Kuni Ishihara

Characterization of the gene for pyruvate,orthophosphate dikinase from rice, a C3 plant, and a comparison of structure and expression between C3 and C4 genes for this protein.

To investigate the molecular changes that might have occurred in genes for pyruvate,orthophosphate dikinase (PPDK) during the evolution of C4 plants from C3 plants, we isolated a full-length cDNA and the corresponding gene for a C4-like PPDK from rice, a C3 gramineous plant and compared their structures and promoter activities to those of the corresponding gene from maize, a C4 gramineous plant. As in maize, there are at least two ppdk genes in rice and one of them was very similar to the maize C4-type ppdk. The deduced amino acid sequence of the rice PPDK was 88% homologous to the maize C4-type PPDK in the mature peptide region and 56% homologous in the transit peptide region. The C4-like ppdk in rice contained 21 exons, which were interrupted by twenty introns, and the positions of the introns were essentially the same as those in the gene from maize, with the except in that the gene from rice had two extra introns. Such extra introns were also found in the C4-type ppdk from a dicot, Flaveria, at the same positions. These results strongly suggest that the two introns were present in an ancestral gene before the divergence of monocot and dicot plants.The C4-like ppdk in rice contained two functionally independent promoters had generated a larger transcript with the transit peptide region and a smaller transcript without this region. The unusual dual-promoter system for transcription has been conserved in the C4-type ppdk gene from maize, indicating that the dual-promoter system is a common feature of ppdk genes in both C3 and C4 plants. The patterns of expression of the two transcripts in rice were different: the larger transcript was expressed exclusively in green leaves at a low level whereas the smaller transcript was expressed in some reproductive organs at a high level. Essentially the same patterns of expression were observed in maize, but the level of expression of the larger transcript in maize green leaves was much higher than that in green leaves of rice. The promoter activities of the rice and maize genes for PPDK were examined directly in a transient expression assay in maize mesophyll protoplasts after electroporation with promoter::β-glucuronidase chimeric genes. The rice promoter for the smaller transcript was very active in the protoplasts but the rice promoter for the larger transcript had relatively low activity. By contrast, both promoters of the maize gene had high activity. Taken together, these results demonstrate that the rice C4-like ppdk is very similar to the maize C4-type ppdk, not only in terms of primary structure but also in terms of the regulation of expression, with the exception that the strength of the maize promoter for the larger transcript is higher. The results strongly suggest that the genetic alterations required to give rise to the C4-type ppdk gene were relatively limited.

CHARACTERISTICS OF PHOTOSYNTHETIC CARBON METABOLISM OF SPIKELETS IN RICE

In lemmas and paleae of rice, the amount of pyruvate, Pi dikinase (PPDK) protein increased dramatically 6 d after anthesis and this change was consistent with that in the activity of PPDK. Since lemmas and paleae at this stage also showed high activities of the other marker enzymes of C4 pathway including phosphot enolpyruvate carboxylase (Imaizumi et al. (1990) Plant Cell Physiol 31: 835–843), photosynthetic carbon metabolism with lemmas at this stage were characterized. In a 14C pulse-12C chase study by photosynthetic CO2 fixation, about 35% and 25% of 14C fixed in lemmas were incorporated initially into 3-phosphoglycerate (3-PGA) and C4 acids, respectively. This suggests that lemmas participate mainly in C3-type photosynthetic metabolism, but that lemmas may also participate in the metabolism of C4 acids to some extent. To clarify this possibility, large amounts of 14C-labeled C4 acids were synthesized in vivo by a light-enhanced dark CO2 fixation (LED) method and the fate of 14C in C4 acids in the light was investigated. The percentage distribution of 14C in C-4 position of malate was about 90% and 83% after 10 s of photosynthetic 14CO2 fixation and 110 s of LED, respectively. Some of the 14C incorporated into C4 acids was transferred into 3-PGA and sugar phosphates. The possibility of direct fixation of CO2 by phosphot enolpyruvate carboxylase and metabolic pathway of CO2 released by decarboxylation of malate produced were discussed.

Studies on the Effects of Some Organic Acids on the Root Hair Formation in the Root of Rice Plants.

It was found by the authors that the root hair formation and their growth in rice roots are accelerated when the water percolates vertically in the paddy soils, and was thought that certain substances responsible for regulating the root hair development are derived from the compost under the irrigated condition, and they are probably eliminated from the paddy soils by water-percolation (KAWATA and ISHIHARA, 1961 a). The present experiment was carried on to find the morphogenetical effects of some organic acids (formic, acetic and butyric acid) on the development of the root hairs and epidermal cells of the roots by using the ordinary or aseptic water culture method. The following results were obtained. When the concentration of acetic acid was kept between 2.5 mmol and 5.0 mmol., which is known to be frequently found in the paddy soils, it inhibited the root hair formation and their growth to a great extent, but it did not affect the root and epidermal cell elongation so much. This morphological appearance of epidermis of the root treated with acetic acid was similar to that of the rice plants grown in the non water-percolated soils. Formic acid (2.5 mmol. and 5.0 mmol.) and butyric acid (2.5 mmol. and 0.50 mmol.) retarded not only the root hair formation and their growth, but also the root and epidermal cell elongation. From these results, especially in the case of the acetic acid, our hypothesis mentioned above seems to be true. As reported in the previous paper (KAWATA and ISHIHARA, 1961 b) the ribonucleic acid, the metabolism of which is closely related to the process of the potassium absorption in rice roots, was found to exist in the root hairs for a long time. It is suggested, on the basis of these facts, that the development of the rice plants in the paddy fields is affected because of the accumulation of these organic acids in the soils, which control the absorption of nutrients.