Kazuichi Yoshida

Differential Expression of Two hemA mRNAs Encoding Glutamyl-tRNA Reductase Proteins in Greening Cucumber Seedlings

The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.

The third member of the hemA gene family encoding glutamyl-tRNA reductase is primarily expressed in roots in Hordeum vulgare

Accumulation of chlorophylls and heme is primarily controlled at the level of 5-aminolevulinate (ALA) synthesis in higher plants. ALA is formed from glutamate in three enzymatic steps in plants. Among them, the reduction of glutamyl-tRNAGluto glutamate-1-semialdehyde (GSA) is likely to be a regulatory point of ALA synthesis. This reaction is catalyzed by glutamyl-tRNA reductase (GTR), which is encoded by a hemA gene. We have isolated a novel isoform of a hemA cDNA clone from barley (Hordeum vulgare) that is the third member of the hemA gene family. mRNA of this isoform is accumulated primarily in roots, suggesting that the isoform is regulated in an organ-specific manner by the demand for heme synthesis rather than chlorophyll. Phylogenetic analysis was done using the deduced amino acid sequences of hemA isoforms from barley, cucumber and Arabidopsis thaliana. The results indicate that the existing gene families in these plants arose after the divergence of monocotyledonous and dicotyledonous plants.

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.

Action Spectrum for Induction of Promoter Activity of Phenylalanine Ammonia‐Lyase Gene by UV in Carrot Suspension Cells

Abstract— The full‐length promoter (−2335) of the carrot (Daucus carota) phenylalanine ammonia‐lyase gene (gDcPALl) fused to the luciferase reporter gene was transiently transformed to carrot protoplasts by electroporation, and the promoter activity induced by monochromatic UV light of various wavelengths was examined. The action spectrum constructed from the fluence–response curves showed a single peak at around 280 nm, suggesting that the activation of the gDcPALl promoter is categorizable as one of the UVB light responses. The same assay system was applied to variously truncated gDcPALl promoters and to CaMV35S promoter fusion with various parts 5′‐upstream of the gDcPALl promoter. The region from ‐396 to ‐190 (relative to the transcription start site) fused to the CaMV35S core (‐90) promoter showed a 280 nm‐dominant response. However, gDcPALl promoters truncated above ‐570 and ‐396, although they contain the region between ‐396 and ‐190, did not show such a typical UVB response, i.e. they responded to 260 nm light as much as to 280 nm light. The promoter truncated to below ‐190 also responded to 260 nm light as much as to 280 nm light. Therefore we assumed that the gDcPALl promoter is composed of three functionally different parts: the upstream above ‐570 (modulator), the region from ‐396 to ‐190 (UVB responsive) and the downstream below ‐190 (UVB and C responsive). The overall UVB response of the gDcPALl full‐length promoter is explained as the result of interaction of these three components.

Action spectra for photogene expression in etiolated pea seedlings

Action spectra (responsivity spectra) for the transcript accumulation of four photogenes, rbcS, rbcL, Cab and psaB, had a major peak in the red region (660 nm). Violet (400 nm) and green (510 nm) light also induced these transcripts, but their effects differed with the gene. The effect of red light was reversed by later exposure to far‐red light, but the effects of violet and green light were not. Non‐reversible photoreactions in addition to the phytochrome‐mediated photoreversible reaction seemed to be involved in the expression of these photogenes.

FLUENCE‐RESPONSE RELATIONSHIP FOR PHOTOGENE EXPRESSION IN ETIOLATED PEA SEEDLINGS†

Fluence‐response relationships in transcript accumulation were investigated at various wavelengths for the following photogenes: the nuclear‐encoded small subunit gene of ribulose bisphosphate carboxylase/oxygenase (rbcS), the nuclear‐encoded chlorophyll alb‐binding protein gene (Cab), the chloroplast‐encoded large subunit gene of ribulose bisphosphate carboxylase/oxygenase (rbch) and the chloroplast‐encoded photosystem I A2 subunit gene (psaB). The fluence‐response curves for all genes obtained at all wavelengths showed a biphasic character suggesting the involvement of both very low fluence response and low fluence response mediated by phytochrome. The proportion responding in the very low fluence range differed with the gene, and was largest for rbcL followed by psaB, Cab and rbcS in this order.

Interaction of Microsomal Cytochrome P-450 s and N-Phenylcarbamates that Induce Flowering in Asparagus Seedlings

n-Propyl N-(3,4-dichlorophenyl)carbamate, which induces flowering while it inhibits a step or steps in the phenylpropanoid metabolism in Asparagus officinalis L. seedlings, was found to retard the conversion of t-cinnamic acid to p-coumaric acid by high-pressure liquid chromatography of the metabolites in the shikimic acid pathway. The concentrations of the metabolites preceding t-cinnamic acid on the pathway in treated and untreated seedlings were the same, but those of p-coumaric acid and later metabolites were significantly lower in treated plants. The carbamate inhibited phenylpropanoid metabolism when used to treat stem segments that included a shoot apex primordium, where flowers are induced, and when added to a 100,000 × g microsomal fraction prepared from such segments. NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases in the 100,000 × g fraction were not inhibited by the carbamate. The results showed that this compound has its site of action on the endoplasmic reticulum and that it inhibits cytochrome P-450s, including t-cinnamic acid 4-hydroxylase. We examined the flower-inducing activity of known cytochrome P-450 inhibitors, and found that piperonyl butoxide also causes flowering.

INHIBITORY EFFECT OF CONTINUOUS FAR‐RED PREIRRADIATION ON CHLOROPHYLL ACCUMULATION OF PHARBITIS NIL COTYLEDONS

Abstract The effect of continuous far‐red (FR) preirradiation on the accumulation of chlorophyll (Chi) during a white light (WL; 500 lx) period was examined using Pharbitis nil cotyledons. The saturation level of accumulated Chi attained after prolonged exposure to WL was always lowered by continuous FR irradiation preceding the WL. The rate of Chi accumulation during the rapid increase phase (operationally defined as the amount of Chi accumulated during a 24‐h WL period) was enhanced by preirradiation with up to 36 h of FR. However, when the FR preirradiation lasted longer, the rate was reduced below the dark control level. Even FR preirradiation of up to 36 h fully reduced the rate of Chi accumulation under WL when 36 h or longer darkness was spaced between the FR and the WL period.

EFFECT OF CONTINUOUS FAR‐RED PREIRRADIATION ON THE LAG PHASE OF CHLOROPHYLL A ACCUMULATION IN PHARBITIS NIL COTYLEDONS

In Pharbitis nil cotyledons, the lag phase of chlorophyll a (Chi a) accumulation which continues for about 2 h after the onset of continuous white light is eliminated by preirradiation with far‐red light (FR) for 24 or 48 h. When the period of FR preirradiation is prolonged to 72 h or more, however, the lag phase is observed again (FR‐induced lag phase) and the rate of Chi a accumulation during the rapid accumulation phase is lowered below the dark control level.

THE SHAPE OF FLUENCE‐RESPONSE CURVES FOR THE LIGHT‐INDUCED TRANSCRIPT ACCUMULATION IN PEA SEEDLINGS

The fluence‐response curves for the transcript accumulation of four light‐regulated genes were investigated at various times after exposure to 5‐min red light ranging from a very low fluence (1.6 × 10−3μmol m−2) to low fluence (3.0 × 103μmol m−2). The fluence‐response curves were monophasic 6 or 12 h after exposure, although they were biphasic 48 h after exposure, as reported elsewhere. The curves determined 18 or 24 h after red light exposure showed a monophasic or slightly biphasic character. That is, the shape of the fluence‐response curves changed from monophasic to biphasic with an increase in the time of darkness after the treatment with 5 min red light.