Significant accumulation of C(4)-specific pyruvate, orthophosphate dikinase in a C(3) plant, rice

Abstract

The C(4)-Pdk gene encoding the C(4) enzyme pyruvate, orthophosphate dikinase (PPDK) of maize (Zea mays cv Golden Cross Bantam) was introduced into the C(3) plant, rice (Oryza sativa cv Kitaake). When the intact maize C(4)-Pdk gene, containing its own promoter and terminator sequences and exon/intron structure, was introduced, the PPDK activity in the leaves of some transgenic lines was greatly increased, in one line reaching 40-fold over that of wild-type plants. In a homozygous line, the PPDK protein accounted for 35% of total leaf-soluble protein or 16% of total leaf nitrogen. In contrast, introduction of a chimeric gene containing the full-length cDNA of the maize PPDK fused to the maize C(4)-Pdk promoter or the rice Cab promoter only increased PPDK activity and protein level slightly. These observations suggest that the intron(s) or the terminator sequence of the maize gene, or a combination of both, is necessary for high-level expression. In maize and transgenic rice plants carrying the intact maize gene, the level of transcript in the leaves per copy of the maize C(4)-Pdk gene was comparable, and the maize gene was expressed in a similar organ-specific manner. These results suggest that the maize C(4)-Pdk gene behaves in a quantitatively and qualitatively similar way in maize and transgenic rice plants. The activity of the maize PPDK protein expressed in rice leaves was light/dark regulated as it is in maize. This is the first reported evidence for the presence of an endogenous PPDK regulatory protein in a C(3) plant.

Figure 1

Constructs used for rice transformation. A, The construct with the maize C₄-Pdk gene. B, The Pdk promoter::cDNA construct with the maize chloroplastic PPDK cDNA fused to the 5′-flanking sequence of the maize C₄-Pdk gene. C, The Cab promoter::cDNA construct with the maize chloroplastic PPDK cDNA fused to the rice Cab promoter. The coding and the 5′- and 3′-non-coding regions are represented by shadowed and hatched boxes, respectively. Top diagram shows the restriction map of the maize C₄-Pdk gene. B, E, S, and X indicate BamHI, EcoRI, SalI, and XbaI sites, respectively. Short horizontal bars in A indicate the positions of the primers used for reverse transcriptase (RT)-PCR analysis, and MCS indicates a multicloning site that includes a BamHI site.

Figure 2

The PPDK activities of leaves in the primary transgenic rice plants. Transgenic plants introduced with the intact maize C₄-Pdk gene construct (A), the Pdk promoter::cDNA construct (B), and the Cab promoter::cDNA construct (C). Enzyme activities are expressed as fold increases over the activity in wild-type rice plants.

Figure 3

Expression of the maize C₄-Pdk gene in leaves of the primary transgenic rice plants. A, RNA gel-blot analysis. RNAs in the same gel stained with ethidium bromide are shown in the bottom. B, SDS-PAGE of leaf-soluble protein. Polypeptide profiles after Coomassie Blue staining (top); immunoblot profiles with an antiserum raised against the maize C₄-specific PPDK (anti-maize PPDK, bottom). M, Maize; R, wild-type rice; 1 and 2, transformants introduced with the Pdk promoter::cDNA construct; 3 and 4, transformants introduced with the Cab promoter::cDNA construct; 5 through 8, transformants introduced with the maize C₄-Pdk gene construct. The PPDK activities of the transformants relative to wild-type rice were 1-, 2-, 2-, and 1-fold (lanes 1–4, respectively) and 8- to 15-fold (lanes 5–8). LSU and SSU, Large and small subunits of Rubisco, respectively.

Figure 4

Correlation of the levels of transcript and protein in leaves with the copy number of transgenes in high-expressing lines of transgenic rice with the intact maize C₄-Pdk gene. A, DNA gel-blot analysis. Five and 15 μg of genomic DNA from rice and maize plants, respectively, were digested with BamHI and probed with a 3.4-kb BamHI fragment of the maize C₄-Pdk gene. B, RNA gel-blot analysis. RNAs in the same gel stained with ethidium bromide are shown in the bottom. C, Polypeptide profiles of leaf-soluble protein after Coomassie Blue staining. D, Plots of the levels of the maize C₄-Pdk transcript (crosses) and the PPDK protein (○) against the copy number of transgenes per haploid. The levels of the 3.5-kb transcript and the 95-kD protein in transgenic rice leaves are normalized to those in maize leaves. The copy number of transgenes was calculated from the ratio of the levels of the rice 3.4-kb band and the maize 5.5-kb band in A, taking into account that the ratio of the genome sizes of maize:rice is 5:1. M, Maize; R, wild-type rice; 1 through 4, homozygous transformants of T₃ generation PD272, PD332, PD259, and PD278, respectively. The PPDK activities of transformants relative to wild-type plants were 7-, 5-, 12-, and 22-fold (lanes 1–4, respectively).

Figure 5

DNA gel-blot analyses of low-expressing lines of transgenic rice plants of T₂ generation. A, Transformants introduced with the maize C₄-Pdk gene construct with PPDK activities in leaves less than 2-fold wild-type levels (lanes 1–5), together with PD259 (lane 6) for comparison. B, Transformants introduced with the Pdk promoter::cDNA construct (lanes 7–10). a, Location within the introduced gene of restriction sites and probes. E, H, and X indicate EcoRI, HindIII, and XbaI sites, respectively. Bidirectional arrows and numbers indicate fragments excised from the introduced gene and their sizes in kilobases, respectively. b, Polypeptide profiles after Coomassie Blue staining (left) and immunoblot profiles with antimaize PPDK (right) of leaf-soluble protein. Arrowheads indicate the positions of the band of the maize PPDK protein. c, DNA gel-blot analysis. Restriction enzymes and probes used were indicated on the bottom side of panels. P1 and P2 in A and B represent the plasmid DNA used for transformation and the probes, respectively, of which amounts corresponded to 10 and one copies, respectively, per haploid genome of rice. M, Maize; R, wild-type rice.

Figure 6

RT-PCR analysis of the two different transcripts derived from the maize C₄-Pdk gene. A, Developing leaves. B, Hulled rice at a milky stage. Total RNA (5 μg from leaves and 2.5 μg from hulled rice) were used for cDNA synthesis, and 2 μL (a and c) and 0.02 μL (b) of cDNAs were used for the PCR reaction using PF-1 and PR-1 (a and b), and PF-2 and PR-1 (c) as primers (see Fig. 1). d, Electrophoretograms of total RNA used for the RT-PCR analysis after staining with ethidium bromide.

Figure 7

Accumulation of the PPDK protein in various organs of wild-type rice and transgenic rice plants PD259 and PD278. Soluble protein was extracted from leaf blade (1 and 7), leaf sheath (2), hulled rice at a milky stage (3), glume (4), rachis branches including rachis (5), stem (6), and root (8). Samples in lanes 7 and 8 were boiled in the SDS-PAGE sample buffer prior to SDS-PAGE. Each lane was loaded with 1.0 μg of protein. Immunoblots with anti-maize PPDK are shown.

Figure 8

Light-dependent regulation of PPDK activity in leaves. Maize and rice plants, which had been grown in a growth chamber on the day/night cycle for 5 weeks, were illuminated for 8 h from the start of the daytime and were then transferred to the dark for 10 h. The uppermost fully expanded leaves were harvested at the end of illumination (L) and after the subsequent dark incubation (D). Top, Polypeptide profiles of leaf-soluble protein after Coomassie Blue staining. Bottom, PPDK activities. The bars represent ses of three measurements.