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. 2018 Jul 17;178(1):72–81. doi: 10.1104/pp.18.00618

Diffusion of CO2 across the Mesophyll-Bundle Sheath Cell Interface in a C4 Plant with Genetically Reduced PEP Carboxylase Activity1,[OPEN]

Hugo Alonso-Cantabrana a, Asaph B Cousins b, Florence Danila a, Timothy Ryan a, Robert E Sharwood a, Susanne von Caemmerer a,2, Robert T Furbank a,3
PMCID: PMC6130029  PMID: 30018172

RNAi reduction of PEPC activity in Setaria viridis results in low CO2 assimilation rates and increased plasmodesmata density at the mesophyll-bundle sheath interface.

Abstract

Phosphoenolpyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C4 photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in Setaria viridis and isolated independent transformants with very low PEPC activities. These plants required high ambient CO2 concentrations for growth, consistent with the essential role of PEPC in C4 photosynthesis. The combination of estimating direct CO2 fixation by the bundle sheath using gas-exchange measurements and modeling C4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO2 diffusion (gbs) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on gbs depending on the technique used to calculate gbs. Anatomical measurements revealed that plants with reduced PEPC activity had reduced cell wall thickness and increased plasmodesmata (PD) density at the mesophyll-bundle sheath (M-BS) cell interface, whereas we observed little difference in these parameters at the mesophyll-mesophyll cell interface. The increased PD density at the M-BS interface was largely driven by an increase in the number of PD pit fields (cluster of PDs) rather than an increase in PD per pit field or the size of pit fields. The correlation of gbs with bundle sheath surface area per leaf area and PD area per M-BS area showed that these parameters and cell wall thickness are important determinants of gbs. It is intriguing to speculate that PD development is responsive to changes in C4 photosynthetic flux.

C4 plants have evolved a CO2-concentrating mechanism that enables the elevation of CO2 around the active sites of Rubisco by a combination of anatomical and biochemical specialization (Hatch, 1987). C4 photosynthesis has evolved independently more than 60 times and provides one of the most widespread and effective solutions for overcoming the catalytic inefficiency of Rubisco (Sage et al., 2012; Christin and Osborne, 2013). The two key carboxylases of the C4 photosynthetic pathway are localized to different cellular compartments. Phosphoenolpyruvate carboxylase (PEPC) is localized to the cytosol of mesophyll cells, and Rubisco is localized to the chloroplasts of bundle sheath cells. C4 acids produced by PEPC diffuse through plasmodesmata (PD) into the bundle sheath cells, where they are decarboxylated, thus supplying CO2 for Rubisco. In order for the CO2-concentrating mechanism to operate effectively, PEPC and C4 cycle activity must exceed Rubisco and C3 cycle activity to balance the leakage of CO2 out of the bundle sheath compartment. This ensures above-ambient bundle sheath CO2 partial pressure (pCO2) but minimizes energetically wasteful overcycling of the mesophyll CO2 pump (Furbank and Hatch, 1987; von Caemmerer and Furbank, 2003).

It has been hypothesized that a low rate of CO2 diffusion across the bundle sheath and mesophyll interface is an essential feature of the C4 photosynthetic CO2-concentrating mechanism (Berry and Farquhar, 1978; Jenkins et al., 1989a, 1989b). However, what anatomical characteristics are essential for low bundle sheath conductance (gbs) is poorly understood (von Caemmerer and Furbank, 2003). Jenkins (1989) demonstrated that, in C4 species, inhibition of PEPC with the PEPC-specific inhibitor 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) propenoate (DCDP) eliminated CO2 assimilation in ambient air. This inhibitor has been used previously to estimate gbs, and values are in the range of 0.6 to 10 mmol m−2 s−1 bar−1 (Jenkins, 1989; Brown and Byrd, 1993; Kiirats et al., 2002).

The temperature dependence of gbs is an important input into models of C4 photosynthesis; however, little is known about this process (von Caemmerer, 2000; Yin et al., 2016). In C3 species, strong temperature dependencies of mesophyll conductance, meaning the allowance of CO2 diffusion from intercellular air space to chloroplasts, have been observed in some species but not in others (von Caemmerer and Evans, 2015). This raises questions about the temperature dependence of gbs and whether similar diversity exists in the temperature dependence of gbs among C4 species. The only estimates of the temperature dependence of gbs so far were made in the PEPC mutant of the C4 dicot Amaranthus edulis, which lacks the C4 isoform of PEPC (Kiirats et al., 2002). It often has been speculated that interspecific diversity in gbs may be due to variation in the presence of secondary thickening and suberization of the bundle sheath cell walls; however, it also has been suggested that diffusion path length and positioning of organelles may be equally important (von Caemmerer and Furbank, 2003). A. edulis is of the NAD-malic enzyme (ME) biochemical type that lacks a suberized lamella at the mesophyll-bundle sheath (M-BS) cell interface and has been reported to have a high gbs (Kiirats et al., 2002). However, other reports do not find significant differences in gbs between C4 species having suberized bundle sheath cell walls and those without (Jenkins et al., 1989; Brown and Byrd, 1993).

The model C4 monocot species Setaria viridis (green foxtail millet), used in this study, is of the NADP-ME type, and its bundle sheath wall contains a suberized lamella (Danila et al., 2016). S. viridis is closely related to agronomically important C4 crops including Setaria italica (foxtail millet), Zea mays (maize), Sorghum bicolor (sorghum), and Saccharum officinarum (sugarcane; Brutnell et al., 2010). It has become a popular model species due to its rapid generation time, small stature, high seed production, diploid status, and small sequenced and publicly available genome (Doust, 2007; Brutnell et al., 2010; Li and Brutnell, 2011). Here, we used a stable genetic transformation system to produce S. viridis harboring an RNA interference (RNAi) construct targeting the cytosolic C4 PEPC isoform and isolated a number of independent transformants with very low PEPC activities. The biochemical properties of S. viridis PEPC and Rubisco are well characterized (Boyd et al., 2015), and this allowed us to estimate gbs in the progeny of these plants in combination with anatomical investigations of the M-BS interface.

RESULTS

Characterization of αPEPC Transformants

Using an RNAi construct targeting the C4 PEPC isoform of S. viridis (Supplemental Figs. S1 and S2), we generated a number of T0 plants that showed either wild-type or very low PEPC activity levels (Fig. 1). The difference in PEPC activity was reflected in differences in CO2 assimilation rates. T0 plants with low PEPC activity had very low CO2 assimilation rates that did not saturate at intercellular pCO2 above 1,200 μbar, in contrast to the wild-type-like T0 plants (Fig. 2). For the T1 progeny of selected T0 plants, in vitro measurements of PEPC and Rubisco activities and CO2 response curves representing CO2 assimilation rates showed that the properties of the T1 progeny mirrored those of the parent plants (Figs. 3 and 4). In plants with a severe reduction in PEPC activity, some reduction in Rubisco activity also was observed; however, the reduction in Rubisco activity was not proportional to the reduction in PEPC activity. A more detailed analysis of αPEPC 4 T1 progeny showed that soluble protein, chlorophyll, carbonic anhydrase, and ME activities, as well as the protein content, also were reduced (Table 1). These are presumably pleiotropic effects associated with the slow growth of the αPEPC 4 progeny even at ambient CO2 of 2% (v/v).

Figure 1.

Figure 1.

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In vitro maximal PEPC activity of individual T0 plants compared with the average activity level of wild-type (wt) plants grown under the same growth conditions (n = 4). Error bars denote se.

Figure 2.

Figure 2.

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CO2 response curves of leaves of wild-type (Wt) and individual T0 plants measured at 1,500 μmol quanta m−2 s−1, leaf temperature of 25°C, and 21% oxygen.

Figure 3.

Figure 3.

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In vitro maximal PEPC and Rubisco activity of wild-type (Wt) plants (average value, n = 4) and plants of the T1 progeny of selected T0 plants (individual values). Error bars denote se.

Figure 4.

Figure 4.

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Leaf CO2 response curves of wild-type plants (average value, n = 4) and individual T1 progeny selected from T0 plants measured at 1,500 μmol quanta m−2 s−1, leaf temperature of 25°C, and 21% oxygen. Error bars denote se.

Table 1. Average in vitro activities of key photosynthetic enzymes and chlorophyll content for wild-type and αPEPC 4 T1 progeny (n > 4 ± se).

In vitro activities were measured as described in “Materials and Methods” at 25°C. Different lowercase letters following the values indicate statistically significant differences (P < 0.05, Tukey-Kramer honestly significant difference).

Parameter Wild Type αPEPC 4
Chlorophyll content (mmol m−2) 0.48 ± 0.10 a,b 0.34 ± 0.02 b
Chlorophyll a:b ratio 5.28 ± 0.21 a 3.40 ± 0.18 b
Rubisco (µmol CO2 m−2 s−1) 25.4 ± 1.90 a 9.2 ± 1.7 b
PEPC (µmol CO2 m−2 s−1) 311.6 ± 25.3 a 1.67 ± 0.53 b
NADP-ME (µmol CO2 m−2 s−1) 36.9 ± 2.4 a,b 27.0 ± 3.5 b
Carbonic anhydrase (µmol CO2 m−2 s−1) 1,703 ± 186 a 928 ± 152 b
Soluble protein (g m−2) 13.7 ± 1.2 a 9.6 ± 1.4 b
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Estimation of gbs to CO2 Diffusion

gbs was estimated by two methods. In the first approach, we assumed that there was a negligible amount of C4 cycle activity (Table 1) and that the leaves fixed CO2 directly via Rubisco in the bundle sheath. This allowed us to estimate a total conductance for CO2 diffusion from intercellular air space to the bundle sheath from the initial slope of the CO2 response curves, as shown in Figure 5. In the second approach, we used the model for C4 photosynthesis (von Caemmerer and Furbank, 1999; von Caemmerer, 2000) and fitted the CO2 response to the enzyme-limited model as described in “Materials and Methods.” For this method, the Vcmax at 25°C was calculated from the measured Rubisco site content of αPEPC 4 T1 progeny of 3 ± 0.1 μmol m−2 and the catalytic turnover rate of 5.44 s−1 as 16.3 μmol m−2 s−1. For the wild type, Rubisco site content was 6.4 ± 0.23 μmol m−2 (Vcmax = 34.7 μmol m−2 s−1). Additionally, Vpmax for each individual plant was determined from the in vitro activity of PEPC at 25°C. We used the temperature dependence of enzyme kinetic parameters for PEPC and Rubisco determined by Boyd et al. (2015) for S. viridis and the temperature dependence of mesophyll conductance determined by Ubierna et al. (2017). The parameters used are given in Supplemental Table S1. Both techniques gave low values of gbs of 2.9 ± 0.14 and 3.8 ± 0.32 mmol m−2 s−1 bar−1 for the two methods at 25°C, respectively, in the αPEPC 4 mutant. The slope method predicted a monotonic increase of gbs from 2.7 ± 0.28 to 4.9 ± 0.14 mmol m−2 s−1 bar−1 at 20°C to 40°C (Fig. 6). However, there was no significant temperature dependence predicted using the C4 model-fitting routine. Estimates of the two techniques differed significantly at 20°C, 25°C, and 40°C. gbs also was estimated in wild-type leaves after feeding with the PEPC inhibitor DCDP (Supplemental Fig. S3) using the slope method. This resulted in an average gbs of 2.2 ± 0.05 mmol m−2 s−1 bar−1 at 25°C, which was significantly less (P < 0.001) than the gbs estimated for the αPEPC 4 mutant.

Figure 5.

Figure 5.

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CO2 response curves of leaves of αPEPC 4 T1 progeny measured at different leaf temperatures of 20°C, 25°C, 30°C, and 35°C (n = 4) or 25°C, 30°C, 35°C, and 40°C (n = 4). Measurements were made at 1,800 μmol quanta m−2 s−1 and 2% oxygen. Error bars denote se. Wt, Wild type.

Figure 6.

Figure 6.

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Estimation of the gbs at different leaf temperatures. Estimations were made either from the initial slope of the CO2 response curves shown in Figure 4 or by fitting the C4 photosynthesis model (von Caemmerer and Furbank, 1999; von Caemmerer, 2000) as described in “Materials and Methods.”

Anatomical Measurements and PD Density

The comparison of leaf anatomical characteristics of wild-type S. viridis with the T1 progeny of αPEPC 12, αPEPC 13, and αPEPC 4 mutants shows that reduction in PEPC activity affected leaf anatomy (Fig. 7). There was a visible decrease in chloroplast content in the bundle sheath cells of all mutant lines. Starch production within chloroplasts of all mutant lines was less than that of the wild type, as shown by the decreased size of starch granules particularly in αPEPC 4. Chloroplasts in αPEPC 4 leaves were smaller in both mesophyll and bundle sheath cells compared with those in the wild type. It is interesting, however, that starch was visible in both mesophyll and bundle sheath chloroplasts in these plants. gbs on a leaf area basis is the product of the conductance across the M-BS interface and the bundle sheath surface area-to-leaf area ratio (Sb). All PEPC mutants had thinner bundle sheath and mesophyll cell walls. There was, however, little difference in Sb, and only αPEPC 4 had slightly lower Sb values of 2.06 ± 0.037 compared with 2.38 ± 0.035 of the wild type (Table 2). C4 acids and inevitably CO2 diffuse through PD, which are distributed in clusters called pit fields in leaves (Danila et al., 2016). There was little difference between the wild type and mutants in terms of PD per pit field area at either the M-BS or the M-M interface. However, the pit field area per M-BS interface area was greater in all mutants compared with that in the wild type, with αPEPC 4 showing almost twice the coverage. This increase in pit field area was not observed at the M-M interface, where there was a decrease in pit field area (Fig. 8; Table 2; Supplemental Fig. S4). An increased number of pit fields drove the increase in pit field area per M-BS interface area, as there were only small changes in the average pit field area.

Figure 7.

Figure 7.

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Light (left) and transmission electron (right) micrographs of transverse leaf sections of wild-type and PEPC RNAi T1 progeny of S. viridis. A, Wild type. B, αPEPC 12. C, αPEPC 13. D, αPEPC 4. BS, Bundle sheath cell; cw, cell wall; M, mesophyll cell; s, starch granule. Light micrograph bars = 20 µm and transmission electron micrograph bars = 5 µm.

Table 2. Leaf anatomical measurements in the wild type and αPEPC mutants of S. viridis.

Statistically significant differences according to posthoc Tukey’s test at P < 0.05 are indicated by different lowercase letters. Values followed by the same letter within a row are not significantly different. BS, Bundle sheath; M, mesophyll; M-M, mesophyll-mesophyll cell interface.

Parameter Wild Type αPEPC 12 αPEPC 13 αPEPC 4
BS cell wall thickness (µm) 0.68 ± 0.029 a 0.29 ± 0.019 b 0.52 ± 0.026 c 0.23 ± 0.009 b
M cell wall thickness (µm) 0.27 ± 0.009 a 0.11 ± 0.004 b 0.22 ± 0.011 c 0.12 ± 0.005 b
Sb (m2 m−2) 2.38 ± 0.035 a 2.33 ± 0.042 a 2.32 ± 0.046 a 2.06 ± 0.037 b
M-BS PD per pit field area (PD µm−2) 50.8 ± 0.49 a 61.6 ± 1.19 b 55.7 ± 0.55 c 51.0 ± 0.57 a
M-BS pit field area per interface area (%) 12.9 ± 0.27 a 16.4 ± 0.27 b 18.9 ± 0.54 c 22.9 ± 0.42 d
M-BS PD per interface area (PD µm−2) 6.6 ± 0.12 a 10.1 ± 0.13 b 10.5 ± 0.10 b 11.7 ± 0.17 c
M-BS average pit field area (µm2) 0.6 ± 0.05 a 0.5 ± 0.04 a 0.7 ± 0.06 a 0.6 ± 0.07 a
M-BS PD area (µm2) 0.008 ± 0.0002 a 0.008 ± 0.0002 a,b 0.007 ± 0.0002 b 0.005 ± 0.0002 c
M-BS PD area per interface area (µm2 µm−2) 0.054 ± 0.0005 a 0.079 ± 0.0007 b 0.077 ± 0.0006 c 0.061 ± 0.0007 d
M-BS PD area per unit of leaf area (m2 m−2) 0.128 ± 0.0007 a 0.184 ± 0.0010 b 0.178 ± 0.0010 c 0.125 ± 0.0009 a
M-M PD per pit field area (PD µm−1) 55.0 ± 1.26 a 62.9 ± 1.14 b 55.0 ± 0.74 a 71.1 ± 0.76 c
M-M pit field area per interface area (%) 6.3 ± 0.15 a 3.4 ± 0.10 b 4.8 ± 0.12 c 6.8 ± 0.16 a
M-M PD per interface area (PD µm−2) 3.5 ± 0.04 a 2.1 ± 0.02 b 2.6 ± 0.04 c 4.9 ± 0.08 d
M-M average pit field area (µm2) 0.6 ± 0.10 a 0.3 ± 0.03 b 0.4 ± 0.04 a,b 0.3 ± 0.04 b
M-M PD area (µm2) 0.010 ± 0.0004 a 0.006 ± 0.0002 b 0.006 ± 0.0001 b 0.004 ± 0.0001 c
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Figure 8.

Figure 8.

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Quantification of PD in wild-type and select αPEPC T1 progeny of S. viridis. A, Number of PD per pit field was estimated from scanning electron microscopy images. B, Percentage pit field area per interface area. C, Number of PD per interface area. Scanning electron microscopy images were used to quantify PD per PD pit field area, and 3D confocal images were used to quantify percentage PD pit field area per interface area as described in “Materials and Methods” and by Danila et al. (2016). Sample images are shown in Supplemental Figure S5.

DISCUSSION

PEPC Is Required for High Rates of C4 Photosynthesis

Using a PEPC RNAi construct targeting the C4 PEPC isoform of S. italica, we isolated several independent transgenic lines with very low PEPC activity in leaves that resulted in low net CO2 assimilation rates at ambient pCO2 (Figs. 2 and 4). Our results confirm that lack of this isoform of PEPC greatly impairs C4 photosynthesis, as has been shown previously in a PEPC knockout mutant of A. edulis (Dever, 1997). The nonsaturating nature of the CO2 response curves of the mutants with very low PEPC activity suggests that CO2 fixation occurred via the direct fixation of CO2 by Rubisco. Despite a CO2 environment of 2% in the growth facility, the PEPC mutants grew more slowly and showed reductions in other photosynthetic enzymes such as Rubisco, NADP-ME, and carbonic anhydrase activity (Table 1). This was not observed in A. edulis PEPC mutants, where Rubisco and carbonic anhydrase activity were not affected (Cousins et al., 2007). A. edulis is an NAD-ME-type C4 species that lacks a suberin lamella at the M-BS interface. It could be that the greater gbs (∼10 mmol m−2 s−1 bar−1) estimated for this species allows for the direct fixation of CO2 by Rubisco and growth at high CO2 concentrations (Kiirats et al., 2002). Furthermore, S. viridis is an NADP-ME species, where the C3 cycle in the bundle sheath relies on NADPH resulting from malate decarboxylation and CO2 assimilation may be more dependent on some C4 cycle activity for C3 cycle operation (Hatch, 1987; Furbank, 2011).

Interaction between Leaf Anatomy, PD Density, and C4 Photosynthetic Biochemistry

It has been suggested from modeling that CO2 diffusion across the M-BS interface can occur both through the PD (57%) and directly through the cell wall (43%; Jenkins et al., 1989b). The availability of 3D imaging technology allows for the visualization of pit fields by confocal microscopy with the use of a callose antibody and the visualization of PD in pit fields by scanning electron microscopy (Danila et al., 2016; Supplemental Fig. S4), providing us with a method to quantify the M-BS PD density in both wild-type and low-PEPC S. viridis. Our data highlight that 57% of the CO2 leakage out of the bundle sheath occurs across 7% to 12% of the M-BS interface area. Furthermore, our examination of three independent lines with low PEPC presents a surprising result. PD density at the M-BS interface is increased substantially, and this is not observed at the M-M interface (Fig. 8). This increase in PD density is driven largely by an increase in the number of pit fields rather than an increase in PD per pit field or the size of pit fields. We have shown previously that PD density was at least doubled in C4 species compared with C3 species, which gave a clear indication of the enhanced expression of PD developmental genes in C4 species (Danila et al., 2016). The reduction of available CO2 in the bundle sheath cells of plants with low PEPC may trigger a compensation mechanism to allow for a higher CO2 flux. However, until now, the genes underpinning PD development remain largely unknown (Brunkard and Zambryski, 2017). In a recent study where chloroplast and mitochondrial development was induced in rice (Oryza sativa) bundle sheath through the constitutive expression of maize GOLDEN2-LIKE genes, increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased intercellular connections (Wang et al., 2017). Genetic studies have revealed that PD development is regulated by intercellular signaling pathways, which may involve stromules from chloroplasts (Stonebloom et al., 2012). In NADP-ME C4 decarboxylation types such as S. viridis, chloroplasts appress the M-BS interface that would facilitate such interactions. It is tempting to speculate that a shift in photosynthetic metabolism and direct CO2 fixation by the bundle sheath chloroplast affects intercellular signaling pathways and PD development.

gbs

gbs is an essential parameter of all models of C4 photosynthesis, but it remains difficult to estimate (Berry and Farquhar, 1978; von Caemmerer and Furbank, 1999; von Caemmerer, 2000; Wang et al., 2014; Bellasio et al., 2016). Our measured values of gbs to CO2 are in the range of values reported in the literature for the NADP-ME species sorghum and maize; however, the previously reported gbs values vary widely, in the range of 1.13 to 60 mmol m−2 s−1 (Brown and Byrd, 1993; He and Edwards, 1996), but less than the values reported for A. edulis. S. viridis has become a popular model C4 grass species, and the availability of species-specific enzyme kinetic constants no doubt improved our ability to estimate gbs. However, the interpretation of the temperature dependencies of gbs obtained here remains challenging. When using the initial slope method, the same approach used by Kiirats et al. (2002), we calculated a temperature dependence not unlike that observed previously with a low Q(10) of ∼1.3. However, when we incorporated a small amount of residual PEPC activity and used the C4 model to estimate gbs, we calculated no temperature dependence. Unfortunately, we cannot resolve this discrepancy. Evans and von Caemmerer (2013) calculated that CO2 diffusion through a liquid phase alone should result in a small decline in conductance with increasing temperature. They hypothesized that it is the membrane diffusion component that is responsible for the observed increase in mesophyll conductance with temperature in many C3 species, depending on the balance between the liquid and membrane diffusion path (von Caemmerer and Evans, 2015). It is difficult to predict this response for an NADP-ME-type C4 species, where CO2 is produced in the bundle sheath chloroplasts and diffuses out across the chloroplast envelope, through the liquid phase of the cytosol and through a membrane/liquid phase in PD, in addition to the complex pathway across a suberized cell wall.

Based on permeability coefficients determined for CO2 in C4 leaves and isolated bundle sheath cells (Furbank et al., 1989; Jenkins et al., 1989a), Jenkins et al. (1989b) suggested that approximately 43% of the CO2 leakage from the bundle sheath occurs via an apoplastic route and 57% via PD. In the αPEPC 4 transgenic line, the PD number at the M-BS interface was almost doubled but PD size was reduced, and this led to a 13% increase in PD area per M-BS interface (Fig. 8; Table 2). When comparing gbs calculated from DCDP-fed wild-type plants with that obtained for this transgenic line, values are around 50% higher on a bundle sheath surface area basis (1.4 mmol m−2 s−1 bar−1 for αPEPC 4 compared with 0.92 mmol m−2 s−1 bar−1 for the wild type). The diffusion paths via PD and the apoplast are in parallel, and the 13% increase in PD area is insufficient to account for the 50% difference. This suggests that reduced cell wall thickness in αPEPC 4 transgenic plants results in a significant increase in conductance via the apoplastic route.

The leakage of CO2 outward from the bundle sheath has long been recognized as a crucial determinant of the efficiency of the C4 pathway (Furbank and Hatch, 1987; Furbank et al., 1990; von Caemmerer and Furbank, 2003). However, the relative importance of the symplastic and apoplastic barriers to diffusion remains somewhat elusive. During the evolution of C4 plants, increases in symplastic connectivity between mesophyll and bundle sheath cells were required to support C4 metabolite flux (Danila et al., 2016), but inevitably, this increase in metabolite conductance would lead to an increase in gbs for CO2. We show here that the symplastic diffusion pathway between mesophyll and bundle sheath cells is not entirely genetically predetermined and that the manipulation of metabolism can affect PD development and cell-to-cell connectivity in C4 leaves.

MATERIALS AND METHODS

Selection of the PEPC Gene

The genome sequence of Setaria italica, obtained from the Phytozome database (https://phytozome.jgi.doe.gov), was used as a reference because no genome sequence was available for Setaria viridis at the time of the experiment (Bennetzen et al., 2012). There are six genes encoding PEPC enzymes in S. italica and seven in S. viridis (Xu et al., 2013). The cytosolic PEPC isoform involved in C4 photosynthesis in S. italica, Si005789m, was identified due to the presence of a conserved substitution of an Ala to a Ser in the C-terminal region of the protein (Supplemental Fig. S1) that has been shown to be common to many C4 isoforms of PEPC in both monocots and dicots (Bläsing et al., 2000). The homolog in S. viridis is Sevir.4G143500.

Plasmid Construction

Total RNA was isolated and purified from wild-type S. viridis A10 leaves using the TRIzol reagent (Invitrogen). cDNA was synthesized using the SuperScript III First-Strand Synthesis kit (Invitrogen) from 1 μg of total RNA. Primers (SiPEPC1F, 5′-CACCCCCGGAGACGGAGTACGGCA-3′; and SiPEPC1R, 5′-GCGGCGATGCCCTTCATGGT-3′) were designed targeting the cytosolic C4 isoform of the PEPC open reading frame from S. italica (GenBank accession no. NP_001267758.1). These were used to amplify via PCR a 658-bp DNA fragment from the S. viridis cDNA library, from positions 2,223 to 2,881 in the Sevir.4G143500 coding sequence. The fragment was ligated subsequently into the pENTR-dTOPO vector (Invitrogen) and sequenced to confirm its identity. The fragment was then inserted via a double Gateway system LR reaction (Invitrogen) into the hairpin RNAi binary vector pSTARGATE (Wesley et al., 2001) to form a stem-loop PEPC region under the control of the ubiquitin promoter/intron and octopine synthase terminator (Supplemental Fig. S1). The resulting pSTARGATE-αPEPC RNAi construct was validated by sequencing and introduced into Agrobacterium tumefaciens strain AGL1.

Callus Induction and Plant Transformation

Stable transformation of S. viridis (accession A10.1) was carried out as described by Brutnell et al. (2010). Seed coats were removed mechanically from mature S. viridis seeds to improve germination. Seeds were sterilized before plating on callus induction medium (CIM; 4.3 g L−1 Murashige and Skoog [MS] salts, pH 5.8, 10 mL L−1 100× MS vitamin stock, 40 g L−1 maltose, 35 mg L−1 ZnSO4O·7H2O, 0.6 mg L−1 CuSO4O·5H2O, 4 g L−1 Gelzan, 0.5 mg L−1 kinetin, and 2 mg L−1 2,4-D). After 4 weeks in the dark at 24°C, any seedling structures or gelatinous calli were removed and remaining calli were transferred to fresh CIM. After a further 2 weeks, good-quality calli or portions of calli were selected based on their white color and dry texture, separated from brown or gelatinous tissue, divided in 2-mm pieces, and replated onto fresh CIM. One week later, transformations were performed.

A. tumefaciens AGL1 containing the construct of interest were grown in the presence of 50 µg L−1 kanamycin and 50 µg L−1 rifampicin at 28°C to OD600 = 0.5 and then resuspended in CIM without Gelzan and hormones. Acetosyringone (200 mm) and synperonic (0.01%, w/v) were added to the A. tumefaciens solution before incubating the calli in the medium for 5 min at room temperature. The calli were blotted dry on sterile filter paper and incubated at 22°C for 3 d in the dark. The calli were then transferred to selective CIM (CIM containing 40 mg L−1 hygromycin and 150 mg L−1 timentin) and incubated in the dark at 24°C for 16 d. Calli were then transferred to selective plant regeneration medium containing 4.3 g L−1 MS salts, pH 5.8, 10 mL L−1 100× MS vitamins, 20 g L−1 Suc, 7 g L−1 Phytoblend, 2 mg L−1 kinetin, 150 mg L−1 timentin, and 15 mg L−1 hygromycin. Calli were maintained at 24°C under a 16-h-light/8-h-dark photoperiod and a light intensity of 60 µmol photons m−2 s−1. Developing shoots were transferred to selective rooting medium containing 2.15 g L−1 MS salts, pH 5.7, 10 mL L−1 100× MS vitamins, 30 g L−1 Suc, 7 g L−1 Phytoblend, 150 mg L−1 timentin, and 20 mg L−1 hygromycin. Shoots that survived and developed roots were genotyped by PCR to amplify the pSTARGATE-PEPC vector using primers (PSIntF1, 5ʹ-TAATGCTAATATAACAAAGCGCAAGATCTA-3ʹ; and PSIntR1, 5ʹ-AAGATCAATGATAACACAATGACATGATCT-3ʹ) or primers directed against the hygromycin phosphotransferase gene (F, 5ʹ-TGGCGTGATTTCATATGCGC-3ʹ; and R, 5ʹ-CGTCAACCAAGCTCTGATAG-3ʹ). Positive transformants were transplanted to soil.

Plant Growth Conditions

Wild-type, T0, and T1 plants were grown in controlled environmental chambers with the following conditions: irradiance of 500 µmol quanta m−2 s−1, 16-h photoperiod, 28°C day temperature, 24°C night temperature, and 2% CO2. Each plant was grown in 2-L pots filled with garden soil mix and fertilized with Osmocote (Scotts). Pots were watered daily.

To promote germination, T1 seeds were first incubated in 5% liquid smoke (Wrights) for 24 h. Treated T1 seeds were then allowed to germinate in garden soil mix fertilized with Osmocote (Scotts) in small containers before being transferred to individual 2-L pots.

Gas-Exchange Measurements

Net CO2 assimilation rate (A) was measured over a range of intercellular pCO2 (Ci) values on the uppermost, fully expanded leaf of 5-week-old S. viridis plants using a portable gas-exchange system (LI-COR 6400XT; LI-COR Biosciences). Measurements were made after leaves had equilibrated at 380 µbar, flow rate of 500 µmol s−1, leaf temperature of 25°C, and irradiance of 1,500 µmol quanta m−2 s−1. CO2 response curves were measured in a stepwise increase (3-min intervals) at pCO2 ranging from 0 to 2,000 µbar while maintaining leaf temperature and irradiance conditions. For estimates of gbs done for T1 progeny of αPEPC 4, measurements were made at 1,800 µmol quanta m−2 s−1 and 2% oxygen at pCO2 ranging from 400 to 2,000 µbar.

Gas-Exchange Measurements after DCDP Feeding of Wild-Type Leaves

The PEPC inhibitor DCDP has been shown to reduce PEPC activity in vivo (Jenkins, 1989; Brown and Byrd, 1993; Kiirats et al., 2002). Detached wild-type leaves whose cut edge was immersed in water were stabilized in the gas-exchange system at 1,800 µmol quanta m−2 s−1 and 2% oxygen, leaf temperature of 25°C, and 400 µbar pCO2. Then, DCDP was added to achieve a final concentration of 2 to 4 mm. After CO2 assimilation rates decreased to a steady very low rate of 0.5 ± 0.15 µmol m−2 s−1, CO2 response curves were measured as described above for the estimation of gbs.

Determination of Enzyme Activities

For carbonic anhydrase activity, leaf discs (0.78 cm2) were collected from the uppermost fully expanded leaf of 5-week-old S. viridis plants and frozen in liquid nitrogen. Soluble protein was extracted by grinding one frozen leaf disc (0.5 cm2) in ice-cold glass homogenizers (Tenbroek) in 500 µL of extraction buffer (50 mm HEPES, pH 7.8, 1% [w/v] polyvinylpyrrolidone, 1 mm EDTA, 10 mm DTT, 0.1% [v/v] Triton X-100, and 2% [v/v] protease inhibitor cocktail [Sigma]). Crude extracts were centrifuged at 4°C for 1 min at 13,000g, and the supernatant was collected for the soluble carbonic anhydrase assay. Activity was measured on a membrane inlet mass spectrometer to measure the rates of 18O exchange from labeled 13C18O2 to H216O at 25°C (Badger and Price, 1989; von Caemmerer et al., 2004). The hydration rates were calculated as described by Jenkins et al. (1989b).

For Rubisco, PEPC, and NADP-ME activities, soluble protein was extracted from fresh leaf discs collected from leaves used for gas-exchange analysis. Spectrophotometric assays were then performed as described previously (Pengelly et al., 2010, 2012; Sharwood et al., 2016).

For the experiment where gbs was estimated for wild-type and αPEPC 4 plants, the Rubisco catalytic site content was measured by stoichiometric binding of [14C]carboxy-arabinitol-P2 as described by Ruuska et al. (1998) and Sharwood et al. (2008). Leaf discs (0.5 cm2) were extracted in extraction buffer as described above. MgCl2 and NaHCO3 were added to final concentrations of 20 and 15 mm, respectively, together with 29 μm [14C]carboxy-arabinitol-P2 (specific radioactivity about 10,6651 cpm nmol−1) to 100-μL aliquots of extracts, which were incubated for 45 min before gel filtration.

Anatomical Measurements

Tissue from the midportion of the third fully expanded leaf was collected from wild-type and transgenic plants. This tissue was used for light microscopy, electron microscopy, and 3D immunolocalization preparations (Danila et al., 2016). The Sb was measured using at least 10 individual small veins from light micrographs of transverse leaf sections (Pengelly et al., 2010). Bundle sheath and mesophyll cell wall thickness were measured using transmission electron micrographs of transverse leaf sections. The quantitation of PD per µm2 of pit field, percentage pit field per interface area, and PD per µm2 of interface was carried out as described (Danila et al., 2016). All measurements were performed using ImageJ software (National Institutes of Health).

Estimation of gbs

gbs was estimated by two methods. In the first approach, it was assumed that there was a negligible amount of C4 cycle activity and that the leaves fixed CO2 via Rubisco in the bundle sheath. Under these conditions a total conductance from intercellular air space to the bundle sheath can be estimated from the initial slope of the CO2 response by using Equation 2.45 from von Caemmerer (2000):

graphic file with name PP_201800618DR1_equ1.jpg (1)

Where gt is the total conductance to CO2 diffusion from the intercellular air space to the site of Rubisco carboxylation in the bundle sheath. dA/dCi is the initial slope of the CO2 response curve of CO2 assimilation rate versus intercellular pCO2 (Ci). Vcmax is the maximal Rubisco activity and Rd is leaf mitochondrial respiration in the light; Kc and Ko are the Michaelis-Menten constants of Rubisco for CO2 and oxygen, respectively, and O is the oxygen partial pressure. Γ* is the chloroplast pCO2, where, in the absence of respiration, Rubisco carboxylation equals the photorespiratory CO2 release (Laisk, 1977; von Caemmerer, 2000, Eq. 2.17), defined as:

graphic file with name PP_201800618DR1_equ2.jpg (2)

Rubisco kinetic constants for S. viridis were taken from Boyd et al. (2015) and are given in Supplemental Table S1.

Total conductance (gt) is a good approximation for gbs, since the conductances are in series and gbs is 2 orders of magnitude less than gm and gbs/gm is small:

graphic file with name PP_201800618DR1_equ3.jpg (3)

In the second approach, we used the model for C4 photosynthesis (von Caemmerer and Furbank, 1999; von Caemmerer, 2000) and fitted the CO2 response to the enzyme-limited model (von Caemmerer, 2000, Eq. 4.21) using the parameters given in Supplemental Table S1 for the fitting routine where only gbs was fitted. Mesophyll pCO2 (Cm) was calculated from the mesophyll conductance values (gm) given in Supplemental Table S1 and measured CO2 assimilation rates (A) as:

graphic file with name PP_201800618DR1_equ4.jpg (4)

We assumed that no oxygen evolution occurs in the bundle sheath cells, as S. viridis is an NADP-ME subtype and does not have functional PSII in the bundle sheath. Therefore, the parameter α, which defines the fraction of oxygen evolution occurring in the bundle sheath, is set to zero.

Statistical Analysis

Statistical analysis of images was carried out using Origin software, and statistical differences according to posthoc Tukey’s test at P < 0.05 were used. One-way ANOVAs with posthoc Tukey’s test analyses were performed for all measurements of gbs with P = 0.05 using the IBM SPSS Statistics 22 package.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number MF967570.

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. Alignment of the PEPC amino acid sequences involved in C4 photosynthesis in maize, sorghum, S. viridis, and S. italica.

  • Supplemental Figure S2. Hairpin RNAi vector pSTARGATE-PEPC used for plant transformation.

  • Supplemental Figure S3. Effect of the PEPC inhibitor DCDP feeding on CO2 assimilation rate in S. viridis wild-type leaves.

  • Supplemental Figure S4. Pit field distribution and pit field size at cell-cell interfaces in leaves of wild-type and αPEPC T1 progeny of S. viridis.

  • Supplemental Table S1. Temperature dependencies of PEPC and Rubisco kinetic constants and mitochondrial respiration used in the calculations of gbs.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

We thank Jasper Pengelly for assisting with construct generation, Xueqin Wang for assisting with S. viridis transformations, Soumi Bala for help with biochemical assays and gas exchange, and Joyce van Eck and Tom Brutnell for helpful discussions regarding S. viridis transformations. We thank Rosemary White for advice and assistance with confocal microscopy and Joanne Lee and the Centre for Advanced Microscopy at the Australian National University for technical assistance with electron microscopy.

Footnotes

1

This research was supported by the Bill and Melinda Gates Foundation’s funding for the C4 Rice consortium and by the Australian Research Council (ARC) Centre of Excellence for Translational Photosynthesis (CE140100015). R.E.S. is funded by ARC DECRA (DE130101760), and A.B.C. was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, Photosynthetic Systems (DE-SC0001685) and the Edward R. Meyer Distinguished Professorship in Sciences.

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