Levels Of rbcL mRNA Research Articles

A day in the life in the dynamic marine environment: how nutrients shape diel patterns of phytoplankton photosynthesis and carbon fixation gene expression in the Mississippi and Orinoco River plumes

This research addresses the hypothesis that environmental conditions affect temporal connectivity between daily transcription of ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco) genes and photosynthetic capacity among phytoplankton. From surface samples collected in the Mississippi River plume (MRP) and Orinoco River plume (ORP), we made size-fractionated measurements of Rubisco (rbcL) mRNA from four phytoplankton groups (heterokonts, haptophytes, Synechococcus, and Prochlorococcus) and chlorophyll-normalized photosynthesis–irradiance parameters (including light-saturated photosynthetic rate—PmaxB), plus nutrient uptake and inorganic carbon. Chlorophyll, photosynthesis, nutrient uptake, and total rbcL mRNA levels were substantially greater in the MRP. Rubisco mRNA and PmaxB exhibited characteristic diel patterns. Regressions with temporally offset data revealed photosynthesis cycles correlated to rbcL mRNA, but with a time lag. This delay was greater in the MRP, and greater among cells >2 μm. At both sites, PmaxB of <2 μm cells exhibited an earlier increase and closer temporal coupling to rbcL mRNA, suggesting carbon fixation is under tighter transcriptional control in picoplankton. The data support our hypothesis such that in the MRP, high productivity and apparent nutrient stress (perhaps including pCO2) resulted in earlier transcription and longer delay to increasing PmaxB among larger phytoplankton. Under stress, larger eukaryotic cells may need longer time to translate adequate Rubisco enzyme because of cellular resource limitation or diversion to nutrient acquisition. In the relatively oligotrophic ORP, phytoplankton may conserve cellular resources by restricting the time for Rubisco translation. These findings provide an interesting glimpse into ways marine algae can modulate the physiology of carbon fixation in response to environmental challenges.

Differences in Rubisco content and its synthesis in leaves at different positions in Eucalyptus globulus seedlings

The dynamics of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) content and turnover during leaf development are not well understood in woody plants. Rubisco synthesis, N influx and the mRNA levels of Rubisco-encoding genes were determined as a function of leaf position in 4.5-month-old Eucalyptus globulus seedlings. Rubisco concentration was slightly higher in the top leaves as leaf expansion progressed and was almost maximal in the uppermost fully expanded leaves. Rubisco concentration remained almost constant in the fully expanded leaves at the top and middle positions and then became slightly low at the lowest positions. Rubisco synthesis was active only in the top leaves. These results suggest that Rubisco turnover rate is low in the middle leaves, leading to the maintenance of Rubisco contents, and that Rubisco degradation primarily occurs in the lowest leaves. Changes in the RBCS and rbcL mRNA levels were roughly parallel with Rubisco synthesis, but N influx was more closely correlated with Rubisco synthesis. These results suggest that N influx rather than the transcript abundance of Rubisco-encoding genes is of primary importance in regulating the rate of Rubisco synthesis. Additionally, expression of RBCS multigene family in E. globulus leaves was discussed.

Differences in Expression of the RBCS Multigene Family and Rubisco Protein Content in Various Rice Plant Tissues at Different Growth Stages

Four out of five members of the RBCS multigene family (OsRBCS2-OsRBCS5) were highly expressed in leaf blades of rice (Oryza sativa L.) irrespective of plant growth stage, whereas accumulation of all RBCS mRNAs in leaf sheaths, roots and developing spikelets was quite low. A highly positive correlation was observed between total RBCS and RBCL mRNA levels and Rubisco content at their maxima, irrespective of tissue and growth stage. The results indicate that the total RBCS mRNA level may be a primary determinant for maximal Rubisco protein content and that Rubisco gene expression is well coordinated through the whole life of rice.

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.

Increased Rubisco Content in Transgenic Rice Transformed with the ‘Sense’ rbcS Gene

Rice (Oryza sativa L.) plants with substantially increased Rubisco content were obtained by Agrobacterium-mediated transformation with the rice rbcS sense gene under the control of the rice rbcS promoter. The primary transformants were screened for the ratio of Rubisco to leaf-N content, and the transformants with >120% wild-type levels of Rubisco were selected. In the progeny of the selected lines of the transformants, the mRNA levels of one member of the rbcS gene family were increased from 3.9- to 6.2-fold, whereas those of other members of the rbcS gene family were unchanged. The total levels of rbcS mRNA were increased from 2.1- to 2.8-fold. The levels of rbcL mRNA were increased from 1.2- to 1.9-fold. Rubisco protein content was significantly increased by 30% on a leaf area basis. The ratio of Rubisco-N to leaf-N was also increased by 10-20%, irrespective of N treatment. The specific activity of Rubisco per unit of enzyme protein was not different. However, light-saturated photosynthesis was not enhanced even when the rate was measured at low [CO2] where Rubisco becomes limiting for photosynthesis. Some lines showed lower photosynthesis at high [CO2] (>60 Pa). We conclude that introduction of additional sense rbcS leads to overexpression of rbcS and that this overexpression slightly up-regulates the gene expression of rbcL at the transcript level and enhances the amount of Rubisco holoenzyme. However, overproduction of Rubisco protein does not improve photosynthesis.

Amount of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) Protein and levels of mRNAs of rbcS and rbcL in the leaves at different positions in transgenic rice plants with decreased content of Rubisco

The differences in the amounts of various photosynthetic components, including ribulose- 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and chlorophyll (Chl), and the activities of chloroplastic fructose-1,6-bisphosphatase (Cp-FBPase), NADP-glyceraldehyde-3- phosphate dehydrogenase (NADP-G3PDH), ribulose-5-phosphate kinase (Ru5P kinase), and sucrose-phosphate synthase (SPS) enzymes were examined in leaf blades at different leaf positions on the main culms of transgenic rbcS antisense rice plants. The transcript levels of rbcS and rbcL mRNAs were also determined. All the leaves were collected when the flag leaves became fully expanded. Although the Rubisco content was much lower in the upper leaves in the rbcS antisense plants than in the wild-type plants, the difference was less pronounced in lower leaves. Whereas the levels of rbcS mRNA in all the leaves were depressed in the antisense plants, the levels of rbcL mRNA did not differ. Both rbcS and rbcL mRNA levels in lower leaves were very low in the wild-type and antisense plants. Chl content and the activities of Cp-FBPase, NADP-G3PDH, Ru5P kinase, and SPS were slightly higher in the antisense plants. However, the changes among the leaves at different positions were similar to those in the wild-type plants. These results suggest that a selectively slow decrease in the Rubisco content in lower leaves occurs in the rbcS antisense plants and that it is caused by the attenuation of degradation and not by the maintenance of Rubisco synthesis.

Analysis of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities by group-specific gene probing

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 149:239-253 (1997) - doi:10.3354/meps149239 Analysis of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities by group-specific gene probing Pichard SL, Campbell L, Carder K, Kang JB, Patch J, Tabita FR, Paul JH To understand the composition and photosynthetic carbon fixing activities of natural phytoplankton communities, we employed group-specific ribulose bisphosphate carboxylase (RubisCO) large subunit gene probes (rbcL)to examine RubisCO gene expression. The rbcL genes from Synechococcus PCC6301 (cyano) and from Cylindrotheca sp. (chromo) were used as probes at select stations to examine levels of rbcL mRNA in specific size fractions (>5 µm, 1-5 µm, <1 µm) in surface waters of the mouth of Tampa Bay (estuarine), West Florida Shelf (coastal), and from the offshore Gulf of Mexico. Using DNA purified from algal isolates, we demonstrated that the cyano probe was specific for the chlorophyte/cyanobacterial RubisCO evolutionary lineage and the chromo probe was specific for the chromophyte evolutionary lineage (diatoms, prymnesiophytes, and other non-green microalgae). For coastal/estuarine environments, both cyano and chromo rbcL mRNA was predominately confined to the >5 µm size fraction, whereas in offshore oligotrophic environments, the cyano mRNA was associated with smaller cells (<1 µm). Similarly, 14C carbon fixation rates and chl a were predominately associated with the >5 µm fraction in coastal/estuarine environments, while in offshore environments, a greater percentage was present in the <1 µm fraction. In profiles through the euphotic zone, cyano rbcL mRNA exhibited maximal values at depths above 65 m at all stations where the waters were dominated by Synechococcus and Prochlorococcus. In contrast, chromo rbcL mRNA increased with depth from undetectable levels in surface waters to its highest levels at or below the subsurface chlorophyll maximum (SCM, 67 m or deeper). Carbon fixation rates were generally elevated in both surface waters and around the SCM. The SCM was dominated by chromophytic picoeucaryotes, as detected by HPLC pigment analysis and flow cytometry. Such analyses are consistent with the rbcL gene probe patterns of euphotic zones of offshore oligotrophic environments. This study demonstrates the utility of group-specific gene probes for examining the expression of carbon fixing genes in phytoplankton and is a first approach to understanding the active phytoplankton community structure and its relationship to the fixation of inorganic carbon in marine environments. RubisCO · mRNA · Flow cytometry · HPLC pigments · Natural phytoplankton Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 149. Publication date: April 10, 1997 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1997 Inter-Research.

Carbon Cycling: Molecular Regulation of Photosynthetic Carbon Fixation

Photosynthetic carbon fixation by phytoplankton is a key component of the global carbon cycle. Our understanding of the types of picoplankton and ultraphytoplankton involved in this process is evolving. However, mechanisms of regulation of photosynthetic carbon fixation in the oceans are poorly understood. All phytoplankton fix CO2 by reductive carboxylation employing the enzyme ribulose bisphosphate carboxylase (RuBPCase). The sequence of the gene encoding the large subunit of the enzyme (rbcL) has been relatively conserved, with two major evolutionary groups among oxygenic photoautrotrophs: the cyanobacteria/green algae/higher plants and the chromophytic algae. Gene probes made from representative members of these groups have been used to study the transcriptional regulation of RuBPCase in natural phytoplankton populations. Levels of rbcL mRNA correlated with rates of photosynthetic carbon fixation. A diel pattern in both carbon fixation and levels of rbcL mRNA was observed, with greatest values for both during daylight hours. This data supports transcriptional regulation as a major mechanism for regulation of carbon fixation in the oceans. This approach can be used to measure expression of conserved genes encoding other important geochemical functions.

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

The effect of light intensities on the transcript level of proteins involved in photosynthesis in mustard plants

Summary The influence of light quantity on the steady-state levels of plastid encoded transcripts was examined during the development of primary leaves from mustard ( Sinapis alba ) plants. RbcL mRNA, petA mRNA, and psbA mRNA, which encode the large subunit (LSU) of Rubisco, Cyt f of the Cyt b6/f complex, and D1 protein of PS II were investigated in leaves grown under high-light (HL) or low-light (LL) conditions. Additionally, the nuclear encoded 25 S rRNA was quantified. As a proportion of total RNA, the levels of 25 S rRNA, rbcL mRNA, petA mRNA, and psbA mRNA did not differ substantially in the HL versus LL plants. During leaf ontogenesis, though, the proportion of psbA mRNA in total RNA increased drastically, in contrast to the other transcripts. The results calculated per unit fresh weight, dry weight, leaf area, and chlorophyll closely resembled each other and gave an idea of the absolute prevalence of the transcripts in the cell. The light regime had a remarkable influence on the absolute levels of 25 S rRNA, rbcL mRNA, and petA mRNA: Paralleling total RNA content, the amounts of the three transcripts per unit fresh weight at the beginning of leaf development were, in HL plants, more than twice the amounts found in LL plants. Furthermore, during leaf ontogenesis the decrease in total RNA, 25 S rRNA, rbcL mRNA, and petA mRNA was more pronounced in HL than in LL, while the psbA mRNA content per unit fresh weight was only slightly higher in HL than in LL leaves and did not decline significantly during HL and LL leaf ontogenesis. Comparison of the absolute levels of rbcL mRNA, petA mRNA, and psbA mRNA with the amounts of the corresponding proteins revealed that the prevalence of the transcripts contributes principally to the differences in the protein content in HL and LL leaves.

Regulation of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities. I. Diel rhythms

Marine phytoplankton fix carbon dloxide primarily through the action of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the first enzyme in the Calvin Cycle. Although the regulation of this enzyme has been studied in algal cultures and higher plants, little is known regarding RubisCO regulation in natural phytoplankton populations. To determine if natural communities of phytoplankton utilize transcriptional regulation to control RubisCO expression, we investigated the diel relationship between ' carbon fixation and RublsCO large subunit (rbcL) transcript levels, rbcL DNA, chlorophyll a, autofluorescent cell counts and bacterial direct counts for natural communities of the southeastern Gulf of Mexico. Studies were performed with natural phytoplankton populations in Lagrangian studies or deck-top incubators as well as with a Prochlorococcus isolate in culture from the North Pacific Ocean. rbcL mRNA and DNA were detected by stringent hybridization using an antisense rbcL RNA gene probe originating from Synechococcus sp. PCC 6301. For natural communities, carbon fixation maxima (-0.1 to 0.7 pg C 1-' h-') occurred from early morning to mld afternoon, with minimal values at nlght. Peak levels of rbcL mRNA (3.6 to 22.2 ng 1-') almost always coincided with the time of maxlmum carbon fixation and were positively correlated. When natural phytoplankton popula t ion~ or the Prochlorococcus culture were maintained in continuous illuminat~on for 24 h, the same die1 pattern of RubisCO regulation was observed, implying that diel entra~ned rhythms in rbcL transcription occurred. The results thus indicate that natural phytoplankton communrties appear to regulate carbon fixatlon at least in part by transcriptional control of RubisCO synthesis and that such patterns appear to be rhythmlc in nature.

Ribulose bisphosphate carboxylase gene expression in subtropical marine phytoplankton populations

Oceanic phytoplankton are known to fix CO2 primarily through the action of the enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase). The amino acid and nucleotide sequence of the large subunit of this enzyme have been conserved across the evolution of the chlorophytic plants (from cyanobacteria to higher plants via green algae) with approximately 80 and 70 % homology at the amino acid and nucleotide levels respectively. To understand the molecular regulation of this enzyme in phytoplankton, we have measured levels of the RuBPCase large subunit (rbcL) mRNA and DNA, in combination with rates of photosynthetic CO2 fixation, autofluorescent cell counts, and chlorophyll a in natural phytoplankton communities of Tampa Bay (Flonda, USA) and the southeastern Gulf of Mexico. We measured rbcL mRNA, rbcL DNA, and the ratio of rbcL mRNA to rbcL DNA by extracting RNA and DNA and probing the extracts with the Synechococcus PCC 6301 rbcL gene as a probe. Additionally, rbcL mRNA was amplified from certain samples using a reverse transcriptase-linked polymerase chain reaction procedure. In a transect from Tampa Bay seaward, levels of rbcL mRNA decreased 3to 8-fold from the estuarine environment to the offshore environment, and followed similar trends as photosynthetic CO2 fixation, picocyanobacterial counts, and chlorophyll a. In an offshore vertical profile, the subsurface maximum in the rbcL mRNA/DNA ratio coincided with the 60 m maximum in photosynthetic assimilation rates. In a die1 study using 150 1 of offshore water in a deck-top incubator, the rbcL mRNA.DNA ratio was nearly an order of magnitude greater (76 ng mRNA ng-' DNA) during the light period than in the dark (17 ng mRNA ng-' DNA). Likewise, the photosynthetic rate (PB) at constant illun~ination was highest during daylight hours (6.2 pg C pg-' chl a h-') and lowest at night (1.9 pg C pg-' chl a h-'). These results indicate that (1) carbon fixation in oceanic phytoplankton may be transcriptionally regulated at the level of the RuBPCase gene and (2) that the quantitatlon of particular target mRNAs and DNAs is an effective means to study the regulation of conserved gene functions in natural water column microbial populations.

Chloroplast transcription is required to express the nuclear genes rbcS and cab. Plastid DNA copy number is regulated independently

RbcL and rbcS mRNA levels and plastid transcription activity are low in the basal meristematic region of barley primary leaves and increase coordinately during leaf cell development with a similar time course in dark-grown or illuminated seedlings. The capacity of light to cause cab mRNA accumulation shows a similar dependence on leaf cell development. These results indicate that the initial activation of chloroplast gene expression and the expression of some nuclear genes encoding plastid proteins are coupled to leaf cell development. RbcL and rbcS mRNA levels and plastid transcription activity decline in older leaf sections of dark-grown or illuminated barley. The decreases in plastid transcription and rbcS and rbcL mRNA levels in older dark-grown seedlings could be reversed by plant illumination. Therefore, while the initial activation of plastid transcription and accumulation of rbcS mRNA are largely light-independent, these events become light-dependent in older leaves of dark-grown barley. If the initial increase in plastid transcription which occurs early in leaf cell development is prevented by tagetitoxin, a specific inhibitor of the plastid RNA polymerase, rbcS mRNA does not accumulate and cab mRNA accumulation cannot be induced by light. The effect of tagetitoxin is selective because this compound does not inhibit barley leaf growth, or the normal accumulation of nuclear-encoded actin and BN3 transcripts and plastid DNA which occurs during chloroplast development. Furthermore, a barley pigment-deficient mutant, alb-f17, and plants containing photo-oxidized plastids show parallel reductions in plastid transcription activity and levels of rbcS and cab mRNA. This suggests that the activation of plastid transcription during the early stages of chloroplast biogenesis is necessary for the expression of rbcS and cab.

Light-induced transcription of chloroplast genes. psbA transcription is differentially enhanced in illuminated barley.

Transcription and Northern dot blot analyses were used to determine the relationship between light-induced changes in RNA levels and transcription in barley and maize plastids. Overall plastid transcription activity is near maximum and largely light independent in apical sections of 4.5-day-old dark-grown barley seedings. With continued development in the dark, transcription activity in barley plastids declined precipitously, but transcription could be reactivated if plants were illuminated. Illumination of 8-day-old dark-grown barley seedlings increased the transcription of psbA, rbcL, and 16 S rDNA; however, the stimulation of psbA transcription was 2-4-fold more than rbcL or 16 S rDNA. Differential enhancement of psbA transcription occurred when 4.5- or 8-day-old dark-grown seedlings were illuminated, and the influence persisted in plants illuminated for 72 h. Illumination of 8-day-old dark-grown barley caused an increase in psbA and rbcL mRNA levels, but the increase in psbA mRNA was greater than that of rbcL. Similarly, illumination of 9-day-old dark-grown maize increased plastid transcription activity and the levels of psbA and rbcL mRNA. These results indicate that light can modulate overall plastid transcription activity and differentially stimulate the transcription of genes including psbA.