Indole-3-acetaldoxime delays root iron-deficiency responses and modify auxin homeostasis in Medicago truncatula.

Iron (Fe) is an essential plant micronutrient, being a major limiting growth factor in calcareous soils. To increase Fe uptake, plants induce lateral roots growth, the expression of a Fe(III)-chelate reductase (FCR), a Fe(II)-transporter and a H+-ATPase and the secretion of flavins. Furthermore, auxin hormone family is involved in the Fe-deficiency responses but the action mechanism remains elusive. In this work, we evaluated the effect of the auxin-precursor indole-3-acetaldoxime (IAOx) on hydroponically grown Medicago truncatula plants under different Fe conditions. Upon 4-days of Fe starvation, the pH of the nutrient solution decreased, while both the FCR activity and the presence of flavins increased. Exogenous IAOx increased lateral roots growth contributing to superroot phenotype, decreased chlorosis, and delayed up to 3-days the pH-decrease, the FCR-activity increase, and the presence of flavins, compared to Fe-deficient plants. Gene expression levels were in concordance with the physiological responses. RESULTS: showed that IAOx was immediately transformed to IAN in roots and shoots to maintain auxin homeostasis. IAOx plays an active role in iron homeostasis delaying symptoms and responses in Fe-deficient plants. We may speculate that IAOx or its derivatives remobilize Fe from root cells to alleviate Fe-deficiency. Overall, these results point out that the IAOx-derived phenotype may have advantages to overcome nutritional stresses.

Prospecting the Resilience of Several Spanish Ancient Varieties of Red Grape under Climate Change Scenarios.

BACKGROUND: Climate change results in warmer air temperatures and an uncertain amount and distribution of annual precipitations, which will directly impact rainfed crops, such as the grapevine. Traditionally, ancient autochthones grapevine varieties have been substituted by modern ones with higher productivity. However, this homogenization of genotypes reduces the genetic diversity of vineyards which could make their ability to adapt to challenges imposed by future climate conditions difficult. Therefore, this work aimed to assess the response of four ancient grapevine varieties to high temperatures under different water availabilities, focusing on plant water relations, grape technological and phenolic maturity, and the antioxidant capacity of the must. METHODS: The study was conducted on fruit-bearing cuttings grown in pots in temperature-gradient greenhouses. A two-factorial design was established where two temperature regimes, ambient and elevated (ambient + 4 °C), were combined with two water regimes, full irrigation and post-veraison deficit irrigation, during fruit ripening. RESULTS: There were significant differences among the ancient varieties regarding plant water relations and fruit quality. CONCLUSION: This research underlines the importance of evaluating the behavior of ancient grapevine varieties that could offer good options for the adaptation of viticulture to future climate conditions.

The importance of the urea cycle and its relationships to polyamine metabolism during ammonium stress in Medicago truncatula.

The ornithine-urea cycle (urea cycle) makes a significant contribution to the metabolic responses of lower photosynthetic eukaryotes to episodes of high nitrogen availability. In this study, we compared the role of the plant urea cycle and its relationships to polyamine metabolism in ammonium-fed and nitrate-fed Medicago truncatula plants. High ammonium resulted in the accumulation of ammonium and pathway intermediates, particularly glutamine, arginine, ornithine, and putrescine. Arginine decarboxylase activity was decreased in roots, suggesting that the ornithine decarboxylase-dependent production of putrescine was important in situations of ammonium stress. The activity of copper amine oxidase, which releases ammonium from putrescine, was significantly decreased in both shoots and roots. In addition, physiological concentrations of ammonium inhibited copper amine oxidase activity in in vitro assays, supporting the conclusion that high ammonium accumulation favors putrescine synthesis. Moreover, early supplementation of plants with putrescine avoided ammonium toxicity. The levels of transcripts encoding urea-cycle-related proteins were increased and transcripts involved in polyamine catabolism were decreased under high ammonium concentrations. We conclude that the urea cycle and associated polyamine metabolism function as important protective mechanisms limiting ammonium toxicity in M. truncatula. These findings demonstrate the relevance of the urea cycle to polyamine metabolism in higher plants.

Assessment of Nutritional and Quality Properties of Leaves and Musts in Three Local Spanish Grapevine Varieties Undergoing Controlled Climate Change Scenarios.

The market demand together with the need for alternatives to withstand climate change led to the recovery of autochthonous grapevine varieties. Under climate change, the summer pruning of vineyards may lead to an increase of vegetative residuals of nutritional and medicinal interest. The objectives of our study were (1) to evaluate the nutritional properties of the leaves of three local Spanish grapevines (Tinto Velasco, TV, Pasera, PAS, and Ambrosina, AMB) when grown under climate change conditions, and (2) to test the potentiality of these grapevines as suitable candidates to be cultivated under climate change scenarios based on the quality of their must. Experimental assays were performed with fruit-bearing cuttings grown in temperature gradient greenhouses that simulate rising CO2 (700 µmol mol-1) and warming (ambient temperature +4 °C), either acting alone or in combination. TV and AMB were the most and the least affected by air temperature and CO2 concentration, respectively. The interaction of elevated CO2 with high temperature induced the accumulation of proteins and phenolic compounds in leaves of TV, thus enhancing their nutritional properties. In PAS, the negative effect of high temperature on protein contents was compensated for by elevated CO2. Warming was the most threatening scenario for maintaining the must quality in the three varieties, but elevated CO2 exerted a beneficial effect when acting alone and compensated for the negative effects of high temperatures. While TV may be a candidate to be cultivated in not very warm areas (higher altitudes or colder latitudes), PAS behaved as the most stable genotype under different environmental scenarios, making it the most versatile candidate for cultivation in areas affected by climate change.

Tuning promoter boundaries improves regulatory motif discovery in nonmodel plants: the peach example.

The identification of functional elements encoded in plant genomes is necessary to understand gene regulation. Although much attention has been paid to model species like Arabidopsis (Arabidopsis thaliana), little is known about regulatory motifs in other plants. Here, we describe a bottom-up approach for de novo motif discovery using peach (Prunus persica) as an example. These predictions require pre-computed gene clusters grouped by their expression similarity. After optimizing the boundaries of proximal promoter regions, two motif discovery algorithms from RSAT::Plants (http://plants.rsat.eu) were tested (oligo and dyad analysis). Overall, 18 out of 45 co-expressed modules were enriched in motifs typical of well-known transcription factor (TF) families (bHLH, bZip, BZR, CAMTA, DOF, E2FE, AP2-ERF, Myb-like, NAC, TCP, and WRKY) and a few uncharacterized motifs. Our results indicate that small modules and promoter window of [-500 bp, +200 bp] relative to the transcription start site (TSS) maximize the number of motifs found and reduce low-complexity signals in peach. The distribution of discovered regulatory sites was unbalanced, as they accumulated around the TSS. This approach was benchmarked by testing two different expression-based clustering algorithms (network-based and hierarchical) and, as control, genes grouped for harboring ChIPseq peaks of the same Arabidopsis TF. The method was also verified on maize (Zea mays), a species with a large genome. In summary, this article presents a glimpse of the peach regulatory components at genome scale and provides a general protocol that can be applied to other species. A Docker software container is released to facilitate the reproduction of these analyses.

Association analysis and molecular tagging of phytochemicals in the endangered medicinal plant licorice (Glycyrrhiza glabra L.).

Licorice (Glycyrrhiza glabra L.) is a medicinal plant species valued in many countries in Asia and Europe for its phytochemical characteristics. Licorice biodiversity is becoming threatened nowadays in Iran due to increasing demand and a drastic decline of its natural habitats. Therefore, licorice domestication would be necessary in the near future, and molecular breeding would help to introduce genotypes suitable for cultivation. The present study was carried out with 170 individual licorice plants sampled in the wild in 59 localizations in 21 provinces of Iran. The association of 436 polymorphic AFLP markers, produced by 15 primer combinations (EcoRI/MseI), with six phenotypic phytochemical traits was studied. The AMOVA analysis show gene diversity among and within localizations. The population structure analysis identified two main sub-populations with significant genetic variation. Significant associations were identified between three markers (E3/M40-4, E34/M4-12 and E12/M31-15) and glycyrrhizin concentration, and between four markers (E11/M34-12, E11/M34-15, E9/M7-29, and E9/M7-30) and phenolic compounds contents. Markers detected can be useful in the domestication of licorice as well as in breeding programs. Licorice sampled in four localizations (KBA1, KBA2, SKh2 and Fa1) were found to be superior in terms of glycyrrhizin and antioxidants content, and therefore they can be considered as elite genotypes which could be included in the domestication process.

Identification of hybridization and introgression between Cinnamomum kanehirae Hayata and C. camphora (L.) Presl using genotyping-by-sequencing.

Cinnamomum kanehirae Hayata and C. camphora (L.) Presl are important tree species in eastern Asia. The wood of C. kanehirae is in increasing demand for culturing Antrodia cinnamomea, a medicinal fungus that naturally grows inside the trunk of C. kanehirae. Putative hybrids between C. kanehirae and C. camphora were previously reported but with no scientific evidence, leading to confusion or misplanting. First, to identify the female parent of putative hybrids, the maternal inheritance InDel (insertion/deletion) markers were developed by using low-coverage sequencing. SNPs were developed by using genotyping-by-sequencing (GBS) approach in C. kanehirae, C. camphora and putative hybrids. The results indicated that the female parent of the studied hybrids was C. camphora. Eight hundred and forty of the 529,006 high-density SNPs were selected and used for analysis. Hybrids were classified as F1 (C. kanehirae × C. camphora), F2 and backcrosses. Hybridization has occurred in the human-developed area of eastern and southwestern Taiwan, and the introgression was bidirectional. For producing pure wood, buffering zones should be established around seed orchards to avoid cross-species pollination and to preserve the genetic purity of C. kanehirae. The DNA markers developed in this study will also be valuable for further wood identification, breeding and evolutionary research.

Is the Tolerance of Commercial Peach Cultivars to Brown Rot Caused by Monilinia laxa Modulated by its Antioxidant Content?

Brown rot, caused by Monilinia spp., provokes pre- and post-harvest damage in peach (Prunus persica (L.) Batsch), which causes an economic impact in the industry. With a view to breeding for increased tolerance to this disease, a screening test based upon artificial fruit inoculation was validated on several parental lines of a peach breeding program during the two-period harvest. In addition, cultivars with different total phenolic contents were included in the two-year study. All physicochemical fruit traits recorded at harvest showed differences among all cultivars. The antioxidant compound content determined using spectrophotometry (to measure ascorbic acid and antioxidant capacity) and UPLC-MS (to measure and identify phenolic compounds) also revealed important differences among all genotypes. The rate of brown rot lesion following fruit inoculation varied widely among cultivars, and it was possible to discriminate between highly and less susceptible cultivars. Cultivars with minimal development of damage were identified as germplasm with the desirable allele combination to increase brown rot tolerance in peach breeding programs. Finally, Pearson's correlation coefficients (r) between pairs of variables were calculated, searching for any biochemical candidate conferring tolerance. The correlation of phytopathological traits with the antioxidant composition, concerning contents of ascorbic, neochlorogenic, and chlorogenic acids and total polyphenols in fruit, is discussed.

Interactional Effects of Climate Change Factors on the Water Status, Photosynthetic Rate, and Metabolic Regulation in Peach.

Environmental stress factors caused by climate change affect plant growth and crop production, and pose a growing threat to sustainable agriculture, especially for tree crops. In this context, we sought to investigate the responses to climate change of two Prunus rootstocks (GF677 and Adesoto) budded with Catherina peach cultivar. Plants were grown in 15 L pots in temperature gradient greenhouses for an 18 days acclimation period after which six treatments were applied: [CO2 levels (400 versus 700 µmol mol-1), temperature (ambient versus ambient + 4°C), and water availability (well irrigated versus drought)]. After 23 days, the effects of stress were evaluated as changes in physiological and biochemical traits, including expression of relevant genes. Stem water potential decreased under drought stress in plants grafted on GF677 and Adesoto rootstocks; however, elevated CO2 and temperature affected plant water content differently in both combinations. The photosynthetic rate of plants grafted on GF677 increased under high CO2, but decreased under high temperature and drought conditions. The photosynthetic rates of plants grafted onto Adesoto were only affected by drought treatment. Furthermore, in GF677-Catherina plants, elevated CO2 alleviated the effect of drought, whereas in those grafted onto Adesoto, the same condition produced acclimation in the rate. Stomatal conductance decreased under high CO2 and drought stress in both grafted rootstocks, and the combination of these conditions improved water-use efficiency. Changes in the sugar content in scion leaves and roots were significantly different under the stress conditions in both combinations. Meanwhile, the expression of most of the assessed genes was significantly affected by treatment. Regarding genotypes, GF677 rootstock showed more changes at the molecular and transcriptomic level than did Adesoto rootstock. A coordinated shift was found between the physiological status and the transcriptomic responses. This study revealed adaptive responses to climate change at the physiological, metabolic, and transcriptomic levels in two Prunus rootstocks budded with 'Catherina'. Overall, these results demonstrate the resilient capacity and plasticity of these contrasting genotypes, which can be further used to combat ongoing climate changes and support sustainable peach production.

Transcriptional Responses in Root and Leaf of Prunus persica under Drought Stress Using RNA Sequencing.

Prunus persica L. Batsch, or peach, is one of the most important crops and it is widely established in irrigated arid and semi-arid regions. However, due to variations in the climate and the increased aridity, drought has become a major constraint, causing crop losses worldwide. The use of drought-tolerant rootstocks in modern fruit production appears to be a useful method of alleviating water deficit problems. However, the transcriptomic variation and the major molecular mechanisms that underlie the adaptation of drought-tolerant rootstocks to water shortage remain unclear. Hence, in this study, high-throughput sequencing (RNA-seq) was performed to assess the transcriptomic changes and the key genes involved in the response to drought in root tissues (GF677 rootstock) and leaf tissues (graft, var. Catherina) subjected to 16 days of drought stress. In total, 12 RNA libraries were constructed and sequenced. This generated a total of 315 M raw reads from both tissues, which allowed the assembly of 22,079 and 17,854 genes associated with the root and leaf tissues, respectively. Subsets of 500 differentially expressed genes (DEGs) in roots and 236 in leaves were identified and functionally annotated with 56 gene ontology (GO) terms and 99 metabolic pathways, which were mostly associated with aminobenzoate degradation and phenylpropanoid biosynthesis. The GO analysis highlighted the biological functions that were exclusive to the root tissue, such as "locomotion," "hormone metabolic process," and "detection of stimulus," indicating the stress-buffering role of the GF677 rootstock. Furthermore, the complex regulatory network involved in the drought response was revealed, involving proteins that are associated with signaling transduction, transcription and hormone regulation, redox homeostasis, and frontline barriers. We identified two poorly characterized genes in P. persica: growth-regulating factor 5 (GRF5), which may be involved in cellular expansion, and AtHB12, which may be involved in root elongation. The reliability of the RNA-seq experiment was validated by analyzing the expression patterns of 34 DEGs potentially involved in drought tolerance using quantitative reverse transcription polymerase chain reaction. The transcriptomic resources generated in this study provide a broad characterization of the acclimation of P. persica to drought, shedding light on the major molecular responses to the most important environmental stressor.

Influence of antioxidant compounds, total sugars and genetic background on the chilling injury susceptibility of a non-melting peach (Prunus persica (L.) Batsch) progeny.

BACKGROUND: To identify genotypes with good organoleptic properties, antioxidant-rich content and low susceptibility to chilling injury (CI), fruits from 130 peach cultivars were studied over three consecutive years. Pomological traits, l-ascorbic acid, flavonoids, total phenolics, relative antioxidant capacity (RAC) and sugars were determined. Major symptoms of CI developed at 5 °C, such as leatheriness, flesh browning, bleeding and loss of flavor, were evaluated. RESULTS: The population exhibited wide phenotypic variation in agronomic and biochemical traits. Six genotypes with high total phenolics, RAC, flavonoids and total sugars were selected. The progeny also showed variability for all evaluated CI symptoms, and 16 genotypes showed considerably lower susceptibility to CI. After 2 weeks of cold storage, leatheriness and bleeding were the main CI symptoms observed, whereas flesh browning was predominant after 4 weeks. CONCLUSION: It was possible to find varieties with high phenolic concentration and relatively low or intermediate CI susceptibility (22, 33, 68, 80, 81, 96 and 120). However, the correlations observed between CI and phenolic contents highlight their potential influence on susceptibility to internal browning. This relationship should be considered in the current breeding programs to select cultivars with high bioactive compound contents, health-enhancing properties and good postharvest performance.

Agronomical parameters, sugar profile and antioxidant compounds of "Catherine" peach cultivar influenced by different plum rootstocks.

The influence of seven plum rootstocks (Adesoto, Monpol, Montizo, Puebla de Soto 67 AD, PM 105 AD, St. Julien GF 655/2 and Constantí 1) on individual and total sugars, as well as on antioxidant content in fruit flesh of "Catherine" peaches, was evaluated for three years. Agronomical and basic fruit quality parameters were also determined. At twelve years after budding, significant differences were found between rootstocks for the different agronomic and fruit quality traits evaluated. The Pollizo plum rootstocks Adesoto and PM 105 AD seem to induce higher sweetness to peach fruits, based on soluble solids content, individual (sucrose, fructose and sorbitol) and total sugars. A clear tendency was also observed with the rootstock Adesoto, inducing the highest content of phenolics, flavonoids, vitamin C and relative antioxidant capacity (RAC). Thus, the results of this study demonstrate the significant effect of rootstock on the sugar profile and phytochemical characteristics of peach fruits. In addition, this work shows the importance of the sugar profile, because specific sugars play an important role in peach flavour quality, as well as the studied phytochemical compounds when looking for high quality peaches with enhanced health properties.

Physiological, biochemical and molecular responses in four Prunus rootstocks submitted to drought stress.

An understanding of the mechanisms that determine plant response to reduced water availability is essential to improve water-use efficiency (WUE) of stone fruit crops. The physiological, biochemical and molecular drought responses of four Prunus rootstocks (GF 677, Cadaman, ROOTPAC 20 and ROOTPAC(®) R) budded with 'Catherina' peach cultivar were studied. Trees were grown in 15-l containers and subjected to a progressive water stress for 26 days, monitoring soil moisture content by time domain reflectometry. Photosynthetic and gas exchange parameters were determined. Root and leaf soluble sugars and proline content were also measured. At the end of the experiment, stressed plants showed lower net photosynthesis rate, stomatal conductance and transpiration rate, and higher intrinsic leaf WUE (AN/gs). Soluble sugars and proline concentration changes were observed, in both root and leaf tissues, especially in an advanced state of stress. The accumulation of proline in roots and leaves with drought stress was related to the decrease in osmotic potential and increase in WUE, whereas the accumulation of sorbitol in leaves, raffinose in roots and proline in both tissues was related only to the increase in the WUE. Owing to the putative role of raffinose and proline as antioxidants and their low concentration, they could be ameliorating deleterious effects of drought-induced oxidative stress by protecting membranes and enzymes rather than acting as active osmolytes. Higher expression of P5SC gene in roots was also consistent with proline accumulation in the tolerant genotype GF 677. These results indicate that accumulation of sorbitol, raffinose and proline in different tissues and/or the increase in P5SC expression could be used as markers of drought tolerance in peach cultivars grafted on Prunus rootstocks.

Changes induced by Fe deficiency and Fe resupply in the root protein profile of a peach-almond hybrid rootstock.

The changes in the root extract protein profile of the Prunus hybrid GF 677 rootstock (P. dulcis × P. persica) grown in hydroponics as affected by Fe deficiency and short-term (24 h) Fe resupply have been studied by 2-dimensional gel electrophoresis-based techniques. A total of 335 spots were consistently found in the gels. Iron deficiency caused above 2-fold increases or >50% decreases in the relative abundance in 10 and 6 spots, respectively, whereas one spot was only detected in Fe-deficient plants. Iron resupply to Fe-deficient plants caused increases and decreases in relative abundance in 15 and 16 spots, respectively, and one more spot was only detected in Fe-resupplied Fe-deficient plants. Ninety-five percent of the proteins changing in relative abundance were identified using nanoliquid chromatography-tandem mass spectrometry. Defense responses against oxidative and general stress accounted for 50% of the changes in Fe-deficient roots. Also, a slight induction of the glycolysis-fermentation pathways was observed in GF 677 roots with Fe deficiency. The root protein profile of 24 h Fe-resupplied plants was similar to that of Fe-deficient plants, indicating that the deactivation of Fe-deficiency metabolic responses is slow. Taken together, our results suggest that the high tolerance of GF 677 rootstock to Fe deficiency may be related to its ability to elicit a sound defense response against both general and oxidative stress.

Two Fe-superoxide dismutase families respond differently to stress and senescence in legumes.

Three main families of SODs in plants may be distinguished according to the metal in the active center: CuZnSODs, MnSOD, and FeSOD. CuZnSODs have two sub-families localized either in plant cell cytosol or in plastids, the MnSOD family is essentially restricted to mitochondria, and the FeSOD enzyme family has been typically localized into the plastid. Here, we describe, based on a phylogenetic tree and experimental data, the existence of two FeSOD sub-families: a plastidial localized sub-family that is universal to plants, and a cytosolic localized FeSOD sub-family observed in determinate-forming nodule legumes. Anti-cytosolic FeSOD (cyt_FeSOD) antibodies were employed, together with a novel antibody raised against plastidial FeSOD (p_FeSOD). Stress conditions, such as nitrate excess or drought, markedly increased cyt_FeSOD contents in soybean tissues. Also, cyt_FeSOD content and activity increased with age in both soybean and cowpea plants, while the cyt_CuZnSOD isozyme was predominant during early stages. p_FeSOD in leaves decreased with most of the stresses applied, but this isozyme markedly increased with abscisic acid in roots. The great differences observed for p_FeSOD and cyt_FeSOD contents in response to stress and aging in plant tissues reveal distinct functionality and confirm the existence of two immunologically differentiated FeSOD sub-families. The in-gel FeSOD activity patterns showed a good correlation to cyt_FeSOD contents but not to those of p_FeSOD. This indicates that cyt_FeSOD is the main active FeSOD in soybean and cowpea tissues. The diversity of functions associated with the complexity of FeSOD isoenzymes depending of the location is discussed.

Effects of nitrogen source and water availability on stem carbohydrates and cellulosic bioethanol traits of alfalfa plants.

Symbiotic association of legumes with rhizobia frequently results in higher photosynthesis and soluble carbohydrates in comparison with nitrate-fed plants, which might improve its potential for biomass conversion into bioethanol. A greenhouse experiment was conducted to examine the effects of nitrogen source and water availability on stem characteristics and on relationships between carbohydrates, phenolic metabolism activity and cell wall composition in alfalfa (Medicago sativa L. cv. Aragón). The experiment included three treatments: (1) plants fed with ammonium nitrate (AN); (2) plants inoculated with rhizobia (R); and (3) plants inoculated with rhizobia and amended with sewage sludge (RS). Two levels of irrigation were imposed: (1) well-watered and (2) drought stress. Under well-watered conditions, nitrogen-fixing plants have increased photosynthesis and stem fermentable carbohydrate concentrations, which result in higher potential for biomass conversion to bioethanol than in AN plants. The latter had higher lignin due to enhanced activities of phenolic metabolism-related enzymes. Under drought conditions, the potential for bioethanol conversion decreased to a similar level in all treatments. Drought-stressed nitrogen-fixing plants have high concentrations of fermentable carbohydrates and cell wall cellulose, but ammonium nitrate-fed plants produced higher plant and stem biomass, which might compensate the decreasing stem carbohydrates and cellulose concentrations.

Evaluation of antioxidant compounds and total sugar content in a nectarine [Prunus persica (L.) Batsch] progeny.

Epidemiological studies suggest that consumption of fruit rich in phenolic compounds is associated with health-protective effects due to their antioxidant properties. For these reasons quality evaluation has become an important issue in fruit industry and in breeding programs. Phytochemical traits such as total phenolics, flavonoids, anthocyanins, L-ascorbic acid, sugar content and relative antioxidant capacity (RAC) were analyzed over four years in flesh fruit of an F1 population "Venus" × "Big Top" nectarines. Other traits such as harvesting date, yield, fruit weight, firmness, soluble solids concentration (SSC), pH, titratable acidity (TA) and ripening index (RI) were also determined in the progeny. Results showed high variability among genotypes for all analyzed traits. Total phenolics and flavonoids showed significant positive correlations with RAC implying that both are important antioxidant bioactive compounds in peaches. We found genotypes with enhanced antioxidant capacity and a better performance than progenitors, and in consequence the best marketability.

Effects of cadmium on cork oak (Quercus suber L.) plants grown in hydroponics.

Cork oak (Quercus suber L.) is an autochthonous tree species that is being used for reforestation in heavy-metal-contaminated areas in Spain. A hydroponics experiment was carried out to characterize the effects of Cd on several morphological and physiological parameters in this species, including shoot length, nutrient concentrations and allocation in different organs, leaf pigment concentrations, photosynthetic efficiency, root ferric chelate reductase (FCR) activity and organic acid concentrations in xylem sap. Four different Cd treatments were applied, adding Cd chelated with EDTA or as chloride salt at two different concentrations (10 and 50 µM Cd). After 1 month of Cd treatment, plant growth was significantly inhibited in all treatments. Results indicate that Cd accumulates in all organs 7- to 500-fold when compared with control plants. The highest Cd concentration was found in the 50 µM CdCl(2) treatment, which led to concentrations of ~30, 123 and 1153 µg Cd g(-1) dry weight in leaves, stems and roots, respectively. In the strongest Cd treatments the concentrations of P and Ca decreased in some plant parts, whereas the Mn leaf concentrations decreased with three of the four Cd treatments applied. The concentrations of chlorophyll and carotenoids on an area basis decreased, whereas the (zeaxanthin plus antheraxanthin)/(total violaxanthin cycle carotenoids) ratio and the non-photochemical quenching increased significantly in all Cd treatments. Cadmium treatments caused significant increases in the activity of the enzyme FCR in roots and in the concentrations of organic acids in xylem sap. Some of the physiological changes found support the fact that Cd induces a deficiency of Fe in cork oak, although the plant Fe concentrations were not reduced significantly. At higher concentrations the effects of Cd were more pronounced, and were more marked when Cd was in the free ion form than when present in the form of Cd-EDTA.

Physiological responses and differential gene expression in Prunus rootstocks under iron deficiency conditions.

Two Prunus rootstocks, the Myrobalan plum P 2175 and the interspecific peach-almond hybrid, Felinem, were studied to characterize their biochemical and molecular responses induced under iron-Deficient conditions. Plants of both genotypes were submitted to different treatments using a hydroponic system that permitted removal of Fe from the nutrient solution. Control plants were grown in 90 µM Fe (III)-EDTA, Deficient plants were grown in an iron free solution, and plants submitted to an Inductor treatment were resupplied with 180 µM Fe (III)-EDTA over 1 and 2 days after a period of 4 or 15 days of growth on an iron-free solution. Felinem increased the activity of the iron chelate reductase (FC-R) in the Inductor treatment after 4 days of iron deprivation. In contrast, P 2175 did not show any response after at least 15 days without iron. The induction of the FC-R activity in this genotype was coincident in time with the medium acidification. These results suggest two different mechanisms of iron chlorosis tolerance in both Strategy I genotypes. Felinem would use the iron reduction as the main mechanism to capture the iron from the soil, and in P 2175, the mechanism of response would be slower and start with the acidification of the medium synchronized with the gradual loss of chlorophyll in leaves. To better understand the control of these responses at the molecular level, the differential expression of PFRO2, PIRT1 and PAHA2 genes involved in the reductase activity, the iron transport in roots, and the proton release, respectively, were analyzed. The expression of these genes, estimated by quantitative real-time PCR, was different between genotypes and among treatments. The results were in agreement with the physiological responses observed.

Metabolic response in roots of Prunus rootstocks submitted to iron chlorosis.

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different responses for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus×Prunus persica), and the more sensitive Barrier (P. persica×Prunus davidiana) were grown hydroponically in iron-sufficient and -deficient conditions over two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, provides complementary information for selection of genotypes tolerant to iron chlorosis.