Analysis of a C4 maize pyruvate, orthophosphate dikinase expressed in C3 transgenic Arabidopsis plants

Plant Science 129 (1997) 57 – 64

Analysis of a C4 maize pyruvate, orthophosphate dikinase expressed in C3 transgenic Arabidopsis plants K. Ishimaru a, H. Ichikawa a, M. Matsuoka b, R. Ohsugi a,* a

National Institute of Agrobiological Resources, Kannondai 2 -1 -2, Tsukuba, Ibaraki 305, Japan b Nagoya Uni6ersity, Nagoya 464 -01, Japan Received 26 March 1997; received in revised form 5 June 1997; accepted 5 June 1997

Abstract Pyruvate,orthophosphate dikinase (PPDK) catalyzes the formation of phosphoenolpyruvate, the initial acceptor of CO2 in the C4 photosynthetic pathway. Transgenic C3 Arabidopsis plants expressing the maize C4 PPDK gene under the control of either the Arabidopsis rbcS promoter or the cauliflower mosaic virus 35S promoter were studied. The level of PPDK protein was quite low in contrast to the high steady-state level of PPDK transcripts in several transgenic plants. A PPDK polypeptide with a similar size to that in maize was found exclusively in the chloroplasts of transgenic Arabidopsis plants. This result indicates that the transit peptide of C4 PPDK in the C4 monocot maize is functional in the chloroplast protein import system of the C3 dicot Arabidopsis. The activities of PPDK in leaf extracts of the transgenic plants were up to four times higher than those in the control nontransgenic plants and the transgenic plants with the b-glucuronidase (GUS) gene, although they were still less than 3% of the PPDK activity in maize. The relative PPDK activity per unit PPDK protein in transgenic Arabidopsis was similar to that in maize. These results suggest that the low PPDK activity in transgenic Arabidopsis plants may be attributed to possible regulation at post-transcriptional and/or translational levels. The modestly increased PPDK activity did not influence the activities of ribulose-1,5-bisphosphate carboxylase and other C4-related enzymes (phosphoenolpyruvate carboxylase, NAD(P)-malic enzyme), and photosynthetic CO2-exchange parameters. © 1997 Elsevier Science Ireland Ltd. Keywords: Arabidopsis; C3–C4 photosynthesis; Pyruvate,orthophosphate dikinase; Transgenic plant

Abbre6iations: CaMV =cauliflower mosaic virus; Cb =carbenicillin; GUS = b-glucuronidase; IDH = isocitrate dehydrogenase; kan = kanamycin; MDH = malate dehydrogenase; ME=malic enzyme; PAGE = polyacrylamide gel electrophoresis; PEP= phosphoenolpyruvate; PEPC=phosphoenolyruvate carboxylase; PPDK = pyruvate,orthophosphate dikinase; PPDKRP = pyruvate,orthophosphate dikinase regulatory protein; RuBPC =ribulose-1,5-bisphosphate carboxylase; SDS =sodium lauryl sulfate; Vm =vancomycin. * Corresponding author.

1. Introduction C4 plants exhibit higher photosynthetic rates and water use efficiency than C3 plants, mainly because they increase the CO2 concentration near RuBPC in bundle sheath cells sufficiently to de-

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press photorespiration. In order to transfer these desirable features to C3 plants, several attempts using gene engineering methods have been reported. There have been four examples in which the introduction of C4 proteins into C3 plants was successful. The maize C4 PEPC gene was expressed in the C3 tobacco plant under the control of either the cab gene promoter [1] or the CaMV 35S promoter [2]. Gehlen et al. reported transgenic potato introduced PEPC genes from various origins [3]. Chloroplastic NADP-MDH from Sorghum was introduced into tobacco and its protein was accumulated in the leaf chloroplasts of transgenic plants [4]. PPDK catalyzes the formation of PEP, the initial acceptor of CO2 in the C4 photosynthetic pathway [5,6]. The in vitro rate of PEP formation by PPDK is the lowest rate for any enzyme in the C4 pathway, being relatively close to the overall rate of photosynthesis, suggesting that this reaction may be the rate-limiting step in the C4 photosynthetic pathway [7,8]. By contrast, C3 plants have very low PPDK activity and this is considered insignificant in photosynthesis, although there is some activity of other enzymes involved in the C4 pathway such as PEPC [9]. PPDK, encoded by a nuclear gene, is translated as an immature preprotein with a transit peptide in the cytoplasm, imported into the chloroplast, and processed to a mature protein lacking the transit peptide [10]. PPDK activity is regulated by phosphorylation and dephosphorylation that are mediated by a bifunctional PPDKRP (reviewed by Edwards et al. [11]). To our knowledge, the present paper reports the first attempt to introduce maize C4 PPDK cDNA into C3 Arabidopsis thaliana under the control of the CaMV 35S or Arabidopsis rbcS promoter. This study was conducted to address two questions: (1) Is there any problem in functional expression of the maize PPDK gene and, if so, which step (such as transcription or translation) regulates expression? (2) Can C4 maize PPDK protein enter the chloroplast of C3 Arabidopsis? A high expression of steady-state PPDK mRNA was detected, whereas the accumulated PPDK protein was low in transgenic plants. This

result suggests that a post-transcriptional and/or translational control mechanism may be involved. On the other hand, the PPDK protein produced in transgenic plants had a relative activity similar to that in maize without post-translational and phosphorylational control.

2. Materials and methods

2.1. Construction of chimeric genes Two different promoters, from the CaMV 35S or Arabidopsis rbcS genes, were used for the construction of chimeric genes containing the coding region of the 3.2 kb maize C4-PPDK cDNA [12] and the nopaline synthase terminator (Fig. 1A). This cDNA included 105 bases of the 5%-untranslated leader sequence before ATG.

2.2. Transformation of Arabidopsis plants The transformation of Arabidopsis plants was done according to the root-transformation method [13]. Roots excised from plants were cultured on callus-inducing medium [basal medium (Murashige-Skoog medium [14] plus B5 vitamins [15], sucrose (20 g/l), 2-(N-morpholino)ethanesulfonic acid (0.5 g/l), and Gelrite (2.5 g/l), pH 5.7) plus 2,4-dichlorophenoxyacetic acid (0.5 mg/l) and kinetin (0.05 mg/l)] for 3 days in the dark. The explants were infected with Agrobacterium tumefaciens (strain EHA101) including binary vector for 2 days in the dark and then transferred to shoot-inducing medium [SIM: basal medium plus indole-3-acetic acid (0.15 mg/l), N 6-(2-isopentenyl)adenine(2ip) (5.0 mg/l)]. The explants were induced in SIM-containing Cb (100 mg/l) and Vm (300 mg/l) for 2 days and then transferred to selection medium [SIM plus Cb (100 mg/l), Vm (300 mg/l) and kan (100 mg/l)] for deriving transgenic shoots (T0 generation). The regenerated plants were transferred to basal medium plus kan (50 mg/l) for shoot elongation, flowering, and obtaining self-fertilized seeds in vitro. On the basis of the segregation pattern of the T3 transgenic seedlings for tolerance to kan, all transgenic plants were homozygotes.

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2.3. Plant growth conditions Sterilized seeds of transgenic plants were germinated and grown on germination medium [basal

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medium plus kan (50 mg/l)]. After 2 weeks, the plants were transferred to boxes containing soil, peat moss and vermiculite, and grown in a chamber with 12 h light at 23°C and 12 h dark at 20°C. The photon flux density using incandescent lamps was 600 mmol m − 2 s − 1 at plant height.

2.4. Isolation of RNA and northern blot analysis Total RNA was isolated from expanded leaf tissue (ca. 100 mg) of T3 plants with buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 5 mM EDTA, 2 mM aurintricarboxylic acid, 2% (w/v) SDS and 2% (w/v) triisopropylnaphthalenesulfonic acid (sodium salt) according to Fromm et al. [16]. For northern blot analysis, total RNA (15 mg), fractionated by electrophoresis (1.1% (w/v) agarose gel containing 6% (v/v) formaldehyde), was blotted onto a nylon membrane. This membrane was then hybridized with a 32 P-labeled probe DNA: the KpnI fragmented coding region of PPDK cDNA (Fig. 1A).

2.5. Western blot analysis

Fig. 1. T-DNA construct of a binary vector containing the chimeric C4 PPDK gene, and the level of transcript relative to PPDK in maize, transgenic and nontransgenic Arabidopsis with the PPDK or GUS genes. (A) The T-DNA construct contains the CaMV 35S or rbcS promoter-maize PPDK gene, which has a nos transcriptional terminator-polyadenation signal (Tnos). The cDNA region encoding the PPDK transit-peptide is lined diagonally. (B) Equal amounts (15 mg) of total RNA were loaded in each lane and separated on 1.1% denaturing gel. (C) Northern blot analysis of RNA from transgenic and nontransgenic Arabidopsis plants and maize. After blotting onto a nylon membrane, the filter was hybridized with a maize C4-PPDK cDNA probe. Wild, nontransgenic control plants. GUS, transgenic plants with GUS. 35P + number, transgenic plants with the PPDK cDNA under the control of the CaMV 35S promoter. RP +number, transgenic plants with the PPDK cDNA under the control of the Arabidopsis rbcS promoter.

Frozen rosette leaf tissues were ground in liquid N2 to a fine powder, and soluble protein was extracted with buffer containing 50 mM Tris– HCl (pH 7.6), 2 mM EDTA and 10% (v/v) 2-mercaptoethanol. After centrifugation at 10 000× g for 5 min, 20 mg soluble leaf protein was subjected to SDS-PAGE on a 12.5% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS. After electrophoresis, proteins were transferred to a nylon membrane and probed with a polyclonal antibody raised against maize C4-PPDK and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G antibody. Protein concentration was determined by the method of Bradford [17] using bovine serum albumin as standard.

2.6. Cell fractionation and protein extraction Leaf protoplasts were prepared essentially according to the method of Siemens et al. [18]. After washing the isolated protoplasts with buffer containing 125 mM CaCl2, 145 mM NaCl, 5 mM

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KCl, 5 mM glucose, pH 5.6, and resuspension in 0.5 M mannitol, they were burst by pulling and pushing a few times through a syringe and immediately fractionated by successive centrifugation at 300×g for 90 s and 15 000 × g for 30 min. The fractions containing chloroplasts, mitochondria and cytoplasm were obtained, respectively, as the 300×g pellet, 15 000× g pellet, and 15 000× g supernatant. The intact chloroplasts were ruptured by adding buffer containing 50 mM Tris – HCl (pH 7.6), 2 mM EDTA, and 10% (v/v) 2-mercaptoethanol. In order to evaluate the purity of the fractions, the chlorophyll content and the activities of RuBPC (as a marker enzyme for chloroplasts), and NAD-IDH (for mitochondria) were measured. About 97% of the total chlorophyll was retained in the 300×g pellet and only 3% in the 15 000 × g pellet. About 95% of the RuBPC activity was observed in the 300 × g pellet (29.5 mmol/mg protein h − 1) and trace activities were also in the 15 000× g supernatant fluid, which included ruptured chloroplasts. All NADIDH activity was found in the 15 000× g pellet (1.92 mmol/mg protein h − 1). For each measurement, the values differed by less than 10%. Each fraction was used for western blot analysis after measuring the quantity of protein by the above mentioned method.

2.7. Enzyme extraction and assay About 1 g rosette leaf tissue was frozen in liquid N2 in the middle of the day and stored at − 80°C before use. The frozen tissues were ground into a powder in liquid N2 using a mortar and pestle. After extraction in buffer containing 50 mM Hepes–KOH (pH 7.5), 0.2 mM EDTA, 7.5 mM MgCl2 and 2.5 mM MnCl2, the crude extract was filtered through two layers of Miracloth (Calbiochem, La Jolla, CA). The filtrate was centrifuged at 10 000× g for 5 min at 4°C and the crude supernatant fluid was used for enzyme assays. RuBPC activity was measured according to Usuda [8], PEPC and PPDK according to Usuda et al. [7], NADP-ME and NAD-ME according to Ueno et al. [19], and NAD-IDH according to Bergman et al. [20]. Chlorophyll content was measured by the method of Arnon [21].

2.8. Calculation of the relati6e PPDK acti6ity per unit PPDK protein The relative activity per unit maize PPDK protein was estimated as the ratio of the increased activity of PPDK to the density of its band in a western blotting analysis. In the transgenic plants, the activity contributed by the introduced maize PPDK was obtained from the difference in the total PPDK activity and the endogenous PPDK activity. The density of the maize PPDK protein in transgenic plants and maize was estimated by the peak height of the bands on the membrane of the western blot analysis obtained by scanning with a densitometer (Shimadzu CS-910 dualwavelength TLC scanner).

3. Results

3.1. Northern blot analysis In order to study the steady-state level of PPDK mRNA, a northern blot analysis was carried out. When the maize PPDK cDNA probe was hybridized to 15 mg of total RNA, no detectable band was observed in leaves of untransformed Arabidopsis and of transgenic plants with GUS cDNA, although there was significant expression in maize leaves (Fig. 1C). This result indicates that the maize PPDK cDNA probe did not cross-react with the endogenous PPDK mRNA in Arabidopsis or that its expression level was quite low. The steady-state levels of maize PPDK transcripts controlled by the CaMV promoter in transgenic leaves were notably higher than those in the maize leaf. On the other hand, the level of PPDK mRNA controlled by the Arabidopsis rbcS promoter was dependent on the lines and lower than that in transgenic plants controlled by the CaMV 35S promoter.

3.2. Western blot analysis To determine if the transgenic Arabidopsis plants have elevated levels of the maize PPDK protein, western blot analysis of leaf extracts was carried out using anti-maize PPDK antibodies. As

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was much lower than that in maize leaves, there was an equal level of PPDK accumulation irrespective of the differences in promoters and steady-state transcript levels (Fig. 1C).

3.3. Immunocytochemical localization To determine if the mature PPDK protein exists in chloroplasts as it does in maize, western blot analysis of proteins was carried out with isolated chloroplastic, mitochondrial and cytoplasmic fractions. As shown in Fig. 2B, the maize PPDK protein was found only in the chloroplastic fraction of transgenic plants (RP148 and 35P21), indicating that the C4-PPDK preprotein was imported into the chloroplast where it was processed to mature protein. Fig. 2. Detection of maize C4-PPDK in transgenic and nontransgenic Arabidopsis plants. Soluble protein extracts were fractionated by SDS-PAGE, transferred to a nylon membrane, and probed with polyclonal antibody raised against maize PPDK. (A) Twenty micrograms soluble protein from Arabidopsis and 0.5 mg from maize leaves (control) were used. The molecular masses of protein markers (in kDa) are indicated on the right. (B) After protoplast fractionation, western blot analysis of the chloroplastic, cytoplasmic and mitochondrial fractions was carried out. Each sample contained 2 mg soluble protein. As a positive control, 1 mg of soluble protein extracted from maize leaves was used. The molecular masses of protein markers are indicated on the right (in kDa). For plant type, see legend to Fig. 1.

shown in Fig. 2A, when 20 mg protein was applied, there was no visible band in leaves of untransformed Arabidopsis plants. Since PPDK activity was detected in wild type Arabidopsis leaves (see below), this result suggests that the level of the Arabidopsis C3-PPDK protein was very low and/or that the antiserum used was highly specific for maize PPDK and did not crossreact with the endogenous PPDK protein. In contrast to the nontransgenic plants and transgenic plants with the GUS gene, all transgenic plants in which maize PPDK transcripts were found exhibited proteins that cross-reacted with the maize PPDK antisera, and the size of the PPDK protein in these plants was similar to that in maize. Although the level of this accumulated protein

3.4. Acti6ities of photosynthetic enzymes Since the two transgenic lines (RP148 and 35P21) had the highest in vitro activity of PPDK, and PPDK protein was found in the chloroplasts (Fig. 2B), they were selected for further biochemical and physiological analyses. The activity of PPDK on a chlorophyll basis was 2.5–4.0 times higher in the transgenic plants than in untransformed plants or those transformed with the GUS gene (Table 1). On the other hand, the activities of RuBPC, PEPC, NAD-ME, and NADP-ME were similar in all leaves examined. Similarly, there was no significant difference in chlorophyll content between the transgenic plants and the controls. These results indicate that the expression of the maize C4 PPDK gene and the resulting increase in PPDK activity did not affect the activities of the other related enzymes examined.

3.5. Comparison of the acti6ity of the PPDK protein and its influence on other processes The relative PPDK activities were 1.65, 1.23 and 1.46 for control maize and the transgenic RP148 and 35P21 plants, respectively. This result indicated that the maize PPDK protein was produced functionally without severe modification in transgenic plants (i.e. phosphorylation). Over-expression of the PPDK gene did not affect the leaf

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Table 1 RuBPC, PPDK, and other C4 enzyme activities, and total chlorophyll content (Chl) in control (wild type) and transgenic Arabidopsis plants (with PPDK or GUS gene) Plant type

RuBPC

PPDK

PEPC

NAD-ME

NADP-ME

Chl

Wild GUS RP148 35P21

184.0 9 24.6a 177.5 9 15.4a 158.0 9 10.5a 155.5 9 11.1a

0.99 0.2b 0.89 0.2b 2.29 0.4a 3.69 0.9a

14.7 9 0.8a 14.49 0.6a 14.0 9 0.7a 13.99 0.8a

14.8 9 0.8b 20.4 9 0.4a 16.5 9 0.5b 15.0 9 0.7b

8.3 91.3a 6.6 90.1a 6.1 90.7a 6.5 90.7a

55.0 93.9a 47.3 9 0.6a 47.5 9 2.3a 53.4 9 0.1a

All values in mmol/mg Chl/h except Chl (mg cm−2). Data are the means9S.E. of three replicates. Means with the same letter in a given column are not significantly different (Duncan’s multiple range test, PB0.05). For plant type, see legend to Fig. 1.

photosynthetic rate or CO2 compensation point (data not shown).

4. Discussion In transgenic Arabidopsis plants with the PPDK gene, PPDK activities increased 2- to 4-fold compared to that of wild type and transgenic Arabidopsis with the GUS gene, although they were still less than 3% of the PPDK activity in maize. There are several potential steps which regulate the expression of PPDK activity in transgenic plants: (1) transcription, (2) post-transcription, (3) translation, (4) import across the chloroplast envelope membrane, and (5) post-translational control such as phosphorylation; however, it is somewhat difficult to clarify the difference between post-transcriptional and translational regulation. Comparison of the steady-state mRNA level and protein quantity showed that the quantity of PPDK protein did not reflect the high steady-state level of PPDK expression in these transgenic plants. This result suggests that expression of the PPDK gene may not be regulated at the transcriptional level but more likely at a post-transcriptional and/or translational step in transgenic plants. To our knowledge, there is no report on a comparison of expression levels between mRNA and protein in transgenic plants with C4 photosynthetic genes. It is reported that the untranslated 5% leader sequence can influence translation, although the position and length of the leader sequence which is required to affect the translational rate are still unclear [22,23]. In the present

experiments, the PPDK cDNA included 105 bp of untranslated leader sequence before the ATG. Since the accumulation of maize PPDK protein in transgenic Arabidopsis was notably low, the untranslated leader sequence of PPDK cDNA may affect the translational rate. Further study using vectors containing different lengths of 5% untranslated leader sequence is needed to clarify this point. The PPDK protein has a transit peptide that is indispensable for the transport of the precursor protein from the cytosol into chloroplasts [10]. As shown in Fig. 2B, PPDK protein was detected only in the chloroplasts of transgenic plants. This result indicates that the sequence encoding the transit peptide of the maize PPDK precursor protein functions in the C3 dicot Arabidopsis as in the C4 monocot maize. Similarly, Gallardo et al. [4] demonstrated that the transit peptide of chloroplastic NADP-MDH in the C4 monocot Sorghum can be recognized and processed in the C3 dicot tobacco. There are several reports of cDNA clones encoding PPDK obtained from various C4 and C3 plants [12]. Although their primary structures are quite similar, there is a wide diversity in the transit peptide sequences [12]. The homology of the deduced amino acid sequences of the transit peptide between the C4 monocot maize and C3 monocot rice is 59%, whereas that between maize and the C4 dicot Fla6eria triner6ia, and between maize and C3 F. pringlei is 19% and 17%, respectively (for review, see Matsuoka [12]). It is, therefore, somewhat surprising that the transit peptide of the C4 monocot PPDK can function in the chloroplast protein import system of the C3 dicot Arabidopsis since the wide variation in the

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transit peptide sequences between monocot and dicot species suggests that these species evolved their requisite protein import systems independently. However, it is possible that the low PPDK activity in transgenic plants in this study may be attributed to some extent, to a low efficiency of transport of the PPDK protein into the C3 chloroplast. The relative PPDK activity per unit PPDK protein in the transgenic plants was similar to that in maize. Since these activities were measured after activation, this result suggests that the maize PPDK protein was functionally expressed in transgenic Arabidopsis without severe modification during translation and import across the chloroplast envelope membrane. Therefore, it is likely that PPDK activity may increase in transgenic plants if PPDK protein was accumulated to a greater extent. In C4 plants, PPDKRP controls PPDK activity by catalyzing Thr-phosphorylation to inactivate the enzyme and Thr-P dephosphorylation to regenerate the active form (reviewed by Edwards et al. [11]). High PPDK activity has been observed in C3 rice panicles [24] and wheat grains [25,26]. It is unclear whether the same PPDK activity control machinery exists in C3 plants as in C4 plants. If it does, it may be necessary to modify the Thr-phosphorylation site of the C4 PPDK protein prior to introduction into C3 plants in order to increase its maximal activity. The levels of other C4-photosynthetic enzyme activities introduced into C3 plants have been reported to be 2–3 times higher (for PEPC [1 – 3] and NADP-MDH [4]) than in wild type plants, thus having levels similar to our transgenic PPDK plants. Gehlen et al. [3] reported that PEPC activity in transgenic potato, using a vector containing 52 bp of the 5% non-coding region of the chalcone synthase gene from Petroselinum hortense, was enhanced 3- to 8-fold compared to that from the vector with the PEPC gene alone. However, the absolute activity was only 3-fold higher than that of wild type potato. Therefore, another possible reason for the low PPDK activity in transgenic plants may be that some kind of negative control on the functional expression of introduced gene may exist to set an upper limit of activity expressed, beyond which it may be deleterious to the transgenic plants.

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In conclusion, the expression of the maize PPDK gene in transgenic Arabidopsis is likely to be regulated at post-transcriptional and/or translational levels. The transit peptide of C4 PPDK in the C4 monocot maize is functional in the chloroplast protein import system of the C3 dicot Arabidopsis. Since PPDK activity in these transgenic C3 plants was still very low compared with that in maize, an attempt to obtain transgenic plants with higher PPDK activity will be necessary to alter carbon metabolism in Arabidopsis.

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