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The pck1 promoter from Urochloa panicoides (a C4 plant) directs expression differently in rice (a C3 plant) and maize (a C4 plant)
Plant Science 165 (2003) 603 /611 www.elsevier.com/locate/plantsci
The pck1 promoter from Urochloa panicoides (a C4 plant) directs expression differently in rice (a C3 plant) and maize (a C4 plant) Shoichi Suzuki a,*, James N. Burnell b a
b
Leaf Tobacco Research Laboratory, Japan Tobacco Inc., 1900 Idei Oyama, Tochigi 323-0808, Japan Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Qld. 4811, Australia Received 27 December 2002; received in revised form 31 March 2003; accepted 12 May 2003
Abstract A chimeric gene using b-glucuronidase (GUS) as a reporter gene under the control of a 1.3 kb 5?-flanking region of pck1 (involved in C4 photosynthesis in Urochloa panicoides ) was introduced into rice and maize. GUS activity was detected in leaf blades, leaf sheaths and roots of transgenic rice plants and was detected at high levels in leaf blades and at low levels in leaf sheaths and roots of transgenic maize plants. The pck1 promoter drove the expression of GUS activity in transgenic maize following 6 h of illumination. In contrast, GUS activity was not induced in transgenic rice even after 24 h illumination. Histochemical analysis revealed that GUS staining was localized to bundle sheath cells and vascular bundles of both rice and maize transformants and GUS activity in bundle sheath cells of transgenic maize was induced by light. These results suggest that the 1.3 kb pck1 promoter contains cis-acting elements for preferential and abundant expression in bundle sheath cells of the leaf blade with light dependence in maize but rice lacks some trans-acting elements required for the expression controlled by pck1. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: C4; PCK; Photosynthetic genes; Transgenic rice; Promoter; Urochloa panicoides
1. Introduction In C4 plants, photosynthesis occurs by the cooperation of two different cell types, mesophyll cells (where CO2 is fixed into 4C compounds) and bundle sheath cells [where CO2, generated by decarboxylation of 4C compounds is fixed by ribulose bisphosphate carboxylase/oxygenase (RubisCO)]. In C3 plants, however, photosynthesis occurs only in the mesophyll cells. The
Abbreviations: A. tumefaciens , Agrobacterium tumefaciens ; bar, phosphinothricin acetyl transferase; GUS, b-glucuronidase; hpt, hygromycinphosphotransferase; ME, malic enzyme; PCK, phoshoenol pyruvate carboxykinase; Pdk, pyruvate, orthophosphate dikinase; PEPC, phosphoenol pyruvate carboxylase; RT-PCR, reverse transcription-PCR; rbcS, RubisCO small subunit; RubisCO, ribulose bisphosphate carboxylase/oxygenase; U. panicoides , Urochloa panicoides . * Corresponding author. Tel.: /81-285-25-5868; fax: /81-285-254460. E-mail address: [email protected] (S. Suzuki).
C4 photosynthetic pathway operates to concentrate CO2 in bundle sheath cells and essentially eliminates photorespiration [1]. The genes encoding the C4 photosynthetic enzymes are expressed exclusively in either the mesophyll or bundle sheath cells of C4 plants [2]. It is believed that C4 plants have arisen polyphyletically from C3 plants in the course of evolution [3,4]. Furthermore, it is suggested that the C4 photosynthetic genes have evolved from a set of pre-existing genes that had not been utilized for photosynthesis in ancestral C3 plants [5]. Modification of the ancestral genes and development of Kranz anatomy with differentiated photosynthetic bundle sheath cells, must have allowed high-level, lightdependent, and cell-specific expression of those genes to facilitate the evolution of C4 plants from their C3 ancestors. For example, the phosphoenol pyruvate carboxylase (PEPC) in C3 plants is expressed at low levels in a variety of tissues but is not involved in photosynthesis. On the other hand, in C4 plants, a large amount of PEPC is found exclusively in mesophyll cells and
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00229-2
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catalyzes the initial fixation of inorganic carbon. Similarly the small subunit of RubisCO (rbcS) is essential for photosynthesis in both C3 and C4 plants, however, cellspecificity of the rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, whereas the C4 rbcS is expressed only in bundle sheath cells. In phosphoenol pyruvate carboxykinase (PCK)-type C4 plants such as Urochloa panicoides , PCK is localized in the cytosol [6,7] and is the major decarboxylating enzyme in bundle sheath cells of the plant [8]. We have previously reported that PCK in U. panicoides is encoded by at least four genes, and that transcripts of pck1 and pck2 are most abundant in leaf tissue, whereas transcripts of pck3 and pck4 are most abundant in root tissue [9]. It has been reported that maize leaves contain appreciable amounts of PCK, though maize is an NADP-malic enzyme-type C4 plant [10]. It has also been reported that the maize PCK gene was specifically expressed in bundle sheath cells [11] and that the maize PCK was involved in the decarboxylation of aspartate in the bundle sheath cells of maize [12]. These results indicate that PCK may play a role in C4 photosynthesis in maize. The specificity of expression of the C4 photosynthetic genes, Ppc, pyruvate, orthophosphate dikinase (Pdk), and rbcS from maize have been investigated in tobacco [13] and rice [14 /17]. These studies indicated that the promoters of these three genes in maize directed lightregulated, organ- and cell-specific expression in C3 plants. These studies also indicated that the promoters of these C4 photosynthetic genes functioned in C3 plants in a similar manner to how they operated in maize and that the mechanisms for the regulation of expression of C4 photosynthetic genes were conserved in C3 plants. Exceptionally, the maize rbcS promoter directed specific expression in mesophyll cells of rice though maize rbcS is localized in bundle sheath cells. The expression of rbcS is coordinated with the expression of rbcL in chloroplasts, and maize rbcS promoter introduced into rice might be affected by the expression of rice rbcL, which is under the control of a rice regulatory system. The leaves of rice plants possess cells that are anatomically designated as bundle sheath cells but are functionally inactive in terms of photosynthesis. PCK is a C4 photosynthetic enzyme specifically expressed in bundle sheath cells of U. panicoides , a C4 plant. In this study, we investigated the regulation of the pck promoter from U. panicoides in a series of transgenic experiments with rice and maize. We found that the pck1 promoter involved in C4 photosynthesis of U. panicoides directed the light-regulated, organ- and cell-specific expression in maize, similar to the expression pattern found in U. panicoides . However, in rice, the expression of pck1 was cell-specific but was neither organ-specific nor regulated by light.
2. Material and methods 2.1. Isolation of total RNA and RT-PCR Total RNA was isolated with a SNAP total RNA Isolation kit (InVitrogen) according to the supplier’s instructions. Primers that are common to all four PCK genes, FW1 (5?-ATCAACGGCGTGCCGTC-3?) and RV2 (5?-CACGGGCAAGATGCAAAG-3?) were prepared for RT-PCR. RT-PCR was carried out using a One shot RT-PCR kit (BRL) according to the manufacturer’s instructions. Total RNA isolated from various organs (30 ng) was used as template for each reaction. To distinguish pck1 and pck2 from pck3 and pck4, the RT-PCR product was digested with Hin dIII (Takara). PCR products were separated on a 5% polyacrylamide gel in 1/ TBE buffer and the products were stained with SYBER Green I or ethidium bromide. Data of these images were read using an FX image analyzer (BioRad) using QUANTITY ONE software (BioRad). 2.2. Northern blot analysis Total RNA, denatured by glyoxal and DMSO, was separated on a 1.2% agarose gel with 10 mM Na-Pi (pH 7) and transferred onto a nylon membrane (GeneScreenPlus, DuPont) by capillary blotting [18]. A DNA fragment digested from pck2 cDNA using Sac I (Takara) was purified from agarose [18] and radiolabeled with [a-32P]dCTP (Pharmacia) by random priming. Hybridization was carried out in accordance with the GeneScreenPlus protocol. The membrane was washed three times with 1 / SSC, 0.1% SDS and once with 0.1 / SSC, 0.1% SDS at 65 8C for 15 min. 2.3. Construction of pck1 promoter/GUS fusion and transformation The pck1 genomic sequence was previously isolated from a lDASHII U. panicoides genomic library [9]. Three clones, l3-2, l3-3 and l13-1 contained the 5?upstream region of pck1. A Sal I fragment containing the 5?-upstream region of pck1 was sub-cloned from l32 and used as a template for PCR. A DNA fragment of the pck1 promoter, ranging from one base upstream from the translation initiation site (/1) and (/1323), was amplified with DNA polymerase (Amplitaq; Takara) (all positions are given relative to the translation start site). This was achieved by using a 5?-oligonucleotide (5?-CGCGTAAGCTTGATCCTGGAGCTTTATTATG-3?) which carried a Hin dIII site and a 3? oligonucleotide (5?-GATAGGATCCTCGTTCGAGCGTCCTGC-3?) which carried a Bam HI site, as primers. The amplified promoter fragment was digested with Hin dIII/Bam HI and exchanged for the 35S promoter of cauliflower mosaic virus in pSB21 [19] to
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generate pSB21pck1. The pSB21pck1 was introduced into Agrobacterium tumefaciens strain LBA4404 (pSB4), by homologous recombination [19]. This step generated A . tumefaciens harboring a pSB plasmid that contained both pck1::GUS and 35S::hpt sandwiched by independent border sequence sets (co-transformation system) [19]. The pSB21pck1 and pSB4 were introduced into rice cultivar, tsukinohikari, through A . tumefaciens LBA4404 [20]. For transformation of maize, a fragment containing phosphinothricin acetyl transferase gene (bar) under the control of the 35S promoter was excised from pSB131 [21] and inserted into the Hin dIII site of pSB21pck1 by blunt-end ligation to yield pSBpck1bar. Agrobacterium-mediated transformation of maize inbred A188 was carried out as described by Ishida et al. [21]. Block structures of the transgenes are shown in Fig. 1.
2.4. Measurement of GUS activity and histochemical staining About 10/200 mg of tissue was homogenized in a mortar and pestle in 0.1 /1 ml of an extraction buffer containing 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 0.1% 2-mercaptoethanol in 50 mM sodium phosphate (pH 7.0) and sand. The homogenate was centrifuged at 15 000 /g for 5 min, at 4 8C and GUS activity [22] and protein content [23] of the supernatant was determined. For the histochemical staining of GUS activity, small section from each sample was transferred to incubation buffer containing 1 mM 5-bromo-4chloro-3-indolyl glucuronide (Sigma), 20% methanol, and 50 mM sodium phosphate (pH 7.0), briefly vacuum infiltrated, and incubated at 37 8C for a few hours to overnight. The stained samples were de-colored and sliced manually or with a micro-slicer (DTK-2000, Dosaka EM, Japan).
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2.5. Light treatment of etiolated leaves T3 seeds from self-pollinated transgenic rice and maize plants were germinated in complete darkness at 25 8C for 2 weeks. The etiolated seedlings were placed under continuous white light (ca. 100 mEm 2 s 1) for the indicated times. The second leaves from ten seedlings were assayed for GUS activity.
3. Results 3.1. Expression of pck1 in U. panicoides The organ-specific transcription of PCK genes in U. panicoides was examined by RT-PCR (for relative abundance) and northern blot analysis (for accumulation of pck transcripts in various organs). RT-PCR was performed with primers that are common to all four PCK genes (Fig. 2). The predicted size of the amplified fragments from pck1, pck2, pck3 and pck4 are 474, 443, 431 and 434 bp, respectively (Fig. 3). Fragments of 582, 553, 517 and 524 bp were amplified from genomic DNA of pck1, pck2, pck3 and pck4, respectively, because all four genes have introns within this region. Total RNA from leaf blades, leaf sheaths, stems and roots was used as template for RT-PCR. Two amplified bands were detected from leaf blades, leaf sheaths and stems (Fig. 3a, lane 2 /4). Only one amplified band was detected from roots (Fig. 3a, lane 5). There is a Hin dIII site in this sequence of pck1 and pck2, but not pck3 and pck4 (Fig. 2). RT-PCR products were digested by Hin dIII to allow pck1 and pck2 to be distinguished from other forms of pck. The size of the bands from leaf blades, leaf sheaths and stems was decreased by Hin dIII digestion (Fig. 3a, lane 6 /8) but that from roots was not (Fig. 3a, lane 9). Nucleotide sequence of the two fragments, the upper and lower one, from leaf blades agreed with that of pck1 and pck2, respectively. In addition, further experiments indicated that both pck1 and pck2 were the
Fig. 1. Structure of chimeric constructs used for transformation. The promoter sequence for pck1 was fused to a GUS coding region, which was followed by the polyadenylation signal from the nopaline synthetase gene (TNOS) for rice transformation (a) and for maize transformation (b). For rice transformation, the selection marker gene (HPT) was introduced by co-transformation. 35S, 35S promoter of cauliflower mosaic virus; HPT, hygromycin phosphotransferase gene; BAR, phosphinothricin acetyltransferase gene; RB, right border; LB, left border.
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Fig. 2. Comparison of the nucleotide sequence of the four U. panicoides PCK genes. The nucleotide sequences of the 3?-ends of the four pcks are shown. Sequences of primers for RT-PCR, FW1 and RV2, and nested PCR, FW2 and RV1, are indicated by the closed squares. The Hin dIII site and the insertion site of the intron are indicated by open squares and closed triangles, respectively. The putative stop codons are underlined.
most abundant forms of pck in leaf blade, leaf sheath and stem and undetectable in root tissue. Light induction of PCK genes was examined by RTPCR with common primers. Only one band undigested by Hin dIII was observed before illumination (Fig. 3b, lane 2/3). After 3 h illumination, a new band corresponding to the amplified fragment from pck1 began to be detected (Fig. 3b, lane 4) and the size of the band was decreased by Hin dIII digestion (Fig. 3b, lane 5).
Another new band, corresponding to pck2, also began to be detected after Hin dIII digestion (Fig. 3b, lane 5). After 6 h illumination, an upper band was detected more strongly (Fig. 3b, lane 6) and most of the lower band was decreased by Hin dIII digestion (Fig. 3b, lane 7). After 12 and 24 h illumination, two bands amplified by RT-PCR were completely digested by Hin dIII (Fig. 3b, lanes 8 /11). The relative abundance of both pck1 and pck2 transcripts was undetectable before illumination
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Fig. 3. Relative abundance of PCK genes in various organs of U. panicoides . (a) Total RNA (30 ng) isolated from leaf blades (lane2, 6), leaf sheaths (lane 3, 7), stems (lane 4, 8) and roots (lane 5, 9) was used for RT-PCR with the FW1 and RV2 primers. RT-PCR products from each tissue were digested with Hin dIII (lanes 6 /9). 0.2, 1, 1 and 5 ml of the products from leaf blades, leaf sheaths, stems and roots, respectively, were separated on a polyacrylamide gel. A 1 kb ladder (BRL) was used as a molecular weight marker (lane 1). (b) Seedlings were grown in the dark for 10 days and then transferred to light for 3, 6, 12 and 24 h. Total RNA (30 ng) isolated from dark leaves (lane 2, 3) and greening leaves for 3 h (lane 4, 5), 6 h (lane 6, 7), 12 h (lane 8, 9) and 24 h (lane 10, 11) was used for RT-PCR. RT-PCR products were digested with Hin dIII. Equal volumes of the products were separated on a polyacrylamide gel. A 1 kb ladder (BRL) was used as a molecular weight marker (lane1) and the band sizes indicated on the left of the figure.
but became the most abundant forms of pck after illumination. These results indicated that both pck1 and pck2 are induced by light. The Sac I-digested fragment of pck2 cDNA (nucleotide 478 /1636), that is 97% identical to pck1 cDNA and 94% identical to the putative coding sequences of both pck3 and pck4 (data not shown), was used as a probe for northern blot hybridization in order to examine the accumulation of the transcripts of all four PCK genes in U. panicoides . A very high level of the transcripts was observed in leaf blades and only low levels of the transcripts were detected in leaf sheaths and stems. The accumulation of the transcripts was undetectable in roots. Combined with the results of RT-PCR, these results indicated that transcripts of both pck1 and pck2 were accumulated mainly in leaf blades, to a lesser extent in leaf sheaths and stem but not in roots. 3.2. The expression of GUS activity in transgenic rice and maize plants Six and 15 of the primary transformants (T1) from rice and maize, respectively, were grown in a greenhouse and four independent lines that gave high GUS activity levels in leaf blades were selected from each of the rice and maize plants. After self-pollination, T2 seeds were collected from the primary transformants and used for various measurements. Distribution of GUS activities in T2 plants was measured in various organs (Table 1). In three of the four transgenic rice lines, leaf blade showed
Table 1 GUS activity controlled by the promoter of pck1 from U. panicoides in different organs of transgenic rice and maize plants Transgenic plant
rice1-1 rice1-2 rice2-1 rice2-2 rice3-1 rice3-2 rice4-1 rice4-2 Non-transformed rice maize1-1 maize1-2 maize2-1 maize2-2 maize3-1 maize3-2 maize4-1 Non-transformed maize
GUS activity (pmol min 1 mg 1 protein) Leaf blade
Leaf sheath
Root
1361 1654 1043 1128 2607 3031 5167 4862 18 2266 6458 3493 3316 27 645 22 002 754 19
3824 3584 371 371 1814 1134 1980 1934 29 405 1548 583 509 1013 2425 453 27
5645 2017 544 190 981 564 2065 640 45 229 675 859 584 37 114 23 24
3-Week-old plants were used for GUS assay.
the highest GUS activity, but significant GUS activity was also expressed in leaf sheath and root. On the other hand, in transgenic maize, the GUS activity in all four lines was much higher in leaf blade than in leaf sheath and root. These results indicate that the pck1 promoter of U. panicoides can control preferential expression in
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leaf blade of maize, but it can not control organ-specific expression in rice.
etiolated U. panicoides showed little expression of pck1 in the dark (Fig. 3, [24]).
3.3. Light induction of pck1-GUS in transgenic rice and maize plants
3.4. Histochemical analysis of pck1-GUS transgenic rice and maize
RT-PCR experiments showed that the expression of pck1 in U. panicoides is positively induced by light at the transcriptional level (Fig. 3b, [9]). We examined whether or not the pck1 promoter directs light-inducible expression in C3 and C4 plants. Self-pollinated T3 seeds were germinated in the dark for 2 weeks and then exposed to light. The second leaf blades of ten seedlings from each transformed line were assayed for GUS activity. Results with four independent lines of transgenic rice and maize are shown in Fig. 4. GUS activity did not increase in the rice lines after plants had been transferred to light, but rather decreased during the first 24 h of illumination (Fig. 4a). On the other hand, GUS activity increased in maize lines after illumination for 6 h and continued to increase almost linearly up to at least 24 h (Fig. 4b). It should be noted that in both transgenic rice and maize plants, the U. panicoides pck1 promoter drove the reporter gene expression in darkness. Importantly
The cell-specific expression driven by the pck1 promoter was examined in transgenic rice and maize by histochemical staining of GUS activity. A similar pattern of GUS staining was observed in more than three independent transformants except for roots of transgenic maize plants. In leaf blades of both transgenic rice and maize plants, heavy staining was observed only in large and small vascular bundles and bundle sheath cells (Fig. 5a and b). Vascular bundles and bundle sheath cells were also stained in leaf sheaths of both transgenic rice and maize plants (Fig. 5c and d). GUS staining was faint in roots of many transgenic maize plants but some of them expressed relatively high GUS activity showing GUS staining in roots. GUS staining was observed in all cells of the stele from both transgenic rice and maize plants but not in the endodermis and cortex cells outside the stele (Fig. 5e and f). These results indicated that cell-specific expression driven by the pck1 promoter was similar in both transgenic rice and maize plants grown in the light. In the leaf blades of dark grown plants GUS expression was limited to the vascular bundles and was not detected in bundle sheath cells of maize (Fig. 5h), whereas in rice, GUS expression was detected in vascular cells, bundle sheath cells and motor cells (Fig. 5). After a week of illumination of etiolated seedlings GUS staining in transgenic maize plants extended to the bundle sheath cells (Fig. 5j), and in rice, the GUS expression in motor cells disappeared (Fig. 5i).
4. Discussion
Fig. 4. Light induction of GUS activity in greening rice (a) and maize (b) seedlings transformed with pck1 promoter::GUS constructs. Seedlings were grown in the dark for 2 weeks and then transferred to the light. GUS activities in etiolated and greening leaves of transgenic plants carrying pck1 promoter::GUS constructs were measured by the formation of 4-methylumbelliferone (4-MU) at the indicated times after transfer to the light. Four independent lines of (a) (rice2, j; rice3-1, ^; rice3-2, '; rice4, m) in (a) and (b) maize (maize1, '; maize2, ^; maize3-1, m; maize3-2, j) and non-transformed maize (k) were analyzed.
It has been suggested that all three C4 photosynthetic genes, Ppc, Pdk and rbcS retained the elements to direct specific expression in mesophyll cells of C3 plants with light dependence, but Pdk and rbcS from C3 plants did not contain the elements to direct cell-specific expression in C4 plants [14 /17]. It is speculated that Ppc, Pdk and rbcS from maize leaves have obtained cis-acting elements for different regulation during the course of the evolution of C4 plants from their C3 ancestors. It is not known how the genes expressed in the bundle sheath cells of C4 monocot plants, other than rbcS, are directed in C3 monocot plants. It would be valuable to understand how pck directs gene expression in rice because rice leaves have cells, which are anatomically similar to the bundle sheath cells of C4 plants. We have previously reported that U. panicoides , a PCK-type C4 plant, has at least four PCK genes [9]. In
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Fig. 5. Histochemical localization of GUS activity controlled by the pck1 promoter in transgenic rice and maize plants. Cross-sections of leaf blade (a), leaf sheath (c), root (e), etiolated leaf of seedling (g) and greening leaf of seedling (i) are from a rice transformant. Cross-sections of leaf blade (b), leaf sheath (d), root (f), etiolated leaf of seedling (h) and greening leaf of seedling (j) are from a maize transformant. Scale bar: 0.1 mm.
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the present study the expression pattern of pck transcripts was examined with leaf blades, leaf sheaths, stems and roots to identify the PCK genes utilized in C4 photosynthesis. The results of the RT-PCR experiments showed that transcripts of both pck1 and pck2 are the most abundant pck transcripts in leaf blades, leaf sheaths, and stems but not in roots (Fig. 3a) and both of the transcripts were induced by light (Fig. 3b). High levels of transcripts were detected in leaf blades while low levels were detected in both leaf blades and stems by northern blot. These data indicated that both pck1 and pck2 are the genes involved in C4 photosynthesis. The product from roots amplified by RT-PCR was reamplified by nested PCR to detect pck3 and pck4 separately (refer to Fig. 2). Two fragments were detected and the nucleotide sequence indicated that they were pck3 and pck4 (data not shown). No accumulation of the transcripts was observed in roots by northern blot. It is likely that transcripts of both pck3 and pck4 are very minor even in roots though they have been detected by RT-PCR. In order to elucidate whether or not the regulatory mechanisms of expression of pck1 (one of the C4 photosynthetic genes) are conserved between C3 and C4 plants, a chimeric pck1 promoter /GUS fusion gene was introduced into rice and maize. The 1.3 kb promoter, 5?-upstream from the translation initiation site, was sufficient to direct the expression of the GUS gene in both rice and maize (Table 1). The pck1 promoter directed different organ-specific expression in rice and maize. In maize, GUS activity was expressed in the leaf blade. GUS activity was also detected in leaf sheaths and roots but the level of activity was only about one fifth and one tenth of that detected in the leaf blades, respectively. The organ-specific expression of pck1 detected in transgenic maize was similar to that in U. panicoides except that the expression of pck1 was observed in the roots of maize but not of U. panicoides (Table 1, Fig. 3a). In rice, leaf blades exhibited the highest levels of GUS activity while the leaf sheath and root showed about 40% of GUS activity detected in the leaf blade. And finally, in one line of the transgenic rice plants the leaf blade did not exhibit the highest level of GUS activity. The organ specificity of pck1 expression was clearly different between rice and U. panicoides . This indicates that the mechanism of organ-specific expression of pck1 from U. panicoides differs from that of Ppc and Pdk from maize because both Ppc and Pdk have shown similar organ specificity in rice to that in maize [14 /16]. GUS activity rapidly increased in leaf blades of transgenic maize following illumination and the pattern of increase was similar to that observed in U. panicoides (Fig. 3b, Fig. 4b). However, in transgenic maize about one third of the GUS activity shown in leaf blade after 24 h illumination was observed prior to the plants being illuminated. These results indicate that the
pck1 promoter in transgenic maize can direct an increase of the expression by light but can not repress the expression in darkness. On the other hand, GUS activity was not induced by light in transgenic rice. This indicates that light regulation of pck1 from U. panicoides is different from that of Ppc and Pdk from maize because they are induced by light in rice [14 /16]. GUS staining in leaf blades and leaf sheaths was observed in the vascular bundle and bundle sheath cell of both rice and maize and was observed in the root stele of both rice and maize (Fig. 5a/f). GUS staining in the etiolated leaves of maize was observed in vascular bundles (Fig. 5h) but in etiolated leaves of rice was observed in vascular bundles, bundle sheath cells and motor cells (Fig. 5g). In maize, illumination induced GUS expression in bundle sheath cells (Fig. 5j). In rice, however, GUS expression in motor cells was decreased following illumination and GUS staining was limited to vascular bundles and bundle sheath cells (Fig. 5i). These data indicate that cell-specific expression of the pck1 promoter is similar in the leaf blades, leaf sheaths and roots of both transgenic rice and maize plants but is regulated differently by light in rice and maize plants. It has been suggested that Ppc, Pdk and rbcS from maize are upregulated by light without change to the cell-specificity in transgenic rice. As for pck1 from U. panicoides , not only the transcription but also the cell-specificity appeared to be regulated by light in rice. It is likely that the increase of GUS activity by illumination in maize has been mainly caused by an increase in GUS activity in bundle sheath cells. On the other hand, it is suggested that the decrease of GUS activity in rice following illumination may be due to GUS activity in motor cells. Motor cells are a specific type of cell found in rice but not in either U. panicoides or maize. It is not apparent why the pck1 promoter directs gene expression in motor cells of etiolated leaves. All of the results suggest that the 1.3 kb pck1 promoter contains cisacting elements for preferential and abundant expression in bundle sheath cells of leaf blades with light dependence but rice lacks some trans-acting elements required for the expression driven by pck1. To date no information has been reported on rice PCK. In an exploratory study a single hybridizing band was detected on a genomic southern blot probed with a pck2 cDNA fragment of U. panicoides . A cDNA fragment about 1 kb in length and 88% identical to pck1 of U. panicoides was obtained by RT-PCR (Suzuki, unpublished data). Accumulation of the transcripts was detected strongly in roots and weakly in green leaves, dark and light seedlings and flowers by northern blot hybridization with the partial rice PCK cDNA fragment as a probe (unpublished data). This suggests that the expression pattern of rice PCK is quite different from that of rbcS. It is possible that this difference causes the different regulation of the expres-
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sion between rbcS and pck1 in the course of evolution from C3 to C4 plants. In this study, we have shown that the U. panicoides pck1 promoter directed gene expression in bundle sheath cells but not in mesophyll cells of rice without light-induction and organ specificity. This suggests that pck1, that is the C4 photosynthetic gene and specific to bundle sheath cells of C4 plants, is regulated by a different system compared with other C4 photosynthetic genes such as Ppc, Pdk and rbcS. In order to fully elucidate how the pck gene has changed the regulation system it is necessary to examine the regulation of rice pck gene in C3 and C4 plants and to identify the regulatory elements.
Acknowledgements S.S. wishes to thank Yuji Ishida for transformation of maize and Mihoko Maruta for transformation of rice. S.S. also would like to thank Dr H. Imaseki for critical reading of the manuscripts.
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[10] R.P. Walker, R.M. Acheson, L.I. Tecsi, R.C. Leegood, Phosphoenolpyruvate carboxykinase in C4 plants: its role and regulation, Aust. J. Plant Physiol. 24 (1997) 459 /468. [11] T. Furumoto, S. Hata, K. Izui, Cloning of cell-type specific genes in maize leaves and characterization of phosphoenolpyruvate carboxykinase, a bundle sheath cell-specific enzyme, Plant Mol. Biol. 41 (1999) 301 /311. [12] A. Wingler, R.P. Walker, Z.H. Chen, R.C. Leegood, Phosphoenolpyruvate carboxykinase is involved in the decarboxylation of aspartate in the bundle sheath of maize, Plant Physiol. 120 (1999) 539 /545. [13] M. Matsuoka, Y. Sanada, Expression of photosynthetic genes from the C4 plant, maize, in tobacco, Mol. Gen. Genet. 225 (1991) 411 /419. [14] M. Matsuoka, Y. Tada, T. Fujimura, Y. Kano-Murakami, Tissue-specific light-regulated expression directed by the promoter of a C4 gene, maize pyruvate, orthophosphate dikinase, in a C3 plant, rice, Proc. Natl. Acad. Sci. 90 (1993) 9586 /9590. [15] M. Matsuoka, J. Kyozuka, K. Shimamoto, Y. Kano-Murakami, The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice), Plant J. 6 (3) (1994) 311 /319. [16] M. Nomura, N. Sentoku, A. Nishimura, J.H. Lin, C. Honda, M. Taniguchi, Y. Ishida, S. Ohta, T. Komari, M. Miyao-Tokutomi, Y. Kano-Murakami, S. Tajima, M.S.B. Ku, M. Matsuoka, The evolution of C4 plants: acquisition of cis-regulatory sequence in the promoter of C4-type pyruvate, orthophosphate dikinase gene, Plant J. 22 (3) (2000) 211 /221. [17] M. Nomura, K. Katayama, A. Nishimura, Y. Ishida, S. Ohta, T. Komari, M. Miyao-Tokutomi, S. Tajima, M. Matsuoka, The promoter of rbcS in a C3 plant (rice) directs organ-specific, lightdependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression, Plant Mol. Biol. 44 (2000) 99 /106. [18] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [19] T. Komari, Y. Hiei, Y. Saito, N. Murai, T. Kumashiro, Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers, Plant J. 10 (1996) 165 /174. [20] Y. Hiei, S. Ohta, T. Komari, T. Kumashiro, Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J. 6 (1994) 271 /282. [21] Y. Ishida, H. Saito, S. Ohta, Y. Hiei, T. Komari, T. Kumashiro, High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens , Nat. Biotech. 14 (1996) 745 /750. [22] R.A. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusion: bglucuronidase as a sensitive and versatile gene fusion marker in higher plants, EMBO J. 6 (1987) 3901 /3907. [23] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248 /254. [24] P.M. Finnegan, J.N. Burnell, Isolation and sequence analysis of cDNA encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides , Plant Mol. Biol. 27 (1995) 365 /376.
The pck1 promoter from Urochloa panicoides (a C4 plant) directs expression differently in rice (a C3 plant) and maize (a C4 plant) Shoichi Suzuki a,*, James N. Burnell b a
b
Leaf Tobacco Research Laboratory, Japan Tobacco Inc., 1900 Idei Oyama, Tochigi 323-0808, Japan Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Qld. 4811, Australia Received 27 December 2002; received in revised form 31 March 2003; accepted 12 May 2003
Abstract A chimeric gene using b-glucuronidase (GUS) as a reporter gene under the control of a 1.3 kb 5?-flanking region of pck1 (involved in C4 photosynthesis in Urochloa panicoides ) was introduced into rice and maize. GUS activity was detected in leaf blades, leaf sheaths and roots of transgenic rice plants and was detected at high levels in leaf blades and at low levels in leaf sheaths and roots of transgenic maize plants. The pck1 promoter drove the expression of GUS activity in transgenic maize following 6 h of illumination. In contrast, GUS activity was not induced in transgenic rice even after 24 h illumination. Histochemical analysis revealed that GUS staining was localized to bundle sheath cells and vascular bundles of both rice and maize transformants and GUS activity in bundle sheath cells of transgenic maize was induced by light. These results suggest that the 1.3 kb pck1 promoter contains cis-acting elements for preferential and abundant expression in bundle sheath cells of the leaf blade with light dependence in maize but rice lacks some trans-acting elements required for the expression controlled by pck1. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: C4; PCK; Photosynthetic genes; Transgenic rice; Promoter; Urochloa panicoides
1. Introduction In C4 plants, photosynthesis occurs by the cooperation of two different cell types, mesophyll cells (where CO2 is fixed into 4C compounds) and bundle sheath cells [where CO2, generated by decarboxylation of 4C compounds is fixed by ribulose bisphosphate carboxylase/oxygenase (RubisCO)]. In C3 plants, however, photosynthesis occurs only in the mesophyll cells. The
Abbreviations: A. tumefaciens , Agrobacterium tumefaciens ; bar, phosphinothricin acetyl transferase; GUS, b-glucuronidase; hpt, hygromycinphosphotransferase; ME, malic enzyme; PCK, phoshoenol pyruvate carboxykinase; Pdk, pyruvate, orthophosphate dikinase; PEPC, phosphoenol pyruvate carboxylase; RT-PCR, reverse transcription-PCR; rbcS, RubisCO small subunit; RubisCO, ribulose bisphosphate carboxylase/oxygenase; U. panicoides , Urochloa panicoides . * Corresponding author. Tel.: /81-285-25-5868; fax: /81-285-254460. E-mail address: [email protected] (S. Suzuki).
C4 photosynthetic pathway operates to concentrate CO2 in bundle sheath cells and essentially eliminates photorespiration [1]. The genes encoding the C4 photosynthetic enzymes are expressed exclusively in either the mesophyll or bundle sheath cells of C4 plants [2]. It is believed that C4 plants have arisen polyphyletically from C3 plants in the course of evolution [3,4]. Furthermore, it is suggested that the C4 photosynthetic genes have evolved from a set of pre-existing genes that had not been utilized for photosynthesis in ancestral C3 plants [5]. Modification of the ancestral genes and development of Kranz anatomy with differentiated photosynthetic bundle sheath cells, must have allowed high-level, lightdependent, and cell-specific expression of those genes to facilitate the evolution of C4 plants from their C3 ancestors. For example, the phosphoenol pyruvate carboxylase (PEPC) in C3 plants is expressed at low levels in a variety of tissues but is not involved in photosynthesis. On the other hand, in C4 plants, a large amount of PEPC is found exclusively in mesophyll cells and
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00229-2
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catalyzes the initial fixation of inorganic carbon. Similarly the small subunit of RubisCO (rbcS) is essential for photosynthesis in both C3 and C4 plants, however, cellspecificity of the rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, whereas the C4 rbcS is expressed only in bundle sheath cells. In phosphoenol pyruvate carboxykinase (PCK)-type C4 plants such as Urochloa panicoides , PCK is localized in the cytosol [6,7] and is the major decarboxylating enzyme in bundle sheath cells of the plant [8]. We have previously reported that PCK in U. panicoides is encoded by at least four genes, and that transcripts of pck1 and pck2 are most abundant in leaf tissue, whereas transcripts of pck3 and pck4 are most abundant in root tissue [9]. It has been reported that maize leaves contain appreciable amounts of PCK, though maize is an NADP-malic enzyme-type C4 plant [10]. It has also been reported that the maize PCK gene was specifically expressed in bundle sheath cells [11] and that the maize PCK was involved in the decarboxylation of aspartate in the bundle sheath cells of maize [12]. These results indicate that PCK may play a role in C4 photosynthesis in maize. The specificity of expression of the C4 photosynthetic genes, Ppc, pyruvate, orthophosphate dikinase (Pdk), and rbcS from maize have been investigated in tobacco [13] and rice [14 /17]. These studies indicated that the promoters of these three genes in maize directed lightregulated, organ- and cell-specific expression in C3 plants. These studies also indicated that the promoters of these C4 photosynthetic genes functioned in C3 plants in a similar manner to how they operated in maize and that the mechanisms for the regulation of expression of C4 photosynthetic genes were conserved in C3 plants. Exceptionally, the maize rbcS promoter directed specific expression in mesophyll cells of rice though maize rbcS is localized in bundle sheath cells. The expression of rbcS is coordinated with the expression of rbcL in chloroplasts, and maize rbcS promoter introduced into rice might be affected by the expression of rice rbcL, which is under the control of a rice regulatory system. The leaves of rice plants possess cells that are anatomically designated as bundle sheath cells but are functionally inactive in terms of photosynthesis. PCK is a C4 photosynthetic enzyme specifically expressed in bundle sheath cells of U. panicoides , a C4 plant. In this study, we investigated the regulation of the pck promoter from U. panicoides in a series of transgenic experiments with rice and maize. We found that the pck1 promoter involved in C4 photosynthesis of U. panicoides directed the light-regulated, organ- and cell-specific expression in maize, similar to the expression pattern found in U. panicoides . However, in rice, the expression of pck1 was cell-specific but was neither organ-specific nor regulated by light.
2. Material and methods 2.1. Isolation of total RNA and RT-PCR Total RNA was isolated with a SNAP total RNA Isolation kit (InVitrogen) according to the supplier’s instructions. Primers that are common to all four PCK genes, FW1 (5?-ATCAACGGCGTGCCGTC-3?) and RV2 (5?-CACGGGCAAGATGCAAAG-3?) were prepared for RT-PCR. RT-PCR was carried out using a One shot RT-PCR kit (BRL) according to the manufacturer’s instructions. Total RNA isolated from various organs (30 ng) was used as template for each reaction. To distinguish pck1 and pck2 from pck3 and pck4, the RT-PCR product was digested with Hin dIII (Takara). PCR products were separated on a 5% polyacrylamide gel in 1/ TBE buffer and the products were stained with SYBER Green I or ethidium bromide. Data of these images were read using an FX image analyzer (BioRad) using QUANTITY ONE software (BioRad). 2.2. Northern blot analysis Total RNA, denatured by glyoxal and DMSO, was separated on a 1.2% agarose gel with 10 mM Na-Pi (pH 7) and transferred onto a nylon membrane (GeneScreenPlus, DuPont) by capillary blotting [18]. A DNA fragment digested from pck2 cDNA using Sac I (Takara) was purified from agarose [18] and radiolabeled with [a-32P]dCTP (Pharmacia) by random priming. Hybridization was carried out in accordance with the GeneScreenPlus protocol. The membrane was washed three times with 1 / SSC, 0.1% SDS and once with 0.1 / SSC, 0.1% SDS at 65 8C for 15 min. 2.3. Construction of pck1 promoter/GUS fusion and transformation The pck1 genomic sequence was previously isolated from a lDASHII U. panicoides genomic library [9]. Three clones, l3-2, l3-3 and l13-1 contained the 5?upstream region of pck1. A Sal I fragment containing the 5?-upstream region of pck1 was sub-cloned from l32 and used as a template for PCR. A DNA fragment of the pck1 promoter, ranging from one base upstream from the translation initiation site (/1) and (/1323), was amplified with DNA polymerase (Amplitaq; Takara) (all positions are given relative to the translation start site). This was achieved by using a 5?-oligonucleotide (5?-CGCGTAAGCTTGATCCTGGAGCTTTATTATG-3?) which carried a Hin dIII site and a 3? oligonucleotide (5?-GATAGGATCCTCGTTCGAGCGTCCTGC-3?) which carried a Bam HI site, as primers. The amplified promoter fragment was digested with Hin dIII/Bam HI and exchanged for the 35S promoter of cauliflower mosaic virus in pSB21 [19] to
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generate pSB21pck1. The pSB21pck1 was introduced into Agrobacterium tumefaciens strain LBA4404 (pSB4), by homologous recombination [19]. This step generated A . tumefaciens harboring a pSB plasmid that contained both pck1::GUS and 35S::hpt sandwiched by independent border sequence sets (co-transformation system) [19]. The pSB21pck1 and pSB4 were introduced into rice cultivar, tsukinohikari, through A . tumefaciens LBA4404 [20]. For transformation of maize, a fragment containing phosphinothricin acetyl transferase gene (bar) under the control of the 35S promoter was excised from pSB131 [21] and inserted into the Hin dIII site of pSB21pck1 by blunt-end ligation to yield pSBpck1bar. Agrobacterium-mediated transformation of maize inbred A188 was carried out as described by Ishida et al. [21]. Block structures of the transgenes are shown in Fig. 1.
2.4. Measurement of GUS activity and histochemical staining About 10/200 mg of tissue was homogenized in a mortar and pestle in 0.1 /1 ml of an extraction buffer containing 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 0.1% 2-mercaptoethanol in 50 mM sodium phosphate (pH 7.0) and sand. The homogenate was centrifuged at 15 000 /g for 5 min, at 4 8C and GUS activity [22] and protein content [23] of the supernatant was determined. For the histochemical staining of GUS activity, small section from each sample was transferred to incubation buffer containing 1 mM 5-bromo-4chloro-3-indolyl glucuronide (Sigma), 20% methanol, and 50 mM sodium phosphate (pH 7.0), briefly vacuum infiltrated, and incubated at 37 8C for a few hours to overnight. The stained samples were de-colored and sliced manually or with a micro-slicer (DTK-2000, Dosaka EM, Japan).
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2.5. Light treatment of etiolated leaves T3 seeds from self-pollinated transgenic rice and maize plants were germinated in complete darkness at 25 8C for 2 weeks. The etiolated seedlings were placed under continuous white light (ca. 100 mEm 2 s 1) for the indicated times. The second leaves from ten seedlings were assayed for GUS activity.
3. Results 3.1. Expression of pck1 in U. panicoides The organ-specific transcription of PCK genes in U. panicoides was examined by RT-PCR (for relative abundance) and northern blot analysis (for accumulation of pck transcripts in various organs). RT-PCR was performed with primers that are common to all four PCK genes (Fig. 2). The predicted size of the amplified fragments from pck1, pck2, pck3 and pck4 are 474, 443, 431 and 434 bp, respectively (Fig. 3). Fragments of 582, 553, 517 and 524 bp were amplified from genomic DNA of pck1, pck2, pck3 and pck4, respectively, because all four genes have introns within this region. Total RNA from leaf blades, leaf sheaths, stems and roots was used as template for RT-PCR. Two amplified bands were detected from leaf blades, leaf sheaths and stems (Fig. 3a, lane 2 /4). Only one amplified band was detected from roots (Fig. 3a, lane 5). There is a Hin dIII site in this sequence of pck1 and pck2, but not pck3 and pck4 (Fig. 2). RT-PCR products were digested by Hin dIII to allow pck1 and pck2 to be distinguished from other forms of pck. The size of the bands from leaf blades, leaf sheaths and stems was decreased by Hin dIII digestion (Fig. 3a, lane 6 /8) but that from roots was not (Fig. 3a, lane 9). Nucleotide sequence of the two fragments, the upper and lower one, from leaf blades agreed with that of pck1 and pck2, respectively. In addition, further experiments indicated that both pck1 and pck2 were the
Fig. 1. Structure of chimeric constructs used for transformation. The promoter sequence for pck1 was fused to a GUS coding region, which was followed by the polyadenylation signal from the nopaline synthetase gene (TNOS) for rice transformation (a) and for maize transformation (b). For rice transformation, the selection marker gene (HPT) was introduced by co-transformation. 35S, 35S promoter of cauliflower mosaic virus; HPT, hygromycin phosphotransferase gene; BAR, phosphinothricin acetyltransferase gene; RB, right border; LB, left border.
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Fig. 2. Comparison of the nucleotide sequence of the four U. panicoides PCK genes. The nucleotide sequences of the 3?-ends of the four pcks are shown. Sequences of primers for RT-PCR, FW1 and RV2, and nested PCR, FW2 and RV1, are indicated by the closed squares. The Hin dIII site and the insertion site of the intron are indicated by open squares and closed triangles, respectively. The putative stop codons are underlined.
most abundant forms of pck in leaf blade, leaf sheath and stem and undetectable in root tissue. Light induction of PCK genes was examined by RTPCR with common primers. Only one band undigested by Hin dIII was observed before illumination (Fig. 3b, lane 2/3). After 3 h illumination, a new band corresponding to the amplified fragment from pck1 began to be detected (Fig. 3b, lane 4) and the size of the band was decreased by Hin dIII digestion (Fig. 3b, lane 5).
Another new band, corresponding to pck2, also began to be detected after Hin dIII digestion (Fig. 3b, lane 5). After 6 h illumination, an upper band was detected more strongly (Fig. 3b, lane 6) and most of the lower band was decreased by Hin dIII digestion (Fig. 3b, lane 7). After 12 and 24 h illumination, two bands amplified by RT-PCR were completely digested by Hin dIII (Fig. 3b, lanes 8 /11). The relative abundance of both pck1 and pck2 transcripts was undetectable before illumination
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Fig. 3. Relative abundance of PCK genes in various organs of U. panicoides . (a) Total RNA (30 ng) isolated from leaf blades (lane2, 6), leaf sheaths (lane 3, 7), stems (lane 4, 8) and roots (lane 5, 9) was used for RT-PCR with the FW1 and RV2 primers. RT-PCR products from each tissue were digested with Hin dIII (lanes 6 /9). 0.2, 1, 1 and 5 ml of the products from leaf blades, leaf sheaths, stems and roots, respectively, were separated on a polyacrylamide gel. A 1 kb ladder (BRL) was used as a molecular weight marker (lane 1). (b) Seedlings were grown in the dark for 10 days and then transferred to light for 3, 6, 12 and 24 h. Total RNA (30 ng) isolated from dark leaves (lane 2, 3) and greening leaves for 3 h (lane 4, 5), 6 h (lane 6, 7), 12 h (lane 8, 9) and 24 h (lane 10, 11) was used for RT-PCR. RT-PCR products were digested with Hin dIII. Equal volumes of the products were separated on a polyacrylamide gel. A 1 kb ladder (BRL) was used as a molecular weight marker (lane1) and the band sizes indicated on the left of the figure.
but became the most abundant forms of pck after illumination. These results indicated that both pck1 and pck2 are induced by light. The Sac I-digested fragment of pck2 cDNA (nucleotide 478 /1636), that is 97% identical to pck1 cDNA and 94% identical to the putative coding sequences of both pck3 and pck4 (data not shown), was used as a probe for northern blot hybridization in order to examine the accumulation of the transcripts of all four PCK genes in U. panicoides . A very high level of the transcripts was observed in leaf blades and only low levels of the transcripts were detected in leaf sheaths and stems. The accumulation of the transcripts was undetectable in roots. Combined with the results of RT-PCR, these results indicated that transcripts of both pck1 and pck2 were accumulated mainly in leaf blades, to a lesser extent in leaf sheaths and stem but not in roots. 3.2. The expression of GUS activity in transgenic rice and maize plants Six and 15 of the primary transformants (T1) from rice and maize, respectively, were grown in a greenhouse and four independent lines that gave high GUS activity levels in leaf blades were selected from each of the rice and maize plants. After self-pollination, T2 seeds were collected from the primary transformants and used for various measurements. Distribution of GUS activities in T2 plants was measured in various organs (Table 1). In three of the four transgenic rice lines, leaf blade showed
Table 1 GUS activity controlled by the promoter of pck1 from U. panicoides in different organs of transgenic rice and maize plants Transgenic plant
rice1-1 rice1-2 rice2-1 rice2-2 rice3-1 rice3-2 rice4-1 rice4-2 Non-transformed rice maize1-1 maize1-2 maize2-1 maize2-2 maize3-1 maize3-2 maize4-1 Non-transformed maize
GUS activity (pmol min 1 mg 1 protein) Leaf blade
Leaf sheath
Root
1361 1654 1043 1128 2607 3031 5167 4862 18 2266 6458 3493 3316 27 645 22 002 754 19
3824 3584 371 371 1814 1134 1980 1934 29 405 1548 583 509 1013 2425 453 27
5645 2017 544 190 981 564 2065 640 45 229 675 859 584 37 114 23 24
3-Week-old plants were used for GUS assay.
the highest GUS activity, but significant GUS activity was also expressed in leaf sheath and root. On the other hand, in transgenic maize, the GUS activity in all four lines was much higher in leaf blade than in leaf sheath and root. These results indicate that the pck1 promoter of U. panicoides can control preferential expression in
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leaf blade of maize, but it can not control organ-specific expression in rice.
etiolated U. panicoides showed little expression of pck1 in the dark (Fig. 3, [24]).
3.3. Light induction of pck1-GUS in transgenic rice and maize plants
3.4. Histochemical analysis of pck1-GUS transgenic rice and maize
RT-PCR experiments showed that the expression of pck1 in U. panicoides is positively induced by light at the transcriptional level (Fig. 3b, [9]). We examined whether or not the pck1 promoter directs light-inducible expression in C3 and C4 plants. Self-pollinated T3 seeds were germinated in the dark for 2 weeks and then exposed to light. The second leaf blades of ten seedlings from each transformed line were assayed for GUS activity. Results with four independent lines of transgenic rice and maize are shown in Fig. 4. GUS activity did not increase in the rice lines after plants had been transferred to light, but rather decreased during the first 24 h of illumination (Fig. 4a). On the other hand, GUS activity increased in maize lines after illumination for 6 h and continued to increase almost linearly up to at least 24 h (Fig. 4b). It should be noted that in both transgenic rice and maize plants, the U. panicoides pck1 promoter drove the reporter gene expression in darkness. Importantly
The cell-specific expression driven by the pck1 promoter was examined in transgenic rice and maize by histochemical staining of GUS activity. A similar pattern of GUS staining was observed in more than three independent transformants except for roots of transgenic maize plants. In leaf blades of both transgenic rice and maize plants, heavy staining was observed only in large and small vascular bundles and bundle sheath cells (Fig. 5a and b). Vascular bundles and bundle sheath cells were also stained in leaf sheaths of both transgenic rice and maize plants (Fig. 5c and d). GUS staining was faint in roots of many transgenic maize plants but some of them expressed relatively high GUS activity showing GUS staining in roots. GUS staining was observed in all cells of the stele from both transgenic rice and maize plants but not in the endodermis and cortex cells outside the stele (Fig. 5e and f). These results indicated that cell-specific expression driven by the pck1 promoter was similar in both transgenic rice and maize plants grown in the light. In the leaf blades of dark grown plants GUS expression was limited to the vascular bundles and was not detected in bundle sheath cells of maize (Fig. 5h), whereas in rice, GUS expression was detected in vascular cells, bundle sheath cells and motor cells (Fig. 5). After a week of illumination of etiolated seedlings GUS staining in transgenic maize plants extended to the bundle sheath cells (Fig. 5j), and in rice, the GUS expression in motor cells disappeared (Fig. 5i).
4. Discussion
Fig. 4. Light induction of GUS activity in greening rice (a) and maize (b) seedlings transformed with pck1 promoter::GUS constructs. Seedlings were grown in the dark for 2 weeks and then transferred to the light. GUS activities in etiolated and greening leaves of transgenic plants carrying pck1 promoter::GUS constructs were measured by the formation of 4-methylumbelliferone (4-MU) at the indicated times after transfer to the light. Four independent lines of (a) (rice2, j; rice3-1, ^; rice3-2, '; rice4, m) in (a) and (b) maize (maize1, '; maize2, ^; maize3-1, m; maize3-2, j) and non-transformed maize (k) were analyzed.
It has been suggested that all three C4 photosynthetic genes, Ppc, Pdk and rbcS retained the elements to direct specific expression in mesophyll cells of C3 plants with light dependence, but Pdk and rbcS from C3 plants did not contain the elements to direct cell-specific expression in C4 plants [14 /17]. It is speculated that Ppc, Pdk and rbcS from maize leaves have obtained cis-acting elements for different regulation during the course of the evolution of C4 plants from their C3 ancestors. It is not known how the genes expressed in the bundle sheath cells of C4 monocot plants, other than rbcS, are directed in C3 monocot plants. It would be valuable to understand how pck directs gene expression in rice because rice leaves have cells, which are anatomically similar to the bundle sheath cells of C4 plants. We have previously reported that U. panicoides , a PCK-type C4 plant, has at least four PCK genes [9]. In
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Fig. 5. Histochemical localization of GUS activity controlled by the pck1 promoter in transgenic rice and maize plants. Cross-sections of leaf blade (a), leaf sheath (c), root (e), etiolated leaf of seedling (g) and greening leaf of seedling (i) are from a rice transformant. Cross-sections of leaf blade (b), leaf sheath (d), root (f), etiolated leaf of seedling (h) and greening leaf of seedling (j) are from a maize transformant. Scale bar: 0.1 mm.
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the present study the expression pattern of pck transcripts was examined with leaf blades, leaf sheaths, stems and roots to identify the PCK genes utilized in C4 photosynthesis. The results of the RT-PCR experiments showed that transcripts of both pck1 and pck2 are the most abundant pck transcripts in leaf blades, leaf sheaths, and stems but not in roots (Fig. 3a) and both of the transcripts were induced by light (Fig. 3b). High levels of transcripts were detected in leaf blades while low levels were detected in both leaf blades and stems by northern blot. These data indicated that both pck1 and pck2 are the genes involved in C4 photosynthesis. The product from roots amplified by RT-PCR was reamplified by nested PCR to detect pck3 and pck4 separately (refer to Fig. 2). Two fragments were detected and the nucleotide sequence indicated that they were pck3 and pck4 (data not shown). No accumulation of the transcripts was observed in roots by northern blot. It is likely that transcripts of both pck3 and pck4 are very minor even in roots though they have been detected by RT-PCR. In order to elucidate whether or not the regulatory mechanisms of expression of pck1 (one of the C4 photosynthetic genes) are conserved between C3 and C4 plants, a chimeric pck1 promoter /GUS fusion gene was introduced into rice and maize. The 1.3 kb promoter, 5?-upstream from the translation initiation site, was sufficient to direct the expression of the GUS gene in both rice and maize (Table 1). The pck1 promoter directed different organ-specific expression in rice and maize. In maize, GUS activity was expressed in the leaf blade. GUS activity was also detected in leaf sheaths and roots but the level of activity was only about one fifth and one tenth of that detected in the leaf blades, respectively. The organ-specific expression of pck1 detected in transgenic maize was similar to that in U. panicoides except that the expression of pck1 was observed in the roots of maize but not of U. panicoides (Table 1, Fig. 3a). In rice, leaf blades exhibited the highest levels of GUS activity while the leaf sheath and root showed about 40% of GUS activity detected in the leaf blade. And finally, in one line of the transgenic rice plants the leaf blade did not exhibit the highest level of GUS activity. The organ specificity of pck1 expression was clearly different between rice and U. panicoides . This indicates that the mechanism of organ-specific expression of pck1 from U. panicoides differs from that of Ppc and Pdk from maize because both Ppc and Pdk have shown similar organ specificity in rice to that in maize [14 /16]. GUS activity rapidly increased in leaf blades of transgenic maize following illumination and the pattern of increase was similar to that observed in U. panicoides (Fig. 3b, Fig. 4b). However, in transgenic maize about one third of the GUS activity shown in leaf blade after 24 h illumination was observed prior to the plants being illuminated. These results indicate that the
pck1 promoter in transgenic maize can direct an increase of the expression by light but can not repress the expression in darkness. On the other hand, GUS activity was not induced by light in transgenic rice. This indicates that light regulation of pck1 from U. panicoides is different from that of Ppc and Pdk from maize because they are induced by light in rice [14 /16]. GUS staining in leaf blades and leaf sheaths was observed in the vascular bundle and bundle sheath cell of both rice and maize and was observed in the root stele of both rice and maize (Fig. 5a/f). GUS staining in the etiolated leaves of maize was observed in vascular bundles (Fig. 5h) but in etiolated leaves of rice was observed in vascular bundles, bundle sheath cells and motor cells (Fig. 5g). In maize, illumination induced GUS expression in bundle sheath cells (Fig. 5j). In rice, however, GUS expression in motor cells was decreased following illumination and GUS staining was limited to vascular bundles and bundle sheath cells (Fig. 5i). These data indicate that cell-specific expression of the pck1 promoter is similar in the leaf blades, leaf sheaths and roots of both transgenic rice and maize plants but is regulated differently by light in rice and maize plants. It has been suggested that Ppc, Pdk and rbcS from maize are upregulated by light without change to the cell-specificity in transgenic rice. As for pck1 from U. panicoides , not only the transcription but also the cell-specificity appeared to be regulated by light in rice. It is likely that the increase of GUS activity by illumination in maize has been mainly caused by an increase in GUS activity in bundle sheath cells. On the other hand, it is suggested that the decrease of GUS activity in rice following illumination may be due to GUS activity in motor cells. Motor cells are a specific type of cell found in rice but not in either U. panicoides or maize. It is not apparent why the pck1 promoter directs gene expression in motor cells of etiolated leaves. All of the results suggest that the 1.3 kb pck1 promoter contains cisacting elements for preferential and abundant expression in bundle sheath cells of leaf blades with light dependence but rice lacks some trans-acting elements required for the expression driven by pck1. To date no information has been reported on rice PCK. In an exploratory study a single hybridizing band was detected on a genomic southern blot probed with a pck2 cDNA fragment of U. panicoides . A cDNA fragment about 1 kb in length and 88% identical to pck1 of U. panicoides was obtained by RT-PCR (Suzuki, unpublished data). Accumulation of the transcripts was detected strongly in roots and weakly in green leaves, dark and light seedlings and flowers by northern blot hybridization with the partial rice PCK cDNA fragment as a probe (unpublished data). This suggests that the expression pattern of rice PCK is quite different from that of rbcS. It is possible that this difference causes the different regulation of the expres-
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sion between rbcS and pck1 in the course of evolution from C3 to C4 plants. In this study, we have shown that the U. panicoides pck1 promoter directed gene expression in bundle sheath cells but not in mesophyll cells of rice without light-induction and organ specificity. This suggests that pck1, that is the C4 photosynthetic gene and specific to bundle sheath cells of C4 plants, is regulated by a different system compared with other C4 photosynthetic genes such as Ppc, Pdk and rbcS. In order to fully elucidate how the pck gene has changed the regulation system it is necessary to examine the regulation of rice pck gene in C3 and C4 plants and to identify the regulatory elements.
Acknowledgements S.S. wishes to thank Yuji Ishida for transformation of maize and Mihoko Maruta for transformation of rice. S.S. also would like to thank Dr H. Imaseki for critical reading of the manuscripts.
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