Atsushi Komamine

A Novel cis -Acting Element in Promoters of Plant B-Type Cyclin Genes Activates M Phase–Specific Transcription

Plant B-type cyclin genes are expressed late in the G2 and M phases of the cell cycle. Previously, we showed that the promoter of a Catharanthus roseus B-type cyclin, CYM, could direct M phase–specific transcription of a β-glucuronidase reporter gene in synchronously dividing BY2 tobacco cells. In this study, we determined the regulatory elements contained within the CYM promoter by using a luciferase reporter gene. Mutational analysis showed that a 9-bp element is essential for M phase–specific promoter activity in synchronized BY2 cells. The CYM promoter contains three other sequences similar to this element. A gain-of-function assay demonstrated that when fused to a heterologous promoter, these elements are sufficient for M phase–specific expression; therefore, we named these elements M-specific activators (MSAs). We found MSA-like sequences in B-type cyclin promoters from tobacco, soybean, and Arabidopsis as well as in the promoters of two M phase–specific genes, NACK1 and NACK2, which encode tobacco kinesin-like proteins. Thus, MSA may be a common cis-acting promoter element that controls M phase–specific expression of cell cycle–related genes in plants.

Cloning and Characterization of Polyphenol Oxidase cDNAs of Phytolacca americana

Two cDNA clones encoding polyphenol oxidases were isolated from a cDNA library constructed from a log-phase suspension culture of Phytolacca americana (pokeweed) producing betalains. The clones exhibit 93 and 86% sequence identity at the nucleotide and deduced amino acid levels, respectively. Both clones contain two copper-binding domains characterized by histidine-rich regions, which are found ubiquitously in all polyphenol oxidases/tyrosinases, and a putative third histidine-rich, copper-binding region, which is common to all plant polyphenol oxidases. One of the Phytolacca cDNA deduced amino acid sequences contains the ubiquitous transit peptide for all proteins targeted to the internal lumen of thylakoid membranes of plastids and is considered to be 98 residues in length based on a proposed sequence cleavage site motif. This would produce a processed peptide of approximately 54 kD. In addition to common features of transit peptides, it was found that an additional conserved region for polyphenol oxidases was located between the hydroxy amino acid-rich region and the thylakoid transfer domain. Spatial and temporal expression was investigated by northern blot analysis of total RNA from various organs of Phytolacca plants. Transcripts of the two clones were found to be 2.1 and 2.3 kb, respectively. Both transcripts were present only at substantial levels in ripening, betalain-containing fruit.

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca2+ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.

Isolation and characterization of homeobox-containing genes of carrot.

Homeodomains (HDs) are DNA-binding domains that have been well characterized in animals, and HD proteins are thought to be regulators of transcription. To investigate the regulation of gene expression during somatic embryogenesis in carrot, an attempt was made to isolate cDNA clones that encode HD proteins. A cDNA library from carrot somatic embryos was screened with a degenerate oligonucleotide probe that corresponded to a conserved amino acid sequence of HDs, and one cDNA clone (CHB1) encoding an HD protein was isolated. The amino acid sequence deduced from the nucleotide sequence of this clone contained a putative leucine zipper motif adjacent to the anticipated HD. The homeodomain/leucine zipper (HD-Zip) sequence of this cDNA was used for further screening, and five additional independent clones (CHB2 through CHB6) were isolated. Although the HD-Zip sequences encoded by these clones were similar to each other, the sequences beyond the HD-Zip regions varied greatly. Transcripts corresponding to CHB1 through CHB6 were expressed at different times during somatic embryogenesis. In particular, transcripts corresponding to CHB2 were expressed in close association with the early development of embryos.

Betacyanins from plants and cell cultures of Phytolacca americana

Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant. Whereas in fruits prebetanin (betanin 6-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components. These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-[(5-O-E-feruloyl)-2-O-beta-D-apiofuranosyl] -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.

Isolation of a carrot gene expressed specifically during early-stage somatic embryogenesis.

We report the first successful isolation by subtractive hybridization of a gene expressed specifically during somatic embryogenesis. Embryogenic cell clusters, 32–50 μm in diameter, were isolated by sieving and density-gradient centrifugation. The cDNA library was constructed from proglobulars which were formed from embryogenic cell clusters 3 days after transfer to auxin-free modified Lin and Stabas medium. For use as probe in screening, the same cDNA used for library construction was enriched for specific sequences using subtractive hybridization. The cDNA used for subtraction was prepared from suspension cultures 5 days after subculturing in auxin-containing medium. Nine independent differentially expressed cDNA clones were obtained from a screen of 150 000 recombinant phages. Northern analysis indicated one of these, CFM6, to be expressed specifically during somatic embryogenesis. In addition, one hybridizing transcript was detected in plantlet cotyledons, and two transcripts were detected in hypocotyls. Two separate and distinct hybridizing transcripts are expressed specifically in hypocotyl tissue. The amino acid sequence deduced from the nucleotide sequence of the CEM6 cDNA indicates that it encodes a glycine-rich protein containing a hydrophobic signal-sequence like domain. Its early embryo-specific expression and sequence characteristics suggest an important role as a cell wall protein in embryogenesis.

EXPRESSION OF EXTENSIN GENES IS DEPENDENT ON THE STAGE OF THE CELL CYCLE AND CELL PROLIFERATION IN SUSPENSION-CULTURED CATHARANTHUS ROSEUS CELLS

To isolate cDNAs expressed at a specific phase of the cell cycle in a higher plant, we performed differential screening of a cDNA library prepared from the S-phase cells of synchronized cultures of Catharanthus roseus. Sequence analysis shows that two of the identified cDNAs, cyc15 and cyc17, encode extensins that represent a family of cell wall hydroxyproline-rich glycoproteins. Protein sequences deduced from the two cDNAs contain the characteristic pentapeptide repeat sequence, Ser-Pro-Pro-Pro-Pro, which is commonly observed in extensins. The protein sequences also share several other extensin characteristics such as the presence of a N-terminal signal peptide and a high content of Tyr and Lys residues. When C. roseus cell suspension cultures were synchronized by phosphate starvation, the mRNAs of both cyc15 and cyc17 were transiently expressed during the S and G2 phases of the cell cycle. However, significant amounts of the mRNAs also accumulated in phosphate-starved cells arrested in the G1 phase. In asynchronous cultures, both genes were expressed during the stationary phase, when cell proliferation ceased. The observed patterns of expression suggest that the extensin genes, cyc15 and cyc17, are under two types of regulation: one that depends on the stage of the cell cycle and another that is induced during the growth arrest. Thus, the products of these genes may function both during the progression through the cell cycle and in the strengthening of the cell wall after cell division.

Plant 21D7 Protein, a Nuclear Antigen Associated with Cell Division, Is a Component of the 26S Proteasome

Previously, we cloned a carrot (Daucus carota L.) cDNA encoding a 45-kD protein, 21D7, located in the nuclei of proliferating cells. The 21D7 protein is similar to the partial sequence of a regulatory subunit of the bovine 26S proteasome, p58 (G. DeMartino, C.R. Moomaw, O.P. Zagnitko, R.J. Proske, M. Chu-Ping, S.J. Afendis, J.C. Swaffield, C.A. Slaughter [1994] J Biol Chem 269: 20878–20884) and to the deduced sequence encoded by the Saccharomyces cerevisiae gene SUN2 (M. Kawamura, K. Kominami, J. Takeuchi, A. Toh-e [1996] Mol Gen Genet 251: [146–152]). In our work, the expression of plant 21D7 cDNA rescued the yeast sun2 mutant. Fractionation of carrot and spinach (Spinacia oleracea L.) crude extracts showed that the 21D7 protein sedimented with the active 26S proteasomes. The cessation of cell proliferation in carrot suspensions at the stationary phase caused 26S proteasome dissociation and, correspondingly, the 21D7 protein sedimented together with the free regulatory complexes of the 26S proteasomes. Large-scale purification of carrot 26S proteasomes resulted in coisolation of the 21D7 protein. Polyacrylamide gel electrophoresis under nondenaturing conditions showed that the 21D7 protein had the same mobility as the 26S proteasome and that proteasome dissociation changed the mobility of the 21D7 protein accordingly. We conclude that the 21D7 protein is a subunit of the plant 26S proteasome and that it probably belongs to the proteasome regulatory complex.

Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to cell division activity in suspension cultures

Regulatory mechanisms of betacyanin biosynthesis in suspension cultures of Phytolacca americana and anthocyanin in Vitis sp. were investigated in relation to cell division activity.

High-frequency somatic embryogenesis from small suspension-cultured clusters of cells of an interspecific hybrid of Oryza

Suspension cultures in which cell clusters were small and had a high capacity for somatic embryogenesis (about 60%) were established from immature panicles of F1 plants from a cross between Oryza sativa and Oryza latifolia The cells were subcultured at seven-day intervals in a modified N6 medium. The cell clusters were quite small (approximately 30–200μm in diameter) after culture for two months in this medium. When small clusters of cells were transferred to N6 medium, that had been diluted with an equal volume of water and supplemented with α-naphthalenacetic acid (53 nM), 4-pyridylurea (2.2 μM) and sucrose (30 gl-1), somatic embryogenesis occurred at high frequency (about 60%). The system established in the present work is useful for biochemical and molecular biological research of the somatic embryogenesis in the Gramineae.