Subunit Of RuBPCase Research Articles

Overexpression of the jasmonate-inducible 23 kDa protein (JIP 23) from barley in transgenic tobacco leads to the repression of leaf proteins

We investigated transgenic tobacco lines which express different amounts of the barley JIP 23. In these plants the amount of several proteins decreased proportionally to increasing amounts of JIP 23 whereas the transcript levels were constant as determined for the small and the large subunit of RuBPCase. However, the translation initiation of the rbcS transcript was found to be less efficient than in the wild type. In contrast, the jip 23 transcript was efficiently initiated, indicating that no unspecific impairment of initiation occurred. The data suggest that the barley JIP 23 leads to discrimination among certain tobacco transcripts during translation initiation.

Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the C02 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledon? ous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific ex? pression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumula? tion and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.

Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant.

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledonous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific expression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumulation and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.

Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5?-triphosphate

Photoaffinity labeling with [(32)P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of (32)P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [(32)P] 8-N3ATP. (32)P-labeling was dependent upon ultraviolet irradiation and occurred with [(32)P] 8-N3ATP labeled in the α-position, indicating covalent incorporation of the photoprobe. Both [(32)P] 8-N3ATP and [(32)P] 8-N3GTP were incorporated to a similar extent into the 53-kilodalton (kDa) "large" subunit (LSu), but incorporation of [(32)P] 8-N3GTP into the 14-kDa "small" subunit (SSu) of RuBPCase was <5% of that measured with [(32)P] 8-N3ATP. Distinct binding sites for 8-N3ATP on the two subunits were indicated by different apparent K D values, 3 and 18 μM for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg(2+), anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N3ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [(32)P] 8-N3ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N3ATP binds to a site on the SSu that has physiological significance.

Ammonium toxicity: description of the syndrome in Sinapis alba and the search for its causation

In many plant species, prolonged application of ammonium (NH4+) as a source of nitrogen results in physiological and morphological disorders (‘ammonium toxicity’). In the mustard (Sinapis alba L.) seedling we have previously observed particularly severe symptoms of ammonium toxicity in the absence of external nitrate (NO3‐) or with increasing NH4+/NO3‐ ratios. In the present investigation we have studied the symptoms of this ‘toxicity’in more depth, i.e. at the morphological, plastidic, enzyme and mRNA levels, in an effort to elucidate the causation of the syndrome.It could be confirmed that the syndrome is specific for ammonium and is not caused by a surplus of nitrogen. The syndrome is caused neither by pH changes in the medium nor by non‐specific osmotic effects. Furthermore, the syndrome is not causally related to the fact that nitrate reductase (NR; EC 1.6.6.1.) is induced by ammonium. Development of the syndrome requires neither photosynthesis nor intact plastids. Nevertheless, the plastids are severely affected by ammonium application as is anthocyanin synthesis.Enzymes are differently affected. Among the plastidic enzymes, levels of ribulose‐1,5‐bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) and NADP‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (NADP‐GPD; EC 1.2.1.13) are strongly reduced and abundance of translatable mRNA of the small subunit of RuBPCase is decreased, whereas nitrite reductase (NIR; EC 1.7.7.1) is not affected. Among extraplastidic enzymes, the level of chalcone synthase (CHS; EC 2.3.1.74) is strongly reduced, the NAD‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (NADGPD; EC 1.2.1.12) level is unaffected, whereas the isocitrate lyase (ICL; EC 4.1.3.1) level is strongly promoted.The fat → carbohydrate transformation seems to be impaired by ammonium: fat degradation is reduced, starch accumulation is strongly inhibited and the levels of glucose and fructose are decreased.It appears from the present data and from results obtained in a companion study (U. Hecht and H. Mohr, in preparation) that the ammonium toxicity syndrome is detectable as soon as ammonium accumulation occurs in the plant. However, the actual mechanism through which the excess ammonium affects metabolism remains unclear at present.

MODE OF COACTION BETWEEN UV‐A AND LIGHT ABSORBED BY PHYTOCHROME IN CONTROL OF APPEARANCE OF RIBULOSE‐1.5‐BISPHOSPHATE CARBOXYLASE IN THE SHOOT OF MILO (Sorghum vulgare Pers.)

Abstract— In shoots of milo (Sorghum vulgare Pers.) appearance of ribulosebisphosphate carboxylase (RuBPCase) and of translatable mRNA for its small subunit is stimulated strongly by red light (R, operating through phytochrome) and UV‐A light (UV‐A). Ultraviolet‐A is more effective than R.The mode of coaction between phytochrome and light absorbed by the blue/UV‐A light photoreceptor (‘cryptochrome’) was analyzed in detail in case of enzyme appearance. Fluence rate dependencies, lagphases and the time course of the response are compatible with the view that UV‐A intensifies a process which is occurring in R alone albeit at a lower rate.With both light qualities the light effect is fully reversible by far‐red light up to 1 h. This means that during this period only phytochrome (Pfr) controls the terminal response, i.e. the actual appearance of RuBPCase. During this 1 h period after the onset of light UV‐A or R have no effect on the level of translatable mRNA for the small subunit of RuBPCase indicating that it requires more than 1 h for the light signal to affect gene expression.When R and UV‐A are given longer onset of escape from full reversibility is observed at the same time for both light qualities in the case of RuBPCase appearance. The extent of the reversible response is greater after UV‐A pretreatment than after a R pretreatment.It is argued that the data are consistent with the concept that phytochrome (Pfr) controls the terminal photoresponse, in the present case appearance of RuBPCase, while light absorbed via cryptochrome leads to an increase in responsiveness of the RuBPCase producing machinery towards Pfr.

Effects of specific inhibitors on the coordination of the concentrations of ribulose-bisphosphate-carboxylase subunits and their corresponding mRNAs in the alga Chlorogonium

Investigations were carried out on the effects of inhibitors of transcription and translation on the concentrations of the subunits of the plastid enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) and their corresponding mRNAs in the unicellular green alga Chlorogonium elongatum Dangeard. The light-induced increase of nuclear-coded small-subunit mRNA was strongly inhibited by α-amanitin while the increase of plastid-coded large-subunit mRNA was only weakly affected: Consequently, the mRNAs of the two subunits were present in very different proportions. Nevertheless, the light-induced increase of both subunits was strongly reduced by α-amanitin to the same degree, and hence the ratio of their concentrations was not affected compared with the untreated control cells. The effect of cycloheximide on the subunit mRNAs was similar to but weaker than that of α-amanitin. Again the increases in the subunit levels were strongly inhibited to the same degree. By contrast, rifampicin and chloramphenicol inhibited the light-induced increase of large-subunit mRNA more strongly than that of small-subunit mRNA, but the differences were less distinct than those caused by α-amanitin and cycloheximide. Again, the increase in both subunits was inhibited almost to the same extent. These results - especially those of the α-amanitin experiments - clearly show that the fine coordination of the RuBPCase subunits occurs posttranscriptionally at the level of translation and-or degradation. This conclusion was confirmed by pulse-chase experiments. Inhibition of the synthesis of the large subunits by chloramphenicol resulted - as also found by other authors-in a degradation of excess small subunits in the plastid. On the other hand, inhibition of the concentration of small subunits caused a proportionate reduction in the synthesis of large subunits, but no rapid degradation of large subunits could be detected. Therefore, the fine coordination of both subunits of RuBPCase is achieved by the degradation of an excess of small subunits, while the level of large subunits is adapted to the small subunit concentration, probably by adjustment of translation of the large-subunit mRNA. Furthermore, our experiments with α-amanitin and cycloheximide allow us to conclude that in the blue-light induction of large-subunit mRNA in the plastid the nucleocytoplasmic compartment is not directly involved.

Immunocytological and chemical studies on the stromacentre-forming protein from Avena plastids

The stromacentre (SC), a particular structure in the plastids of Avena, was isolated from etioplasts of Avena sativa by density gradient centrifugation and then analyzed and compared with ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) from A. sativa, with pyrenoids of Chlorella vulgaris and with the "stromacentre" of Opuntia. Purified SC-elements consisted of protein subunits with a relative molecular weight of 63 kDa, different from the isolated RuBPCase of A. sativa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After peptide mapping, the proteolytic cleavage patterns of the 63-kDa protein were also found to be different from those of the RuBPCase. Antibodies against SC-elements, RuBPCase, and the large subunit of RuBPCase were produced. Ouchterlony double immunodiffusion tests did not give crossreactions between the SC-elements and RuBPCase or the large subunit of this enzyme. Immunogold labelling of ultrathin sections showed that antibodies against the SC-elements marked the stromacentre in Avena, but not the pyrenoids in Chlorella. Antibodies against the large subunit of RuBPCase, however, did not label the SC, but labelled the stroma of the plastids in Avena and the pyrenoids of Chlorella. In Opuntia, a comparable structure described as an SC was not labelled by any of the antisera. Immunoelectrophoretical investigations demonstrated a strong correlation between the presence of the 63-kDa protein and the occurrence of the SC in different Avena species with and without SC.

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.

Ribulose-1,5-bisphosphate carboxylase/oxygenase expression in melon plants infected with Colletotrichum lagenarium

Ribulose-1,5-bisphosphate carboxylase/oxygelase (RuBPCase) was studied in melon leaves infected by Colletotrichum lagenarium, a fungal pathogen of melons. Electrophoretic analysis of melon leaf proteins indicated a strong effect of infection on RuBPCase, the subunits of which gradually disappeared during the different stages of infection. Enzyme activity also declined 4 d after inoculation and its content, measured by immunoelectrophoresis, decreased to a similar extent. Synthesis of the large and small subunits of RuBPCase was followed by in-vivo pulse-labeling experiments. A drastic decrease in the rate of RuBPCase-subunit synthesis occurred 3 d after inoculation and preceded the appearance of disease symptoms. There was an apparent coordination of the synthesis of the two subunits under these conditions.

Time course of competence in phytochrome-controlled appearance of nuclear-encoded plastidic proteins and messenger RNAs

The phytochrome-controlled expression of genes coding for plastidic proteins was studied in mustard (Sinapis alba L.) seedling cotyledons in continuous red (R) and far-red (FR) light, i.e. under steady-state conditions with regard to phytochrome, and in darkness over a time span of 8 d after sowing (25° C). (i) The time courses of the levels of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBPCase) and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GPD) were found to be optimum curves. The time at which the optimum (peak) occurred was - independent of fluence rate - the same in R (strong phytochrome action, chlorophyll accumulation and photosynthesis) and FR (strong phytochrome action but no significant chlorophyll accumulation and no photosynthesis). The starting point (first detectable inccrease of enzyme level) was also endogenously fixed and not affected by light. However, the two enzymes differed insofar as the peak was at 4 d after sowing for RuBPCase activity and 4.5 d for GPD. Western blots of the small (SSU) and large (LSU) subunits of RuBPCase showed that enzyme activity and protein levels were correlated. It was concluded that a dramatic change of competence towards phytochrome had occurred and that this change was endogenous. This conclusion was confirmed by short-term induction experiments. In constant darkness (D) the low enzyme levels were saturation rather than optimum curves, presumably because enzyme turnover was lacking. (ii) The time course of accumulation of membrane components showed that chlorophyll and LHCP (light-harvesting chlorophyll a/b-binding protein of photosystem II) levels were closely correlated in R until 6 d after sowing. Thereafter the levels remained constant. The accumulation of membrane components was not related to the accumulation of Calvin-cycle enzymes. (iii) Time courses of the levels of translatable mRNAs, particularly SSU mRNA and LHCP mRNA were determined. In the case of SSU the maximum mRNA-level was found in R, FR and D around 3 d. This was compatible with the in-situ protein accumulation rate. Induction experiments with FR showed that accumulation of SSU mRNA followed the same rise and fall (peak at 3 d) as would be expected from the time course of mRNA levels and from enzyme-induction experiments. In the case of LHCP mRNA the peak was between 3 and 4 d in R, and was not well correlated with in-situ protein accumulation. Translatable LHCP mRNA was also formed in FR and in D-with a peak between 3 and 4 d-although LHCP protein was not detectable under these circumstances (because of the lack of chlorophyll). The data indicate that competence of gene expression towards phytochrome is determined endogenously. However, in the case of LHCP its appearance is not only limited by mRNA but also depends on the availability of chlorophyll.

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS-polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [ 32P]orthophosphate. Autoradiographic analysis of 32P-labelled RuBPCase on SDS-PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.

The specific activity of ribulose-1,5-bisphosphate carboxylase in relation to genotype in wheat

The specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) in crude extracts of leaves from euploid, amphiploid and alloplasmic lines of wheat fell into high or low categories (3.75 or 2.70 μmol·mg(-1)·min(-1), 30°C). For the alloplasmic lines, where the same hexaploid nuclear genome was substituted into different cytoplasms, the specific activity of RuBPCase was consistent with the type of cytoplasm (high for the B and S cytoplasms and low for the A and D cytoplasms). There was no evidence from the euploid and amphiploid lines that small subunits encoded in different nuclear genomes influenced the specific activity. High specific activity was conferred by possession of the chloroplast genome of the B-type cytoplasm which encodes the large subunit of RuBPCase. All lines with a cytoplasm derived from the Sitopsis section of wheat, with the exception of Aegilops longissima and A. speltoides 18940, had RuBPCase with high specific activity. In contrast with the euploid lines of A. longissima, the alloplasmic line containing A. longissima cytoplasm from a different source had RuBPCase with high specific activity. The difference in specific activity found here in-vitro was not apparent in-vivo when leaf gas exchange was measured.

Ribulose Bisphosphate Carboxylase from Three Chlorophyll c-Containing Algae

Distinctive properties are identified in the molecular structure of ribulose, 1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing algae (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrotheca fusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common.

Phytochrome Control of Levels of mRNA Complementary to Plastid and Nuclear Genes of Maize

The involvement of phytochrome in the control of the levels of RNA transcribed from maize plastid and nuclear genes was examined. The effects of illumination with red light, far-red light, or red light followed by far-red light on relative amounts of RNAs complementary to maize plastid genes for the large subunit of ribulose bisphosphate carboxylase (RuBPCase); the 32-kilodalton thylakoid membrane triazine herbicide binding B protein of photosystem II; the alpha, beta, and epsilon subunits of CF(1); subunit III (proton-translocating) of CF(0); the reaction center proteins A1 and A2 of photosystem I; two other light-induced genes for membrane proteins of photosystem II (ORFs 353 and 473); and one gene for an unidentified membrane protein (UORF 443) were measured by hybridization of labeled DNA probes to samples of leaf RNA. Transcripts of two nuclear-encoded genes, the genes for the small subunit of RuBPCase and the light-harvesting chlorophyll a/b binding protein, were studied in the same way. The levels of RNA complementary to all of these light-induced genes were significantly increased within 3 to 6 hours after brief illumination with red light. The stimulatory effects of red light were largely reversed by subsequent illumination with far-red light. It is concluded that phytochrome controls increases in the levels of mRNAs complementary to certain plastid and nuclear genes in dark-grown maize seedlings.

Proteolysis of Endogenous Substrates in Senescing Oat Leaves

Proteolysis of ribulose bisphosphate carboxylase (RuBPCase) during senescence was monitored using oat leaf segments (Avena sativa cv Victory), kept in the dark. We here report the development of a novel approach for measuring protein degradation of endogenous substrates both in situ and in vitro in crude extracts using specific antibodies against highly purified polypeptides. The proteolytic products were separated on sodium dodecyl sulfate-gels. They were then electrotransferred onto nitrocellulose paper and identified with specific antibodies to both the large and small subunits of RuBPCase. We could show differences in pH optima between two proteases degrading the subunits of RuBPCase. While both subunits were best hydrolyzed in acid and basic pH, they degraded differently at neutral pH. Furthermore, the large subunit displayed a different pattern of degradative products at the different pH levels. Older leaf segments, which were incubated in darkness, underwent enhanced proteolysis, as compared with young ones. These results show the advantages of the assay in demonstrating: (a) in situ proteolysis of specific substrates in crude extracts without further purification; (b) in vitro differential proteolysis of endogenous substrates during senescence.

Characterization of Peptide Hydrolase Activity Associated with Thylakoids of the Primary Leaves of Wheat

Summary Intact chloroplasts, free of vacuolar contamination, can be isolated from mesophyll protoplasts prepared from the primary leaves of 8–10 day old wheat seedlings ( Triticum aestivum L.). Thylakoids prepared from these chloroplasts exhibited peptide hydrolase activity. Activity was detected by following the degradation of the large sub-unit of surface-associated ribulose hisphosphate carboxylase (RuBPCase) using the technique of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit of RuBPCase was degraded with a pH optimum of 4.8 by an enzyme which was sensitive to sulfhydryl-group inhibitors. At pH 4.8 there was little activity against any of the thylakoid proteins. The surface-associated RuBPCase was easily removed by washing the thylakoids with sodium pyrophosphate. With repeated washing, the peptide hydrolase activity was also removed from the thylakoids. A second type of peptide hydrolase activity was detected only after treatment of the thylakoids with sodium dodecyl sulfate. In this instance, the α and β sub-units of coupling factor, a major thylakoid protein, were degraded. Degradation was optimal at pH 6.2 and was inhibited with sulfhydryl-group inhibitors.

Increasing the variability of Lycopersicon peruvianum Mill. by protoplast fusion with Petunia hybrida L.

Somatic hybridization of Lycopersicon peruvianum and Petunia hybrida was carried out to transfer cytoplasmic male sterility from Petunia to Lycopersicon. Cytological, morphological and biochemical analyses were performed to characterize the regenerated plants. Two regenerated plants, R3 and R6, were male sterile. R3 possessed chromosomes morphologically similar to those of both parental types. Leaf morphologies of these two plants and a third plant, R7, were intermediate between the two parents. The stability of RUBPCase was verified during parental plant development and after in vitro culture. Plant R7 presented a new form of the large subunit of RUBPCase.

Heat modification of ribulose-1,5-bisphosphate carboxylase/oxygenase by temperature pretreatment of wheat (Triticum aestivum L.) seedlings

The effect of low-, ambient- and high-temperature pretreatments (48 h at 4° C, 20° C or 36° C) of wheat seedlings (spring wheat Triticum aestivum L., cv. Kolibri) on the solubility properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase; EC 4.1.1.39) was studied. The extractable protein moiety of heat-pretreated plants exhibited increased solubility in dilute buffer (50 mM k-phosphate, pH 6.8), compared with protein extracted from 4° C- or 20° C-plants. The salting-out characteristics for ammonium-sulfate precipitation confirmed this finding since a delayed precipitation of extractable protein from 36°C-plants was observed. Using polyacrylamide gel electrophoresis, the in-vivo temperature-induced differences in protein solubility could be traced back to a change in the solubility of RuBPCase. The RuBPCase was purified from wheat seedlings, and the purified enzyme also exhibited differential solubility. In order to evaluate this further, purified RuBPCase was subjected to probing for conformational properties. A decrease of fluorescence of the RuBPCase 1-anilino-8-naphtalene sulfonate complex revealed that the RuBPCase from 36° C-plants had a more hydrophilic protein surface. Titration of the sulfhydryl groups of native RuBP-Case with 5,5'-dithiobis (2-nitrobenzoic acid) pointed to a reduced accessibility of the R-SH groups in the case of the 36° C-type of RuBPCase. The large subunit of RuBPCase from 4° C/20° C-plants tended to give rise to an artificial lower-molecular-weight polypeptide which could not be found in crude or purified RuBPCase from heat-pretreated wheat seedlings.

Ribulose Bisphosphate Carboxylase Synthesis in Barley Leaves

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.