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Engineering bioenergy crops to accumulate coproducts in planta can increase the value of lignocellulosic biomass and enable a sustainable bioeconomy. In this study, we engineered sorghum with a bacterial gene encoding a chorismate pyruvate-lyase (ubiC) to reroute the plastidial pool of chorismate from the shikimate pathway into the valuable compound 4-hydroxybenzoic acid (4-HBA). A gene encoding a feedback-resistant version of 3-deoxy-d-arabino-heptulonate-7-phosphate synthase (aroG) was also introduced in an attempt to increase the carbon flux through the shikimate pathway. At the full maturity and senesced stage, two independent lines that co-express ubiC and aroG produced 1.5 and 1.7 dw% of 4-HBA in biomass, which represents 36- and 40-fold increases compared to the titer measured in wildtype. The two transgenic lines showed no obvious phenotypes, growth defects, nor alteration of cell wall polysaccharide content when cultivated under controlled conditions. In the field, when harvested before grain maturity, transgenic lines contained 0.8 and 1.2 dw% of 4-HBA, which represent economically relevant titers based on recent technoeconomic analysis. Only a slight reduction (11-15%) in biomass yield was observed in transgenics grown under natural environment. This work provides the first metabolic engineering steps toward 4-HBA overproduction in the bioenergy crop sorghum to improve the economics of biorefineries by accumulating a value-added coproduct that can be recovered from biomass and provide an additional revenue stream.

Industrial biotechnology based on yeast fermentation is a promising strategy that can alleviate global warming and climate change. However, Saccharomyces cerevisiae , widely used in bioprocesses, releases a large amount of carbon dioxide (CO 2) during fermentation. This study developed a mixotrophic CO 2 -fixing S. cerevisiae to achieve carbon neutrality and sustainability in bioprocess. A CO 2 -fixation pathway was constructed in a xylose-utilizing S. cerevisiae by heterologous expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) and phosphoribulokinase (PRK). Furthermore, a delta-integration strategy was utilized, and the RuBisCO gene copy number was increased to 10 copies to improve the efficiency of CO 2 -fixation. An additional Cas9-based genome editing was performed to overexpress other CO 2 -fixation related genes. The resulting CO 2 -fixing yeast, SJ03, exhibited the highest RuBisCO activity. During anaerobic xylose fermentation, ethanol concentration was increased by 17% and ethanol yield was increased by 16% compared to the control strain. In addition, CO 2 emissions decreased by 7%. These results suggest that overexpression of the CO 2 -fixation pathway coupled with xylose utilization in S. cerevisiae might reduce CO 2 emission in bioprocesses. [Display omitted] ● A CO 2 -fixation pathway was constructed in a xylose-utilizing S. cerevisiae. ● Delta-integration strategy was undertaken to overexpress the RuBisCO. ● CO 2 -fixation significantly increased ethanol yields and reduced CO 2 emission during fermentation. [ABSTRACT FROM AUTHOR]

An endangered and rare species Aloe pseudorubroviolacea from the plant family Asphodelaceae which is presently recorded as endangered in Saudi Arabia collected from Al-Baha region of Saudi Arabia its GPS Latitude and Longitude coordinates 19.8345, 41.5481. The chloroplast matK and rbcL gene was considered in this study based on molecular identification the size is about 571 and 664 bp respectively. From the sequence analysis the gene matK and rbcL confirm that this species is very much closely related with A. rubroviolacea and also inter related with the species Astroloba rubriflora, Chrysopogon gryllus, Chortolirion angolense shows about 98.7% sequence homology. The partial matK and rbcL gene sequence discriminate Aloe pseudorubroviolacea from the closely related plant species, A. rubroviolacea. The gene sequence of rbcL discriminates the species from Chrysopogon gryllus and Chortolirion angolense, demonstrates the nucleotide variations in 3 different sites (623C/T; 653C/T; 700C/A). This study showed that matK and rbcL sequence region of chloroplast gene used to authenticate the samples of A. pseudorubroviolacea and which provide to help in correct identification and conservation process of this medicinally valuable endangered plant species.

The Earth's crust hosts a subsurface, dark, and oligotrophic biosphere that is poorly understood in terms of the energy supporting its biomass production and impact on food webs at the Earth's surface. Dark oligotrophic volcanic ecosystems (DOVEs) are good environments for investigations of life in the absence of sunlight as they are poor in organics, rich in chemical reactants and well known for chemical exchange with Earth's surface systems. Ice caves near the summit of Mt. Erebus (Antarctica) offer DOVEs in a polar alpine environment that is starved in organics and with oxygenated hydrothermal circulation in highly reducing host rock. We surveyed the microbial communities using PCR, cloning, sequencing and analysis of the small subunit (16S) ribosomal and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO) genes in sediment samples from three different caves, two that are completely dark and one that receives snow-filtered sunlight seasonally. The microbial communities in all three caves are composed primarily of Bacteria and fungi; Archaea were not detected. The bacterial communities from these ice caves display low phylogenetic diversity, but with a remarkable diversity of RubisCO genes including new deeply branching Form I clades, implicating the Calvin-Benson-Bassham (CBB) cycle as a pathway of CO2 fixation. The microbial communities in one of the dark caves, Warren Cave, which has a remarkably low phylogenetic diversity, were analyzed in more detail to gain a possible perspective on the energetic basis of the microbial ecosystem in the cave. Atmospheric carbon (CO2 and CO), including from volcanic emissions, likely supplies carbon and/or some of the energy requirements of chemoautotrophic microbial communities in Warren Cave and probably other Mt. Erebus ice caves. Our work casts a first glimpse at Mt. Erebus ice caves as natural laboratories for exploring carbon, energy and nutrient sources in the subsurface biosphere and the nutritional limits on life.

The CO2 assimilation rate, N content, Rubisco activation state, and protein distribution to Rubisco and Rubisco activase in the leaf were analyzed for four soybean genotypes at two growth stages under field conditions. The CO2 assimilation rate was largely constant except for one genotype during the reproductive stage. Rubisco activation state significantly decreased with the increase of N content during this period in three of four genotypes. When standardized by total soluble protein amount, Rubisco activase amount was constant during this period in three genotypes. The results in the present study showed the deactivation of Rubisco with the N accumulation, which can result in the constant rate of CO2 assimilation during the reproductive stage of soybean. The deactivation of Rubisco was attributed to an unknown mechanism other than to the deficiency of Rubisco activase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO2 assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO2 are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol(ethanol+acetate+pyruvate)/molglucose. However, RuBisCO is known for its low kcat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO2 recycling, where CO2 produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO2 recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO2 recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1–2.3 mol(ethanol+acetate+pyruvate)/molglucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO2 recycling in E. coli.

An endangered and rare species Aloe pseudorubroviolacea from the plant family Asphodelaceae which is presently recorded as endangered in Saudi Arabia collected from Al-Baha region of Saudi Arabia its GPS Latitude and Longitude coordinates 19.8345, 41.5481. The chloroplast matK and rbcL gene was considered in this study based on molecular identification the size is about 571 and 664 bp respectively. From the sequence analysis the gene matK and rbcL confirm that this species is very much closely related with A. rubroviolacea and also inter related with the species Astroloba rubriflora, Chrysopogon gryllus , Chortolirion angolense shows about 98.7% sequence homology. The partial matK and rbcL gene sequence discriminate Aloe pseudorubroviolacea from the closely related plant species, A. rubroviolacea. The gene sequence of rbcL discriminates the species from Chrysopogon gryllus and Chortolirion angolense , demonstrates the nucleotide variations in 3 different sites (623C/T; 653C/T; 700C/A). This study showed that matK and rbcL sequence region of chloroplast gene used to authenticate the samples of A. pseudorubroviolacea and which provide to help in correct identification and conservation process of this medicinally valuable endangered plant species. [ABSTRACT FROM AUTHOR]

The carbon sequestration potential of different crops in their mature period in Northern China was analyzed in order to promote the selection of green species. Carbon dioxide infrared gas detection, three-dimensional fluorescence, fixed carbon enzyme activity analysis and other methods were used to test the carbon sequestration capacity of nine representative mature crops in the Beijing area. Results show that the carbon sequestration capacity of the nine crops was: corn > sorghum > wheat > tomato > cucumber > cabbage > celery > eggplant > pepper. Among them, the carbon sequestration capacity of corn and sorghum which are C4 crops was stronger than that of other crops. Meanwhile, the three-dimensional fluorescence spectra of corn were closer to sorghum, and there were significant differences in the characteristics of the three-dimensional fluorescence spectra of C3 crops. Based on the analysis of the activity of the fixed carbon enzyme—Phosphoenolpyruvate Carboxylase (PEP) carboxylase and Ribulose-1, 5-Bisphosphate (RuBP) Carboxylase of these plants show that the carbon sequestration capacity of C4 crops was much larger than C3 crops because of its higher PEP carboxylase activity. The carbon sequestration capacity of C3 crops was positively correlated with the activity of RuBP carboxylase in addition to cabbage. Therefore, the carbon sequestration capacity of crops can be measured by using the portable carbon dioxide infrared analyzer. [ABSTRACT FROM AUTHOR]

ABSTRACT Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis ( Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: In our previous study, the feasibility of Rubisco-based engineered E. coli (that contains heterologous phosphoribulokinase (PrkA) and Rubisco) for in situ CO2 recycling during the fermentation of pentoses or hexoses was demonstrated. Nevertheless, it is perplexing to see that only roughly 70 % of the carbon fed to the bacterial culture could be accounted for in the standard metabolic products. This low carbon recovery during fermentation occurred even though CO2 emission was effectively reduced by Rubisco-based engineered pathway. Results: In this study, the heterologous expression of form I Rubisco was found to enhance the accumulation of pyruvate in Escherichia coli MZLF [E. coli BL21(DE3) [DELTA]zwf, [DELTA]ldh, [DELTA]frd]. This may be attributed to the enhanced glycolytic reaction supported by the increased biomass and the ethanol/acetate ratio. Besides, it was found that the transcription of arcA (encodes the redox-dependent transcriptional activators ArcA that positively regulates the transcription of pyruvate formate-lyase) was down-regulated in the presence of Rubisco. The enhanced accumulation of pyruvate also occurs when PrkA is co-expressed with Rubisco in E. coli MZLF. Furthermore, E. coli containing Rubisco-based engineered pathway has a distinct profile of the fermentation products, indicating CO2 was converted into fermentation products. By analyzing the ratio of total C-2 (2-carbon fermentation products) to total C-1 (1-carbon fermentation product) of MZLFB (MZLF containing Rubisco-based engineered pathway), it is estimated that 9 % of carbon is directed into Rubisco-based engineered pathway. Conclusions: Here, we report for the first time the complete profile of fermentation products using E. coli MZLF and its derived strains. It has been shown that the expression of Rubisco alone in MZLF enhances the accumulation of pyruvate. By including the contribution of pyruvate accumulation, the perplexing problem of low carbon recovery during fermentation by E. coli containing Rubisco-based engineered pathway has been solved. 9 % of glucose consumption is directed from glycolysis to Rubisco-based engineered pathway in MZLFB. The principle characteristics of mixotroph MZLFB are the high bacterial growth and the low CO2 emission. [ABSTRACT FROM AUTHOR]

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering. [ABSTRACT FROM AUTHOR]

Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 ( GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air-grown ggt1 mutants showed retarded growth and development, that was not observed at high CO2 (3000 μL L−1). When compared to wild-type ( WT) plants, air-grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ ADP and NADP+/ NADPH ratios. When compared to WT plants, their net CO2 assimilation rates (An) were 50% lower and this mirrored decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBis CO) contents. High CO2-grown ggt1 plants transferred to air revealed a rapid decrease of An and photosynthetic electron transfer rate while maintaining a high energetic state. Short-term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBis CO content, activity and activation state were not altered after a short-term transfer while the ATP/ ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBis CO levels were both reduced in ggt1 leaves after a long-term (12 days) acclimation to air from high CO2 when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low An limits nitrogen-assimilation, this decreases leaf RuBis CO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants. [ABSTRACT FROM AUTHOR]

Most plants contain two isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a chloroplast protein that maintains the activity of Rubisco during photosynthesis. The longer (α-) Rca isoform has previously been shown to regulate the activity of Rubisco in response to both the ADP:ATP ratio and redox potential via thioredoxin-f. We have characterized the arrangement of the different spinach (Spinacia oleracea) isoforms in solution, and show how the presence of nucleotides changes the oligomeric state. Although the shorter (ß-) isoform from both tobacco (Nicotiana tabacum) and spinach tend to form a range of oligomers in solution, the size of which are relatively unaffected by the addition of nucleotide, the spinach a-isoform assembles as a hexamer in the presence of adenosine 5'-[γ-thio]triphosphate (ATPγS). These hexamers have significantly higher heat stability, and may play a role in optimizing photosynthesis at higher temperatures. Hexamers were also observed for mixtures of the two isoforms, suggesting that the a-isoform can act as a structural scaffold for hexamer formation by the ß-isoform. Additionally, it is shown that a variant of the tobacco ß-isoform acts in a similar fashion to the a-isoform of spinach, forming thermally stable hexamers in the presence of ATPy S. Both isoforms had similar rates of ATP hydrolysis, suggesting that a propensity for hexamer formation may not necessarily be correlated with activity. Modelling of the hexameric structures suggests that although the N-terminus of Rca forms a highly dynamic, extended structure, the C-terminus is located adjacent to the intersubunit interface. [ABSTRACT FROM AUTHOR]

Although photorespiration is known to be regulated in response to temperature and light conditions, comprehensive systems-level information concerning such responses has not yet been recorded. For this reason, we here describe metabolic, transcriptional, protein, and physiological response to either independently increasing or decreasing light or temperature conditions from the growth conditions of Arabidopsis. The collated data suggest that the response to both treatments is similar and that it is not mediated at the level of transcription but rather by the inherent metabolic capacities of the leaf.Although a considerable increase in our knowledge concerning the importance of metabolic adjustments to unfavorable growth conditions has been recently provided, relatively little is known about the adjustments which occur in response to fluctuation in environmental factors. Evaluating the metabolic adjustments occurring under changing environmental conditions thus offers a good opportunity to increase our current understanding of the crosstalk between the major pathways which are affected by such conditions. To this end, plants growing under normal conditions were transferred to different light and temperature conditions which were anticipated to affect (amongst other processes) the rates of photosynthesis and photorespiration and characterized at the physiological, molecular, and metabolic levels following this transition. Our results revealed similar behavior in response to both treatments and imply a tight connectivity of photorespiration with the major pathways of plant metabolism. They further highlight that the majority of the regulation of these pathways is not mediated at the level of transcription but that leaf metabolism is rather pre-poised to adapt to changes in these input parameters. [ABSTRACT FROM AUTHOR]

Background: Redox-cofactor balancing constrains product yields in anaerobic fermentation processes. This challenge is exemplified by the formation of glycerol as major by-product in yeast-based bioethanol production, which is a direct consequence of the need to reoxidize excess NADH and causes a loss of conversion efficiency. Enabling the use of CO2 as electron acceptor for NADH oxidation in heterotrophic microorganisms would increase product yields in industrial biotechnology. Results: A hitherto unexplored strategy to address this redox challenge is the functional expression in yeast of enzymes from autotrophs, thereby enabling the use of CO2 as electron acceptor for NADH reoxidation. Functional expression of the Calvin cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase (Rubisco) in Saccharomyces cerevisiae led to a 90% reduction of the by-product glycerol and a 10% increase in ethanol production in sugar-limited chemostat cultures on a mixture of glucose and galactose. Co-expression of the Escherichia coli chaperones GroEL and GroES was key to successful expression of CbbM, a form-II Rubisco from the chemolithoautotrophic bacterium Thiobacillus denitrificans in yeast. Conclusions: Our results demonstrate functional expression of Rubisco in a heterotrophic eukaryote and demonstrate how incorporation of CO2 as a co-substrate in metabolic engineering of heterotrophic industrial microorganisms can be used to improve product yields. Rapid advances in molecular biology should allow for rapid insertion of this 4-gene expression cassette in industrial yeast strains to improve production, not only of 1st and 2nd generation ethanol production, but also of other renewable fuels or chemicals. [ABSTRACT FROM AUTHOR]

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for 15N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBis CO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type ( WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (ir LOX3) and phenolamide biosynthesis (ir MYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein ( TSP) and RuBis CO was accompanied by an increase in defense metabolites, ir LOX3 showed a similar, albeit attenuated, pattern, whereas ir MYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBis CO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBis CO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, 15N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBis CO turnover. [ABSTRACT FROM AUTHOR]

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]- x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose- 1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of 'misfire' products of Rubisco. [ABSTRACT FROM AUTHOR]

An indica variety Takanari is known as one of the most productive rice varieties in Japan and consistently produces 20–30% heavier dry matter during ripening than Japanese commercial varieties in the field. The higher rate of photosynthesis of individual leaves during ripening has been recognized in Takanari. By using pot-grown plants under conditions of minimal mutual shading, it was confirmed that the higher rate of leaf photosynthesis is responsible for the higher dry matter production after heading in Takanari as compared with a japonica variety, Koshihikari. The rate of leaf photosynthesis and shoot dry weight became larger in Takanari after the panicle formation and heading stages, respectively, than in Koshihikari. Roots grew rapidly in the panicle formation stage until heading in Takanari compared with Koshihikari. The higher rate of leaf photosynthesis in Takanari resulted not only from the higher content of leaf nitrogen, which was caused by its elevated capacity for nitrogen accumulation, but also from higher stomatal conductance. When measured under light-saturated conditions, stomatal conductance was already decreased due to the reduction in leaf water potential in Koshihikari even under conditions of a relatively small difference in leaf–air vapour pressure difference. In contrast, the higher stomatal conductance was supported by the maintenance of higher leaf water potential through the higher hydraulic conductance in Takanari with the larger area of root surface. However, no increase in root hydraulic conductivity was expected in Takanari. The larger root surface area of Takanari might be a target trait in future rice breeding for increasing dry matter production. [ABSTRACT FROM AUTHOR]

Net photosynthetic rate ( P) measured at elevated CO concentration ( C), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and nitrogen (N) content in rice leaves decreased significantly after exposure to long term C. The reduction in P, Rubisco, and leaf N at C was similar for the last fully expanded leaf blade (LFELB) and expanding leaf blade (ELB). Spatial leaf N content in the ELB was highest in the zone of cell division, sharply declined as cell expansion progressed and gradually increased with cell maturation. Maximum reduction in spatial leaf N and Rubisco content was found at C only within cell expansion and maturation zones. The spatial leaf N content correlated well with the amount of Rubisco synthesized during leaf expansion, suggesting that N deposition into the expanding leaf blade may be the key for Rubisco synthesis and possibly photosynthetic acclimation to C. [ABSTRACT FROM AUTHOR]

The article discusses the water-soluble extract from Kale and its immunostimulatory in Vitro and in Vivo effects. It mentions that the water-soluble fraction of kale has immunoglobulin (Ig) production stimulating activity in human peripheral blood lymphocytes and human hybridoma HB4C5 cells. It notes that the primary active substance in kale is heat-stable protein with a molecular weight higher than 50 kDa.

Using 'Jinyou 3' cucumber seedlings as test materials, this paper studied their photo-synthetic rate (Pn) , Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase ( RCA) activities, and gene expression of Rubisco and RCA under optimal temperature and weak light (WL: 25°C/18 °C , 100 μmol ∙ m-2 ∙ s-1) , suboptimal temperature and weak light (ST+WL: 18°C/12°C , 100 μmol ∙ m-2∙s-1) , and low temperature and weak light (LT+WL: 10 °C/5°C, 100 μmol ∙ m-2 ∙ s-1). Comparing with the control (25 °C/18 °C, 400 μmol ∙ m-2 ∙ s-1) , treatments WL, ST+WL, and LT+WL all led to a remarkable decrease in leaf area and dry matter mass. At initial stage, the Pn, Rubisco activity, rbcL and rbcS expression, RCA activity, and CsRCA expression in the three treatments declined by a big margin; 5-7 days later, these parameters tended to be less changed in treatment WL, ascended slowly in treatment ST+WL, and decreased continuously in treatment LT+WL. These results suggested that the photosynthetic apparatus of test cucumber seedlings could gradually adapt to weak light or suboptimal temperature and weak light. The Rubisco and RCA activities and the gene expression of Rubisco and RCA showed the similar responses to low temperature and weak light as the Pn, suggesting that the decline in Rubisco and RCA activities and gene expression in cucumber seedlings under low temperature and weak light could be the important reason leading to the decrease of Pn. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to quantify the effect of the pollutant, trifluoroacetate (TFA), on growth and photosynthesis of Phaseolus vulgaris (C3) and Zea mays (C4) in order to elucidate the physiological and biochemical basis of its inhibitory action. In whole plant studies, photosynthetic gas exchange, fast phase fluorescence kinetics and Rubisco activity were measured in parallel over a 14-day period in plants cultivated in a water culture system with NaTFA added at concentrations ranging from 0.625 to 160mgl−1. Although initial stimulation of some photosynthetic parameters was observed at low TFA concentrations early on in the experiment, marked inhibition occurred at higher concentrations. In general Z. mays was affected more severely than P. vulgaris showing a large TFA-induced decrease in both apparent carboxylation efficiency (ACE) and in vitro Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) activity. Analysis of photosynthetic gas exchange revealed that besides constraints on mesophyll processes such as Rubisco activity, stomatal limitation also increased with increasing TFA concentration, especially in P. vulgaris. In depth analysis of the fast phase fluorescence transients pointed at TFA-induced uncoupling of the oxygen evolving complex (OEC) and inhibition of electron transport beyond Q a including possible constraints on the reduction of end electron acceptors of photosystem I. [Copyright &y& Elsevier]

Abstract: As a Brassicaceae, Arabidopsis thaliana constitutes an excellent model system to investigate oil biosynthesis in seeds. Extensive tools for the genetic and molecular dissection of this model species are now available. Together with analytical procedures adapted to its tiny seeds, these tools have allowed major advances in isolating and characterising the factors that participate in the metabolic and developmental control of seed filling. Once the biochemical pathways producing storage lipids, namely triacylglycerols, were elucidated, the question of the regulation of this metabolic network has arisen. The coordinated up regulation of genes encoding enzymes of the fatty acid biosynthetic pathway observed at the onset of seed maturation suggests that the pathway may be subjected to a system of global transcriptional regulation. This has been further established by the study of master regulators of the maturation program like LEAFY COTYLEDON2 and the characterisation of the WRINKLED1 transcription factor. These factors have been shown to participate in a regulatory cascade controlling the induction of the genes involved in fatty acid biosynthesis at the onset of the maturation phase. Although much remains to be elucidated, the framework of the regulatory system controlling fatty acid biosynthesis in Arabidopsis seeds is coming into focus. [Copyright &y& Elsevier]

Abstract: The present study investigated the possible mediatory role of salicylic acid (SA) in protecting plants from cadmium (Cd) toxicity. The exposure of pea plants to increasing Cd concentrations (0.5, 1.0, 2.0 and 5.0μM) during early stages of their establishment, caused a gradual decrease in shoot and root fresh weight accumulation, the rate of CO2 fixation and the activity of ribulose-1,5-bisphosphate carboxylase (RuBPC, E.C. 4.1.1.39), the effect being most expressed at higher Cd concentrations. In vivo the excess of Cd-induced alterations in the redox cycling of oxygen-evolving centers and the assimilatory capacity of the pea leaves as revealed by changes in thermoluminescence emission after flash illumination. The levels of some important parameters associated with oxidative stress, namely lipid peroxidation, electrolyte leakage and proline production were increased. Seed pretreatment with SA alleviated the negative effect of Cd on growth, photosynthesis, carboxylation reactions, thermoluminescence characteristics and chlorophyll content, and led to decrease in oxidative injuries caused by Cd. The data suggest that the beneficial effect of SA during an earlier growth period could be related to avoidance of cumulative damage upon exposure to cadmium thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity. In addition, the observed high endogenous levels of SA after treatment with Cd suggests that SA may act directly as an antioxidant to scavenge the reactive oxygen species and/or indirectly modulate redox balance through activation of antioxidant responses. Taken together these evidences could explain at some extend the protective role of SA on photochemical activity of chloroplast membranes and photosynthetic carboxylation reactions in Cd-stressed pea plants. [Copyright &y& Elsevier]

Summary: Photosynthetic responses to increasing temperatures play important roles in regulating heat tolerance. The objectives of this study were to determine photosynthetic acclimation to increasing temperatures for creeping bentgrass (Agrostis stolonifera L.) and to examine changes in major photosynthetic components (photosynthetic pigments, photochemical efficiency, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activity, and activation state of rubisco) involved in heat responses of photosynthesis. ‘Penncross’ was exposed to 20, 25, 30, and 35°C for 7d at each temperature (acclimated) before being exposed to 40°C for 28d or directly exposed to 40°C for 28d from 20°C (non-acclimated) in growth chambers. Leaf net photosynthetic rate (Pn), photochemical efficiency, rubisco activity, rubisco activation state, chlorophyll content, and carotenoid content decreased when grasses were subjected to severe heat stress at 40°C for 28d. The declines in rubisco activity and activation state were most dramatic among different photosynthetic components examined in this study. Heat-acclimated plants were able to maintain significantly higher Pn, the content of chlorophyll and carotenoid, and the level of rubisco activity and activation state during subsequent exposure to severe heat stress, compared to non-acclimated plants. These results suggested that photosynthetic acclimation to increasing temperatures contributed to creeping bentgrass tolerance to severe heat stress, which was associated with the maintenance of both higher light-harvesting capacity and carbon fixation activity during heat stress. [Copyright &y& Elsevier]

Abstract: Glyphosate is one of the most widely used herbicides in cereal-growing regions worldwide. In the present work, the protein expression profile of rice leaves exposed to glyphosate was analyzed in order to investigate the alternative effects of glyphosate on plants. Two-week-old rice leaves were subjected to glyphosate or a reactive oxygen species (ROS) inducing herbicide paraquat, and total soluble proteins were extracted and analyzed by two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. A total of 25 differentially expressed proteins were identified from the glyphosate treated sample, wherein 18 proteins were up-regulated and 7 proteins were down-regulated. These proteins had shown a parallel expression pattern in response to paraquat. Results from the 2-DE analysis, combined with immunoblotting, clearly revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit was significantly decreased by the treatment of both herbicides. An increased accumulation of antioxidant enzymes including ascorbate peroxidase, glutathione S-transferase, thioredoxin h-type, nucleoside diphosphate kinase 1, peroxiredoxin and a superoxide dismutase [Cu–Zn] chloroplast precursor in the glyphosate-treated sample suggests that a glyphosate treatment possibly generates oxidative stress in plants. Moreover, a gene expression analysis of five antioxidant enzymes by Northern blot confirmed their mRNA levels in the rice leaves. A histo-cytochemical investigation with DAB (3,3-diaminobenzidine) to localize H2O2 and increases of the thiobarbituric acid reactive substances (TBARS) concentration revealed that the glyphosate application generates ROS, which resulted in the peroxidation and destruction of lipids in the rice leaves. [Copyright &y& Elsevier]

Abstract: Limonium bicolor, a halophytic species of Plumbaginaceae, can thrive in saline or saline-alkali (sodic) soil, demonstrating that it has developed an efficient saline-alkali resistance system, and is an ideal material for the study of saline-alkali tolerance. In order to identify and characterize the complexity of this adaptation, expressed sequence tags (ESTs) analysis and real-time reverse transcriptase–polymerase chain reaction (RT–PCR) were conducted. We constructed a cDNA library of L. bicolor exposed to 0.4M NaHCO3 for 48h, and obtained 2358 ESTs, representing 1735 unique genes. A BLASTX search revealed that 1393 ESTs, representing 873 unique genes, showed significant similarity (E-values <10−4) to protein sequences in the non-redundant database. These ESTs were further grouped into 12 functional categories according to their functional annotation. The most abundant categories were metabolism (18.74%), photosynthesis (14.86%), unknown classification (12.20%), defense (12.20%), and transport facilitation (10.19%). In total, 286 putative abiotic stress related transcripts, representing 121 unique genes, were identified. Among them, the two most abundant genes encoded metallothionein (EH794553) and lipid transfer protein (EH794695), each of which accounted for 1.4% of the total ESTs. The expression of 18 putative stress-related genes were further analyzed in roots and leaves of L. bicolor using real-time RT–PCR, and 14 genes were differentially expressed by more than 2-fold as a result of the NaHCO3 stress. The results of this study may contribute to our understanding of the molecular mechanism of saline-alkali tolerance in L. bicolor. [Copyright &y& Elsevier]

Summary: Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphophate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level. [Copyright &y& Elsevier]

Abstract: The in vitro protein import experiment is one of the most important techniques for determining protein localization. For chloroplastic proteins, proteins of interest are incubated with isolated chloroplasts in the presence of energy sources. Radio-labeled proteins synthesized either in vitro or in vivo have been widely used as substrate proteins. Here we report our development of the protein import assay system in which non-radio-labeled proteins, overexpressed in Escherichia coli, were applied. In this system, substrate proteins were designed to carry epitope-tags, thus allowing analysis of imported proteins by SDS-PAGE, followed by immunoblotting to detect these tags. Furthermore, the imported proteins were found to be incorporated into their native form. These observations indicated that recombinant proteins were imported into chloroplasts and folded correctly. Therefore, this assay system could represent another valuable tool for determining protein localization. [Copyright &y& Elsevier]

Abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO2 into the biosphere. At the same time Rubisco is an extremely inefficient catalyst and its carboxylase activity is compromised by an opposing oxygenase activity involving atmospheric O2. The shortcomings of Rubisco have implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. This review uses the information provided in these structures in a structure-based sequence alignment and discusses Rubisco function in the context of structural variations at all levels – amino acid sequence, fold, tertiary and quaternary structure – with an evolutionary perspective and an emphasis on the structural features of the enzyme that may determine its function as a carboxylase. [Copyright &y& Elsevier]

Abstract: To identify the effects of auxin on rice root formation, proteins induced by exogenous addition of auxin to rice seedlings were analyzed by a proteomic approach. Root formation by rice seedlings was promoted by 0.45μM 2,4-dichlorophenoxyacetic acid (2,4-D) and repressed by 60μM p-chlorophenoxyisobutyric acid (PCIB). Proteins extracted from the basal part of leaf sheaths of rice seedlings treated with 2,4-D or PCIB for 48h were labeled with Cy3 and Cy5, and separated by two-dimensional polyacrylamide gel electrophoresis. Out of nine proteins up-regulated by 2,4-D and down-regulated by PCIB, five proteins showing significant difference in abundance were used for expression analysis at the transcript abundance level. Transcript abundance of the mitochondrial complex I subunit slightly increased with 2,4-D treatment and were repressed by PCIB. The transcript abundance of EF-1β′, myosin heavy chain and mitochondrial [Mn]SOD increased with 2,4-D treatment but did not decrease with PCIB. The transcript abundance of aldehyde dehydrogenase was not effected by 2,4-D or PCIB. These results indicate that mitochondrial complex I subunit is part of the downstream signal cascade of PCIB, whereas myosin heavy chain, mitochondrial [Mn]SOD and EF-1β′ are involved in the 2,4-D signal cascade but are probably upstream of PCIB. [Copyright &y& Elsevier]

• Here, the kinetic properties and immunolocalization of phospho enolpyruvate carboxylase (PEPC) and ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. • The activity, optimal pH andl-malate sensitivity of PEPC, and the Michaelis-Menten constant ( Km) for phospho enolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. • Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mm and a IC50 forl-malate (thel-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mm. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. • This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway). [ABSTRACT FROM AUTHOR]

To elucidate early drought responses in needles of Norway spruce (Picea abies (Karst.) L.), we subjected 1-year-old seedlings to gradual desiccation for 6 weeks. Four weeks of drought treatment caused a small but significant decrease in photosystem II quantum yield of light-adapted needles (Φa) compared with that of well-watered controls. Six weeks of drought treatment reduced Φa and the photosystem II quantum yield of dark-adapted needles (Φ) by 50 and 8%, respectively, and reduced shoot water potential by 0.7 MPa, but had no measurable effect on needle relative water content. After two weeks of drought treatment, and before there was a discernible effect of drought on Φ or a statistically significant effect on shoot water potential, needles were analyzed for changes in protein composition. Five out of several hundred detected proteins in needles of drought-treated plants showed consistent changes compared with control leaves. The proteins were identified by LC-MS/MS as components of the oxygen-evolving complex (oxygen evolving enhancer protein 2), ribulose-1,5-bisphosphate carboxylase/oxgenase large subunit, and one protein of unknown function, whose mRNA was found in a previous screen of wound- and methyl-jasmonate-induced bark proteins. [ABSTRACT FROM AUTHOR]

Abstract: To evaluate the effects of low atmospheric pressure on leaf photosynthesis, we compared the photosynthesis of identical leaves of Fagus crenata at lowland (0m a.s.l.) and at highland (2360m a.s.l.). At the high altitude, the atmospheric pressure and partial pressure of CO2 at intercellular air spaces in the leaf (C i360) decreased to 77% and 78% of those at the low altitude, respectively. On the other hand, the efficiency of photosynthetic CO2-utilization was apparently higher at the high altitude because of a mitigation of the O2-inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) under low ambient partial pressure of O2. This stimulation of photosynthesis partly compensated a photosynthetic depression due to the low C i360, and the net assimilation rate (A n360) at the high altitude retained 94% of the value at the low altitude. A theoretical model indicated that the stimulation of photosynthesis at high altitudes depend on internal conductance (g i) and/or on Rubisco content. The model demonstrated that low atmospheric pressure at high altitudes caused severe restrictions of photosynthesis when leaves had a small g i and/or a large amount of Rubisco, whichever are repeatedly reported in alpine plants. [Copyright &y& Elsevier]

The distributions of bacterial form IA and form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were investigated using Lowes Cove intertidal mudflat and Damariscotta Lake littoral sediments by PCR amplification of 492–495 bp fragments of the large subunit RuBisCO gene, cbbL. Genomic extracts for amplification were obtained from lake surface (upper 2 mm), mudflat surface (upper 2 mm), subsurface (5–7 cm), and soft-shell clam ( Mya arenaria) burrow-wall sediments, as well as from a sulfide-oxidizing mat. Phylogenetic analyses of cbbL clone libraries revealed that Lowes Cove sediments were dominated by form IA cbbL-containing sequences most closely related to cbbL genes of sulfur-oxidizing bacteria or sulfide-oxidizing mats. In contrast, Damariscotta Lake cbbL clones contained primarily form IC cbbL sequences, which typify aerobic CO- and hydrogen-oxidizing facultative chemolithotrophs. Statistical analyses supported clear differentiation of intertidal and lake chemolithotroph communities, and provided evidence for some differentiation among intertidal communities.amova andlibshuff analyses of Lowes Cove libraries suggested that M. arenaria burrow-wall sediments did not harbour distinct communities compared with surface and subsurface sediments, but that surface and subsurface libraries displayed moderate differences. The results collectively support a conceptual model in which the relative distribution of form IA- and IC-containing bacterial chemolithotrophs depends on sulfide availability, which could reflect the role of sulfate reduction in sediment organic matter metabolism, or the presence of geothermal sulfide sources. [ABSTRACT FROM AUTHOR]

We examined the effects of leaf age and mutual shading on the morphology, photosynthetic properties and nitrogen (N) allocation of foliage of an evergreen understory shrub, Daphniphyllum humile Maxim, growing along a natural light gradient in a deciduous Fagus crenata-dominated forest in Japan. Seedlings in high-light environments were subject to greater mutual shading and 1-year-old foliage survival was lower than in seedlings in low-light environments, indicating that the survival rates of foliage were related to the degree of mutual shading. Although specific leaf area (SLA) in current and 1-year-old foliage was curvilinearly related to daily photosynthetic photon flux (PPF), SLA was unaffected by leaf age, indicating that foliage in D. humile may not acclimate morphologically to annual changes in light caused by mutual shading. Light-saturated net photosynthetic rates (Pmax) were correlated with daily PPF in current-year foliage. In addition, a strong, positive relationship was found between nitrogen concentration per unit leaf area and Pmax. In contrast, the relationship among PPF, N and photosynthetic parameters in 1-year old foliage was weak because of the strong remobilization of N from older leaves to current-year foliage in plants growing in high light. However, the relationship between daily PPF and both photosynthetic N-use efficiency and the ratio of maximum electron transport rate to maximum carboxylation rate did not differ between current-year and 1-year-old foliage, suggesting that these responses help maintain a high photosynthetic efficiency even in older foliage. We conclude that D. humile maximizes whole-plant carbon gain by maintaining a balance among photosynthetic functions across wide ranges of leaf ages and light environments. [ABSTRACT FROM AUTHOR]

To identify the leaf characteristics associated with the difference in altitudinal distribution of Polygonaceae plants, we investigated the seasonal changes in leaf characteristics of co-occurring Aconogonum weyrichii and Reynoutria japonica at 2250 m a.s.l. on Mt. Fuji. Aconogonum weyrichii is distributed in the alpine region and R. japonica from the lowlands to the highlands. At the beginning of the growing period, leaves of A. weyrichii had larger amounts of nitrogen, Rubisco and higher photosynthetic capacity than R. japonica. The relationship between the amounts of nitrogen and Rubisco did not significantly differ between the species, but a higher Rubisco activity per unit Rubisco protein content was found in A. weyrichii, and was thought to be responsible for the high photosynthetic capacity of this species in summer. Activity of ascorbate peroxidase, a key enzyme of the hydrogen peroxide scavenging system against oxidative stress under chilling and light conditions, increased significantly in R. japonica late in the growing period, but did not significantly change in A. weyrichii. In A. weyrichii, rapid maturation of leaves and a high photosynthetic capacity are thought to be adaptive features for the short growing season in alpine regions. On the other hand, the slower accumulation of nitrogen and Rubisco during the early stage of the growing period in R. japonica resulted in insufficient utilization of the growing period for photosynthetic production and is thought to be one of the factors restricting the upper limit of its distribution. [ABSTRACT FROM AUTHOR]

Abstract: In the green alga Chlamydomonas reinhardtii, an L290F substitution in the chloroplast-encoded large-subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) causes decreases in carboxylation V max, CO2/O2 specificity, and thermal stability. Analysis of photosynthesis-competent revertants selected at the 35°C restrictive temperature identified a rare C65S suppressor substitution in the nuclear-encoded small subunit. C65S enhances catalysis and CO2/O2 specificity in the absence of other wild-type small subunits, and restores thermal stability in vivo. C65S, C65A, and C65P mutant strains were created. C65S and C65A enzymes have normal catalysis, but C65P Rubisco, which contains land-plant Pro, has decreases in carboxylation V max/K m and CO2/O2 specificity. In contrast to other small-subunit substitutions that affect specificity, Cys-65 contacts the large subunit, and the C65P substitution does not cause a decrease in holoenzyme thermal stability in vivo or in vitro. Further analysis of the C65P protein may identify structural alterations that influence catalysis separate from those that affect stability. [Copyright &y& Elsevier]

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae ( Oscillochloris trichoides DG6T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA ( rrs), ribulose-1,5-bisphosphate carboxylase/oxygenase ( cbbL), and nitrogenase ( nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed a lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the “red-like” form I RuBisCO were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated a lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their allocation as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes. [ABSTRACT FROM AUTHOR]

Abstract: We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO2 fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO2 fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO2 fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO2 also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO2 fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein. [Copyright &y& Elsevier]

Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of the plastid ribulose-1,5-bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR-RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR-RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR-RFLP analyses suggested that two strains, C-32 and 90-02, were cultivated P. tenera and that the other five strains, C-24, C-28, C-29, C-30 and M-1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C-32 and 90-02 were further studied by sequencing their RuBisCo spacer and ITS-1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS-1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C-32 and 90-02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR-RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra. [ABSTRACT FROM AUTHOR]

Abstract: The short and long-term responses of flag leaf stomatal conductance (g s) and rate of photosynthesis (An) to elevated CO2 (757μmolmol−1), 4°C warmer temperatures and N supply were investigated in spring wheat (Triticum aestivum L. cv. Alcalá) crops grown in two seasons in field conditions under temperature gradient tunnels in a Mediterranean environment. Plants grown at elevated CO2 had lower g s and An measured at 700μmolmol−1 CO2 than ambient CO2-grown plants, indicating acclimatory responses to elevated CO2. N supply, which was lower in 2003 than 2002, reduced this acclimation in both years. Warmer temperatures at high nitrogen supply also decreased An acclimation. Acclimatory responses of An and g s were highly correlated. The analysis of the g s–An relationship indicated that both parameters are probably linked and respond in parallel to elevated CO2 at ambient temperatures, but non-parallel responses were observed at above-ambient temperatures. [Copyright &y& Elsevier]

Abstract: A poplar plantation has been exposed to an elevated CO2 concentration for 5 years using the free air CO2 enrichment (FACE) technique. Even after such a long period of exposure, leaves of Populus x euramericana have not shown clear signs of photosynthetic acclimation. Only at the end of the growing season for shade leaves was a decrease of maximum velocity of carboxylation (V cmax) observed. Maximum electron transport rate (J max) was increased by FACE treatment in July. Assimilation rates at CO2 partial pressure of 400 (A 400) and 600 (A 600) μmol mol−1 were not significantly different under FACE treatment. Most notably FACE significantly decreased stomatal conductance (g s) both on upper and lower canopy leaves. N fertilization increased N content in the leaves on mass basis (N m) and specific leaf area (SLA) in both CO2 treatments but did not influence the photosynthetic parameters. These data show that in poplar plantations the long-term effects of elevated CO2 on photosynthesis do not differ considerably from the short-term ones even with N deposition. [Copyright &y& Elsevier]