Reversion of fruit-dependent inhibition of flowering in Citrus requires sprouting of buds with epigenetically silenced CcMADS19.

In Citrus, the response to environmental floral inductive signals is inhibited by the presence of developing fruits. The mechanism involves epigenetic activation of the CcMADS19 locus (FLC orthologue), encoding a floral repressor. To understand how this epigenetic regulation is reverted to allow flowering in the following season, we have forced precocious sprouting of axillary buds in fruit-bearing shoots, and examined the competence to floral inductive signals of old and new leaves derived from them. We have found that CcMADS19 is enriched in repressive H3K27me3 marks in young, but not old leaves, revealing that axillary buds retain a silenced version of the floral repressor that is mitotically transmitted to the newly emerging leaves, which are able to induce flowering. Therefore, we propose that flowering in Citrus is necessarily preceded by vegetative sprouting, so that the competence to respond to floral inductive signals is reset in the new leaves.

Fruit-dependent epigenetic regulation of flowering in Citrus.

In many perennial plants, seasonal flowering is primarily controlled by environmental conditions, but in certain polycarpic plants, environmental signals are locally gated by the presence of developing fruits initiated in the previous season through an unknown mechanism. Polycarpy is defined as the ability of plants to undergo several rounds of reproduction during their lifetime, alternating vegetative and reproductive meristems in the same individual. To understand how fruits regulate flowering in polycarpic plants, we focused on alternate bearing in Citrus trees that had been experimentally established as fully flowering or nonflowering. We found that the presence of the fruit causes epigenetic changes correlating with the induction of the CcMADS19 floral repressor, which prevents the activation of the floral promoter CiFT2 even in the presence of the floral inductive signals. By contrast, newly emerging shoots display an opposite epigenetic scenario associated with CcMADS19 repression, thereby allowing the activation of CiFT2 the following cold season.

Gibberellin reactivates and maintains ovary-wall cell division causing fruit set in parthenocarpic Citrus species.

Citrus is a wide genus in which most of the cultivated species and cultivars are natural parthenocarpic mutants or hybrids (i.e. orange, mandarin, tangerine, grapefruit). The autonomous increase in GA1 ovary concentration during anthesis was suggested as being the stimulus responsible for parthenocarpy in Citrus regardless of the species. To determine the exact GA-role in parthenocarpic fruit set, the following hypothesis was tested: GA triggers and maintains cell division in ovary walls causing fruit set. Obligate and facultative parthenocarpic Citrus species were used as a model system because obligate parthenocarpic Citrus sp (i.e. Citrus unshiu) have higher GA levels and better natural parthenocarpic fruit set compared to other facultative parthenocarpic Citrus (i.e. Citrus clementina). The autonomous activation of GA synthesis in C. unshiu ovary preceded cell division and CYCA1.1 up-regulation (a G2-stage cell cycle regulator) at anthesis setting a high proportion of fruits, whereas C. clementina lacked this GA-biosynthesis and CYCA1.1 up-regulation failing in fruit set. In situ hybridization experiments revealed a tissue-specific expression of GA20ox2 only in the dividing tissues of the pericarp. Furthermore, CYCA1.1 expression correlated endogenous GA1 content with GA3 treatment, which stimulated cell division and ovary growth, mostly in C. clementina. Instead, paclobutrazol (GA biosynthesis inhibitor) negated cell division and reduced fruit set. Results suggest that in parthenocarpic citrus the specific GA synthesis in the ovary walls at anthesis triggers cell division and, thus, the necessary ovary growth rate to set fruit.

Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W.

This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured "in vitro" in media with 50 or 400 µM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation.

Non-targeted metabolite profiling of citrus juices as a tool for variety discrimination and metabolite flow analysis.

BACKGROUND: Genetic diversity of citrus includes intrageneric hybrids, cultivars arising from cross-pollination and/or somatic mutations with particular biochemical compounds such as sugar, acids and secondary metabolite composition. RESULTS: Secondary metabolite profiles of juices from 12 commercial varieties grouped into blonde and navel types, mandarins, lemons and grapefruits were analyzed by LC/ESI-QTOF-MS. HCA on metabolite profiling data revealed the existence of natural groups demarcating fruit types and varieties associated to specific composition patterns. The unbiased classification provided by HCA was used for PLS-DA to find the potential variables (mass chromatographic features) responsible for the classification. Abscisic acid and derivatives, several flavonoids and limonoids were identified by analysis of mass spectra. To facilitate interpretation, metabolites were represented as flow charts depicting biosynthetic pathways. Mandarins 'Fortune' and 'Hernandina' along with oranges showed higher ABA contents and ABA degradation products were present as glycosylated forms in oranges and certain mandarins. All orange and grapefruit varieties showed high limonin contents and its glycosylated form, that was only absent in lemons. The rest of identified limonoids were highly abundant in oranges. Particularly, Sucrenya cultivar showed a specific accumulation of obacunone and limonoate A-ring lactone. Polymethoxylated flavanones (tangeritin and isomers) were absolutely absent from lemons and grapefruits whereas kaempferol deoxyhexose hexose isomer #2, naringin and neohesperidin were only present in these cultivars. CONCLUSIONS: Analysis of relative metabolite build-up in closely-related genotypes allowed the efficient demarcation of cultivars and suggested the existence of genotype-specific regulatory mechanisms underlying the differential metabolite accumulation.

Carbon utilization by fruit limits shoot growth in alternate-bearing citrus trees.

Fruit load in alternate-bearing citrus trees is reported to alter shoot number and growth during spring, summer, and autumn flushes, and the source-sink balance, which affects the storage and mobilization of reserve nutrients. The aim of this work was to assess the extent of shoot growth inhibition resulting from the presence of fruits in 'Moncada' mandarin trees loaded with fruit (ON) or with very light fruit load (OFF), and to identify the role of carbohydrates and nitrogenous compounds in the competition between fruits and shoots. Growth of reproductive and vegetative organs was measured on a monthly basis. (13)C- and (15)N-labeled compounds were supplied to trace the allocation of reserve nutrients and subsequent translocation from source to sink. At the end of the year, OFF trees produced more abundant flushes (2.4- and 4.9-fold higher in number and biomass, respectively) than ON trees. Fruits from ON trees accumulated higher C amounts at the expense of developing flushes, whereas OFF trees exhibited the opposite pattern. An inverse relationship was identified between the amount of C utilized by fruits and vegetative flush growth. (13)C-labeling revealed an important role for mature leaves of fruit-bearing branches in supporting shoot/fruit growth, and the elevated sink strength of growing fruits on shoots. N availability for vegetative shoots was not affected by the presence or absence of fruits, which accumulated important amounts of (15)N. In conclusion, our results show that shoot growth is resource-limited as a consequence of fruit development, and vegetative-growth inhibition is caused by photoassimilate limitation. The competence for N is not a decisive factor in limiting vegetative growth under the experimental conditions of this study.

Proteomic study of 'Moncada' mandarin buds from on- versus off-crop trees.

A proteomic analysis of buds from mandarin trees with contrasting fruit load (on- and off-crop trees) was carried out during the onset of low-temperature induction. The aim of the study was to find out more about the molecular mechanism relating to alternate bearing in Citrus and its relationship with flowering. The 'Moncada' variety (Clementine 'Oroval'x'Kara' mandarin), displaying remarkable behaviour in alternate production, was used in this study. From 2D DIGE gel, 192 spots were isolated: 97 showed increased expression in the off-crop buds as compared to the on-crop buds, while 95 exhibited enhanced expression in the on-crop buds versus the off-crop buds. These spots were identified by MALDI-MS or LC-MS-MS. The largest groups of proteins up-expressed in the off-crop buds were the proteins involved in carbohydrate and amino acid metabolism, and the proteins expressed in response to stimuli such as reactive oxygen species. The largest groups of proteins up-expressed in the on-crop buds were related to primary metabolism, oxidative stress and defence responses. Depending on their function, some of these proteins can stimulate the flowering, such as fructose-bisphosphate aldolase or leucine-rich repeat transmembrane protein kinase, while others can inhibit it, such as cytochrome c oxidase subunit II. Twenty-two other proteins with unknown functions were up-expressed in the on- or off-crop buds.

Metabolic responses to iron deficiency in roots of Carrizo citrange [Citrus sinensis (L.) Osbeck. x Poncirus trifoliata (L.) Raf].

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. × 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.

Bicarbonate blocks iron translocation from cotyledons inducing iron stress responses in Citrus roots.

The effect of bicarbonate ion (HCO3(-)) on the mobilization of iron (Fe) reserves from cotyledons to roots during early growth of citrus seedlings and its influence on the components of the iron acquisition system were studied. Monoembryonic seeds of Citrus limon (L.) were germinated "in vitro" on two iron-deprived media, supplemented or not with 10mM HCO3(-) (-Fe+Bic and -Fe, respectively). After 21d of culture, Fe concentration in seedling organs was measured, as well as gene expression and enzymatic activities. Finally, the effect of Fe resupply on the above responses was tested in the presence and absence of HCO3(-) (+Fe+Bic or +Fe, respectively). -Fe+Bic seedlings exhibited lower Fe concentration in shoots and roots than -Fe ones but higher in cotyledons, associated to a significative inhibition of NRAMP3 expression. HCO3(-) upregulated Strategy I related genes (FRO1, FRO2, HA1 and IRT1) and FC-R and H(+)-ATPase activities in roots of Fe-starved seedlings. PEPC1 expression and PEPCase activity were also increased. When -Fe+Bic pre-treated seedlings were transferred to Fe-containing media for 15d, Fe content in shoots and roots increased, although to a lower extent in the +Fe+Bic medium. Consequently, the above-described root responses became markedly repressed, however, this effect was less pronounced in +Fe+Bic seedlings. In conclusion, it appears that HCO3(-) prevents Fe translocation from cotyledons to shoot and root, therefore reducing their Fe levels. This triggers Fe-stress responses in the root, enhancing the expression of genes related with Fe uptake and the corresponding enzymatic activities.

Use or abuse of bioinformatic tools: a response to Samach.

In a recent paper, we described for the first time the effects of fruit on the expression of putative homologues of genes involved in flowering pathways. It was our aim to provide insight into the molecular mechanisms underlying alternate bearing in citrus. However, a bioinformatics-based critique of our and other related papers has been given by Samach in the preceding Viewpoint article in this issue of Annals of Botany. The use of certain bioinformatic tools in a context of structural rather than functional genomics can cast doubts about the veracity of a large amount of data published in recent years. In this response, the contentions raised by Samach are analysed, and rebuttals of his criticisms are presented.

Self-pollination and parthenocarpic ability in developing ovaries of self-incompatible Clementine mandarins (Citrus clementina).

This study aimed to determine if self-pollination is needed to trigger facultative parthenocarpy in self-incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). 'Marisol' and 'Clemenules' mandarins were selected, and self-pollinated and un-pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self-pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time-course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA1 and GA4 ), auxin (IAA) and abscisic acid (ABA) content in the ovary. 'Clemenules' showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9 days after pollination in both cultivars. Self-pollination did not stimulate parthenocarpy, whereas both un-pollinated and self-pollinated ovaries set fruit regardless of the cultivar. On the other hand, 'Marisol' un-pollinated flowers showed greater parthenocarpic ovary growth than 'Clemenules' un-pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of 'Marisol' paralleled higher levels of GA1 in the ovary (+34% at anthesis). 'Marisol' ovary also showed higher hexoses and starch mobilization, but lower ABA levels (-64% at anthesis). Self-pollination did not modify carbohydrates or GA content in the ovary compared to un-pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination-independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self-incompatible mandarins is constitutively regulated.

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.

Proteomic analysis of "Moncada" mandarin leaves with contrasting fruit load.

A proteomic approach was used to know more about the molecular mechanism related to Citrus alternate bearing. To this end, we researched protein expression differences between on-crop and off-crop "Moncada" [Clementine 'Oroval' (Citrus clementina Hort ex Tanaka) x 'Kara' mandarin (Citrus unshiu Marc. x Citrus nobilis Lou.)] mandarin leaves. This variety usually shows a remarkable behaviour in alternate production. Samples were collected in the period during which the fruit affect flowering induction. From 2D DIGE gel, 110 spots were isolated: 43 showed increased expression in the off-crop samples compared to on-crop samples, while 67 showed increased expression in the on-crop samples against off-crop samples. These spots were identified by MALDI-MS or LC-MS-MS. According to the up-expressed proteins in off-crop leaves such as proteins related to nutrient reservoir activity or to the pentose phosphate pathway, the primary metabolism was more active in off-crop trees than in on-crop trees. In contrast, the proteins up-expressed in on-crop samples such as catalase were related to the oxidoreductase activity and, therefore, the redox state seemed different for off-crop and for on-crop leaves. Other proteins with unknown functions were isolated, which could be also related to the alternate bearing and to the flowering induction.

Fruit load modulates flowering-related gene expression in buds of alternate-bearing 'Moncada' mandarin.

BACKGROUND AND AIMS: Gene determination of flowering is the result of complex interactions involving both promoters and inhibitors. In this study, the expression of flowering-related genes at the meristem level in alternate-bearing citrus trees is analysed, together with the interplay between buds and leaves in the determination of flowering. METHODS: First defruiting experiments were performed to manipulate blossoming intensity in 'Moncada' mandarin, Citrus clementina. Further defoliation was performed to elucidate the role leaves play in the flowering process. In both cases, the activity of flowering-related genes was investigated at the flower induction (November) and differentiation (February) stages. KEY RESULTS: Study of the expression pattern of flowering-genes in buds from on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (CiFT), TWIN SISTER OF FT (TSF), APETALA1 (CsAP1) and LEAFY (CsLFY) were negatively affected by fruit load. CiFT and TSF activities showed a marked increase in buds from off trees through the study period (ten-fold in November). By contrast, expression of the homologues of the flowering inhibitors of TERMINAL FLOWER 1 (CsTFL), TERMINAL FLOWER 2 (TFL2) and FLOWERING LOCUS C (FLC) was generally lower in off trees. Regarding floral identity genes, the increase in CsAP1 expression in off trees was much greater in buds than in leaves, and significant variations in CsLFY expression (approx. 20 %) were found only in February. Defoliation experiments further revealed that the absence of leaves completely abolished blossoming and severely affected the expression of most of the flowering-related genes, particularly decreasing the activity of floral promoters and of CsAP1 at the induction stage. CONCLUSIONS: These results suggest that the presence of fruit affects flowering by greatly altering gene-expression not only at the leaf but also at the meristem level. Although leaves are required for flowering to occur, their absence strongly affects the activity of floral promoters and identity genes.

Flooding affects uptake and distribution of carbon and nitrogen in citrus seedlings.

Soil flooding has been widely reported to affect large areas of the world. In this work, we investigated the effect of waterlogging on citrus carbon and nitrogen pools and partitioning. Influence on their uptake and translocation was also studied through ¹5N and ¹³C labeling to provide insight into the physiological mechanisms underlying the responses. The data indicated that flooding severely reduced photosynthetic activity and affected growth and biomass partitioning. Total nitrogen content and concentration in the plant also progressively decreased throughout the course of the experiment. After 36 days of treatment, nitrogen content of flooded plants had decreased more than 2.3-fold compared to control seedlings, and reductions in nitrogen concentration ranged from 21 to 55% (in roots and leaves, respectively). Specific absorption rate and transport were also affected, leading to important changes in the distribution of this element inside the plant. Additionally, experiments involving labeled nitrogen revealed that ¹5N uptake rate and accumulation were drastically decreased at the end of the experiment (93% and 54%, respectively). ¹³CO2 assimilation into the plant was strongly reduced by flooding, with δ¹³C reductions ranging from 22 to 37% in leaves and roots, respectively. After 36 days, the relative distribution of absorbed ¹³C was also altered. Thus, ¹³C recovery in flooded leaves increased compared to controls, whereas roots exhibited the opposite pattern. Interestingly, when carbohydrate partitioning was examined, the data revealed that sucrose concentration was augmented significantly in roots (37-56%), whereas starch was reduced. In leaves, a marked increase in sucrose was detected from the first sampling onwards (36-66%), and the same patter was observed for starch. Taken together, these results indicate that flooding altered carbon and nitrogen pools and partitioning in citrus. On one hand, reduced nitrogen concentration appears to be a consequence of impaired uptake and transport. On the other hand, the observed changes in carbohydrate distribution suggest that translocation from leaves to roots was reduced, leading to significant starch accumulation in leaves and further decreases in roots.

Fruit regulates seasonal expression of flowering genes in alternate-bearing 'Moncada' mandarin.

BACKGROUND AND AIMS: The presence of fruit has been widely reported to act as an inhibitor of flowering in fruit trees. This study is an investigation into the effect of fruit load on flowering of 'Moncada' mandarin and on the expression of putative orthologues of genes involved in flowering pathways to provide insight into the molecular mechanisms underlying alternate bearing in citrus. METHODS: The relationship between fruit load and flowering intensity was examined first. Defruiting experiments were further conducted to demonstrate the causal effect of fruit removal upon flowering. Finally, the activity of flowering-related genes was investigated to determine the extent to which their seasonal expression is affected by fruit yield. KEY RESULTS: First observations and defruiting experiments indicated a significant inverse relationship between preceding fruit load and flowering intensity. Moreover, data indicated that when fruit remained on the tree from November onwards, a dramatic inhibition of flowering occurred the following spring. The study of the expression pattern of flowering-genes of on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (FT), SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), APETALA1 (AP1) and LEAFY (LFY) were negatively affected by fruit load. Thus, CiFT expression showed a progressive increase in leaves from off trees through the study period, the highest differences found from December onwards (10-fold). Whereas differences in the relative expression of SOC1 only reached significance from September to mid-December, CsAP1 expression was constantly higher in those trees through the whole study period. Significant variations in CsLFY expression only were found in late February (close to 20 %). On the other hand, the expression of the homologues of TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS C (FLC) did not appear to be related to fruit load. CONCLUSIONS: These results suggest for the first time that fruit inhibits flowering by repressing CiFT and SOC1 expression in leaves of alternate-bearing citrus. Fruit also reduces CsAP1 expression in leaves, and the significant increase in leaf CsLFY expression from off trees in late February was associated with the onset of floral differentiation.

Root signalling and modulation of stomatal closure in flooded citrus seedlings.

In this work, we studied the sequence of responses induced by flooding in citrus plants, with the aim of identifying the signals that lead to stomatal closure. One-year-old seedlings of Carrizo citrange, grown in sand under greenhouse conditions, were waterlogged for 35 d and compared with normally watered well-drained plants. Significant decreases in stomatal conductance and transpiration were detected between flooded and control seedlings from a week after the beginning of the experiment. However ABA concentration in leaves only started to increase after three weeks of flooding, suggesting that stomata closed in the absence of a rise in foliar ABA. Therefore, stomatal closure in waterlogged seedlings does not appear to be induced by ABA, at least during the early stages of flood-stress. The low levels of ABA detected in roots and xylem sap from flooded seedlings indicated that it is very unlikely that the ABA increase in the leaves of these plants is due to ABA translocation from roots to shoots. We propose that ABA is produced in old leaves and transported to younger leaves. Flooding had no effect on water potential or the relative water content of leaves. Soil flooding reduced root hydraulic conductance in citrus seedlings. This effect was already evident after a week of waterlogging, and at the end of the experiment, flood-stressed seedlings reached values of root hydraulic conductance below 12% of that of control plants. This reduction was related to down-regulation of the expression of PIP aquaporins. In addition, whole plant transpiration was reduced by 56% after 35 d under flooding conditions. Flood-stress also decreased the pH of sap extracted from citrus roots. Evidence is presented suggesting that acidosis induced by anoxic stress in roots causes gating of aquaporins, thereby decreasing hydraulic conductance. Additionally, stomatal closure finely balances-out low pH-mediated losses of root hydraulic conductance therefore maintaining stable leaf hydration.

Plant phenotype demarcation using nontargeted LC-MS and GC-MS metabolite profiling.

The characterization of the metabolome is a critical aspect in basic research and plant breeding. In this work, the putative application of metabolomics for phenotyping closely related genotypes has been tested. Crude extracts were profiled by LC-MS and GC-MS, and mass data extraction was performed with XCMS software. Result validation was achieved with principal component analysis (PCA). The ability of the profiling methodologies to discriminate plant genotypes was assessed after hierarchical clustering analysis (HCA). Cluster robustness was assessed by a multiscale bootstrap resampling method. A better performance of LC-MS profiling over GC-MS was evidenced in terms of phenotype demarcation after PCA and HCA. Citrus demarcation was similarly achieved independently of the environmental conditions used to grow plants. In addition, when all different locations were pooled in a single experimental design, it was still possible to differentiate the three closely related genotypes. The presented methodology provides a fast and nontargeted workflow as a powerful tool to discriminate related plant phenotypes. The novelty of the technique relies on the use of mass signals as markers for phenotype demarcation independent of putative metabolite identities and the relatively simple analytical strategy that can be applicable to a wide range of plant matrices with no previous optimization.

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.