Plant Physiol. (1998) 118: 191-197

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C₄ Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable1¹

Lucy H. Smith², Jane A. Langdale, and Raymond Chollet^*

Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, Lincoln, Nebraska 68588-0664 (L.H.S., R.C.); and Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom (J.A.L.)

	ABSTRACT

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We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C₄ phosphoenolpyruvate carboxylase (PEPC) by its Ca²⁺-insensitive protein-serine/threonine kinase (PEPC kinase) in the C₄ mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 µmol m² s¹ photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO₂-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.

	INTRODUCTION

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In illuminated C₄ leaf tissue, PEPC (EC 4.1.1.31) is primarily involved in photosynthetic carbon fixation. This cytosolic C₄ enzyme has been shown to be phosphorylated and thus up-regulated by a reversibly light-activated, Ca²⁺-independent protein-Ser/Thr kinase (PEPC kinase). (For recent reviews on the regulation of plant PEPC by reversible protein phosphorylation, see Nimmo, 1993; Chollet et al., 1996; and Vidal and Chollet, 1997.) To determine how light causes increased PEPC kinase activity in C₄ leaf tissue, the effect of several chemical inhibitors of photosynthesis, such as DL-glyceraldehyde and DCMU, on the phosphorylation of PEPC was examined in detached sorghum and maize (Zea mays) leaves (Bakrim et al., 1992; Jiao and Chollet, 1992). The results implicated a functional Calvin cycle as part of the PEPC kinase light-signal transduction chain, and were the "prelude" to subsequent cellular studies involving isolated C₄ mesophyll protoplasts and cells.

Early experiments using isolated sorghum mesophyll protoplasts indicated that Ca²⁺ and alkaline conditions in the mesophyll cytosol were necessary for PEPC kinase activity to increase upon illumination (Pierre et al., 1992). Later work used isolated mesophyll cells and protoplasts from a different NADP-ME-type C₄ plant (Digitaria sanguinalis), and showed that some component of the Calvin cycle, likely 3-PGA, was also a necessary signaling element (Duff et al., 1996; Giglioli-Guivarc'h et al., 1996). These in situ observations are consistent with the whole-leaf studies summarized above (Bakrim et al., 1992; Jiao and Chollet, 1992).

The present working model for C₄ PEPC kinase up-regulation in the light was formulated mainly from these collective findings. It was proposed that 3-PGA (originating from the Calvin cycle in the bundle sheath) enters the mesophyll cytosol, where it is partially protonated to its dianionic (2) form, and then enters the mesophyll chloroplast by the Pi translocator in a light-dependent manner (Fig. 1). Protonation of 3-PGA in the mesophyll cell cytosol would cause an increase in cytosolic pH. This, combined with a subsequent increase in cytosolic [Ca²⁺] (presumed to come from the vacuole), would ultimately cause an increase in PEPC kinase protein synthesis and, thus, an increase in PEPC kinase activity (Giglioli-Guivarc'h et al., 1996; Vidal and Chollet, 1997). Results from another group using an in vitro translation technique to study apparent changes in PEPC kinase mRNA levels in whole maize leaves showed that the light-induced increase in PEPC kinase activity on a protein level was correlated with an increase in translatable PEPC kinase mRNA (Hartwell et al., 1996). These findings are also incorporated into the model.

View larger version (54K): [in this window] [in a new window]   Figure 1. Current working model of the organization of signaling elements in the C4 PEPC phosphorylation cascade upon illumination (modified from Vidal and Chollet, 1997). CaM, Calmodulin; CDPK, calmodulin-like domain (Ca2+ dependent) protein kinase; 3-PGA(H)2, partially protonated (2) form of 3-PGA; pHc, cytosolic pH; PP2A, type 2A protein phosphatase; PS, photosystems.

Consistent with this working model, it is generally assumed that in C₄ leaf tissue there must be some form of cross-talk between the mesophyll and bundle-sheath cells to ensure efficient metabolic regulation during C₄ photosynthesis (Hatch, 1987; Nelson and Langdale, 1992). For this reason alone, experiments using whole-leaf tissue to elucidate the chain of events leading from illumination of a C₄ leaf to increased PEPC kinase activity are in many ways preferable to those involving isolated mesophyll cells or protoplasts, because no separation of the two photosynthetic cell types is involved. The bundle sheath defective2-mutable1 (bsd2-m1) mutation of maize disrupts C₄ differentiation in bundle-sheath cells, such that Rubisco protein is absent and the bundle-sheath chloroplast structure is aberrant (Roth et al., 1996). Despite the absence of a functional Calvin cycle, the mesophyll cells of bsd2-m1 mutant leaves appear no different from those of the wild type (Roth et al., 1996). In certain genetic backgrounds, bsd2-m1 is a uniformly pale-green mutant that is unable to fix atmospheric CO₂ and, thus, depends on its seed reserves until these are depleted and the seedling dies (within 15 d of planting) (Roth et al., 1996; R. Roth, S. Rolfe, L.H. Smith, J.A. Langdale, and R. Chollet, unpublished observations). We have used bsd2-m1 mutant seedlings in a noninvasive manner to further address the question of whether some component or derivative of the Calvin cycle is indispensable for the light activation of C₄ PEPC kinase activity in maize leaf tissue.

	MATERIALS AND METHODS

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Chemicals

Plant Material and Growth Conditions

Seedlings were grown in soil in a growth chamber maintained at 28°C with a 16-h light (100-130 µmol m² s¹ [400-700 nm])/8-h dark cycle. Third leaves were harvested 11 d after sowing just before the end of the dark period, 2 h into the normal low-light photoperiod, or after an additional 2 h under a more moderate light regime (400-450 µmol m² s¹). W22 seedlings (seeds obtained from the University of Wisconsin-Madison) were used as internal controls for the low-light growth conditions, and these were grown under conditions identical to those used for bsd2-m1 mutant plants. Additional external controls were also used in some experiments; these consisted of greenhouse-grown wild-type tissue (cv Golden Bantam) placed into the growth chamber at the start of the 8-h dark period before leaf harvest. All leaf tissue was harvested into liquid N₂ and stored at 70°C until used for extraction.

Gas-Exchange Analysis

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Preparation of Leaf Extracts for Enzyme Assays

Enzyme Assays

RuBP-dependent fixation of ¹⁴C into acid-stable products by Rubisco was assayed as follows: 10 µL of desalted leaf extract was preincubated in a serum-stoppered vial for 10 min at 30°C in assay medium containing 100 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 0.1 mM EDTA, 0.5 mg mL¹ BSA, and 25 mM [¹⁴C]NaHCO₃ (4 µCi), in a total volume of 500 µL. RuBP (0.5 mM) or buffer alone was then injected to start the reaction. The assays were terminated after 60 s at 30°C by the addition of 100 µL of acetic acid. The contents were dried at 80°C, and 500 µL of distilled water was added to each vial, along with 5 mL of a biodegradable scintillant. ¹⁴C incorporated into acid-stable products was determined as disintegrations per minute in a liquid-scintillation counter. Malate sensitivity of PEPC was assayed spectrophotometrically at 340 nm and at 30°C under suboptimal conditions (pH 7.3, 2.5 mM PEP) in the presence and absence of 0.5 mM L-malate (see Bakrim et al., 1992; Jiao and Chollet, 1992; Duff et al., 1996).

Immunoblot Analysis

Miscellaneous Assays

	RESULTS

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bsd2-m1 Mutants Lack a Functional Calvin Cycle

View larger version (37K): [in this window] [in a new window]   Figure 2. Immunoblot analysis of total soluble proteins extracted from greenhouse-grown control maize and chamber-grown W22 and bsd2-m1 leaves harvested after approximately 8 h of darkness (D), after 2 h of low-light (LL) illumination (approximately 100-130 µmol m2 s1), and after a further 2 h of illumination at a more moderate light (ML) intensity (approximately 400-450 µmol m2 s1). It should be noted that the trace amounts of Rubisco large subunit depicted for bsd2-m1 in this figure were observed infrequently and were not functional in in vitro, 14C-based Rubisco assays (see Table II and ``Discussion''). Mcyt, Mesophyll-cell specific, located in the cytosol; Mchl, mesophyll-cell specific, located in the chloroplast; BSchl, bundle-sheath-cell specific, located in the chloroplast; LSU, large subunit.

Traces of Rubisco polypeptides were seen approximately 25% of the time. Because of this finding, we had to be sure that the mutant was not capable of fixing atmospheric carbon in the light. Thus, we determined net CO₂ exchange by the mutant and controls under both low and moderate light intensities (approximately 100 and 400 µmol m² s¹, respectively), and also under greenhouse light for the corresponding control only (approximately 1300 µmol m² s¹).

When net CO₂ uptake by bsd2-m1 leaves was determined under low light, a negative value resulted (Table I). Therefore, at the low light intensity at which the bsd2-m1 and W22 seedlings were grown, there was no net carbon fixation by the mutant. Under a more moderate light intensity, however, a small but positive value was obtained from the bsd2-m1 leaf tissue, amounting to less than 5% of the corresponding W22 control value. Whether this low apparent rate was a true indication of carbon fixation by Rubisco occurring in bsd2-m1 was determined by in vitro RuBP-dependent C assays performed with desalted extracts from the same leaves used for CO₂-exchange analysis. The results from these sensitive radiometric assays showed that the bsd2-m1 sample had no acid-stable C products and was not fixing carbon (Table II). As expected, extracts from W22 were able to fix carbon in a RuBP-dependent manner, with a rate of about 0.4 µmol min¹ mg¹ soluble leaf protein.

C₄ Enzyme Activities

Changes in the Sensitivity of PEPC to L-Malate Inhibition and in PEPC Kinase Activity in bsd2-m1

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View larger version (64K): [in this window] [in a new window]   Figure 3. In vitro assays of PEPC kinase activity. A, Determination of PEPC kinase activity in desalted crude extracts from greenhouse-grown control maize and chamber-grown W22 and bsd2-m1 leaves harvested after 8 h of darkness (D), after 2 h of low light (LL), and after a further 2 h of illumination at a more moderate light (ML) intensity. Ten micrograms of total soluble protein was assayed for PEPC kinase activity in the presence of [-32P]ATP/Mg (8 µCi), 0.5 mM EGTA, 0.1 µM MC-LR, and 10 µg of purified dephospho (dark form) wild-type maize PEPC. The arrow denotes the approximately 110-kD PEPC polypeptide on this representative phosphorimage. An unknown contaminating phosphoprotein may also be seen (denoted by the asterisk). This phosphoprotein was neither detected by Coomassie blue staining of the gel before phosphorimaging (data not shown) nor derived from the exogenous PEPC substrate (see lane 2 in B). B, Substrate and phosphorylation-site specificity of bsd2-m1 kinase activity. Low-light (LL)-illuminated bsd2-m1 leaves were extracted and assayed for PEPC kinase activity (10 µg of desalted soluble protein) as described for A, with or without 10 µg of purified exogenous recombinant wild-type (Ser-8) or phosphorylation-site mutant sorghum C4 PEPC in the in vitro phosphorylation medium. Lane 1, Plus Ser-8 PEPC; lane 2, minus exogenous PEPC (control); lane 3, plus S8D PEPC; lane 4, plus S8Y PEPC; and lane 5, plus S8T PEPC. The arrow denotes the position of the PEPC polypeptide on this representative phosphorimage.

At the highest light intensity examined (approximately 400 µmol m² s¹), the largest increase in kinase activity for the greenhouse-grown control occurred, with a much smaller increase in kinase activity for W22. Notably, for extracts prepared from bsd2-m1 after darkness or after exposure to low or moderate light, there was an increase in kinase activity with increasing light intensity (Fig. 3A). On average, the PEPC kinase activity in darkened bsd2-m1 samples amounted to approximately 25% of that detected in moderate light, and the kinase activity in low-light samples was approximately 80% of that in moderate light, as determined by phosphorimager analysis. In comparison, the PEPC kinase activity in the W22 leaf samples ranged from <1% (darkness) to approximately 85% (low light) of that in moderate light. These trends are generally consistent with the related changes seen in the sensitivity of PEPC to malate inhibition (Table II).

To ensure that "authentic" PEPC kinase activity was being measured in these in vitro phosphorylation experiments, we repeated the kinase assays using desalted leaf extracts from low-light-exposed bsd2-m1 in the presence and absence of various sorghum recombinant C₄ PEPCs as substrate to document that the Ca²⁺-independent PEPC kinase activity we observed was specific for Ser/Thr residues at position 8. Figure 3B shows the results of a representative set of kinase assays using the different PEPC substrates. It is clear that the kinase activity from bsd2-m1 leaf tissue phosphorylates wild-type sorghum C₄ PEPC (Ser-8) and the recombinant S8T form to a similar extent, but that it cannot phosphorylate the recombinant S8D or S8Y PEPCs. In these important respects the Ca²⁺-insensitive PEPC kinase activity in bsd2-m1 behaves identically to the kinase activity from wild-type maize leaves (Li et al., 1997).

	DISCUSSION

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In this study we set out to examine the light activation of PEPC kinase and its target enzyme in the Rubisco-deficient maize mutant bsd2-m1 (Roth et al., 1996). Because bsd2-m1 leaf tissue is sensitive to high PPFD (Roth et al., 1996), the seedlings were grown under a low-light regime (approximately 130 µmol m² s¹). Wild-type W22 seedlings were grown under these same conditions to serve as an internal control. In certain genetic backgrounds, bsd2-m1 is a uniformly pale-green mutant with reduced contents of total soluble protein and chlorophyll compared with the wild type. However, when total soluble leaf protein was subjected to SDS-PAGE and analyzed by immunoblotting, the only major difference between W22 and bsd2-m1 extracts was in the amount of Rubisco protein (Fig. 2). This bundle-sheath-specific stromal enzyme was frequently undetectable in bsd2-m1 leaves (Roth et al., 1996; also see above), which likely accounts for much of the approximately 50% decrease in total soluble protein in the tissue.

In bsd2-m1 leaves both the large- and small-subunit transcripts of Rubisco are present (Roth et al., 1996) and translated, but the polypeptides are unstable in planta (R. Roth and J.A. Langdale, unpublished data). This would account for the lack of both net CO₂ fixation by bsd2-m1 (Table I) and RuBP-dependent carbon fixation in extracts from bsd2-m1 leaf tissue (Table II), even when trace amounts of the subunit polypeptides were detected on an immunoblot (e.g. Fig. 2), generally only 25% of the time. In addition, the comparative enzyme protein and activity findings summarized in Figure 2, Table II, and above are consistent with previous observations on decreased Rubisco levels (approximately 5%-50% of wild type) in certain hcf mutants of maize (Edwards et al., 1988) and in a C₄ Flaveria species transformed with an antisense RNA construct targeted to RbcS (Furbank et al., 1996). In all cases, the level of C₄ pathway enzymes was not correlated with that of Rubisco, including another bundle-sheath stromal enzyme, NADP-ME.

Although bsd2-m1 lacks functional Rubisco and, therefore, an operative Calvin cycle, PEPC becomes less sensitive to malate inhibition with an increase in light intensity, and correspondingly there is an increase in Ca²⁺-independent protein-Ser/Thr kinase activity (see Table II, Fig. 3, and comments above). These data are seemingly inconsistent with the current working hypothesis (Vidal and Chollet, 1997; Fig. 1) that light activation of C₄ PEPC kinase involves metabolic cross-talk between the neighboring bundle-sheath and mesophyll cells because some component of the Calvin cycle (most likely 3-PGA) is necessary for alkalinization of the mesophyll cytosol, thereby initiating the chain of events resulting in up-regulation of the kinase and the concomitant phosphorylation of PEPC.

Use of bsd2-m1 mutant plants has allowed us to examine changes in PEPC kinase activity in planta after illumination in a seemingly "isolated" mesophyll tissue, without having to disrupt C₄ leaf structure before the start of the experiment. However, our results may not necessarily indicate what actually occurs in normal C₄ leaf tissue but, rather, may simply reflect the functional redundancy or flexibility seen in so many aspects of plant metabolism. For example, it is conceivable that in the mutant 3-PGA is provided by the mobilization of starch/Suc reserves during early seedling growth rather than directly from the Calvin cycle. A similar nonphotosynthetic origin of this presumed signaling element may also possibly occur in darkened leaves performing starch degradation/malate accumulation during CAM and in legume root nodules for the up-regulation of PEPC kinase activity (see Carter et al., 1991; Hartwell et al., 1996; Zhang and Chollet, 1997). Indeed, there is a precedent for such an involvement of reserve materials in providing signaling metabolites during the light activation of maize leaf Suc-P synthase under nonphotosynthetic conditions in an N₂ atmosphere (Huber et al., 1987).

One other notable finding has come from this study. Previous experiments showed that light activation of PEPC in sorghum leaf tissue required a minimum light intensity of approximately 350 µmol m² s¹ (Bakrim et al., 1992). In our study, we had to grow bsd2-m1 plants under low light to prevent photooxidative damage of leaf tissue. When illuminated at approximately 130 µmol m² s¹, PEPC kinase activity increased in both bsd2-m1 and similarly grown W22 plants (see Fig. 3A and remarks above). Therefore, under a low-light growth regime, the sensitivity of PEPC to malate inhibition (Table II) and PEPC kinase activity (Fig. 3A) decreases and increases, respectively, as light intensity increases from darkness to this low PPFD. In contrast, the light activation of PEPC kinase in the greenhouse-grown control seedlings was most evident at the more moderate light intensity of approximately 400 µmol m² s¹ (Fig. 3A), in agreement with the results of Bakrim et al. (1992). This brings yet another factor into the problem of elucidating the regulation of PEPC kinase activity in C₄ (and probably C₃; Li et al., 1996) leaves: the relative light intensity during growth and kinase activation.

Additional experiments will need to be conducted with bsd2-m1 to determine whether alkalinization of the mesophyll cytosol and/or increases in [3-PGA] indeed occur in the light, thus verifying two basic tenets of the model depicted in Figure 1. Although related in situ experiments have been performed with isolated C₄ leaf mesophyll protoplasts and cells (Pierre et al., 1992; Duff et al., 1996; Giglioli-Guivarc'h et al., 1996), these should be repeated using bsd2-m1 cellular preparations to determine how the mutant is able to up-regulate PEPC kinase and its target enzyme in the light in the absence of a metabolite signal originating directly from the Calvin cycle. It would also be worthwhile to further investigate how the up-/down-regulation of PEPC kinase and, thus, PEPC in C₄ and C₃ leaves varies under different light regimes used during growth.

	FOOTNOTES

   Received April 6, 1998; accepted June 8, 1998.

	ABBREVIATIONS

Abbreviations: MC-LR, microcystin-LR. NADP-MDH, NADP-specific malate dehydrogenase. NADP-ME, NADP-specific malic enzyme. PEPC, PEP carboxylase. 3-PGA, 3-phosphoglyceric acid. PPDK, pyruvate,orthophosphate dikinase. RuBP, ribulose-1,5-bisphosphate.

	ACKNOWLEDGMENTS

We thank Audrey Carl and, most especially, Shirley Condon for their excellent technical assistance, and Drs. S. Madhavan and J. Vidal for their generous gift of antibodies.

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