Haruhiko Teramoto

Light-Intensity-Dependent Expression of Lhc Gene Family Encoding Light-Harvesting Chlorophyll-a/b Proteins of Photosystem II in Chlamydomonas reinhardtii

Excessive light conditions repressed the levels of mRNAs accumulation of multiple Lhc genes encoding light-harvesting chlorophyll-a/b (LHC) proteins of photosystem (PS)II in the unicellular green alga, Chlamydomonas reinhardtii. The light intensity required for the repression tended to decrease with lowering temperature or CO2concentration. The responses of six LhcII genes encoding the major LHC (LHCII) proteins and two genes (Lhcb4 andLhcb5) encoding the minor LHC proteins of PSII (CP29 and CP26) were similar. The results indicate that the expression of theseLhc genes is coordinately repressed when the energy input through the antenna systems exceeds the requirement for CO2 assimilation. The Lhc mRNA level repressed under high-light conditions was partially recovered by adding the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting that redox signaling via photosynthetic electron carriers is involved in the gene regulation. However, the mRNA level was still considerably lower under high-light than under low-light conditions even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Repression of theLhc genes by high light was prominent even in the mutants deficient in the reaction center(s) of PSII or both PSI and PSII. The results indicate that two alternative processes are involved in the repression of Lhc genes under high-light conditions, one of which is independent of the photosynthetic reaction centers and electron transport events.

Nucleotide Sequences of cDNA Clones Encoding Ferrochelatase from Barley and Cucumber

Ferrochelatase is the enzyme that catalyzes the last step in the heme biosynthetic pathway. It catalyzes the insertion of iron (Fez+) into the tetrapyrrole ring of protoporphyrin IX to generate protoheme. Recently, we isolated mutants of Escherichia coli that were sensitive to visible light (Miyamoto et al., 1991; Nakahigashi et al., 1991), and these mutants [designated visA (=hemH) mutants] were shown to be the result of a mutation in the visA (=hemH) gene, which encodes ferrochelatase. The nucleotide sequences of genes that encode ferrochelatases from six different sources are available (E. coli, Miyamoto et al., 1991; Bacillus subtilis, Hansson and Hederstedt, 1992; Bradyrhizobium japonicum, Frustaci and O’Brian, 1992; yeast, Labbe-Bois, 1990; mouse, Taketani et al., 1990; human, Nakahashi et al., 1990), but none have been reported from higher plants. Here we report the isolation and the sequences of cDNAs that encode a ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) from barley (Hordeum vulgare L. cv Svalof‘s Bonus) and a ferrochelatase from cucumber (Cucumis sativus L. cv Aonagajibai). Both cDNAs were isolated from libraries in phage vector Xgtll. To screen the clones, we used a deletion mutant of E. coli (strain VSZOO), in which part of the gene for ferrochelatase had been deleted. Because VS200 mutants produce no heme, they cannot grow well even on complete medium. Therefore, we isolated large colonies of VS200 cells that formed among the tiny colonies after infection by the clones in the cDNA libraries of barley and cucumber. Phages obtained from the large colonies were tested for their ability to improve the poor growth of VS200 bacteria. To determine the nucleotide sequence of the cDNA insert in each selected phage clone, an EcoRI fragment was recloned into plasmid vector pUCl18 and serial-deletion clones were constructed by the step-wise method of Yanisch-Perron et al. (1985). The insert of a phage clone from the barley cDNA library was 1728 bp long and included a single open reading frame that encoded a polypeptide of 484 amino acids (Table I). Coincidentally, the insert from the cucumber cDNA library

Noncoding RNA for CR20, a cytokinin-repressed gene of cucumber

The CR20 gene was identified as a cytokinin-repressed gene in excised cotyledons of cucumber. We determined the sequences of some CR20 cDNAs with different structures and sequenced genomic clones for CR20. This gene consisted of three exons, and there were at least three types of transcript, which seemed to be generated by alternative splicing of the second intron. None of the CR20 transcripts included a long open reading frame (ORF). We isolated a cDNA of Arabidopsis thaliana with cucumber CR20 cDNA as a probe. This cDNA for a gene designated AtCR20-1 also lacked a long ORF. A region of 180 nucleotides was conserved in the CR20 RNA of cucumber and the AtCR20-1 RNA of Arabidopsis, although the homology was relatively low when the entire sequences were compared. Each conserved region consisted of seven elements, and seems to form stable secondary structure. These suggest that CR20 RNA may function as an RNA that is not translated into a protein.

Correlated Changes in the Activity, Amount of Protein, and Abundance of Transcript of NADPH:Protochlorophyllide Oxidoreductase and Chlorophyll Accumulation during Greening of Cucumber Cotyledons

Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the 1st h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening.

Isolation of a cDNA clone for a cytokinin-repressed gene in excised cucumber cotyledons.

Rapid changes in gene expression were studied during incubation of cucumber (Cucumis sativus L.) cotyledons with cytokinins in darkness. Complementary-DNA clones for mRNAs whose levels decreased within 4 h of treatment with N6-benzyladenine (BA) were isolated by differential hybridization. One of them (CR9) was sequenced. It is 588 bp long, and would encode a protein consisting of 137 amino-acid residues and having a molecular mass of 15 kDa. The sequence shows a high homology with a light-induced gene from rice. Northern blot analysis of the CR9 transcript showed the level of the mRNA (0.7 kb) to decrease tenfold within 4 h of BA treatment, i.e. well before BA-induced cotyledon expansion was observed. The repression became greater with increasing concentration of BA (10−8–10−5 M). The expression of the CR9 gene was repressed specifically by cytokinins (BA, isopentenyladenine andt-zeatin), but not by adenine or 2,4-dichlorophenoxyacetic acid (auxin). The results are discussed in relation to the primary action of cytokinin.

Changes in expression of two cytokinin-repressed genes, CR9 and CR20, in relation to aging, greening and wounding in cucumber

The expression of two cytokinin-repressed genes, CR9 and CR20, was investigated in cucumber (Cucumis sativus L.). In excised cotyledons, the level of the CR20 transcript markedly decreased in the early periods of treatment with N6-benzyladenine (BA), i.e. well before BA-induced expansion of cotyledons was observed. The repression of CR20 was BA-dose dependent and highly specific for cytokinins. These features were similar to those of CR9 reported previously (H. Teramoto et al., 1994, Planta 193, 573–579). Furthermore, levels of the CR9 and CR20 transcripts decreased during the early phase of greening and soon after wounding of cotyledons. The levels were much lower in young leaves than in mature or senescent leaves. Because cytokinins are thought to control greening and aging of leaves, and to mediate response to wounding, the expression of these two genes could be closely related to the action of such hormones. However, there were some differences between the expression of CR9 and CR20, i.e. the pattern of diurnal changes and the transcript levels in roots. Therefore, some other factors in addition to cytokinins appear to differentially affect the expression of these two genes. Several transcripts of CR20 of different lengths (0.8–2.3 kb) were detected by Northern blot analysis. No long open reading frames could be detected in two CR20 cDNAs with different structures.

Isolation and characterisation of cDNAs for cytokinin-repressed genes

As an approach to the primary action of cytokinins, we studied the repression of gene expression which occurs shortly after the application of this hormone. First we studied the changes in the translatable mRNA population during dark incubation of etiolated cucumber cotyledons with benzyladenine (BA). Two dimensional gel electrophoresis of basic and neutral proteins showed that several spots changed 1 or 2 h after BA application. Among them, three were markedly repressed. Next we isolated cDNA clones for the cytokinin-repressed genes CR9 and CR20 by differential screening. The CR9 cDNA is 588 bp long, and would encode a protein consisting of 137 amino-acid residues, having a molecular mass of 15 kDa. The composition of amino-acid residues indicates that the protein is either neutral or weakly acidic. The hydropathy plot showed that it is probably soluble rather than associated with membranes. The deduced amino-acid sequence shows that it contains two similar sequences of 18 amino-acid residues, each containing two conserved cysteines at an interval of 7 residues. It shows 48% identity with lir1, a light-induced gene from rice [24]. The CR9 transcript began to decrease as early as 1 h after BA application, reaching an extremely low level at 4 h, preceding the initiation of BA-induced cotyledon expansion, although, it began to recover after 8 h. The repression is BA-dose dependent, and highly specific for cytokinins. The CR9 transcript was abundant in mature and senescent leaves, but was not found in roots or young leaves. Wounding and illumination also caused a transient decrease in the CR9 transcript level. When seedlings were grown under a light/dark cycle, expression of CR9 exhibited diurnal fluctuation with an increase in the light period. Expression of another cytokinin-repressed gene, CR20, showed the same pattern of changes as that of CR9 in terms of cytokinin-repression, BA-dose response, cytokinin-specificity, wounding and light effects, although it showed a broad organ specificity. It also exhibited diurnal changes, but opposite to those observed with CR9, showing an increase in the dark period. The nucleotide sequence of CR20 cDNA is quite different from that of CR9 and shows no significant homology with any sequences in the databases. There are many stop codons, hence no long open reading frame to encode a polypeptide. Possible roles of CR9 and CR20 in cytokinin-induced physiological changes are discussed.