Yukiko Sasaki

Plant Acetyl-CoA Carboxylase: Structure, Biosynthesis, Regulation, and Gene Manipulation for Plant Breeding

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Heteromeric ACCase composed of four subunits is usually found in prokaryotes, and homomeric ACCase composed of a single large polypeptide is found in eukaryotes. Most plants have both forms, the heteromeric form in plastids, in which de novo fatty acids are synthesized, and the homomeric form in cytosol. This review focuses on the structure and regulation of plant heteromeric ACCase and its manipulation for plant breeding.

The Compartmentation of Acetyl-Coenzyme A Carboxylase in Plants

Although the biochemical pathways for fatty acid synthesis are more or less similar in plants and animals (Harwood, 1988), there is a major cell biological difference between these two groups of eukaryotes. In plants, the major site of fatty acid synthesis is the plastid, an organelle absent from the animal cell. Many aspects of plastid biology, including fatty acid synthesis, reflect the organelles origins as a prokaryotic symbiont. The synthesis of fatty acids, such as palmitic acid, the prototype 16-carbon fatty acid, requires one molecule of acetyl-CoA and seven molecules of malonyl-CoA, which are added sequentially with the addition of two carbons to the growing fatty acid chain and the release of CO2 at each step. These reactions are catalyzed by fatty acid synthase, an enzyme complex known to exist in a prokaryotic and a eukaryotic form (Wakil et al., 1983; Harwood, 1988). The prokaryotic form (type II) of fatty acid synthase is found in plants. The synthase is composed of several dissociable proteins, whereas the eukaryotic form (type I) found in animals and yeasts is composed of one or two large multifunctional, nondissociable proteins. For either form, the synthesis requires malonyl-CoA, which is supplied by ACCase in the following reaction:

Gas chromatography of trimethylsilylated bases and nucleosides

Abstract Simultaneous separation and the possibility of quantification of the trimethylsilylated (TMS) bases and nucleosides using gas-liquid chromatography are described. The TMS bases and nucleosides were sufficiently volatile for gas chromatography and readily regenerated the original molecules on very mild hydrolysis. The TMS derivatives were isolated from the trimethylsilylation products of bases and nucleosides, and the structures were established by analyses, NMR spectra, and infrared spectra. The structures of the individual components of gas chromatographic peaks are discussed.

Light regulates the gene expression of ribulosebisphosphate carboxylase at the levels of transcription and gene dosage in greening pea leaves

Ribulosebisphosphate carboxylase, composed of large and small subunits, is induced by light in pea leaves. During induction, the synthesis rate of the two mRNAs and the gene dosage were measured. The relative rates of synthesis of the two mRNAs changed with the time of illumination, while the relative gene dosage changed only for the large subunit. The increase in the synthesis rate of the large subunit mRNA was shown to be at least partly due to an increase in gene dosage. These results indicate that the light induction of ribulosebisphosphate carboxylase in the pea is controlled at the levels of transcription and, for the large subunit, also of gene dosage.

Chloroplast envelope protein encoded by chloroplast genome

The gene product of an open reading frame of chloroplast genome, ORF 231 in pea, was immunochemically detected in chloroplast and etioplast envelopes. This is the first protein of a Chloroplast Envelope Membrane encoded by a chloroplast genome. It was named CEM A and the gene, cem A. CEM A is an acidic protein having an apparent molecular mass of 34 kDa on SDS‐PAGE, and a minor component detected in the fractionated inner envelope.

Sequence and transcriptional analysis of the gene cluster trnQ-zfpA-psaI-ORF231-petA in pea chloroplasts

SummaryA 5.1 kb segment of pea chloroplast DNA containing the upstream region of petA was sequenced. RNAs produced from this DNA were characterized. This region encodes putative genes for psbK, trnQ, zfpA, psaI, ORF231, and petA. These genes are all on the same reading strand except for psbK. The gene organization is somewhat different from that of tobacco, rice, and liverwort, which lack the psbK-trnQ genes in this region and contain ORF184/185. Northern blot and primer extension analysis show that the pea transcript covers the zfpA-psaI-ORF231-petA gene cluster and trnQ. These results indicated that the psbK-trnQ genes have been rearranged and a new transcription unit was formed.

Regulation of gene expression of ribulose bisphosphate carboxylase in greening pea leaves

Ribulose bisphosphate carboxylase/oxygenase (RuBisCO) is composed of two subunits, the chloroplastcoded large subunit (LS) and the nuclear-coded small subunit (SS). The effects of different light doses on the levels of the large subunit gene (rbcL), the rbcL and the rbcS mRNAs, and the rate of synthesis of RuBisCO were followed during greening. The rbcL gene dosage changed in response to light whereas the level of the rbcL mRNA changed independently of the gene dosage. This suggests that the expression of the rbcL gene is transcriptionally regulated and that the change in gene dosage only partially contributes to the increase in the mRNA. It appeared that the RuBisCO synthesis rate was proportional to the rbcS mRNA level rather than rbcL mRNA level. These results, taken together with the earlier observations of many researchers, suggest that RuBisCO synthesis in greening pea leaves is controlled primarily at the level of transcription of both genes and is fine-tuned at the post-transcriptional level in chloroplasts, so that the amount of LS is almost stoichiometric to that of SS.

The solubilization and partial characterization of pea RNA polymerases

Abstract A technique has been developed for solubilizing RNA polymerase from pea seedling, which employs the sonication of frozen cells and the addition of insoluble polyvinylpyrrolidone(Polyclar AT) to prevent inactivation of the enzyme. RNA polymerases from peas have properties similar to those of other eucaryotes.

Effect of polyribonucleotides on eukaryotic DNA-dependent RNA polymerases.

Abstract Effects of natural RNAs and synthetic polyribonucleotides on DNA-dependent RNA polymerases (Nucleosidetriphosphate: RNA nucleotidyltransferases, EC 2.7.7.6) from various eukaryotes (rat liver, pea and cauliflower) have been investigated. 1. 1. Poly(G), poly(I), poly(U), yeast RNA and cotton seed tRNA were found to inhibit Enzyme II from all tissues tested and Enzyme I from rat liver and pea. Homoribopolymers were more potent inhibitors than natural RNAs. Poly(A) also inhibited the enzymes except Enzyme I from pea and cauliflower. In contrast, poly(C) had no effect on any enzyme but cauliflower Enzyme I. 2. 2. Enzyme I from cauliflower responded to additions of polyribonucleotides quite differently from other enzymes. The activity was stimulated by polyribonucleotides. 3. 3. With pea Enzyme II, polyribonucleotides inhibited activity competitively with DNA, indicating that there is competitive binding of the polyribonucleotide and DNA on the enzymes surface. The inhibitory effect of polyribonucleotides was partially reduced by preincubation of the enzyme with DNA. The appearance of an enzyme—DNA complex resistant to the inhibitors was independent of the preincubation temperature but was dependent on the ionic strength and MnCl 2 concentration.

Phytochrome-mediated accumulation of chloroplast DNA in pea leaves

Pea seedlings grown for 5 days in the dark were treated with red light for 5 min and grown for 2 more days in the dark. Effects of the red light on chloroplast DNA levels in the pea leaves were examined using probe DNA of the chloroplast‐coded large subunit and nuclear‐coded small subunit of ribulosebisphosphate carboxylase/oxygenase. The gene dosage of the large subunit, but not of the small subunit, was increased by red light. The increase was inhibited by subsequent far‐red light treatment. These results indicate that accumulation of chloroplast DNA in the cell is mediated by phytochrome. Probably the replication of chloroplast DNA is mediated by phytochrome.