What is malate in plants

This light inhibition of respiration has been directly linked to mitochondrial photorespiratory reactions, resulting in increased redox state and adenylate phosphorylation level in the mitochondrial matrix Bykova et al. The effect is understood to include restrictions at the pyruvate dehydrogenase complex and at other reactions within the TCA cycle.

The mutant plants studied here also show light inhibition of TCA cycle-linked respiration. What we note here that is different is the clear elevation of respiratory rate in terms of direct measurements of oxygen consumption and CO 2 evolution in darkened leaves and calculations of leaf CO 2 respiration in light in the mmdh1mmdh2 plants Figs.

This implies that in wild-type Arabidopsis plants, mMDH has a degree of metabolic control in limiting respiratory rate in leaves. This connection is confirmed by the analysis of the complemented mmdh1mmdh2 , where respiratory rates are returned to wild-type levels by the expression of mMDH1 Fig.

Notably, this is a leaf-specific effect, as analysis of root respiration suggests no change in intact tissue respiratory rates Fig. S2 , it appears that this respiratory change has broad downstream consequences for plant biomass production. However, this result of an elevated respiration by mMDH removal is not, at face value, consistent with reports on antisense inhibition of mMDH in tomato.

Work on isolated mitochondria from fruits from the same plants showed a decrease in TCA cycle flux when analyzed by NMR but no change in respiratory capacity when measured as oxygen consumption. The same workers analyzed root respiration rates and showed dramatic reductions in respiratory rate van der Merwe et al.

However, direct gas-exchange measurements of respiration in tomato plants are not reported, and although the antisense clearly reduces mMDH, it is unclear how much residual mMDH was present in the tomato antisense plants to compare with the single or double mutant Arabidopsis data set.

So, while it is not clear how to directly reconcile the differences noted at this time, the hint that the phenotype in tomato is very conditional on daylength might show that substantial dynamics operate in the translation of mMDH changes to resultant changes in respiration rate and plant growth Nunes-Nesi et al.

Our data in Arabidopsis, irrespective of daylength, are most similar to those reported in tomato for short-day-grown rather than long-day-grown plants Nunes-Nesi et al. Metabolic schemes of photorespiration note that to balance the pathway as a whole, the reducing equivalents from Gly oxidation in mitochondria need to be returned to the peroxisome to allow hydroxpyruvate reduction to generate glycerate for reentry to the chloroplast.

Recently, we noted that PMDH double mutants are not classical photorespiratory mutants, as they can grow in normal air, albeit with a slight decrease in photosynthetic rates compared with the wild type Cousins et al.

However, we did report alterations of the rate of photorespiration and the CO 2 PIB, suggesting that the ratio of CO 2 release to ribulose 1,5-bisphosphate oxygenation was altered. S3 and S4. This clearly implies some role of mMDH in photorespiration, perhaps mainly through a limitation on Gly oxidation rate. A role of plant mitochondria in ascorbate synthesis and metabolism was noted with the discovery that the terminal step of the main biosynthetic pathway, GLDH, is a mitochondrial inner membrane protein Siendones et al.

The report that antisense of mMDH can elevate ascorbate pool size in tomato leaves and increase GL-dependent ascorbate biosynthesis rate has been interpreted as linked to these respiratory chain connections and the redox poise of the mitochondrial matrix Nunes-Nesi et al.

It has been reported that the abundance and activity of GLDH is not well correlated with foliar ascorbate levels Bartoli et al. Interestingly, a recent report has shown the capacity for GLDH to be redox regulated, particularly by glutathionylation of a particular amino acid residue Leferink et al. We observed here that mMDH loss is associated with a decrease in the abundance of GLDH in mitochondria and elevated abundance of total cellular ascorbate.

Currently, this does not directly explain the tantalizing connection between the TCA cycle and ascorbate content per se, but it does provide a clear molecular linkage between the machinery of the two pathways. Hence, the ascorbate link to mMDH may be part of a shift in the steady-state abundance of enzymes to maintain the redox poise of the mitochondrial matrix in the absence of mMDH. Overall, a complex and respiration-controlling role for mMDH is revealed by this mutant analysis in Arabidopsis.

Changes in the abundance of isoforms of mMDH in mitochondria between tissues in different plants Bardel et al.

This may provide new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms underpinning to multifaceted link between respiration and photosynthesis in plants. Seedlings were first covered with transparent acrylic hoods and regularly sprayed. After 1 week, the hoods were removed and the seedlings were transplanted into individual pots filled with the same perlite:soil mix. Seeds were germinated indoors and transferred to pots with soil at the seedling stage and then moved to the outdoor garden.

For the wild type, mmdh1 , and mmdh2 , 40 plants were used, and for mmdh1mmdh2 , 30 plants were used. The mmdhmmdh2 double mutant was transformed via the floral dip method with the reconstructed pGreen binary vector p harboring a double cauliflower mosaic virus 35S promoter, full-length mMDH1 cDNA, and a cauliflower mosaic virus terminator cassette Pracharoenwattana et al.

Seeds collected from 35S : mMDH1 and 35S:empty vector-transformed plants were screened for hygromycin resistance. PCR products were visualized on an agarose gel stained with ethidium bromide. All measurements were done after at least 2 h of illumination. One fully developed leaf per plant was enclosed in a 6-cm 2 leaf chamber.

Data were recorded after the leaf acclimated in the leaf chamber to the desired conditions of light, temperature, relative humidity, and CO 2 concentration. Data were adjusted to the enclosed leaf area determined with a LI leaf area meter Li-Cor. At each step, the rate of CO 2 evolution was recorded after steady-state conditions were reached. The rate of mitochondrial respiration in the light day respiration [R d ] was determined using the Laisk method as described by Laisk or more recently by Villar et al.

This method determines the internal CO 2 concentration C i at which the rate of photosynthesis equals that of photorespiration. The rate of mitochondrial respiration in the dark R n was determined by measuring the rate of CO 2 evolution in the dark. Dark respiration was also measured on detached leaf discs using a Clark-type oxygen electrode Hansatech Instruments. Gas-exchange parameters and CO 2 responses curves were determined as described above, feeding the gas-exchange system with N 2 containing only traces of oxygen less than 0.

All relevant Arabidopsis genotypes were grown and maintained in exact accordance with a previously published protocol Lee et al. Shoot mitochondria were isolated from 3-week-old hydroponically grown Arabidopsis using a method adapted from Lee et al. Approximately g of shoot material was homogenized with a Polytron blender Kinematica in mL of cold grinding medium 0.

The homogenate was filtered through four layers of Miracloth and centrifuged at 1, g for 5 min, and the resulting supernatant was then centrifuged at 24, g for 15 min. The organelle pellet was washed by repeating the 1, g and 24, g centrifugation steps twice in Suc wash medium 0.

The gradient was then centrifuged at 40, g for 45 min. The mitochondrial band was seen as a yellow-brown band near the bottom of the centrifuge tube.

The upper layers of the density gradient were removed, and the mitochondrial band was collected. The mitochondrial fraction was diluted approximately 5-fold with Suc wash medium devoid of BSA and centrifuged at 24, g for 10 min. Mitochondria formed a white-yellow band near the top of the gradient and were carefully removed by pipette, with maximal effort exercised to avoid coremoval of the adjacent green band containing chloroplasts and plastids.

The mitochondria obtained were then diluted 5-fold in wash buffer no BSA and centrifuged at 24, g for 10 min. This step was repeated three times, with the final pellet retained in a small volume of wash buffer for usage in later experiments. All oxygen consumption measurements were performed using a Clarke-type oxygen electrode, and the data feed was collected by Oxygraph Plus version 1. The electrode was calibrated via the addition of excess sodium dithionite for depletion of all oxygen in a 1-mL volume of deionized water housed in the electrode chamber.

KCN 0. Note that Glu was used as a respiratory substrate to prevent the accumulation of OAA to drive the reaction in favor of malate oxidation. Rehydration of the strips and the first isoelectric focusing IEF dimension electrophoresis were performed on an IPGphor Unit GE Healthcare using the following settings: 12 h at 30 V rehydration step , 1 h at V, 1-h gradient from to 1, V, 1-h gradient from 1, to 3, V, 2-h gradient from 3, to 8, V, and 5 h at 8, V.

The reaction was stopped by the addition of Lys 10 m m for 10 min on ice in the dark. Rehydration of the strips and the first IEF dimension electrophoresis were performed on an IPGphor Unit GE Healthcare using the following settings: 12 h at 30 V rehydration step , 1 h at V, 1-h gradient from to 1, V, 1-h gradient from 1, to 3, V, 2-h gradient from 3, to 8, V, and 5 h at 8, V.

Following separation, gels were scanned using the Typhoon Trio Variable Mode Imager at a resolution of pixel size , and the photomultiplier tube was set to V. Proteins were processed quantification using the DeCyder 2-D Differential Analysis software version 6. In order to get statistical significance from these experiments, three sets of proteins from three independent experiments were labeled and subjected to electrophoresis. After electrophoresis, proteins were visualized by colloidal Coomassie Brilliant Blue G staining.

The aim of the standard gel is to allow the identification of proteins by excision of gel spots followed by mass spectrometry. Gel spots to be analyzed were cut from colloidal Coomassie Brilliant Blue-stained gels.

Proteins were digested with trypsin trypsin sequencing grade; Roche Diagnostic according to Lee et al. The samples were analyzed with an Agilent series capillary liquid chromatography system and an Agilent Technologies XCT Ultra IonTrap with an electrospray ionization source equipped with a low-flow nebulizer in positive mode controlled by Chemstation Rev B.

Samples were loaded with an Agilent series capillary liquid chromatography system onto a 0. Details of matches are shown in Supplemental Table S1. In accordance with a previously established protocol Ritte et al. The supernatant designated crude extract was quantified and used for the determination of total MDH activity. NADH oxidation rates before and after the addition of 0. Metabolites were extracted from frozen Arabidopsis leaves that had been harvested at the times indicated according to a method adapted from that described by Roessner-Tunali et al.

Detailed methods are given by Sappl et al. Raw gas chromatography GC -MS data preprocessing and statistical analysis were performed using Metabolome-Express software Carroll et al. Full data are available via the aforementioned Web site, and processed data are provided in Supplemental Tables S2 and S3.

Ascorbic acid content was measured by the ascorbate oxidase assay Rao and Ormrod, In accordance with an established protocol Zhang et al. Total ascorbate represents the addition of reduced and oxidized ascorbate concentrations. Gel electrophoresis was performed at 25 mA per gel for 3 h.

Polyacrylamide gels were then incubated in a transfer solution for 1 h. Transferred proteins were probed with primary antibodies against mtHSP70 from T. Chemiluminescence detection linked to horseradish peroxidase was used to visualize a secondary antibody, and quantitative light emission was recorded using ImageQuant-RT ECL Amersham Biosciences.

The following materials are available in the online version of this article. Supplemental Figure S1. Supplemental Figure S2. Additional phenotypic characterization of mmdh mutants. Supplemental Figure S3. Kinetics of CO 2 evolution during the postillumination period in mmdh mutants.

Supplemental Figure S4. Supplemental Figure S5. Supplemental Table S1. Supplemental Table S2. Supplemental Table S3. Plant Physiol : — Google Scholar. Science : — Proteomics 2 : — Plant Cell Environ 28 : — J Biol Chem : — Planta : — BMC Bioinformatics 11 : — Chew O Whelan J Millar AH Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants.

Interestingly, malate was the only organic acid showing differences in its subcellular distribution. Whereas citrate, isocitrate, and fumarate were found predominantly in the vacuoles, malate was reduced significantly in the vacuoles, although increases in malate were observed in the cytosol of the mutant lines Supplemental Table S5.

We next decided to perform a detailed analysis of the primary metabolism in leaves and in guard cell-enriched epidermal fragments by using the established GC-MS approach Lisec et al. This analysis revealed that, among the 48 successfully annotated compounds, considerable changes in amino acids, as well as in both tricarboxylic acid cycle and photorespiratory intermediaries, were observed Fig. By analyzing individual amino acids, we observed significant increases in leaves for both lines in Asn, Asp, and Lys levels as well as in the branched chain amino acids Leu and Ile, and the aromatic amino acid Tyr also was increased in the tdt plants.

Notably, glycolate and Gly, intermediates of the photorespiratory pathway, were decreased significantly in leaves, whereas Gln levels increased in mutant plants in both leaves and guard cells. The levels of some organic acids found in the first half of the tricarboxylic acid cycle citrate only in leaves and isocitrate in both leaves and guard cells were increased strongly, while succinate, fumarate, and malate were reduced in mutant lines only in leaves.

Other changes of note observed in the metabolite profile were the significant increases in myoinositol and reductions in maltose levels in leaves of both lines. Intriguingly, significant increases were observed in the levels of Glc, Fru, and trehalose in guard cells. Heat map representing the changes in relative metabolite contents in leaves and guard cell-enriched epidermal fragments GC from wild-type WT and tdt plants.

The full data sets from these metabolic profiling studies are available in Supplemental Table S6. The color code of the heat map is given at the log 2 following the scale above the diagram. Data are normalized with respect to the mean response calculated for the wild type to allow statistical assessment, individual plants from this set were normalized in the same way.

Gray cells indicate metabolites that were not detected or could not be annotated. We next evaluated whether the metabolic perturbations observed were accompanied by changes in the activity of important enzymes in leaves, which are associated with glycolysis and carbohydrate metabolism Table III. Interestingly, the maximum activities of PGK, pyruvate kinase, and aldolase significantly only for tdt-2 were higher in tdt than in wild-type plants. There were no changes in the activities of hexokinase, phosphofructokinase, enolase, or TPI.

Similarly, trans-aldolase and G6PDH, both related to the pentose phosphate pathway, and Suc synthase were unaltered in tdt plants. However, the activity of acid invertase was decreased in the mutant lines. Activities were determined in whole 5-week-old rosettes harvested at the middle of the light period. We decided to directly assess the respiration rate by performing two complementary approaches. First, we directly evaluated the rate of light respiration in the mutant lines by measuring the 14 CO 2 evolution following incubation of leaf discs with positionally labeled [ 14 C]Glc molecules to assess the relative rate of flux through the tricarboxylic acid cycle.

For this, we incubated leaf discs under light supplied with either [1- 14 C]Glc or [3,4- 14 C]Glc over a period of 6 h.

During that period, we collected the 14 CO 2 evolved at hourly intervals. CO 2 can be released from the C1 position by the action of enzymes that are not associated with mitochondrial respiration, but CO 2 released from the C3,4 positions of Glc cannot Nunes-Nesi et al. Therefore, the ratio of CO 2 evolution from C3,4 to C1 positions provides a reliable indication of the relative rate of the tricarboxylic acid cycle versus other carbohydrate oxidation processes.

By comparing the 14 CO 2 release from mutant lines and wild-type plants, we observed that significant increases occurred only for the tdt-2 line after 5 h of incubation with [1- 14 C]Glc Fig. Furthermore, the higher dark respiration, measured by using an infrared gas analyzer system, revealed higher rates of CO 2 evolution in the leaves of tdt plants than in the wild type Fig.

Respiration parameters in leaf discs from wild-type WT and tdt plants. A and B, 14 CO 2 evolution from isolated leaf discs was determined under light conditions. The 14 CO 2 released was captured at hourly intervals in a KOH trap, and the amount of radiolabel released was quantified subsequently by liquid scintillation counting. C, Ratio of carbon dioxide evolution from C3,4 to C1 positions of Glc in leaves of tdt plants. D, Dark respiration measurements performed on 5-week-old plants.

To evaluate the reasons underlying the growth impairment observed in tdt plants under short-day conditions Table I ; Supplemental Fig. S1 , we decided to investigate whether the impaired organic acid accumulation affected stomatal function and, thereby, photosynthetic capacity in these plants.

We were somewhat surprised to find that the growth phenotype was independent of changes in stomatal density, stomatal index, and photosynthetic capacity Tables I and II ; Supplemental Fig. S6 ; Supplemental Tables S2—S4. Collectively, these results indicate that guard cell function is not highly affected in tdt plants Fig. Notably, although tDT is essential for mediating correct compartmentation of the dicarboxylates, tdt plants still exhibit residual malate-importing activity Emmerlich et al.

It has been suggested that tDT is the major transporter responsible for malate and fumarate through the tonoplast in mesophyll cells Hurth et al. Because this channel does not exhibit sufficient activity to accumulate dicarboxylates at concentrations required for normal metabolic functioning, it may not be able to fully compensate for the absence of tDT in mesophyll cells Hurth et al.

Furthermore, ALMT9 was first observed to mediate malate and fumarate currents directed into the vacuole; it was later shown to mediate malate-induced chloride current, which also is important for stomatal opening Kovermann et al. Consistent with the lack of change in stomatal function, in this study, we did not observe any change in apoplastic levels of fumarate, malate, and citrate Fig. In keeping with this, it is highly tempting to suggest that, although malate and fumarate cannot be accumulated properly in the vacuoles due to the lack of a functional tDT transporter, the majority of these compounds produced need to be redistributed further within the cell.

This would support the proper stomatal function by the maintenance of apoplastic concentrations of organic acids even with decreased total amounts in the leaves Fig. Moreover, it also indicates that these compounds are highly metabolized by the tricarboxylic acid cycle Fig. Thus, it seems that mitochondrial metabolism, especially of those pathways associated with malate, has great potential to improve photosynthesis, and growth ultimately, most likely through a better control of stomatal movements Nunes-Nesi et al.

That said, it remains to be elucidated whether the functional redundancy in the vacuolar organic acid transport in guard cells is responsible for the lack of stomatal phenotype in tdt plants. A detailed photosynthetic characterization revealed that the lower vegetative growth in tdt plants was not due to an impaired photosynthetic capacity.

This analysis was necessary despite the lack of change in stomatal behavior, since the rate of CO 2 diffusion through the stomata is not the only constraint to the photosynthetic performance in plants, and the pathway to CO 2 diffusion from stomata to the Rubisco carboxylation sites in the chloroplasts can become an important limiting factor to the photosynthetic process as well as the Rubisco carboxylic capacity Gerhardt et al.

Our results demonstrated an invariable instantaneous net CO 2 assimilation in tdt plants under both growth irradiance and light saturation Table II ; Supplemental Table S2.

S6 ; Supplemental Tables S3 and S4. Arabidopsis plants with highly reduced levels of malate and fumarate due to the overexpression of a maize Zea mays plastidic NADP-malic enzyme exhibited smaller rosettes with decreased biomass accumulation and thinner leaves when compared with wild-type plants.

This was almost certainly the consequence of a reduced photosynthetic performance under short-day conditions in these plants Zell et al. Interestingly, these findings were not observed when these plants were grown under long-day conditions Fahnenstich et al.

Indeed, the rates of starch and organic acid usage during the night correlate with one another and with the relative growth rate, indicating that, although these two carbon sources are regulated independently, their utilization is highly coordinated Fahnenstich et al. Although many of the molecular details concerning the connection between starch and organic acid metabolism in governing plant growth are being revealed Figueroa et al. We showed here that tdt plants were impaired in their growth under short-day conditions, which can be explained, at least partially, by the reduced malate and fumarate content in the leaves of these plants across the entire diurnal cycle Fig.

Moreover, starch accumulation in tdt mutant lines in our growth conditions was negatively affected, with reduced values at the end of the light period Fig. The impaired malate exchange observed in tdt plants has been proposed previously to be able to provoke unknown regulatory reactions at the expense of cytosolic energy equivalents Emmerlich et al.

This assumption was further reinforced by the demonstration that radiolabeled malate fed into mutant leaf discs entered the tricarboxylic acid cycle much faster than in wild-type tissues Emmerlich et al.

Furthermore, the observation that tdt leaf discs exhibited both an increased respiratory activity and increased respiratory quotient Hurth et al. Here, we provide compelling evidence that the absence of tDT strongly affects mitochondrial metabolism in vivo. By using complementary approaches, we further confirmed that the slower growth in tdt plants was accompanied by enhanced dark and light respiration Fig. Tomato plants exhibiting either an antisense inhibition of fumarase Nunes-Nesi et al.

In these transgenic plants, the flux through the tricarboxylic acid cycle was clearly reduced; however, whereas deficiency in fumarase led to lower CO 2 assimilation and reduction in growth Nunes-Nesi et al.

That respiratory metabolism was affected in these lines is by no means surprising, given that they are affected directly in the tricarboxylic acid cycle. That the tdt lines also are affected is highly interesting, since it suggests that the tricarboxylic acid cycle is, to a considerable extent, fueled directly by malate supply, which is accumulated in the cytosol in these plants Supplemental Table S5.

Moreover, it is in keeping with previous suggestions of a noncyclic flux mode of the tricarboxylic acid cycle in leaves under light conditions Sweetlove et al. This scenario is further supported by the steady-state levels of the intermediates of the tricarboxylic acid cycle in leaves observed here Fig.

It is important to highlight that the levels of succinate, fumarate, and malate were decreased in leaves, but not in guard cells, of mutant lines Fig. This observation suggests a different functional importance of the tDT transporter in mesophyll and guard cells, which is in agreement with the differential expression pattern of tDT , being more expressed in mesophyll cells than in guard cells Bates et al.

Moreover, the high expression of ALMT6 at the guard cell tonoplast seems to compensate for the lack of tDT, at least regarding the proper storage of malate and fumarate in those cells Fig. Curiously, since we observed a strong accumulation of citrate in leaves and isocitrate in both leaves and guard cells and this accumulation is addressed occurring within the vacuole Supplemental Table S5 , it is tempting to speculate that tDT also might be somehow involved with the compartmentalization of these organic acids.

Although we were not able to ascertain in this study which organic acids are effectively transported by the tDT, it will be interesting to investigate in future studies whether the mitochondrial metabolism in guard cells also is affected when tDT is repressed. Collectively, our results suggest that the impaired accumulation of malate and fumarate as a consequence of nonfunctional tDT affects the cellular homeostasis in mesophyll cells by changing mitochondrial metabolism, without negative impacts to the stomatal and photosynthetic behaviors.

When the relative concentrations of the apoplastic and subcellular malate pools are considered Gerhardt et al. Additionally, transporting the increased cytosolic malate pools for the maintenance of apoplastic levels could be a mechanism by which tdt plants maintain stomatal function.

This observation is thus consistent with our previous studies, both suggesting that apoplastic malate levels play a crucial role in stomatal function. All Arabidopsis Arabidopsis thaliana plants used here were of the Wassilewskija ecotype background. Whole rosettes from 5-week-old plants were harvested, and the rosette fresh and dry weight, LA, and SLA were measured.

LA was measured by a digital image method using a scanner Hewlett Packard Scanjet G , and the images were processed using ImageJ software Schindelin et al. SLA was calculated as described by Hunt et al. Nail polish copies were made using a colorless glaze Von Groll et al. The measurements were performed on the images using AxionVision software Carls Zeiss. Stomatal density and stomatal index the ratio of stomata to stomata plus other epidermal cells were determined in at least 10 fields of 0.

After 2 h of incubation, the stomatal aperture was evaluated. The leaves were gently dried, and the adaxial epidermis was carefully fixed to an autoclave tape. The abaxial surface of the leaves was then peeled off by fixing an adhesive film tesafilm crystal clear; Tesa , and the images were taken immediately Azoulay-Shemer et al.

Six leaves from different plants were evaluated, and the aperture of at least 20 stomata per leaf was measured, giving a total of at least stomata per genotype. The isolation of guard cell-enriched epidermal fragments was performed as described previously Pandey et al. Briefly, fully expanded leaves from five rosettes per sample were blended for 1 min plus 1 min twice for 30 s using a Waring blender Phillips; RI with an internal filter to clarify the epidermal fragments of mesophyll and fibrous cells.

For mesophyll cell protoplast isolation, approximately 20 fully expanded leaves per replicate were harvested at the middle of the light period.

Detailed primer information is described in Supplemental Table S1. The leaf apoplastic fluid was collected as described previously with few modifications Madsen et al.

Briefly, six completely expanded leaves were cut with a razor blade and submerged immediately in deionized water to remove any surface contaminants at the middle of the light period.

Afterward, the leaves were submerged in the washing solution deionized water. After vacuum infiltration, leaf surfaces were completely and gently dried. Leaves were placed on a Parafilm sheet, which was folded in such way that the leaves were stacked between layers of Parafilm. Finally, this leaf-Parafilm sandwich was mounted as described Madsen et al.

The apoplastic washing solutions were dried in a lyophilizer. By using standards for citrate, malate, and fumarate, we were able to quantify the absolute amount of these organic acids in the apoplastic fraction using an established GC-MS approach Lisec et al. Epidermis extraction was done by using intact epidermis imaging instead of blending , whereas the abaxial side of leaves was gently pressed onto a coverslip with a very thin coating of medical adhesive Hollister.

Upper cell layers were carefully removed using a razor blade. Gas-exchange parameters were determined simultaneously with chlorophyll a Chl a fluorescence measurements using the same gas-exchange system described above.

All the Chl a fluorescence parameters were measured exactly as described by Medeiros et al. Simultaneously, Chl a fluorescence parameters were obtained Yin et al.

The levels of starch, Suc, Fru, and Glc in the leaf tissues were determined as described previously Fernie et al. Malate and fumarate were determined as detailed by Nunes-Nesi et al. The photosynthetic pigments were determined as described Porra et al. The metabolite profiling was carried out in samples harvested at the middle of the day for both leaves Lisec et al.

Specifically, after isolation, the guard cell-enriched epidermal fragments were snap frozen in liquid nitrogen and lyophilized for 1 week. Approximately 30 mg of lyophilized guard cell-enriched epidermal fragments was disrupted by shaking together with metal balls. The followed extraction and derivatization procedure was performed exactly as described Daloso et al.

Peaks were annotated manually, and ion intensity was determined by the aid of TagFinder software Luedemann et al. NAF was performed as described Arrivault et al. The pellet was resuspended in 7 mL of heptane and divided into six aliquots of equal volume. Prior to analysis, the dried pellets were homogenized with the appropriate extraction buffer by the addition of one steel ball bearing and shaking at 25 Hz for 1 min in a ball mill Retsch MM; Retsch.

Enzyme and metabolite markers ADP Glc pyrophosphorylase and Rubisco activities for the chloroplast, phospho enol pyruvate carboxylase and uridine diphosphate Glc pyrophosphorylase activities for the cytosol, and acid invertase activity and nitrate amounts for the vacuole were determined as described by Arrivault et al.

Malate and fumarate were quantified via coupled enzymatic assays Cross et al. Absorbance was monitored at nm until OD stabilized, 0. The other metabolites were measured using the GC-MS method also detailed above. Determination of subcellular distribution was performed using BestFit software Klie et al. The enzymatic extract was prepared as described previously Gibon et al.

Then, the maximum activities of PGK, pyruvate kinase, phosphofructokinase, aldolase, G6PDH, and acid invertase were determined as described by Gibon et al. Estimations of the tricarboxylic acid cycle flux on the basis of 14 CO 2 evolution were performed following the incubation of isolated leaf discs in 10 m m MES-KOH, pH 6.

The results were interpreted following Rees and Beevers The data were obtained from the experiments using a completely randomized design using three genotypes, with the exception of the stomatal opening and closing kinetics, which were performed in a randomized block design.

All the statistical analyses were performed using the algorithm embedded into Microsoft Excel. The Arabidopsis Genome Initiative locus numbers for the major gene discussed in this article is as follows: tDT At5g The following supplemental materials are available. Supplemental Figure S1. Supplemental Figure S2. Growth phenotypes of wild-type and tdt plants. Supplemental Figure S3.

Transcriptome data in leaves and guard cells dissected manually from Arabidopsis leaves. Supplemental Figure S4. Relative transcript levels of tDT. Supplemental Figure S5. Relative transcript levels of genes involved in organic and inorganic ion transport in guard cells.

Supplemental Figure S6. A N curves in response to C i or C c in wild-type and tdt plants. Supplemental Figure S7. Supplemental Figure S8. Sugar content in wild-type and tdt plants. Supplemental Table S1.

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