Interactions of C₄ Subtype Metabolic Activities and Transport in Maize Are Revealed through the Characterization of DCT2 Mutants

Generation of DCT2 Mutant Lines by Targeted Ac Mutagenesis

Mutant alleles of DCT2 were recovered through insertional mutagenesis that used the Ac transposable element (McClintock, 1948). Transposition events containing an insertion into or around DCT2 were identified by reverse genetics using bti99221::Ac as a donor transposon that was close in physical and genetic proximity to DCT2 (401.2 kb, 0.44 cM). Selections were performed for increased copy number of the Ac element that enrich for transposition events as previously described (Brutnell and Conrad, 2003; Kolkman et al., 2005). Genetic screens of 1050 progeny identified four insertion sites that were fine mapped to the first exon (dct2-1::Ac and dct2-2::Ac), the 5′ untranslated region (dct2-3::Ac), and the second intron (dct2-4::Ac) of DCT2 (Figure 1A). Self-pollination resulted in homozygous lines for further analysis (referred to hereafter as dct2 without the Ac designation). Plants, homozygous for insertions dct2-3 and dct2-4, did not show a mutant phenotype and were deemed too far upstream to affect DCT2 transcription (dct2-3) or were efficiently spliced out of the transcript (dct2-4). Nine-day-old plants, homozygous for the dct2-1 and dct2-2 alleles, were phenotypically similar, with pale-yellow leaves (Figures 1B and 1C), low net photosynthetic rates (Figure 1F), and low chlorophyll content (Figure 1G). The mutants lacked the typical yellow-to-green color gradient observed in developing wild-type leaves (Figure 1C) and had low concentrations of Calvin cycle intermediates (Supplemental Table 1). In addition, quantitative real-time PCR failed to detect DCT2 transcripts in homozygous dct2-1 and dct2-2 lines (Figure 1E). Plants homozygous for these strong loss-of-function alleles developed slowly, resulting in thinner leaves and shorter plants that produced less biomass and infrequently generated seeds (Figure 1D). The metabolic features of dct2-1 were further characterized to assess the molecular basis underlying these phenotypes.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Molecular and Physiological Data for Wild-Type and dct2 Lines.

Insertional mutagenesis of DCT2 resulted in pale leaves and loss of carbon assimilation gradient.

(A) Ac.bti99221, located 401.2 kb (0.44 cM) 5′ of DCT2, relocated creating four insertions in and around the gene: two in the first exon (dct2-1 and dct2-2), one in the 5′ untranslated region (dct2-3), and one in the second intron (dct2-4). Red triangles indicate the 5′ end of the Ac insert.

(B) Wild-type (WT) and mutant plants (dct2-1 and dct2-2) 9 d after sowing. Red demarcations indicate a metric ruler with labeled centimeter increments.

(C), (F), and (G) Nine-day-old leaves of the wild type and dct2-1 showing loss of color gradient. Sections A, B, and C were tested for net CO₂ assimilation (F) with an infrared gas analyzer (LI-6400). Segments −4, −1, +4, and +10 were sampled for RNA-seq and metabolite quantification, including chlorophyll levels (G).

(D) Five-week-old wild-type and dct2-1 and dct2-2 plants showing the impeded development of mutant plants compared with the wild-type.

(E) Quantitative real-time PCR of DCT2 transcripts in the wild type (black). dct2-1 (white) and dct2-2 (gray) show no detectable transcripts of DCT2. Target/ref ratio calculated as DCT2/tubulin expression level. Error bars in (C), (E), and (F) represent sd (n = 3).

Leaf Development Is Impaired in Homozygous dct2-1 Plants

To characterize the phenotype of the mutant plants at the cellular level, histological surveys of dct2-1 and wild-type plants were assessed by transmission electron microscopy (TEM). Samples from four segments were collected along a developing third leaf at 9 d after sowing, as previously described (Li et al., 2010). The segments were referenced relative to the position of the ligule of leaf 2, as this point represents the source-sink boundary in a wild-type leaf, and demarcates the transition from shaded to fully exposed leaf tissue. Thus, segment −4 was defined by location at the base of the leaf blade that is photosynthetically inactive, −1 was taken at the sink-to-source transition zone (just below the point of leaf emergence from the whorl), segment +4 was from the mid-leaf, a region that is exposed to light and photosynthetically active (maturation zone), and +10 was from the tip of the leaf that is the terminally differentiated tissue (Figure 1C). The ultrastructure of the dct2-1 leaf in all segments suggested delayed development compared with wild-type plants (Figure 2). In the −4 segment of the leaf, both BS and M cells of dct2-1 had a large number of vacuoles (Figures 2C and 2D), indicating a lag in development compared with the fused vacuoles in wild-type plants (Figures 2A and 2B). In addition, dct2-1 BS plastids were small and regularly dividing, which is consistent with hindered leaf development and a sign of an immature tissue (Figures 2C and 2D). In the −1 segment, the chloroplasts in both BS and M cells of dct2-1 were also smaller with poorly developed granum lamellae (Figures 2E to 2H). In the +4 segment, both the wild type and dct2-1 showed the typical NADP-ME centrifugal arrangement of plastids. The thylakoid membrane of wild-type chloroplasts thickened in both BS and M cells, and the BS chloroplasts exhibited a small degree of granal stacking (Figures 2I and 2J). dct2-1 BS chloroplasts remained round, similar to their shape at the −1 segment, and in both BS and M chloroplasts, the thylakoid membranes did not show a significant thickening compared with the −1 segment (Figures 2K and 2L). Chloroplasts in both BS and M cells from the wild type were large by the +10 segment and showed an accumulation of starch granules and stacking in BS chloroplasts (Figures 2M and 2N), whereas those from dct2-1 remained in the same state as in the +4 segment and did not have starch granules or display granal stacks (Figures 2O and 2P). This impaired plastid development in dct2-1 plants may be a consequence of carbon starvation or due to a defect in related metabolic pathways.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

TEM of Wild-Type and dct2-1 Leaf Segments.

TEM analysis of the ultrastructure of four segments sampled along a developing third leaf at 9 d after sowing suggests impeded development of photosynthetic cells in dct2-1. The four segments correspond to four rows of panels in the figure and indicate position along the leaf inspected from the base of the leaf (also called −4) to the tip (also called +10) as stated along the y axis. For both the wild type and dct2-1, two columns are presented that show varying degrees of magnification to emphasize salient features described in the text. In particular, the panels emphasize a differing number of vacuoles in −4, differing grana structure and chloroplast size in −1 and +4, and reduced starch and granal stacking in +10. Ch, chloroplast; Vc, vacuole; sg, starch. Bars = 20 µm in (A), (G), (I), (K), (M), and (O), 1 µm in (B), (H), and (J), 10 µm in (C) and (E), and 2 µm in (D), (F), (L), (N), and (P).

DCT2 Transports Malate into the BS Chloroplast during C₄ Photosynthesis

DCT2 encodes a passive-mediated transporter that is minimally capable of exchanging malate for 2-oxoglutarate (2-OG), glutamate, or oxaloacetate (Taniguchi et al., 2004). The malate transport activities of BS chloroplasts were compared between wild-type and dct2-1 plants (Figures 3A and 3B). Due to low-yield and low density of intact dct2-1 BS chloroplasts, the direct uptake of malate into this organelle could not be assessed with traditional silicone-oil centrifugation fractionation methods. However malate-dependent pyruvate formation by maize BS chloroplasts has previously been described (Boag and Jenkins, 1985), as an indirect assay of malate import activity by measuring the conversion of exogenous malate to pyruvate in BS chloroplasts with a stromal NADP-ME. Organelle preparations using a Percoll gradient resulted in ∼89% wild-type and 83% dct2-1 intact isolated chloroplasts, respectively, as indicated by NADP-ME activity before and after chloroplast lysis (Figure 3B). Chlorophyll content per chloroplast was approximately 3 times lower in dct2-1 than in wild-type plastids. Therefore, activities were normalized by chloroplast number (µmol [10⁹ chloroplasts]⁻¹ h⁻¹), and then the transport capacity was compared relative to maximal NADP-ME activity from intact chloroplasts (Figure 3B). This assay revealed that pyruvate was produced at a slower rate in dct2-1 chloroplasts relative to the wild type. Furthermore, chloroplast lysis with detergent resulted in elevated pyruvate formation rates for both the wild type and dct2-1, suggesting a significant transport limitation in both lines (Boag and Jenkins, 1985). Comparison of the lysed chloroplast values using either the NADP-ME activity assay (i.e., 846/1828; ∼46%) or pyruvate formation (i.e., 391/826; ∼47%) measurements showed a similar decrease in activity in dct2-1. The normalized pyruvate formation rate was a modest 2.3% (wild type) or 0.7% (dct2-1) relative to NADP-ME activity with the wild type value being comparable to other reports (Boag and Jenkins, 1985; Jenkins and Boag, 1985; Boag and Jenkins, 1986). The limited pyruvate formation observed in dct2-1 plants was likely due to either some malate import or leakage of the isolated plastids. Regardless, the assay demonstrated compromised malate import associated with the dct2-1 mutation. Malate-dependent pyruvate formation generated from wild-type chloroplasts was enhanced in the presence of aspartate (Figures 3A and 3B), as has been previously reported (Boag and Jenkins, 1985, 1986; Jenkins and Boag, 1985), but the enhancement was not observed in the mutant chloroplasts, suggesting that aspartate is unable to stimulate a malate-specific import process in dct2 plants (discussed further below).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Malate Transport in the Wild Type and dct2-1.

Malate-dependent pyruvate formation, NADP-ME activities, and 2,3,3-²H-malate labeling of BS chloroplasts were measured in 9-d-old wild-type and dct2-1 plants.

(A) Time course of malate-dependent pyruvate formation in BS chloroplasts of wild-type (square symbols) and dct2-1 (round and triangle symbols) plants. BS chloroplasts were suspended in 10 mM malate with or without 10 mM aspartate and the amount of pyruvate formed was quantified spectrophotometrically (se, n = 3).

(B) NADP-ME activity and pyruvate (PYR) formation rate in BS chloroplasts of wild-type and dct2-1 plants (se, n = 3). NADP-ME activity was based on spectral detection of NADP at 340 nm. The rates of pyruvate formation in chloroplasts with or without aspartate were calculated from the slopes in (A) (2 to 5 min). Calculations for the intact chloroplast NADP-ME activity, chloroplast intactness (%), and pyruvate formation in intact chloroplasts normalized to NADP-ME activity (%) are defined in the gray box. Intact chloroplast NADP-ME activity indicates the formation of pyruvate that is not transport limited; thus, normalization by this value provides a comparison specific to the transport function. The percentages in parentheses for pyruvate formation from lysed chloroplasts indicate the pyruvate formation rate relative to maximal NADP-ME activity (e.g., 826/1828; 45%).

(C) Quantification of malate labeling in BS chloroplasts after 1 h of incubation with 2,3,3-²H-malate (purple box, deuteriums indicated by “D” in the small red boxes). LC-MS/MS chromatograms indicate the peaks of unlabeled ([M]⁺) and triple labeled ([M+3]⁺) malate. Malate pool sizes in dct2-1 BS chloroplasts was 42% of the wild type; therefore, twice the amount was injected onto LC-MS/MS for labeling quantification in the spectra. Table: fractional abundance of mass isotopomers in 2,3,3-²H-malate standard, wild-type, and dct2-1 BS chloroplasts after labeling (sd, n = 3). [M+i]⁺, the i^th-labeled mass isotopomer. Labeling data were corrected for natural isotope abundance.

Malate uptake into the BS chloroplasts was further characterized with 2,3,3-²H malate that was supplied to isolated wild-type and dct2-1 BS chloroplasts (Figure 3C). Because the stable isotope incorporation is relative to the entire unlabeled pool, mass spectrometry can judge the absolute amount of malate transport relative to its pool size and additional normalizations were unnecessary. The measurement of ²H-malate was therefore a dynamic, complementary evaluation of metabolism relative to enzyme assays, though requiring additional isolated dct2-1 chloroplasts for sensitive measurement. Significantly more triply labeled malate (malate with three hydrogens replaced by three deuteriums [M+3]⁺) was present in chloroplasts from wild-type (60.3% ± 0.8%) than dct2-1 plants (18.7% ± 4.0%) (Figure 3C) and was qualitatively comparable to the NADP-ME activity assays. The levels of malate in BS cells and in isolated chloroplasts were measured to check that the differences in labeling were not merely a consequence of the preexisting pools. The level of malate in washed chloroplasts was ∼1% of the level in BS cells for both the wild type and dct2-1, consistent with other subcellular measurements of malate (Weiner and Heldt, 1992); however, dct2-1 chloroplasts had reduced malate concentrations (0.49 ± 0.01 nmol gFW⁻¹ BS) relative to the wild type (1.16 ± 0.20 nmol gFW⁻¹ BS). The smaller pools in dct2-1 would be expected to turnover more quickly with isotope (i.e., they would be more labeled) if the transport rate indeed was similar to that of the wild type. Since this was not observed, the differences in isotope incorporation were a consequence of impaired malate transport in dct2-1 chloroplasts and not of changes in pool size. Based on the calculated absolute incorporation of malate labeling in the pool of the wild type and dct2-1, direct malate movement into the chloroplast in dct2-1 could account for ∼13% of the wild-type activity, but could also reflect leakiness in the isolated chloroplast system.

dct2-1 Plants Have Reduced Calvin Cycle Flux Relative to the Wild Type

Metabolic changes in the Calvin-Benson cycle and related pathways that resulted from the loss of DCT2 function were characterized with carbon isotope labeling experiments. Air containing ¹³CO₂ substituted for ¹²CO₂ at near ambient levels (∼330 ppm) was provided to leaf segments (i.e., proximal one-third of the leaf from the tip; sections B+C in Figure 1C) of 9-d-old wild-type and dct2-1 plants, resulting in the labeling of primary intermediates including sugar phosphates, amino acids, and organic acids. Mass isotopomers were quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) of samples extracted at 7 to 11 time points during the labeling period. The average ¹³C enrichment of each metabolite was calculated after correction for natural abundance. ¹³C labeling was more pronounced in Calvin cycle intermediates, including 3-PGA, ribulose-1,5-bisphosphate, and sedoheptulose-7-phosphate from the wild type relative to dct2-1 (Figure 4) and approached isotopic steady state within 3 min (∼85% ¹³C). Similar levels of isotope incorporation have been observed in C₃ plants (Ma et al., 2014), though requiring a longer labeling duration (∼15 min). By contrast, ¹³C enrichment was limited (∼20% ¹³C) in the dct2-1 line. Metabolites that are derived from 3-PGA, including pyruvate, PEP, and alanine (Supplemental Figure 1), were also labeled more rapidly in the wild type and reflected the asymmetrical isotopic pattern of Rubisco-based assimilation (Supplemental Figure 1B). In the wild type, the combined enrichment of all three carbons from alanine was greater than a measurement of the second and third carbons, indicating that the first carbon was most highly labeled through the Calvin cycle. This labeling description is consistent with ¹³C incorporation into the first carbon position of 3-PGA through photosynthetic assimilation.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

¹³CO₂ Labeling of Metabolites.

Data were collected from ¹³CO₂ (∼330 ppm) labeled leaf segments (sections B+C in Figure 1C) of 9-d-old wild-type (solid symbols) and dct2-1 plants (open symbols) at a light intensity of 500 µmol m⁻² s⁻¹ and analyzed for labeling of Calvin cycle intermediates including 3-PGA (A), ribulose-1,5-bisphosphate (RuBP) (B), and sedoheptulose-7-phosphate (S7P) (C). Labeled samples were collected over a 3-min interval at 20, 40, 60, 90, 120, 150, and 180 s. Average ¹³C enrichment (%) was calculated using the formula defined in the gray box, where N is the number of carbon atoms in the metabolite and Mi is the fractional abundance (%) of the i^th mass isotopomer. Mi data were corrected for natural isotope abundance (sd, n = 3). Additional mass isotopomer data are reported in Supplemental Data Set 2.

Malate and Aspartate Labeling Indicate Altered C₄ Subtype Pathway Activities

The simultaneous use of malate and aspartate as substrates for carbon concentration in the BS occurs in both NADP-ME and PEPCK C₄ plants (Hatch, 1971; Chapman and Hatch, 1981; Furbank, 2011; Pick et al., 2011; Wang et al., 2014a). Modeling efforts have also suggested coordination between coexisting C₄ pathways (Wang et al., 2014b). Here, the movement of carbon in wild-type maize and dct2-1 plants was tracked by quantifying the absolute ¹³C incorporation into malate and aspartate pools over the 3-min ¹³CO₂ time course (Figure 5). ¹³CO₂ incorporated with PEP by PEP-carboxylase resulted in singly labeled OAA that is converted to either malate or aspartate (Figure 5A). The relative abundance (%) of the singly labeled mass isotopomer [M+1]⁺ (M, the base mass; 1, one carbon labeled) in malate and aspartate (Figure 5B) was combined with pool size information (Figure 5C) to determine the initial rate of labeling of these metabolites (Figure 5D). The slopes of the initial time points were used to compare the flux into the first step of each C₄ pathway. Due to rapid incorporation of isotope in the wild type, additional time points were added for the early part of the labeling period. ¹³C incorporation into malate was more rapid in the wild type than in dct2-1, and in the wild type, enrichment leveled off within 60 s, suggesting that the singly labeled pool had achieved a maximum value (Figure 5B). In dct2-1, the labeling increased slowly and continued with time, indicating that malate accumulated in other subcellular locations (e.g., vacuoles) not intimately tied to assimilation, resulting in a larger overall pool size (Figure 5C).

• Download figure
• Open in new tab
• Download powerpoint

Figure 5.

¹³CO₂ Labeling of Malate and Aspartate in Leaf Segments of 9-d-Old Wild-Type and dct2-1 Plants.

Leaf segments (sections B + C in Figure 1C) were labeled with 330 ppm ¹³CO₂ at a light intensity of 500 µmol m⁻² s⁻¹ as previously described and analyzed for malate and aspartate labeling.

(A) Simplified metabolic network indicating the position of the singly labeled mass isotopomer [M+1]⁺ of malate and ASP in C₄ photosynthesis. Other intermediates involved in the Calvin cycle can be more extensively labeled ([M+2]⁺ or [M+7]⁺) based on the number of carbon atoms. [M+i]⁺ is ith mass isotopomer. M is the base mass and i is the number of labeled carbons.

(B) Relative abundance (%) of [M+1]⁺ in malate and aspartate (sd, n = 3). Wild-type leaf segments were labeled for 1, 2, 5, 10, 20, 40, 60, 90, 120, 150, and 180 s . dct2-1 leaf segments were labeled for 20, 40, 60, 90, 120, 150, and 180 s. Solid (wild type) and dotted (dct2-1) lines indicate the linear rate of incorporation.

(C) Absolute pool sizes (nmol g FW⁻¹) of malate and aspartate (se, n = 3) in leaf segments of wild-type and dct2-1 measured on LC-MS/MS. Samples were taken from unlabeled leaf segments.

(D) Initial incorporation rate of [M+1]⁺ carbon (nmol g FW⁻¹ s⁻¹) in malate and aspartate calculated from the pool sizes in (B) and the slopes in (C). Asterisk indicates that malate metabolism in dct2-1 was not determined (nd) from ¹³CO₂ leaf labeling due to its accumulation in other places.

In wild-type plants, the rate of ¹³C incorporation into aspartate was significantly slower than into malate (2.9-fold; Figure 5D), consistent with published radiolabeling studies that described higher NADP-ME than PEPCK pathway activities (Hatch, 1971). Aspartate and malate labeling in the wild type plateaued within 60 to 90 s (Figure 5B), whereas these labeled metabolites in dct2-1 plants continued to increase at a much slower rate (Figure 5B). In the wild type, ¹³CO₂ incorporation into the backbone of PEP through Rubisco-based assimilation resulted in aspartate mass isotopomers (i.e., [M+2]⁺ through [M+4]⁺; Supplemental Figure 2A). The higher masses were a measureable percentage of total labeling in the wild type, whereas aspartate remained mostly unlabeled or singly labeled in dct2-1 (Supplemental Figure 2B). In dct2-1, the ¹³C incorporation rate into aspartate was 1.1 nmol g FW⁻¹ s⁻¹ (Figure 5D), which is ∼6% of the wild type. This difference was consistent with the low photosynthetic rates measured in leaf sections B and C (3% or less of the wild type in dct2-1; Figures 1C and 1F). The changes were likely a consequence of the inability of dct2-1 to regenerate PEP, efficiently balance carbon and nitrogen across cell types, and maintain high rates of C₄ metabolism, due to the disruption of the NADP-ME pathway. Previously, Wang et al. (2014b) suggested that the PEPCK pathway does not operate without other C₄ pathways. Such a reliance on the PEPCK pathway would severely compromise photosynthesis and may partially explain the limited growth phenotype of dct2-1.

The contribution of the PEPCK pathway to carbon metabolism was further examined by combining the infrared gas analyses (IRGA; Figure 1F) with isotopic labeling rates of malate and aspartate (Figure 5D). The combined rate of assimilation in the wild type estimated from malate and aspartate isotope incorporation was 67.1 nmol gFW^-1s⁻¹ (Figure 5D). The dct2-1 rate of CO₂ assimilation in leaf section C as determined by IRGA was 3% of the wild type (Figure 1F). For similar leaf thicknesses between lines, this would correspond to a total CO₂ incorporation of 2.1 nmol gFW⁻¹ s⁻¹ (i.e., 3% of 67.1). Given that the aspartate label incorporation was measured to be 1.1 nmol gFW⁻¹ s⁻¹ (Figure 5D), the remainder (∼1.0 nmol gFW⁻¹ s⁻¹) comes from malate that was transported by a mechanism other than through DCT2 (consistent with Figure 3C) or by conversion to aspartate for import to the chloroplast as described below. Thus, PEPCK pathway activity is presumed to be enhanced in the dct2-1 line in a relative sense (∼55% of total net assimilation rate in dct2-1 versus 25% in the wild type). However, despite the apparent increase in PEPCK pathway activity, it is nonetheless insufficient to restore growth as dct2-1 PEPCK-based assimilation was ∼1.5% of the total wild-type assimilation rate (Figure 1F).

BS Cells Convert Malate to Aspartate and Alanine More Readily in dct2-1

The ¹³C experiments indicated that Calvin-Benson cycle activity was decreased in dct2-1 and reflected metabolic reprogramming. As the location and existence of certain C₄ steps in the BS are unclear (Pick et al., 2011; Wang et al., 2014b), ¹⁴C studies with isolated BS cells were conducted to further probe the steps of C₄ metabolism (Figure 6). The ¹⁴C studies indicated the routes of CO₂ delivery to the chloroplast without requiring an intact C₄ cycle. In wild-type BS cells, ¹⁴C-malate was taken up and converted to ¹⁴C-pyruvate, aspartate, and alanine (Figure 6A) and detected after separation on HPLC by scintillation counting. The separation of BS strands from M cells resulted in an incomplete NADP-ME cycle such that pyruvate accumulated following the decarboxylation of malate. Due to the deficiency of malate import in the dct2-1 plants, little pyruvate was detected ; however, two amino acids, aspartate and alanine, were more labeled in dct2-1 than the wild type (Figure 6A). Aspartate could serve as an alternative metabolite to transport carbon within the BS cells when the import of malate into the BS chloroplasts is disrupted. OAA and aspartate generated from malate may partially complement the malate transport deficiency and account for the production of labeled aspartate and alanine in dct2-1. In partial support of such a mechanism, mitochondrial and chloroplast-localized aspartate aminotransferases have been identified (Majeran et al., 2010), though the identification of an analogous alanine aminotransferases remains elusive.

• Download figure
• Open in new tab
• Download powerpoint

Figure 6.

¹⁴C-Malate, Aspartate, and Glutamate Labeling of BS Cells.

Wild-type (solid symbols) and dct2-1 (open symbols) plants were ¹⁴C labeled to describe BS photosynthetic operation (sd, n = 3). Inset shaded plots emphasize the initial 5-min incorporation.

(A) Time-course incorporation of ¹⁴C-malate into pyruvate, aspartate (ASP), and alanine (ALA).

(B) Time-course incorporation of ¹⁴C-ASP into PEP (square symbols) and ALA (red triangle symbols). Solid (wild-type) and dotted (dct2-1) lines indicated the linear incorporations after lag period.

(C) Time-course incorporation of ¹⁴C-glutamate into polar metabolites and 2-OG.

A comparable set of experiments that involved provision of ¹⁴C-aspartate to BS cells circumvented the requirement for an intact PEPCK pathway and were used to test the role of aspartate in providing carbon to the Calvin-Benson cycle. The linear rates of labeling in most cellular intermediates of the BS cells were similar in the wild type and dct2-1 (Supplemental Figure 3), with the exceptions of 3-PGA (Supplemental Figure 3D), PEP, and alanine (Figure 6B). PEP and 3-PGA were more rapidly labeled after a 5-min lag in the wild type, whereas labeling in alanine was greater in dct2-1. The increased radioactivity in 3-PGA and PEP reflected higher overall photosynthetic activities in the wild type, consistent with the net CO₂ assimilation rate, ¹³C data, and differences in growth. The changes in nitrogen movement across the chloroplast envelope associated with aspartate use were likely balanced by alanine production and export and could account for the enhanced alanine labeling.

²H-Malate Labeling in BS Cells Supports Limited Mitochondrial Pathway Activity

2,3,3-²H-malate labeling experiments were used to further confirm aspects of aspartate movement in BS cells (Figure 7). ²H-malate was rapidly taken up and incorporated into fumarate (Figures 7A and 7B) in both the wild type and dct2-1. The steady state levels (∼15%) indicated the active pool of fumarate was quickly saturated. Both fumarate and OAA could be double labeled at different positions within each molecule and, as shown, would contribute to different combinations of single and double labeling in malate as well as aspartate (Figure 7A). Consistent with ¹⁴C-malate experiments, the provision of ²H-malate isotopically labeled alanine at low levels. The results support a mechanism where malate is used to generate OAA and aspartate in the mitochondria. The exchange between the organic acids would enable the removal of several deuteriums in this process, creating singly labeled aspartate that could be exported to the chloroplast and converted to alanine. Aspartate was ∼5 to 10% enriched by 5 min, whereas alanine was closer to 1%, though the labeling patterns were similar (Figure 7B). Singly labeled aspartate and alanine were more abundant than double-labeled mass isotopomers, suggesting ²H-malate is converted to fumarate or other intermediates prior to generation of the two amino acids. Similar to the ¹⁴C-labeling studies (Figures 6A and 6B), absolute 2,3,3-²H-malate incorporation into aspartate and alanine was higher in dct2-1 (Figure 7C). Other organic acids, including succinate and citrate, were also more labeled (Supplemental Figure 4A) in dct2-1, which suggests that mitochondrial involvement was enhanced relative to the wild type.

• Download figure
• Open in new tab
• Download powerpoint

Figure 7.

Time Course 2,3,3-²H-Malate Labeling of BS Cells in Wild-Type and dct2-1 Plants.

(A) Metabolites labeled from provision of 2,3,3-²H-malate (purple box) in BS cells. [M]⁺ is the unlabeled mass isotopomer. [M+1]⁺, [M+2]⁺, and [M+3]⁺ contain one to three deuterium atoms. Orange and blue lines indicate reactions that could lead to [M+1]⁺ and [M+2]⁺ mass isotopomers, respectively, of alanine and aspartate. Only directly impacted heteroatoms (i.e., hydrogens) are presented.

(B) Labeling pattern of fumarate (FUM), aspartate, and alanine in the wild type (black/gray) and dct2-1 (red/dotted). All data were corrected for natural isotope abundance (sd, n = 4).

(C) Quantification of deuterium isotope incorporation (nmol gFW⁻¹) into aspartate and alanine from labeled malate (sd, n = 4). Data were calculated from pool sizes of aspartate and alanine in BS cells and the summed relative labeling data in (B). Solid (wild type) and dotted (dct2-1) lines reflected the linear incorporation rates that were quantified as slopes (nmol gFW⁻¹ s⁻¹).

Conversion of Glutamate to 2-Oxoglutarate Is Unaffected by dct2-1 Mutation

In Arabidopsis thaliana, dicarboxylate transporters OMT1 and DCT1 were shown to be essential for photorespiration (Kinoshita et al., 2011); At-DCT2 (the ortholog of Zm-DCT2), however, is not expressed in photosynthetic leaf tissue in Arabidopsis (Renné et al., 2003). To examine the potential involvement of DCT2 in photorespiration in maize, we measured photorespiratory pool sizes and quantified isotopic labeling from ¹³CO₂ in dct2-1 and wild-type plants (Supplemental Figure 5). Although the 2-phosphoglycolate (2-PG) pool size was reduced in dct2-1, glycine and serine concentrations were elevated and less labeled. The reduced 2-PG pool and increased amino acid concentrations do not support a limitation in nitrogen substrate availability for the related reactions (Supplemental Figure 5A).

Photorespiratory links with malate transport and nitrogen metabolism were further probed by decoupling the pathway and providing the source of nitrogen exogenously. ¹⁴C-glutamate was supplied to isolated BS cells and incorporation of label into polar metabolites was quantified. As shown in Figure 6C, the linear incorporation of ¹⁴C into polar metabolites including 2-OG was similar or slightly less in dct2-1 relative to the wild type and suggested that nitrogen exchange in BS cells was not significantly compromised by the mutation. Although the malate availability for counter-port activity could be compromised in dct2-1 plants, alternative reactions catalyzed by glutamine or asparagine synthetase, glutamate synthase, and glutamate dehydrogenase outside of the chloroplast could also be involved in ammonia recycling (Becker et al., 2000; Miflin and Habash, 2002; Valadier et al., 2008). Transcriptionally upregulated asparagine synthetase (Supplemental Data Set 1) and the accumulation of asparagine (Supplemental Table 1) were observed in dct2-1.

Transcriptome Analysis Indicates Extensive Transcriptional Response to the Loss of DCT2

RNA-seq analysis was performed to inspect global gene expression changes in dct2-1 plants. The mRNA profiles of mutant and wild-type individuals were assessed at the −4, −1, +4, and +10 leaf developmental stages (Figure 1C). The difference in gene expression in dct2-1 compared with the wild type was calculated for each gene as the log₂ (dct2-1 fragments per kilobase of transcript per million mapped reads [FPKM]/wild-type FPKM), using CuffDiff (see Methods), with a false discovery rate of 0.05. Of the 30,634 genes expressed in the leaf tissue, 13,439 (44%) showed a significant difference in expression for at least one of the tested segments (Supplemental Table 3) and were subject to further analysis. To identify metabolic pathways that were affected at the transcriptional level, gene IDs were assigned to pathways using the MapMan pathway annotation for maize (Thimm et al., 2004; Usadel et al., 2009).

Many pathways, including photosynthesis, cell wall metabolism, transport, hormone, signaling, transcriptional regulation, and lipid metabolism, were affected in the mutant plants (Supplemental Table 3). In agreement with the labeling data, photosynthesis genes, such as photosystem I and II; transport genes, including DCT1 and OMT1; Calvin cycle genes; and other C₄ genes, including NADP-ME and PEPCK pathways, showed lower transcript levels (Figures 8A to 8D; Supplemental Table 3). In addition, the levels of transcripts encoding chlorophyll biosynthesis proteins were reduced, reflecting the pale green leaf phenotype of dct2-1 (Figure 8E; Supplemental Data Set 1). Starch synthesis transcripts also appeared to be lower and starch degradation increased in the mutants, explaining the lack of starch granules in dct2-1 leaves (Figures 8F and 8G; Supplemental Data Set 1). Transcripts for genes related to photorespiration were either slightly reduced or unchanged in their expression pattern (Figure 8H; Supplemental Data Set 1). Transcripts for an aspartate aminotransferase gene (GRMZM2G094712), predicted to be localized in mitochondria (Majeran et al., 2010), and two alanine aminotransferase genes (GRMZM5G828630 and GRMZM2G088064) accumulated to higher levels in dct2-1, consistent with the higher levels of aspartate and alanine identified by the isotopic labeling experiments. To date, amino acid transporters in plants remain largely uncharacterized, though the labeling results suggest a heightened need for such transporters during C₄ photosynthesis in dct2-1. Two more genes that showed elevated transcript levels in the mutants may be candidates to move aspartate and alanine during C₄ photosynthesis: GRMZM2G042933, a putative amino acid transporter predicted to be localized in the chloroplast (Majeran et al., 2010), and GRMZM2G149619, a putative bile acid sodium symporter family protein predicted to be localized in the chloroplast (Majeran et al., 2010) (Supplemental Table 3).

• Download figure
• Open in new tab
• Download powerpoint

Figure 8.

Heat Map Representation of the Differences in Gene Expression between dct2-1 and Wild-Type Plants.

RNA-seq was performed on four segments along the developmental gradient of 9-d-old plants. Values represent log₂ (dct2-1 FPKM/wild-type FPKM). Red indicates upregulation in dct2-1 relative to the wild type, and green indicates downregulation in dct2-1 relative to the wild type. Each heat map is accompanied by a significance plot: black squares indicate a statistically significant difference in gene expression in dct2-1 relative to the wild type in a specific segment; gray squares represent unsignificant differences. Squares in each row, moving from left to right, represent segments −4, −1, +4, and +10 (as defined in Figure 1C).

Several large transcription factor families showed significant changes in expression; in some cases, the differences between dct2-1 and the wild type exceeded 100-fold. For example, HB (homeobox) transcription factors, known to be involved in cell differentiation, control of cell growth, and patterning, showed large changes in all segments, as did GATA transcription factors that are known to be involved in light signaling (Supplemental Data Set 1). Though beyond the scope of this report, the transcriptomic data suggest several lines of further study to characterize regulatory factors that may be responsive to altered C₄ metabolism.