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.

Wild Olive Genotypes as a Valuable Source of Resistance to Defoliating Verticillium dahliae.

Resistance to the defoliating pathotype of Verticillium dahliae has been evaluated in a pool of 68 wild genotypes of olive belonging to the SILVOLIVE collection. Resistance was evaluated by assessing symptom severity using a 0-4 rating scale, estimating the relative area under the disease progress curve (RAUDPC), determining the percentage of dead plants (PDP), and measuring the evolution of morphological parameters in inoculated plants over time. In addition, the density levels of V. dahliae in the stem of root-inoculated genotypes have been quantified by means of quantitative real-time PCR at 35 and 120 days after inoculation (dai). Fifteen genotypes (22%) were cataloged as resistant to V. dahliae (i.e., disease parameters did not significantly differ from those of the resistant cultivar Frantoio, or were even lower). Resistant genotypes are characterized by presenting fewer symptoms and a lower amount of V. dahliae DNA at 120 dai than at 35 dai, indicating their ability to control the disease and reduce the density of the pathogen. The rest of the evaluated genotypes showed variable levels of susceptibility. Overall analysis of all genotypes showed high correlation between symptomatology and the amount of V. dahliae DNA in the stem of inoculated genotypes at 120 dai, rather than at 35 dai. However, correlation at 120 dai was not observed in the set of resistant genotypes, suggesting that resistance to defoliating V. dahliae in olive is based on the occurrence of different mechanisms such as avoidance or tolerance. These mechanisms are valuable for designing breeding programs and for the identification of target genes and resistant rootstocks to better control Verticillium wilt in the olive grove.

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.

Hydraulic Traits Emerge as Relevant Determinants of Growth Patterns in Wild Olive Genotypes Under Water Stress.

The hydraulic traits of plants, or the efficiency of water transport throughout the plant hydraulic system, could help to anticipate the impact of climate change and improve crop productivity. However, the mechanisms explaining the role of hydraulic traits on plant photosynthesis and thus, plant growth and yield, are just beginning to emerge. We conducted an experiment to identify differences in growth patterns at leaf, root and whole plant level among four wild olive genotypes and to determine whether hydraulic traits may help to explain such differences through their effect on photosynthesis. We estimated the relative growth rate (RGR), and its components, leaf gas exchange and hydraulic traits both at the leaf and whole-plant level in the olive genotypes over a full year. Photosynthetic capacity parameters were also measured. We observed different responses to water stress in the RGRs of the genotypes studied being best explained by changes in the net CO2 assimilation rate (NAR). Further, net photosynthesis, closely related to NAR, was mainly determined by hydraulic traits, both at leaf and whole-plant levels. This was mediated through the effects of hydraulic traits on stomatal conductance. We observed a decrease in leaf area: sapwood area and leaf area: root area ratios in water-stressed plants, which was more evident in the olive genotype Olea europaea subsp. guanchica (GUA8), whose RGR was less affected by water deficit than the other olive genotypes. In addition, at the leaf level, GUA8 water-stressed plants presented a better photosynthetic capacity due to a higher mesophyll conductance to CO2 and a higher foliar N. We conclude that hydraulic allometry adjustments of whole plant and leaf physiological response were well coordinated, buffering the water stress experienced by GUA8 plants. In turn, this explained their higher relative growth rates compared to the rest of the genotypes under water-stress conditions.

Coping With Water Shortage: An Update on the Role of K+, Cl-, and Water Membrane Transport Mechanisms on Drought Resistance.

Drought is now recognized as the abiotic stress that causes most problems in agriculture, mainly due to the strong water demand from intensive culture and the effects of climate change, especially in arid/semi-arid areas. When plants suffer from water deficit (WD), a plethora of negative physiological alterations such as cell turgor loss, reduction of CO2 net assimilation rate, oxidative stress damage, and nutritional imbalances, among others, can lead to a decrease in the yield production and loss of commercial quality. Nutritional imbalances in plants grown under drought stress occur by decreasing water uptake and leaf transpiration, combined by alteration of nutrient uptake and long-distance transport processes. Plants try to counteract these effects by activating drought resistance mechanisms. Correct accumulation of salts and water constitutes an important portion of these mechanisms, in particular of those related to the cell osmotic adjustment and function of stomata. In recent years, molecular insights into the regulation of K+, Cl-, and water transport under drought have been gained. Therefore, this article brings an update on this topic. Moreover, agronomical practices that ameliorate drought symptoms of crops by improving nutrient homeostasis will also be presented.

Silent S-Type Anion Channel Subunit SLAH1 Gates SLAH3 Open for Chloride Root-to-Shoot Translocation.

Higher plants take up nutrients via the roots and load them into xylem vessels for translocation to the shoot. After uptake, anions have to be channeled toward the root xylem vessels. Thereby, xylem parenchyma and pericycle cells control the anion composition of the root-shoot xylem sap [1-6]. The fact that salt-tolerant genotypes possess lower xylem-sap Cl(-) contents compared to salt-sensitive genotypes [7-10] indicates that membrane transport proteins at the sites of xylem loading contribute to plant salinity tolerance via selective chloride exclusion. However, the molecular mechanism of xylem loading that lies behind the balance between NO3(-) and Cl(-) loading remains largely unknown. Here we identify two root anion channels in Arabidopsis, SLAH1 and SLAH3, that control the shoot NO3(-)/Cl(-) ratio. The AtSLAH1 gene is expressed in the root xylem-pole pericycle, where it co-localizes with AtSLAH3. Under high soil salinity, AtSLAH1 expression markedly declined and the chloride content of the xylem sap in AtSLAH1 loss-of-function mutants was half of the wild-type level only. SLAH3 anion channels are not active per se but require extracellular nitrate and phosphorylation by calcium-dependent kinases (CPKs) [11-13]. When co-expressed in Xenopus oocytes, however, the electrically silent SLAH1 subunit gates SLAH3 open even in the absence of nitrate- and calcium-dependent kinases. Apparently, SLAH1/SLAH3 heteromerization facilitates SLAH3-mediated chloride efflux from pericycle cells into the root xylem vessels. Our results indicate that under salt stress, plants adjust the distribution of NO3(-) and Cl(-) between root and shoot via differential expression and assembly of SLAH1/SLAH3 anion channel subunits.

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.

Physiological and gene expression responses of sunflower (Helianthus annuus L.) plants differ according to irrigation placement.

To investigate effects of soil moisture heterogeneity on plant physiology and gene expression in roots and leaves, three treatments were implemented in sunflower plants growing with roots split between two compartments: a control (C) treatment supplying 100% of plant evapotranspiration, and two treatments receiving 50% of plant evapotranspiration, either evenly distributed to both compartments (deficit irrigation - DI) or unevenly distributed to ensure distinct wet and dry compartments (partial rootzone drying - PRD). Plants receiving the same amount of water responded differently under the two irrigation systems. After 3 days, evapotranspiration was similar in C and DI, but 20% less in PRD, concomitant with decreased leaf water potential (Ψleaf) and increased leaf xylem ABA concentration. Six water-stress responsive genes were highly induced in roots growing in the drying soil compartment of PRD plants, and their expression was best correlated with local soil water content. On the other hand, foliar gene expression differed significantly from that of the root and correlated better with xylem ABA concentration and Ψleaf. While the PRD irrigation strategy triggered stronger physiological and molecular responses, suggesting a more intense and systemic stress reaction due to local dehydration of the dry compartment of PRD plants, the DI strategy resulted in similar water savings without strongly inducing these responses. Correlating physiological and molecular responses in PRD/DI plants may provide insights into the severity and location of water deficits and may enable a better understanding of long-distance signalling mechanisms.

Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production.

Whole-genome duplication, or polyploidy, is common in many plant species and often leads to better adaptation to adverse environmental condition. However, little is known about the physiological and molecular determinants underlying adaptation. We examined the drought tolerance in diploid (2x) and autotetraploid (4x) clones of Rangpur lime (Citrus limonia) rootstocks grafted with 2x Valencia Delta sweet orange (Citrus sinensis) scions, named V/2xRL and V/4xRL, respectively. Physiological experiments to study root-shoot communication associated with gene expression studies in roots and leaves were performed. V/4xRL was much more tolerant to water deficit than V/2xRL. Gene expression analysis in leaves and roots showed that more genes related to the response to water stress were differentially expressed in V/2xRL than in V/4xRL. Prior to the stress, when comparing V/4xRL to V/2xRL, V/4xRL leaves had lower stomatal conductance and greater abscisic acid (ABA) content. In roots, ABA content was higher in V/4xRL and was associated to a greater expression of drought responsive genes, including CsNCED1, a pivotal regulatory gene of ABA biosynthesis. We conclude that tetraploidy modifies the expression of genes in Rangpur lime citrus roots to regulate long-distance ABA signalling and adaptation to stress.

Cl- homeostasis in includer and excluder citrus rootstocks: transport mechanisms and identification of candidate genes.

To reveal specific Cl(-) transport activities in the symplastic pathway, uptake, long-distance transport and distribution of Cl(-) have been investigated in the citrus rootstocks Carrizo citrange (CC, Cl(-) includer) and Cleopatra mandarin (CM, Cl(-) excluder). Using an external concentration of 4.5 mm Cl(-) , both species actively transported Cl(-) to levels that exceeded the critical requirement concentration by one and two orders of magnitude in the excluder and the includer rootstocks, respectively. Both CC and CM modulated Cl(-) influx according to the availability of the nutrient as uptake capacity was induced by Cl(-) starvation, but inhibited after Cl(-) resupply. Net Cl(-) uptake was higher in the includer CC, an observation that correlated with a lower root-to-shoot transport capacity in the excluder CM. The patterns of tissue Cl(-) accumulation indicated that chloride exclusion in the salt-tolerant rootstock CM was caused by a reduced net Cl(-) loading into the root xylem. Genes CcCCC1, CcSLAH1 and CcICln1 putatively involved in the regulation of chloride transport were isolated and their expression analysed in response to both changes in the nutritional status of Cl(-) and salt stress. The previously uncharacterized ICln gene exhibited a strong repression to Cl(-) application in the excluder rootstock, suggesting a role in regulating Cl(-) homeostasis in plants.

Shared and novel molecular responses of mandarin to drought.

Drought is the most important stress experienced by citrus crops. A citrus cDNA microarray of about 6.000 genes has been utilized to identify transcriptomic responses of mandarin to water stress. As observed in other plant species challenged with drought stress, key genes for lysine catabolism, proline and raffinose synthesis, hydrogen peroxide reduction, vacuolar malate transport, RCI2 proteolipids and defence proteins such as osmotin, dehydrins and heat-shock proteins are induced in mandarin. Also, some aquaporin genes are repressed. The osmolyte raffinose could be detected in stressed roots while the dehydrin COR15 protein only accumulated in stressed leaves but not in roots. Novel drought responses in mandarin include the induction of genes encoding a new miraculin isoform, chloroplast beta-carotene hydroxylase, oleoyl desaturase, ribosomal protein RPS13A and protein kinase CTR1. These results suggest that drought tolerance in citrus may benefit from inhibition of proteolysis, activation of zeaxanthin and linolenoyl synthesis, reinforcement of ribosomal structure and down-regulation of the ethylene response.

Membrane transporters and carbon metabolism implicated in chloride homeostasis differentiate salt stress responses in tolerant and sensitive Citrus rootstocks.

Salinity tolerance in Citrus is strongly related to leaf chloride accumulation. Both chloride homeostasis and specific genetic responses to Cl(-) toxicity are issues scarcely investigated in plants. To discriminate the transcriptomic network related to Cl(-) toxicity and salinity tolerance, we have used two Cl(-) salt treatments (NaCl and KCl) to perform a comparative microarray approach on two Citrus genotypes, the salt-sensitive Carrizo citrange, a poor Cl(-) excluder, and the tolerant Cleopatra mandarin, an efficient Cl(-) excluder. The data indicated that Cl(-) toxicity, rather than Na(+) toxicity and/or the concomitant osmotic perturbation, is the primary factor involved in the molecular responses of citrus plant leaves to salinity. A number of uncharacterized membrane transporter genes, like NRT1-2, were differentially regulated in the tolerant and the sensitive genotypes, suggesting its potential implication in Cl(-) homeostasis. Analyses of enriched functional categories showed that the tolerant rootstock induced wider stress responses in gene expression while repressing central metabolic processes such as photosynthesis and carbon utilization. These features were in agreement with phenotypic changes in the patterns of photosynthesis, transpiration, and stomatal conductance and support the concept that regulation of transpiration and its associated metabolic adjustments configure an adaptive response to salinity that reduces Cl(-) accumulation in the tolerant genotype.

Identification and functional characterization of cation-chloride cotransporters in plants.

Chloride (Cl(-)) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation-Cl(-) cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub-family of animal K(+):Cl(-) cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co-ordinated symport of K(+), Na(+) and Cl(-), and this transport activity was inhibited by the 'loop' diuretic bumetanide, a specific inhibitor of vertebrate Na(+):K(+):Cl(-) cotransporters, indicating that At CCC encodes for a bona fide Na(+):K(+):Cl(-) cotransporter. Analysis of At CCC promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T-DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl(-) homeostasis under high salinity, as they accumulated higher and lower Cl(-) amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long-distance Cl(-) transport. Compelling evidence is provided on the occurrence of cation-chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl(-) homeostasis.