A novel insight into the regulation of light-independent chlorophyll biosynthesis in <Emphasis Type="Italic">Larix decidua</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis> seedlings

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment–protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.     

Assembly of cyanobacterial and higher plant ribulose bisphosphate carboxylase subunits into functional homologous and heterologous enzyme molecules in Escherichia coli

The genes for the large (rbcL) and small (rbcS) subunits of ribulose-1,5 bisphosphate carboxylase-oxygenase (RuBPCase) from the cyanobacterium Synechococcus PCC 6301, and the rbcS gene of wheat, have been expressed in Escherichia coli in order to study homologous and heterologous enzyme assembly. Synechococcus L subunits expressed in E. coli in the absence of S subunits assemble into oligomeric structures without detectable enzyme activity. Co-expression of L and S subunits, achieved after infection with an M13 recombinant phage containing the rbcS gene, restores enzyme activity, thus demonstrating the essential role of S in the formation of an active RuBPCase. The S subunit, however, is neither required for the solubility nor for the assembly of the L subunits into oligomeric forms. The specific activity of the homologous Synechococcus RuBPCase can be modulated by changing the intracellular pool size of S by phage infection. Heterologous assembly between L subunits of Synechococcus and S subunits of wheat can be demonstrated and results in a functional enzyme. The hybrid RuBPCase has approximately 10% of the activity of the homologous Synechococcus enzyme.     

Cell-free synthesis of glyoxysomal malate dehydrogenase

Polyadenylated mRNA was isolated from germinating watermelon cotyledons and translated in a wheat germ protein synthesizing system. The synthesis of glyoxysomal malate dehydrogenase was detected by direct immunoprecipitation and electrophoretic analysis of the precipitate. In addition to a small amount of the authentic isoenzyme (subunit molecular weight = 33 000), the major part of the incorporated [35S] methionine was observed in a polypeptide with a molecular weight of 38 000. The possible role of the larger molecule as a precursor of glyoxysomal malate dehydrogenase is discussed.     

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana

By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions.     

Identification and Properties of the Messenger RNA Activity in Chlamydomonas reinhardi Coding for the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase

Properties of the mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi were determined. Large subunit synthesis, directed by RNA from partially purified whole cell extracts, was detected by specific immunoprecipitation of polypeptide products synthesized in a heterologous translation system derived from Escherichia coli. Large subunit synthesis showed sharp RNA concentration dependence in an E. coli translation system, and at optimal RNA concentrations, immunoprecipitable large subunit synthesis accounted for 2% of the total incorporation. Large subunit messenger activity sedimented at 12 to 14S on nondenaturing sucrose gradients and did not bind to oligo(dT)-cellulose suggesting the mRNA is not polyadenylated. The immunoprecipitable products translated in vitro are not complete polypeptide chains, but are smaller peptides identifiable as large subunit fragments by tryptic fingerprint analysis. No immunoprecipitable product was obtained when similar RNA fractions were tested in a wheat germ translation system.     

Ribulose bisphosphate carboxylase synthesis in barley leaves: a developmental approach to the question of coordinated subunit synthesis

The coordination of the synthesis of the large and small subunits of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was studied in young light-grown barley (Hordeum vulgare L. var. UC566) leaves. Since a barley leaf is a continuum of different aged cells with the youngest cells at the base and the oldest at the tip, developmental changes could be investigated by comparing different leaf regions. The rate of total cytoplasmic protein synthesis increased to a maximum before the rate of total organelle protein synthesis. The different positions of the maxima suggested that the synthesis of the small RuBPCase subunit on cytoplasmic ribosomes and the large RuBPCase subunit on chloroplast ribosomes might not be coupled during barley leaf development. However, measurements of the amounts and rates of synthesis of the subunits showed that they were coupled. Although the amounts of the RuBPCase subunits increased from the younger to the older leaf regions, the subunits were present in an equimolar ratio. While the rates of synthesis of both subunits increased to a maximum in a midleaf region and then declined, the ratio of the rates remained constant. That the subunit amounts remained equimolar and the synthetic rates proportional while total RuBPCase synthesis was changing indicated that the synthesis of the subunits was closely coordinated during leaf development. A close coordination was also supported by the kinetics of the inhibition of subunit synthesis in the presence of cycloheximide.     

Effect of tagetitoxin on the levels of ribulose 1,5-bisphosphate carboxylase, ribosomes, and RNA in plastids of wheat leaves

Growth of wheat seedlings in the presence of the phytotoxin tagetitoxin produces pigment-deficient leaves of normal size and morphology whose cells contain only rudimentary plastids. We could not detect the accumulation of either the plastid-encoded large subunit or the nuclear-encoded small subunit of the chloroplast stromal enzyme ribulose 1,5-bisphosphate carboxylase (RuBPCase) in western blots of protein extracted from leaves of such seedlings. Sucrose gradient centrifugation profiles showed that plastid ribosomes were essentially absent in toxin-treated leaf tissue while cytoplasmic ribosomes were relatively unaffected. Northern blot analysis of RNA in toxin-treated leaves showed a deficiency of plastid ribosomal RNA (16S and 23S) as well as reduced levels of plastid mRNAs for the large subunit of RuBPCase and for the 32 kilodalton thylakoid Q_B polypeptide. Northern analysis also showed that the nuclear-encoded rbcS mRNA for the small subunit of RuBPCase is present in only trace amounts in toxin-treated leaves.     

Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K_m at 38 C of 1.8 × 10⁻⁷ m. The K_m for casein was 1.1 × 10⁻⁶ m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.     

Effects of light fluence and wavelength on expression of the gene encoding cucumber hydroxypyruvate reductase.

We have investigated the regulation of cucumber (Cucumis sativus) hydroxypyruvate reductase mRNA abundance in response to white-, red-, and far-red-light treatments. Following irradiation of dark-adapted cucumber seedlings with 15 min to 4 h of either white or red light and return to darkness, the mRNA level for the gene encoding hydroxypyruvate reductase (Hpr) in cotyledons peaks in the darkness 16 to 20 h later. The response of the Hpr mRNA level to total fluence of white light depends more directly on irradiation time than on fluence rate. In addition to this time-dependent component, a phytochrome-dependent component is involved in Hpr regulation in dark-adapted green cotyledons as shown by red-light induction and partial far-red-light reversibility. Parallel measurements of mRNA levels for the ribulose bisphosphate carboxylase/oxygenase small subunit and for the chlorophyll a/b-binding protein show that Hpr is the most responsive to short (about 60 min) white- and red-light treatments and that each mRNA has a characteristic pattern of accumulation in dark-adapted cotyledons in response to light.     

Effects of ribosomal wash factors and spermidine on endogenous and exogenous mRNA stimulated protein synthesis in the wheat germ cell-free system.

Differential effects of Mg2+, spermidine, and reticulocyte ribosomal wash factors on the translation of endogenous, myeloma, and globin mRNA's have been observed in studies with the wheat germ cell-free protein synthesizing system. Spermidine stimulated globin mRNA translation but not the translation of endogenous wheat germ messages, and the polyamine actually inhibited the translation of myeloma mRNA. Ribosomal wash factors, on the other hand, stimulated endogenous and myeloma mRNA dependent protein synthesis in an Mg2+-dependent fashion but inhibited globin mRNA translation. The combination of ribosomal wash factors and spermidine was either stimulatory or inhibitory depending on the Mg2+ concentration and the message. It was further observed that translation of exogenous myeloma mRNA proceeded for only 60 min at 25 degrees C under all conditions tested in this study, while translation of endogenous wheat germ messages continued for longer periods of time. No differential effects of spermidine on the synthesis of high molecular weight myeloma proteins were observed.     

In vitro synthesis of a putative precursor of serine: pyruvate aminotransferase of rat liver mitochondria

Serine:pyruvate aminotransferase [EC 2.6.1.51] of rat liver, an enzyme induced by glucagon in mitochondria, was synthesized in cell-free protein synthesizing systems derived from nuclease-treated rabbit reticulocyte lysate and wheat germ extract as a putative precursor which was approximately 2,000 daltons larger than the subunit of mature enzyme. The hepatic level of translatable messenger RNA coding for the putative precursor was approximately 40 times higher in rats received a glucagon administration 3.5 h before sacrifice than in control animals.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: I. Accumulation of delta-Aminolevulinic Acid in Greening Plant Tissues

δ-Aminolevulinic acid dehydrase activity in cucumber (Cucumis sativus L. var. Alpha green) cotyledons did not change as the tissue was allowed to green for 24 hours. δ-Aminolevulinic acid accumulated in greening cucumber cotyledons, and barley (Hordeum sativum L. var. Numar) and bean (Phaseolus vulgaris L. var. Red Kidney) leaves incubated in the presence of levulinic acid, a specific competitive inhibitor of δ-aminolevulinic acid dehydrase. The rate of δ-aminolevulinic acid accumulation in levulinic acid-treated cucumber cotyledons paralleled the rate of chlorophyll accumulation in the controls, and the quantity of δ-aminolevulinic acid accumulated compensated for the decrease in chlorophyll accumulation. When levulinic acid-treated cucumber cotyledons were returned to darkness, δ-aminolevulinic acid accumulation ceased.     

Cytokinin-controlled ribulose 1,5-bisphosphate carboxylase gene expression in pumpkin cotyledons

Treatment of etiolated and excisedCucurbita cotyledons with exogenous cytokinin (benzyladenine) in darkness or light results in a marked stimulation of Rubisco activity, content of enzyme protein, and incorporation of labelled precursors into it, indicating cytokinin-stimulatedde novo synthesis of the enzyme. Cell-free translations of RNA in the wheat germ andE. coli systems show an increase in both large and small subunit mRNA amounts relative to the increase of total RNA under the influence of the phytohormone and light. This increase in the level of translatable RNA is confirmed by RNA hybridization with the Rubisco large subunit gene of spinach. In addition, our results demonstrating additive effects of benzyladenine and light in cotyledon and chloroplast development suggest that the two factors co-act independently in the causal sequence of Rubisco gene expression. The data are discussed in a general view of cytokinin action in gene expression steps. Parts of the results have been obtained by cooperation with Drs. N. L. Klyachko, E. Romanko, and O. N. Kulaeva, Institute Plant Physiology, Acad. Sci. USSR, Moscow (cf. Lerbset al. 1984).     

The effect of cytokinins on nitrate reductase activity

Cytokinins in addition to nitrate induce nitrate reductase activity (NRA) in some plants. Effects of cytokinins onNRA was investigated in stem pith parenchyma of kale, intact wheat and barley seedlings and isolated cucumber cotyledons. The most profound effect onNRA was found in barley and wheat seedlings.NRA in seedlings sprayed with 100 μM 6-benzylaminopurine (BAP) for three subsequent days was increased in leaves and decreased in roots. These changes were further enhanced in seedlings grown in nutrient solution lacking nitrate:NRA in wheat and barley leaves was increased by 57% and 202%, respectively, in plants supplied with nitrate theNRA increase was not significant: in wheat and barley leaves by 22% and 9%, respectively. Similar effect of BAP and kinetin was found in kale stem parenchyma and cucumber cotyledons. The cytokinin kinetin or BAP alone increasedNRA about twice in kale and three times in cucumber. Addition of nitrate to the medium enhanced the effect of kinetin in kale discs, but the two effects were not additive. Additive effect of nitrate and BAP onNRA was found in cucumber cotyledons in light. In general NRA was more affected by cytokinins in intact seedlings of wheat and barley as compared to explanted tissue of kale and cucumber, and lack of nitrogen made their effect more expressive.