoenolpyruvate carboxylase (PEPC) enzyme for two hybrid taxa derived from native Spartina maritima (Curtis) Fernald and invasive Spartina densiflora Brongn. in comparison with their parental species. Parental species showed contrasted strategies at the PEPC level to cope with salinity. Spartina maritima showed its physiological optimum at 10 to 40 ppt salinity, with high PEPC activity (per unit leaf soluble protein), in contrast to the lower salinity optimum of 0.5 and 10 ppt for S. densiflora, where highest levels of PEPC apparent specific activity coincided with high light-induced activation of PEPC. Both hybrids showed constant PEPC apparent specific activity from fresh water to hypersalinity and exhibited higher net photosynthesis rates in fresh water than their parents. Spartina maritima × densiflora presented three transgressive PEPC-related traits, being the only taxon able to increase its PEPC activation in darkness at high salinity. Spartina densiflora × maritima showed most PEPC-related traits intermediate between its parents. Inheritance types operating differently in reciprocal hybrids determine key functional traits conditioning their ecological performance.

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.