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This work evaluates new citrus hybrid tolerance to salt stress compared to Carrizo citrange. Growth parameters, ion concentration, photosynthetic parameters, leaf water relation, plant damage and proline concentration were assessed in plants irrigated with 0 (control) or 60 mM of NaCl for 60 days. Some hybrids presented marked salt-stress symptomatology like sharp decreases in plant biomass and RGR parameter, and high leaf burn and abscission percentages. While Cl− accumulation in most salinised genotypes was recorded in all the plant fractions, accumulation occurred in some hybrids only in their aerial organs. Stomatal conductance also lowered under salt conditions in all the hybrids, whereas the internal CO2 concentration (Ci) tended to increase. Salt stress lowered the leaf water potentials in all the 0501 hybrids. As a response, the salinised plants induced proline accumulation. The main trait that improved salt tolerance in some genotypes is apparently linked with the imbalance among Cl−, Na+ and K+ ions, especially enhanced K+ uptake and transport rates which delay plant damage.

This work compared the expression of the main B transport-related genes (some members of the aquaporin family -NIP5, TIP5 and PIP1- and some efflux-B transporters -BOR1 and BOR4-), and the response of some physiological parameters in two citrus species [Citrus macrophylla W. (CM) and Citrus aurantium L. (CA)] under moderate and low boron (B) conditions. Seedlings were cultured “in vitro” in media supplemented with 50 or 0 μM H3BO3. NIP5, BOR1 and PIP1 expressions were enhanced by low B levels in both genotypes. TIP5 was down-regulated in the roots and leaves of the CA0 seedlings, and in the roots of CM0. BOR4 also lowered in the roots of both species at 0 μM H3BO3. Consequently, citrus species showed a common tolerance mechanism to low B conditions based on the synergism among transport channel NIP5, non-selective aquaporin PIP1 and transporter BOR1, and the impairment of genes TIP5 and BOR4 related with tolerance responses to B-toxic conditions. However, the CA genotype displayed low B symptoms earlier than CM (reduced plant biomass, length, relative growth rate and chlorophyll content). Proline concentration was higher in CM0 than in CA0 leaves, while the latter also enhanced malonaldehyde content. Although both plants had similar B concentrations, they differed in B content and B partitioning fractions. Whereas the CA genotype was more affected by lack of B treatment as more B was needed inside the cell, the more minimal need of cell B in CM favoured its allocation in the insoluble fraction and allowed growth in this genotype. In conclusion, B compartmentalisation seems critical in tolerance to low B level in citrus.

Background The citrus genus comprises a number of sensitive tropical and subtropical species to cold stress, which limits global citrus distribution to certain latitudes and causes major economic loss. We used RNA-Seq technology to analyze changes in the transcriptome of Valencia delta seedless orange in response to long-term cold stress grafted on two frequently used citrus rootstocks: Carrizo citrange (CAR), considered one of the most cold-tolerant accessions; C. macrophylla (MAC), a very sensitive one. Our objectives were to identify the genetic mechanism that produce the tolerant or sensitive phenotypes in citrus, as well as to gain insights of the rootstock-scion interactions that induce the cold tolerance or sensitivity in the scion. Results Plants were kept at 1 ºC for 30 days. Samples were taken at 0, 15 and 30 days. The metabolomic analysis showed a significant increase in the concentration of free sugars and proline, which was higher for the CAR plants. Hormone quantification in roots showed a substantially increased ABA concentration during cold exposure in the CAR roots, which was not observed in MAC. Different approaches were followed to analyze gene expression. During the stress treatment, the 0-15-day comparison yielded the most DEGs. The functional characterization of DEGs showed enrichment in GO terms and KEGG pathways related to abiotic stress responses previously described in plant cold adaption. The DEGs analysis revealed that several key genes promoting cold adaption were up-regulated in the CAR plants, and those repressing it had higher expression levels in the MAC samples. Conclusions The metabolomic and transcriptomic study herein performed indicates that the mechanisms activated in plants shortly after cold exposure remain active in the long term. Both the hormone quantification and differential expression analysis suggest that ABA signaling might play a relevant role in promoting the cold hardiness or sensitiveness of Valencia sweet orange grafted onto Carrizo citrange or Macrophylla rootstocks, respectively. Our work provides new insights into the mechanisms by which rootstocks modulate resistance to abiotic stress in the production variety grafted onto them.

In this study, different pepper rootstocks are tested for their ability to overcome heat stress situations. This work aims to evaluate: (i) the physiological mechanisms that occur during long heat stress periods (7 days) under controlled conditions in a pepper variety grafted onto accessions; (ii) the heat stress behaviour of these grafted pepper plants under greenhouse conditions in terms of marketable yields. For this purpose, plants of Lamuyo-type sweet pepper ‘Herminio F1’ (VA), grafted onto six accessions (VA/A25, VA/A31, VA/A34, VA/A52, VA/A57, VA/A6), and a self-grafted variety (VA/VA) were grown under controlled conditions in growth chambers (28/24 °C, day/night temperatures and 38/24 °C for control and heat stress, respectively) and under greenhouse conditions (38/24 °C). For the controlled conditions, relative growth rate, leaf area, electrolyte leakage, chlorophyll a fluorescence and heat shock proteins were determined. For the greenhouse conditions, fresh and dry weigh, electrolyte leakage and fruit yield were determined. Our results confirmed that grafting a pepper cultivar onto appropriate rootstocks such as A6, A25 and A57 can overcome the negative effects of heat stress conditions with a higher relative growth rate, leaf area and Fv/Fm, and lower electrolyte leakage under the controlled conditions, and with higher marketable yields under the greenhouse conditions.

This work quantifies Fe uptake in young citrus trees, its partitioning among plant compartments, and the contribution of the Fe absorbed from fertilizer to the development of new tissues. A soil pot experiment was conducted using 4-year-old clementine trees (Citrus clementina Hort ex Tan), and a dose of 240 mg Fe was applied by labeled fertilizer (92% atom 57Fe excess). Plants were uprooted at five different phenologic states: end of flowering (May 15), end of fruit setting and fruit drop (July 1), two fruit growing moments (August 1 and October 15), and at complete fruit maturity (December 10). The Fe accumulated in the root system exceeded 90% of the total Fe content in the plant. All organs progressively enriched with 57Fe (8.5&ndash, 15.5% and 7.4&ndash, 9.9% for young and old organs, respectively). Reproductive ones reached the highest increase (111% between May and October). 57Fe enrichment from woody organs reflects an increasing gradient to sink organs. The root system accumulated 80% of the Fe absorbed from the fertilizer, but the young organs accumulated relatively more Fe uptake during flowering and fruit setting (15.6% and 13.8%, respectively) than old organs (around 9.8%). Although iron derived from fertilizer (Fedff) preferably supplied young organs (16.7&ndash, 31.0%) against old ones (2.5&ndash, 14.9%), it only represented between 13.8% and 21.4% of its content. The use efficiency of the applied Fe (FeUE) barely exceeded 15%. The lowest FeUE were found in young and old organs of the aerial part (1.1&ndash, 1.8% and 0.7&ndash, 1.2%, respectively). Since the pattern of the seasonal absorption of Fe is similar to the monthly distribution curve of the supplied Fe, it is recommended that the application of Fe chelates in calcareous soils should be performed in a similar way to that proposed in this curve.

Low temperature is one of the abiotic stresses that most limits crop production, growth and distribution. Currently climate change weather alters temperature worldwide, which produces both high and low temperatures. This work aimed to know how citrus responds to long-term low temperatures after being submitted to 1 °C day-night during six weeks. Physiological parameters were analyzed and it was obtained that photosynthesis and growth drastically decreased, water potential increased and osmotic potential did not change. Fv/Fm (maximum quantum yield of PSII) lowered, and degradation of chlorophylls a and b took place. The proline concentration in leaves moderately increased. The expression of two genes from the glutamate synthesis route (P5CS1 and P5CS2) and one gene from the ornithine route (dOAT) was quantified. The results reveal that the dOAT transcripts from the proline synthesis gene underwent greater induction than P5CS1 and P5CS2. A marked increase in the expression of proline degradative genes (PDH and P5CDH), a drop in starch, a decrease in the concentration of soluble sugars, and higher total nitrogen quantification in leaves were also observed. Citrus plants stopped growing, but mobilized reserve substances to survive cold stress conditions.

Solanum pimpinellifolium and Solanum lycopersicum var. cerasiforme represent a valuable tool for tomato breeding, particularly for tolerance to abiotic stresses. Water stress and salinity are major constraints to tomato&rsquo, s cultivation, and for which limited genetic variability has been reported within the cultivated species. We evaluated four accessions of S. pimpinellifolium and four of S. l. var. cerasiforme for their adaptation to water deficit and salinity. The CO2 assimilation rate, stomatal conductance, substomatal CO2 concentration, transpiration rate, and leaf chlorophyll concentration were evaluated, as well as morphological and agronomic traits. The accessions showed a remarkable inter- and intra-species response variability to both stresses. Two S. pimpinellifolium accessions and one S. l. var. cerasiforme showed unaltered physiological parameters, thus indicating a good adaptation to water deficit. Two S. l. var. cerasiforme accessions showed an interesting performance under salt stress, one of which showing also good adaptation to water stress. In general, both stresses showed a negative impact on leaf size and fruit fresh weight, especially in the big-sized fruits. However, flowering, fruit setting and earliness remained unaltered or even improved when compared to control conditions. Stressed plants yielded fruits with higher &deg, Brix. Response to stresses seemed to be linked to origin environmental conditions, notwithstanding, variability was observed among accessions of the same region.

Tetraploid (4x) citrus seedlings may be more tolerant to salt stress than diploid (2x) genotypes. Genome duplication in citrus changes both plant physiology and anatomy, leading plants to acquire differentiated capacities to uptake and transport mineral elements. To provide insight into this behaviour, 2x and 4x Citrus macrophylla (CM), seedlings were grown at moderate (40 mM NaCl) and high salinity (80 mM NaCl) for a period of 30 days. Moderate salinity reduced the biomass of 4x plants, but did not affect the 2x plants, even when diploids accumulated more chloride (Cl−) and sodium (Na+) ions in leaves than tetraploids. The leaf K+ concentration descended in 2xCM leaves but not in the tetraploids. These differences were correlated to variations in uptake and transport rates between the genotypes. Leaf osmotic potential in salinized plants was lower in 2x than in 4x leaves, but water potential and turgor were similar between them. Tetraploid plants had a stronger decrease in their gas exchange parameters than diploids when subjected to moderate salt stress. Leaf damage was only observed in 2x plants subjected to high salinity media, and was correlated with higher Cl− leaf concentration than 4x plants, while Na+ did not differ between them. Taken together, our results suggest that genome duplication improves the tolerance to saline toxicity in CM, because the lower Cl− accumulation in leaves delays the damage. This effect may be linked to the reduced transpiration rate of the 4x genotype.

[EN] In this work, we study the capacity to biosynthesize gibberellins (GA) of ovules (either fertilised or unfertilised), developing seeds and pericarp from fruitlets and their relation with fruit set capacity. Experiments were performed in adult, 12-year-old trees of seeded (Pineapple) and seedless parthenocarpic (Washington navel) sweet orange [Citrus sinensis L. Osbeck] cultivars. The activity of GA20-, GA3- and GA2-oxidases and gibberellin levels were measured in the ovules and pericarp of fruitlets in different development states. The results indicate that ovules are the main sites of gibberellin synthesis in fruitlets during the post-anthesis period. The most intense GA(1) synthesis-coincident with the highest expression of GA20ox2, GA3ox1 and GA2ox1-was detected in the ovules of the seeded cultivar, probably induced by fecundation and associated with low early fruitlet abscission rates. By contrast, the low activity detected in the sterile cultivar appears to be rather developmentally or constitutively regulated. As a fruitlet develops, the GA(1) concentration is augmented in the pericarp in comparison to ovules or developing seeds, and levels therein did not exhibit noticeable differences between varieties. Furthermore, developing seeds from pineapple had higher GA(1) content than the unfertilised abortive ovules from Washington navel. Taken together, data suggest a main role for this hormone in the control of fruitlet abscission, and also demonstrate a function in seed development., We thank Drs. Isabel Lopez-Diaz and Esther Carrera for the hormone quantification carried out at the Plant Hormone Quantification Service, IBMCP, Valencia, Spain. Thanks are due to Teresa Sabater from the IBMCP, for their help. This work has been supported by two research projects, RTA2013-00024-CO2-01 from INIA (Ministerio de Economia y Competitividad, Spain) and IVIA-5423 from Conselleria de Agricultura (Generalitat Valenciana, Valencia, Spain).

Iron (Fe) chlorosis is one of the commonest problems in calcareous soils and plant tolerance is determined mainly by rootstocks. This work evaluates new citrus hybrids’ tolerance to Fe chlorosis compared with the most widely used rootstocks in Spain: Citrus macrophylla (CM, Fe chlorosis-tolerant) and Carrizo citrange (CC, Fe chlorosis-sensitive). Growth parameters, Fe concentration, photosynthetic parameters, ferric chelate reductase (FC-R) activity and proton (H + ) extrusion capacity were assessed in plants irrigated with 20 (control, Ct) or 0 (Fe-deficient, -Fe) μM FeEDDHA. Some—Fe hybrids presented marked Fe chlorosis symptomatology reflected by the root:shoot ratio and Chl a and b concentrations sharply dropping, and an increase in the Chl a/b ratio. These effects were very strong in the 050119, 050124-B and 050110—Fe species. The net CO 2 assimilation rate significantly lowered in the 05019, 050131, 050125, 050112 and 05013—Fe species. The 050120, 050125, 050112 and 050124-B—Fe plants presented lower stomatal conductance than the Ct ones. Conversely, the internal CO 2 concentration (Ci) tended to increase in Fe-deprived plants. Fe-deficiency increased FC-R activity and H + extrusion in some 0501 species. Both responses were significantly induced in the CM, 050114, 05019, 050131 and 050125—Fe plants. Fe 2+ accumulation in—Fe plants related inversely with FC-R activity. Most—Fe species with the greatest FC-R activity also accumulated the most Fe 3+ ions and, therefore, the Fe pool was much larger in the root apoplast. Collectively, the main trait that determined Fe-chlorosis tolerance among these genotypes was the ability to: (1) boost Fe 3+ reduction in response to Fe-deficiency; (2) acidify root media; (3) benefit the Fe amounts stored in the root apoplast.

Background and aimsThis work evaluates the regulation of iron uptake responses in Citrus leaves and their involvement in the Fe paradox effect.MethodsExperiments were performed in field-grown ‘Navelina’ trees grafted onto two Cleopatra mandarin ×Poncirus trifoliata(L.) Raf. hybrids with different Fe-chlorosis symptoms: 030146 (non-chlorotic) and 030122 (chlorotic).ResultsChlorotic leaves were smaller than non-chlorotic ones for both dry weight (DW) and area basis, and exhibited marked photosynthetic state affection, but reduced catalase and peroxidase enzymatic activities. Although both samples had a similar total Fe concentration on DW, it was lower in chlorotic leaves when expressed on an area basis. A similar pattern was observed for the total Fe concentration in the apoplast and cell sap and in active Fe (Fe2+) concentration.FRO2gene expression and ferric chelate reductase (FC-R) activity were also lower in chlorotic samples, whileHA1andIRT1were more induced. Despite similar apoplasmic pH, K+/Ca2+was higher in chlorotic leaves, and both citrate and malate concentrations in total tissue and apoplast fluid were lower.Conclusion(1) The rootstock influences Fe acquisition system in the leaf; (2) the increased sensitivity to Fe-deficiency as revealed by chlorosis and decreased biomass, was correlated with lower FC-R activity and lower organic acid level in leaf cells, which could cause a decreased Fe mobility and trigger other Fe-stress responses in this organ to enhance acidification and Fe uptake inside cells; and (3) the chlorosis paradox phenomenon in citrus likely occurs as a combination of a marked FC-R activity impairment in the leaf and the strong growth inhibition in this organ.

10 Pags., 3 Figs., 3 Tabls., The effects of iron (Fe) deficiency on the low-molecular-weight organic acid (LMWOA) metabolism have been investigated in Carrizo citrange (CC) [Citrus sinensis (L.) Osb. x Poncirus trifoliata (L.) Raf.] roots. Major LMWOAs found in roots, xylem sap and root exudates were citrate and malate and their concentrations increased with Fe deficiency. The activities of several enzymes involved in the LMWOA metabolism were also assessed in roots. In the cytosolic fraction, the activities of malate dehydrogenase (cMDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes were 132 and 100% higher in Fe-deficient conditions, whereas the activity of pyruvate kinase was 31% lower and the activity of malic enzyme (ME) did not change. In the mitochondrial fraction, the activities of fumarase, MDH and citrate synthase enzymes were 158, 117 and 53% higher, respectively, in Fe-deficient extracts when compared with Fe-sufficient controls, whereas no significant differences between treatments were found for aconitase (ACO) activity. The expression of their corresponding genes in roots of Fe-deficient plants was higher than that measured in Fe-sufficient controls, except for ACO and ME. Also, dicarboxylate-tricarboxylate carrier (DTC) expression was significantly increased in Fe-deficient roots. In conclusion, Fe deficiency in CC seedlings causes a reprogramming of the carbon metabolism that involves an increase of anaplerotic fixation of carbon via PEPC and MDH activities in the cytosol and a shift of the Krebs cycle in the mitochondria towards a non-cyclic mode, as previously described in herbaceous species. In this scheme, DTC could play an important role shuttling both malate and reducing equivalents between the cytosol and the mitochondria. As a result of this metabolic switch malate and citrate concentrations in roots, xylem sap and root exudates increase., This work was supported by a grant from the Instituto Nacional de Investigaciones Agrarias, Spain (project RTA 04-064).

Iron chlorosis is one of the main abiotic stress factors in plants as it participates in some life-sustaining processes, e.g., respiration and photosynthesis, and is involved in electron transfer through Fe2+/Fe3+ redox reactions.

Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport. Highlights Tetraploidy enhances B excess tolerance in citrange Carrizo Expression of NIP5 and BOR1 transporters and cell wall-bounded B are similar between ploidies B tolerance is attributed to root anatomical modifications induced by genome duplication The rootstock 4x citrange carrizo may prevent citrus trees from B excess.

This work studied the hydraulic characteristics and physiological behavior of two trifoliate orange (Poncirus trifoliata L. Raft) varieties-Flying Dragon (FD) and Rubidoux (RT)-with contrasting size-controlling potential when used as rootstocks for citrus trees. Thus, Valencia orange scions growing on RT root system develop about 40 % more biomass than scions on FD. The anatomical study of xylem root tissue of both rootstocks showed that the number of vessels per cross-sectional area in RT almost doubled that found in FD, whereas diameter distribution did not vary significantly. Hydraulic resistance determined in rootstocks, and bud union segments were, respectively, 2- and 3.4-fold higher in trees on FD than in trees on RT. Root systems accounted for 46.5 and 55.2 % of whole-plant hydraulic resistance, whereas bud union segments represented 7.5 and 14.6 % of this parameter, the dwarfing rootstock (FD) having the highest values. Reduced hydraulic conductance in plants on FD rootstock diminished water potential in high evaporative demand periods, causing a reduction in stomatal conductance with respect to plants on RT. This leads to lower net photosynthetic CO2 assimilation, which may affect biomass production. Translocation of C-13-labeled photoassimilates from leaves to roots was lower in plants on FD than in plants on RT, indicating that in the dwarfing rootstock (FD) there may be a vascular resistance to sucrose transport at the budding union level. Findings show that reduced hydraulic conductance may be the main cause of rootstock-induced dwarfing in citrus grafted onto FD.

Tetraploid citrus seedlings are more tolerant to salt stress than diploid genotypes. To provide insight into the causes of differences in salt tolerance due to ploidy and thus to better understand Cl − exclusion mechanisms in citrus, diploid and tetraploid seedlings of Carrizo citrange (CC) were grown at 0 (control) and 40 mM NaCl (salt-treated) medium for 20 days. Chloride uptake and root-to-shoot translocation rates were on average 1.4-fold higher in diploid than in tetraploid salt-treated plants, which resulted in a greater (1.6-fold) Cl − build up in the leaves of the former. Root hydraulic conductance and leaf transpiration rate were 58% and 17% lower, respectively, in tetraploid than in diploid control plants. Differences remained after salt treatment which reduced these parameters by 30–40% in both genotypes. Morphology of the root system was significantly influenced by ploidy. Tetraploid roots were less branched and with lower number of root tips than those of diploid plants. The cross-section diameter and area were lower in the diploid, and consequently specific root length was higher (1.7-fold) than in tetraploid plants. The exodermis in sections close to the root apex was broader and with higher deposition of suberin in cell walls in the tetraploid than in the diploid genotype. Net CO 2 assimilation rate in tetraploid salt-treated seedlings was 1.5-fold higher than in diploid salt-treated plants, likely due to the loss of photosynthetic capacity of diploid plants induced by Cl − toxicity. Leaf damage was much higher, in terms of burnt area and defoliation, in diploid than in tetraploid salt-treated plants (8- and 6-fold, respectively). Salt treatment significantly reduced (37%) the dry weight of the diploid plants, but did not affect the tetraploids. In conclusion, tetraploid CC plants appear more tolerant to salinization and this effect seems mainly due to differences in morphological and histological traits of roots affecting hydraulic conductance and transpiration rate. These results may suggest that tetraploid CC used as rootstock could improve salt tolerance in citrus trees.

Large quantities of crop residue and animal manure from agricultural and livestock activities are annually produced worldwide. With proper management, these residues are potentially valuable sources of plant nutrients, mainly N. Recycling such subproducts in sustainably-based agricultural systems can minimise the use of mineral fertilisers, and hence reduce the potential risk of surface and groundwater pollution. Therefore, the purpose of this study was to obtain (small scale) two liquid labelled-organic fertilisers, an animal- and a vegetal-based organic (AO and VO, respectively) fertiliser, to be used as organic N sources in subsequent fertigation studies. Forage maize (Zea mays L.) grown under 15N-labelled fertiliser supply was used as raw material for VO fertiliser production, and also as 15N-labelled sheep feed to obtain 15N-labelled manure. The labelled faeces fraction was used as raw material for the AO fertiliser. The VO fertiliser was obtained after an acidic and an enzyme-driven hydrolysis. The AO fertiliser was obtained after acidic hydrolysis. The VO liquid fertiliser presented an N concentration of 330 mg·L-1, 85% of total N was organic, while ammonium and nitrate N accounted for 55% and 45% of the mineral nitrogen fraction, respectively. This fertiliser also exhibited high K, Ca and S concentrations and notable values for the remaining macro- and micronutrients. The AO liquid fertiliser had a similar total N concentration (496 mg·L-1, 82% of total N in an organic form) to that of VO, but its mineral N fraction significantly differed, which came in a predominantly (95%) ammonia form. It also had a high content of N, P, K and other macronutrients, and sufficient Fe, Zn, Mn, Cu and B levels, which suggests its suitability as a potential fertiliser. The percentage of 15N enrichment in both VO and AO liquid fertilisers exceeded 2% 15N atom excess, which enabled their use in subsequent assays run to assess nitrogen uptake efficiency.

Fig 6, “Boron concentration ([Bf], μg g-1 DW) and boron content (Bf, μg) in (A) soluble in water, (B) soluble in organic solvents and (C) insoluble fractions measured in roots and leaves of Citrus macrophylla seedlings grown for 25 days in B-normal (50 μM, Ct) and B-toxic (400 μM, +B) nutrient solutions,” appears incorrectly. Please see the corrected Fig 6 here. Fig 6 Boron concentration ([Bf], μg g-1 DW) and boron content (Bf, μg) in (A) soluble in water, (B) soluble in organic solvents and (C) insoluble fractions measured in roots and leaves of Citrus macrophylla seedlings grown for 25 days in B-normal ...

Plants possess a number of mechanisms to cope with sodium (Na+) under salt stress conditions that include minimizing Ne influx, maximizing efflux back to the growth medium or to apoplastic spaces via Na+/H+. antiporters in the plasma membrane, intracellular compartrnentation of Na+ into the vacuole, as well as recirculation of Na+ out of the shoot via the phloem. Na+ transport in plants constitutes a complex system, in which different Na+ transporters are closely related and their functions are matched tightly. The fact that in citrus under salt stress does chloride appear to be the more toxic ion has led to little attention being paid to Na+ uptake and transport mechanisms in citrus. The aim of this study was to provide insight into the links between the expression levels of candidate Na+ transporter genes (SOS1, NHX1, HKT1), as well as tonoplast proton pumps (V-ATPase, V-PPiase), and Na+ tolerance in two citrus rootstocks, Cleopatra mandarin and trifoliate orange, differing in their Na+ exclusion capacity under salt stress. According to the results of this preliminary study, we hypothesize that higher root Na+ concentration in trifoliate orange genotype, and thus lower allocation of this ion in the shoots, is the result of an enhanced retrieval of Na+ from xylem stream, and an impaired translocation to the shoot tissues, probably as a consequence of the overexpression of putative SOS1 (in roots) and HKT1 (both in roots and shoots). Moreover, the higher transcriptional levels of putative NHX1 found in roots and shoots of trifoliate orange plantlets compared to those of Cleopatra mandarin, together with the enhanced activity of the tonoplast proton pumps in the former, might reveal the preferential sequestration into vacuole of retrieved Na+ from xylem mainly in roots. (C) 2014 Published by Elsevier B.V.

The expression of iron (Fe) acquisition-related genes in roots was studied in roots of two different citrus seedlings, namely, Carrizo citrange (CC, Fe chlorosis-sensitive) and Cleopatra mandarin (CM, Fe chlorosis-tolerant), growing either with (control) or without (-Fe) Fe in the nutrient solution. Fe-deficiency increased expression of the gene HA] coding for proton-ATPase (H+-ATPase) enzyme in both genotypes, although no differences were observed between treatments among rootstocks. Furthermore, while the gene expression levels of FRO2 - which encodes the Ferric Chelate Reductase (FC-R) enzyme-, increased under -Fe condition in both genotypes, CM always recorded the highest activity. CC showed the greatest induction of genes IRT1 and IRT2 encoding two iron transporters, however only IRT1 was significantly induced by Fe starvation. Analysis of the enzymatic activities (H+-ATPase and FC-R) regulated by the aforementioned genes confirmed these results. Thus, in agreement with the acidification pattern registered, H+-ATPase activities were higher in -Fe plants than in controls, although no significant differences were detected between each treatment among rootstocks. Fe starvation also induced FC-R activity; however, this was greater in CM than in CC roots. Interestingly, root Fe-57 uptake rates from Fe-57-EDDHA solutions were increased by Fe-deficiency, especially in the CM genotype, and CM accumulated a much larger Fe pool in the root apoplast than CC. Taken together, the main trait determining Fe-chlorosis tolerance among these genotypes is the ability to boost Fe3+ reduction in response to Fe-deficiency through enhanced FRO2 gene expression. Moreover, Fe chlorosis resistance in these plants could be related to the amount of Fe stored in the root apoplast. (C) 2013 Elsevier B.V. All rights reserved.

This work studies the effect of bicarbonate on plant performance and the iron acquisition system of Forner-Alcaide 5 (FA-5) seedlings, a citrus genotype known for its tolerance to calcareous soils. Plants were irrigated for 6 weeks with or without 10 mM NaHCO3. Treatment significantly decreased shoot growth, photosynthetic levels and iron concentration in shoots and roots. o,o-(57)FeEDDHA experiments indicated that Fe-57 uptake by roots was inhibited in treated plants. Moreover, those seedlings accumulated more Fe-57 in roots, and enhanced mRNA accumulation of ferric reductase genes FRO1 and FRO2 and FC-R activity in roots. H+-ATPase activity and HA1 gene expression were also increased, while HA2 was not affected. In addition, expression of the iron transporter gene IRT1 was increased, while IRT2 was not significantly affected. Finally, according to PEPC enzymatic activity, PEPC1 gene expression was higher in treated roots. In conclusion, it appears that bicarbonate prevents medium acidification by roots, thus reducing Fe2+ uptake. Accordingly, Fe deficiency enhanced the expression of some genes related with the Fe acquisition system (IRT1, FRO1, FRO2, HA1 and PEPC1) and the activity of the corresponding enzymes, which appear to constitute an adaptive mechanism of FA-5 in these soils.

The aim of this work is to study the effect of elevated concentrations of either Zn2+ or Mn2+ in the culture media on the components of the system of iron acquisition by roots from Fe-deficient and Fe-supplied citrus seedlings. Seeds of Citrus aurantium (L.) were germinated in vitro. Nutrient solutions without iron, zinc or manganese were prepared for assays and then combinations of Fe-EDDHA, ZnSO4 center dot 7H(2)O or MnSO4 center dot H2O were added to obtain the following media: -Fe-Zn, +Fe-Zn, -Fe+Zn, +Fe+Zn, -Fe-Mn, +Fe-Mn, -Fe+Mn and +Fe+Mn. Data indicated that when either 100 mu M Zn2+ or 250 mu M Mn2+ were added to culture media containing 20 mu M Fe, iron concentration in roots and shoots was reduced with respect to the seedlings cultured in the same medium lacking Zn2+ or Mn2+. Reciprocally, the presence of iron in the medium lowered Zn and Mn levels in the plant. Addition of either 100 mu M Zn2+ or 250 mu M Mn2+ to the medium enhanced the transcription levels of FRO2, IRT1 and NRAMP3 genes in roots, this effect being more marked in Fe-starved plants. FC-R activity followed the same behaviour as the gene FRO2. Both Zn2+ and Mn2+ stimulated the activity of some genes (FRO2, IRT1 and NRAMP3) which was also induced by Fe-deficiency, although these effects appear to be independent of the inhibition of Fe uptake by these cations.