Rubisco Large Subunit Gene Research Articles

Composition and temporal dynamics of the phytoplankton community in Laizhou Bay revealed by microscopic observation and rbcL gene sequencing

Laizhou Bay, a major breeding ground for economic marine organisms in the northern waters of China, is facing rapid environmental degradation. In this study, field surveys in this area were conducted in the spring, summer, and autumn of 2020. Microscopic observation and RuBisCO large subunit (rbcL) gene analysis were employed to understand the community structure and temporal dynamics of phytoplankton. The phytoplankton community structures detected by the two methods showed significant differences. Microscopic observation revealed the dominance of dinoflagellates in spring that shifted to the dominance of diatoms in summer and autumn. However, rbcL gene sequencing consistently identified diatoms as dominant throughout all three seasons, with their relative abundance showing an increasing trend. Conversely, the relative abundance of the second- and third-most abundant taxa, namely, haptophytes and ochrophytes, decreased as the seasons transitioned. rbcL gene sequencing annotated more species than microscopy. It could detect haptophytes and cryptophytes, which were overlooked by microscopy. In addition, rbcL gene sequencing detected a remarkable amount of Thalassiosira profunda, which was previously unidentified in this sea area. However, it appeared to underestimate the contribution of dinoflagellates considerably, with most taxa being only identified through microscopic identification. The two methods jointly identified 28 harmful algal bloom taxa with similar detection quantities but substantial differences in species composition. Phytoplankton communities were influenced by temperature, salinity, and nutrients. The results of this work suggest that a combination of multiple techniques is necessary for a comprehensive understanding of phytoplankton.

Screening for genetic variability in photosynthetic regulation provides insights into salt performance traits in forage sorghum under salt stress

BackgroundSorghum (Sorghum bicolor) is a promising opportunity crop for arid regions of Africa due to its high tolerance to drought and heat stresses. Screening for genetic variability in photosynthetic regulation under salt stress can help to identify target trait combinations essential for sorghum genetic improvement. The primary objective of this study was to identify reliable indicators of photosynthetic performance under salt stress for forage yield within a panel of 18 sorghum varieties from stage 1 (leaf 3) to stage 7 (late flowering to early silage maturity). We dissected the genetic diversity and variability in five stress-sensitive photosynthetic parameters: nonphotochemical chlorophyll fluorescence quenching (NPQ), the electron transport rate (ETR), the maximum potential quantum efficiency of photosystem II (FV/FM), the CO2 assimilation rate (A), and the photosynthetic performance based on absorption (PIABS). Further, we investigated potential genes for target phenotypes using a combined approach of bioinformatics, transcriptional analysis, and homologous overexpression.ResultsThe panel revealed polymorphism, two admixed subpopulations, and significant molecular variability between and within population. During the investigated development stages, the PIABS varied dramatically and consistently amongst varieties. Under higher saline conditions, PIABS also showed a significant positive connection with A and dry matter gain. Because PIABS is a measure of plants’ overall photosynthetic performance, it was applied to predict the salinity performance index (SPI). The SPI correlated positively with dry matter gain, demonstrating that PIABS could be used as a reliable salt stress performance marker for forage sorghum. Eight rubisco large subunit genes were identified in-silico and validated using qPCR with variable expression across the varieties under saline conditions. Overexpression of Rubisco Large Subunit 8 increased PIABS, altered the OJIP, and growth with an insignificant effect on A.ConclusionsThese findings provide insights into strategies for enhancing the photosynthetic performance of sorghum under saline conditions for improved photosynthetic performance and potential dry matter yield. The integration of molecular approaches, guided by the identified genetic variability, holds promise for genetically breeding sorghum tailored to thrive in arid and saline environments, contributing to sustainable agricultural practices.

Effects of Cu2O nanoparticles on the kinetic characteristics of rapid chlorophyll fluorescence induction and related genes in wheat seedlings.

Metal nanoparticles could be accumulated in soils, which threatens the ecological stability of crops. Investigating the effects of cuprous oxide nanoparticles (Cu2O-NPs) on photosystem Ⅱ (PSⅡ) of wheat seedling leaves holds considerable importance in comprehending the implications of Cu2O-NPs on crop photosynthesis. Following the hydroponic method, we investigated the effects of 0, 10, 50, 100, and 200 mg·L-1 Cu2O-NPs on chlorophyll fluorescence induction kinetics and photosynthetic-related genes in wheat seedlings of "Zhoumai 18". The results showed that, with the increases of Cu2O-NPs concentrations, chlorophyll contents in wheat leaves decreased, and the standardization of the OJIP curve showed a clearly K-phase (ΔK＞0). Cu2O-NPs stress increased the parameters of active PSⅡ reaction centers, including the absorption flux per active RC (ABS/RC), the trapping flux per active RC (TRo/RC), the electron transport flux per active RC (ETo/RC), and the dissipation flux per active RC (DIo/RC). Cu2O-NPs stress decreased the parameters of PSⅡ energy distribution ratio including the maximum quantum yield of PSⅡ (φPo), the quantum yield of electron transport from QA (φEo), and the probability that a trapped exciton moved an electron further than QA (Ψo), while increased the quantum ratio for heat dissipation (φDo). Moreover, there was a decrease in photosynthetic quantum yield Y(Ⅱ), photochemical quenching coefficient (qP), net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of leaves with the increases of Cu2O-NPs concentration. Under Cu2O-NPs stress, the expression levels of genes which included PSⅡ genes (PsbD, PsbP, Lhcb1), Rubisco large subunit genes (RbcL), cytochrome b6/f complex genes (PetD, Rieske), and ATP synthase genes (AtpA, AtpB, AtpE, AtpI) were downregulated. These results indicated that Cu2O-NPs stress altered the activity and structure of PSⅡ in wheat seedlings, affected the activity of PSⅡ reaction centers, performance parameters of PSⅡ donor and acceptor sides. PSⅡ related genes were downregulated and exhibited significant concentration effects.

A rapid field-ready electrical biosensor consisting of bismuthine-derived Au island decorated BiOCl nanosheets for Raphidiopsis raciborskii detection in freshwater.

Cyanobacteria play an essential role in nutrient cycling in aquatic ecosystems. However, certain species adversely affect the environment and human health by causing harmful cyanobacterial algal blooms (cyanoHABs) and producing cyanotoxins. To address this issue, continuous cyanoHAB monitoring has been considered; however, a gold standard has not yet been established. In this study, we aimed to develop a dual DNA-targeting capacitive-type biosensor for rapid field-ready monitoring of Raphidiopsis raciborskii, a causative species of cyanoHAB. To enhance the sensing signal, a plate-like Au-BiOCl nanocomposite was synthesized using a spontaneous carbonation process without additional additives. The alternating-current electrothermal flow (ACEF) technique was applied to enable rapid DNA and probe binding within 10 min. The limits of detection (LODs) for R. raciborskii RubisCO large subunit (rbcL) and RNA polymerase beta subunit (rpoB) genes diluted in deionized (DI) water were 4.89 × 10-17 and 3.89 × 10-17 M, respectively. Furthermore, the LODs of R. raciborskii rbcl and rpoB diluted in freshwater containing HAB were 2.55 × 10-16 and 3.84 × 10-16 M, respectively, demonstrating the field-ready applicability of the device. The fabricated cyanobacterial DNA-sensing platform enabled powerful species-specific detection using a small sample volume and low target concentration without a nucleic acid amplification step, dramatically reducing the detection time. This study has considerable implications for detecting HABs, early warning systems, and species-specific environmental monitoring technology.

Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Effect of Enhanced Ultraviolet-B Radiation on Fruit Maturity and Quality and Leaf Photosynthesis in ‘Guifei’ Mango

To investigate the characteristics of photosynthetic physiological changes in leaves of Mangifera indica L. cv. Guifei under enhanced ultraviolet (UV)-B radiation, natural light-exposed trees were used as controls and 96 kJ·m−2·d−1 enhanced UV-B radiation was artificially simulated in the field. The changes in fruit maturity and quality, the leaf net photosynthetic rate (Pn), photosynthetic pigment contents, photochemical reactions, the activities of photosynthetic enzymes and related gene expression levels were determined. Compared with the control, the percentage of mature fruits under the treatment significantly increased, and fruit quality improved. The net photosynthetic rate (Pn), photosynthetic pigment content, Hill reaction activity, and photochemical quenching coefficient (qP) of the treated leaves showed significantly higher values than those of the control leaves. The activities of Rubisco and Rubisco activating enzyme (RCA) and the expression levels of the Rubisco large subunit and Rubisco small subunit were significantly increased. Treatment with 96 kJ·m−2·d−1 enhanced ultraviolet-B radiation improved Rubisco activity by increasing the expression of the Rubisco large and small subunit genes, thereby enhancing the CO2-fixing capacity and dark reaction capacity of leaves. Thus, the net photosynthetic rate of leaves increased, which promoted the early maturity of ‘Guifei’ mango by the rapid accumulation of photosynthetic products.

Improving photosynthesis through the enhancement of Rubisco carboxylation capacity.

Rising human population, along with the reduction in arable land and the impacts of global change, sets out the need for continuously improving agricultural resource use efficiency and crop yield (CY). Bioengineering approaches for photosynthesis optimization have largely demonstrated the potential for enhancing CY. This review is focused on the improvement of Rubisco functioning, which catalyzes the rate-limiting step of CO2 fixation required for plant growth, but also catalyzes the ribulose-bisphosphate oxygenation initiating the carbon and energy wasteful photorespiration pathway. Rubisco carboxylation capacity can be enhanced by engineering the Rubisco large and/or small subunit genes to improve its catalytic traits, or by engineering the mechanisms that provide enhanced Rubisco expression, activation and/or elevated [CO2] around the active sites to favor carboxylation over oxygenation. Recent advances have been made in the expression, assembly and activation of foreign (either natural or mutant) faster and/or more CO2-specific Rubisco versions. Some components of CO2 concentrating mechanisms (CCMs) from bacteria, algae and C4 plants has been successfully expressed in tobacco and rice. Still, none of the transformed plant lines expressing foreign Rubisco versions and/or simplified CCM components were able to grow faster than wild type plants under present atmospheric [CO2] and optimum conditions. However, the results obtained up to date suggest that it might be achievable in the near future. In addition, photosynthetic and yield improvements have already been observed when manipulating Rubisco quantity and activation degree in crops. Therefore, engineering Rubisco carboxylation capacity continues being a promising target for the improvement in photosynthesis and yield.

First Molecular Record and Distribution of Padina pavonica (Linnaeus) Thivy from the Southern Black Sea

Padina is a brown algal genus widely distributed in warm-temperate and tropical regions. Species-level identification of specimens is often difficult due to intraspecific and interspecific morphological plasticity. This study reveals the results of the phylogenetic relationships of specimens, by analyzing the cytochrome oxidase subunit 3 (Cox3) and RuBisCO large subunit (rbcL) gene sequences. A total of 144 new sequences were analyzed together with other Padina sequences obtained from GenBank. Phylogenetic inference identified one well-resolved clade. All of these analyses showed that this genus has only one species in the Black Sea. This is the first study on the molecular diagnosis of macroalgae in the Black Sea, and it contains the first molecular records of Padina in the Black Sea Region.

Rubisco production in maize mesophyll cells through ectopic expression of subunits and chaperones.

C4 plants, such as maize, strictly compartmentalize Rubisco to bundle sheath chloroplasts. The molecular basis for the restriction of Rubisco from the more abundant mesophyll chloroplasts is not fully understood. Mesophyll chloroplasts transcribe the Rubisco large subunit gene and, when normally quiescent transcription of the nuclear Rubisco small subunit gene family is overcome by ectopic expression, mesophyll chloroplasts still do not accumulate measurable Rubisco. Here we show that a combination of five ubiquitin promoter-driven nuclear transgenes expressed in maize leads to mesophyll accumulation of assembled Rubisco. These encode the Rubisco large and small subunits, Rubisco assembly factors 1 and 2, and the assembly factor Bundle sheath defective 2. In these plants, Rubisco large subunit accumulates in mesophyll cells, and appears to be assembled into a holoenzyme capable of binding the substrate analog CABP (carboxyarabinitol bisphosphate). Isotope discrimination assays suggest, however, that mesophyll Rubisco is not participating in carbon assimilation in these plants, most probably due to a lack of the substrate ribulose 1,5-bisphosphate and/or Rubisco activase. Overall, this work defines a minimal set of Rubisco assembly factors in planta and may help lead to methods of regulating the C4 pathway.

The relative abundance of wheat Rubisco activase isoforms is post-transcriptionally regulated

Diurnal rhythms and light availability affect transcription–translation feedback loops that regulate the synthesis of photosynthetic proteins. The CO2-fixing enzyme Rubisco is the most abundant protein in the leaves of major crop species and its activity depends on interaction with the molecular chaperone Rubisco activase (Rca). In Triticum aestivum L. (wheat), three Rca isoforms are present that differ in their regulatory properties. Here, we tested the hypothesis that the relative abundance of the redox-sensitive and redox-insensitive Rca isoforms could be differentially regulated throughout light–dark diel cycle in wheat. While TaRca1-β expression was consistently negligible throughout the day, transcript levels of both TaRca2-β and TaRca2-α were higher and increased at the start of the day, with peak levels occurring at the middle of the photoperiod. Abundance of TaRca-β protein was maximal 1.5 h after the peak in TaRca2-β expression, but the abundance of TaRca-α remained constant during the entire photoperiod. The redox-sensitive TaRca-α isoform was less abundant, representing 85% of the redox-insensitive TaRca-β at the transcript level and 12.5% at the protein level. Expression of Rubisco large and small subunit genes did not show a consistent pattern throughout the diel cycle, but the abundance of Rubisco decreased by up to 20% during the dark period in fully expanded wheat leaves. These results, combined with a lack of correlation between transcript and protein abundance for both Rca isoforms and Rubisco throughout the entire diel cycle, suggest that the abundance of these photosynthetic enzymes is post-transcriptionally regulated.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

Rubisco Engineering by Plastid Transformation and Protocols for Assessing Expression.

The assimilation of CO2 within chloroplasts is catalyzed by the bifunctional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multigene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master line and analyzing leaf Rubisco content.

Variation in the photosynthetic activities of the dinoflagellate Akashiwo sanguinea during formation of resting cysts

Resting cysts represent the dormant life stage produced by sexual reproduction of dinoflagellates; it generally maintain a limited level of metabolic activities. In the present study, changes in the photosynthetic apparatus of Akashiwo sanguinea during the encystment process in different nitrogen (N) conditions were characterized, and the transcriptional responses of nine photosynthesis genes were further evaluated. The photochemical efficiency decreased with elevation of N deprivation and growth of algae, and the resting cysts maintained limited photosynthetic activity. Meanwhile, the transcription of photosystem II genes (psbA and psbD) were down-regulated, especially the levels of the psbA gene, suggesting the inactivation of photosystem II under N limitation. Moreover, genes involved in cytochrome f (petA) and ATP synthase (atpA, atpB) were also transcribed at low levels in N-deprivation cultures. In contrast, the transcription of the RubisCO large subunit gene (rbcL) was up-regulated, indicating the maintenance of carbon fixation in N-limited conditions. The transcription levels of photosystem I genes (psaA and psaB) showed no significant correlation with the N condition. Further, most of the photosynthesis genes were down-regulated in the resting cysts, which was coincided with the inactive physiological status of the dormant stage. The results demonstrated that photosynthesis genes are differentially regulated in response to varying N conditions and life cycles. The findings will provide fundamental knowledge for better understanding the physiological characteristics and molecular regulation mechanisms in the developmental life cycle of marine dinoflagellates.

Effects of overexpression of the Rubisco small subunit gene under the control of the Rubisco activase promoter on Rubisco contents of rice leaves at different positions

ABSTRACT Photosynthetic activity is expected to be maintained during leaf senescence if the amount of Rubisco is maintained. As the mRNA level of the Rubisco gene decreases rapidly just before the stage of full expansion, its overexpression after full expansion would be effective. We previously found that the mRNA level of the Rubisco activase gene (RCA) is maintained during leaf senescence. In the present study, the Rubisco small subunit gene (RBCS) was overexpressed under the control of the promoter of RCA in rice. Although Rubisco content increased in the uppermost, fully expanded leaves of transgenic plants in comparison with that in wild-type plants, in the penultimate leaves it decreased to a level similar to that of the wild-type plants. In expanding leaves in transgenic plants, the mRNA levels of the transgene RBCS increased further, leading to increases in the mRNA levels of total RBCS and the Rubisco large subunit gene (RBCL). In the uppermost, fully expanded leaves and the penultimate fully expanded leaves, however, the mRNA levels of RBCS2 and total RBCS decreased drastically, and those of RBCL were similar to the wild-type levels. These results indicate that Rubisco content was not maintained during leaf senescence in the transgenic plants. One reason for this is that the expression of RBCS by the RCA promoter was weak in leaves at low positions. We discuss the possible reasons for this unexpected phenomenon.

Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts

A long-term strategy to enhance global crop photosynthesis and yield involves the introduction of cyanobacterial CO2-concentrating mechanisms (CCMs) into plant chloroplasts. Cyanobacterial CCMs enable relatively rapid CO2 fixation by elevating intracellular inorganic carbon as bicarbonate, then concentrating it as CO2 around the enzyme Rubisco in specialized protein micro-compartments called carboxysomes. To date, chloroplastic expression of carboxysomes has been elusive, requiring coordinated expression of almost a dozen proteins. Here we successfully produce simplified carboxysomes, isometric with those of the source organism Cyanobium, within tobacco chloroplasts. We replace the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco large and small subunit genes, along with genes for two key α-carboxysome structural proteins. This minimal gene set produces carboxysomes, which encapsulate the introduced Rubisco and enable autotrophic growth at elevated CO2. This result demonstrates the formation of α-carboxysomes from a reduced gene set, informing the step-wise construction of fully functional α-carboxysomes in chloroplasts.

Morphology and molecular phylogeny of epizoic araphid diatoms on marine zooplankton, including Pseudofalcula hyalina gen. & comb. nov. (Fragilariophyceae, Bacillariophyta).

Some diatoms are able to colonize as epibionts on their potential zooplankton predators. Here, we report Pseudohimantidium pacificum living on the copepod Corycaeus giesbrechti and as a new finding on Oithona nana, Protoraphis atlantica living on the copepod Pontellopsis brevis, Protoraphis hustedtiana on the cypris larvae of barnacles, and Falcula hyalina on the copepod Acartia lilljeborgii. The epizoic diatoms were able to grow as free-living forms under culture conditions. Pseudohimantidium pacificum and P.atlantica appeared as the most derived species from their benthic diatom ancestors. The mucilage pad or stalk of the strains of these species showed important morphological distinction when compared with their epizoic forms. Barnacle larvae explore benthic habitats before settlement, and epibiosis on them is an example where P.hustedtiana profits from the host behavior for dispersal of its benthic populations. Molecular phylogenies based on the SSU rRNA and RuBisCO large subunit (rbcL) gene sequences revealed F.hyalina as an independent lineage within the Fragilariales (Tabularia, Catacombas, and others), consistent with its morphological distinction in the low number of rows (≤6) in the ocellulimbus, among other features. We propose the transfer of F.hyalina to the genus Pseudofalcula gen. nov. Molecular phylogeny suggests a single order for the members of the Cyclophorales and the Protoraphidales, and that the epibioses of araphid diatoms on marine zooplankton have been independently acquired several times. These clades are constituted of both epizoic and epiphytic/epilithic forms that evidence a recent acquisition of the epizoic modus vivendi.

RuBisCO large subunit gene primers for assessing the CO2-assimilating planktonic community structure in Jiaozhou Bay, China.

The protein coding genes (rbcL/cbbL/cbbM) for RuBisCO large subunit, the most abundant protein on earth that drives biological CO2 fixation, were considered as useful marker genes in characterizing CO2-assimilating plankton. However, their community specificity has hindered comprehensive screening of genetic diversity. In this study, six different rbcL/cbbL/cbbM primers were employed to screen clone libraries to identify CO2-assimilating plankton in Jiaozhou Bay. The following community compositions were observed: the community components in Form I A/B rbcL/cbbL clone library mainly comprised Chlorophyta and Proteobacteria, Form ID2 and ID3 libraries consisted of Bacillariophyta, Form II cbbM library consisted of Proteobacteria and Alveolata, and both Form I green and red libraries included Proteobacteria, respectively. At the genus taxonomic level, no overlaps among these clone libraries were observed, except for ID2 and ID3. Overall, the phytoplankton in Jiaozhou Bay mainly consists of Bacillariophyta, Chlorophyta, Cryptophyta, Haptophyceae and Alveolata. The CO2-assimilating prokaryotes mainly consist of Proteobacteria. Considering the high-sequence specificities of these marker genes, we propose that the joint use of multiple primers may be utilized in unveiling the diversity of CO2-assimilating organisms. In addition, designing novel RuBisCO gene primers that generate longer amplicons and have broader phylogenetic coverage may be necessary in the future.

Sustained Photosynthesis in Tobacco Leaves by Fast CO<sub>2</sub>-Fixing Enzymes Encapsulated in Micro-Compartments

Improvement of yield potential in global food crops has hit a breeding road-block which could be solved through genetic engineering. One promising strategy is the introduction of cyanobacterial CO2-concentrating-mechanisms (CCMs) into plant chloroplasts to enhance photosynthesis and yield. CCMs actively accumulate CO2 within specialized microcompartments called carboxysomes, where the enzyme Rubisco rapidly fixes CO2. Here we replaced the endogenous Rubisco large subunit gene with cyanobacterial Form-1A Rubisco and key structural proteins of α- carboxysomes in tobacco chloroplasts. Transformed plants produced purifiable carboxysomes, isometric with those of the source organism Cyanobium marinum. Carboxysomes encapsulated the introduced Rubisco and enabled autotrophic growth at elevated CO2. Our results demonstrate the formation of α-carboxysomes from a minimal gene set, and that cyanobacterial Form-1A Rubisco supports C3 photosynthesis at high CO2. This major advance informs the step-wise construction of fully-functional α-carboxysomes in chloroplasts which, along with other CCM components, are expected to improve crop plant performance.

Identification of Endogenous Reference Genes for RT-qPCR Expression Analysis in Urochloa brizantha Under Abiotic Stresses

Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4 A, Elongation factor-1 alpha, FTSH protease 4, U2 auxiliary fator, Succinol Co-enzyme A, Tubulin alfa-5, Tubulin beta-6, Ubiquitin conjugating enzyme. Total RNA was extract from leaf tissues of U. brizantha subjected to 6, 12 and 24 h of cold and heat stresses (10 and 45 °C, respectively), and drought, including moderate (−0.5 to −0.7 MPa), severe (−1.1 to −1.8 MPa) and recovery after re-watering. The RefFinder web-based tool was used to rank the most stable reference genes for each stress. Elongation factor-1 alpha, Elongation factor-1 alpha or Ubiquitin conjugating enzyme, and Eukaryotic initiation factor 4 A were the most stable genes for heat, cold and drought stress, respectively. The expression of Rubisco large subunit gene was normalized against the most stable gene selected by ReFfinder for each stress.

High photosynthetic capability observed in the wheat germplasm with rye chromosomes

Improving photosynthetic capability is one of the most important factors for increasing wheat yield potential. The photosynthetic capability of wheat germplasm with different alien chromosomes was investigated and compared with bread wheat cultivars (BC) in this study, including wheat addition lines (CA), hexaploid triticale (HT), octoploid triticale (OT), and synthetic hexaploid wheat lines (SHW). Results indicated that HT, OT, and SHW produced significantly higher biomass plant−1(BMPP), with HT displaying the highest grain yield plant−1 (GYPP). Distinct superiority of net photosynthetic rate (Pn) and carboxylation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was observed in HT and OT. Meanwhile, OT showed the highest expression of the Rubisco large subunit gene (rbcL) in the flag leaves at heading and grain-filling stages, though the coding region of rbcL was highly conserved in all investigated materials. Further analysis indicated that OT and Chinese Spring-rye disomic addition lines displayed higher expression of Rubisco small subunit gene (rbcS). Correlation analysis revealed significant and positive correlations between Pn and the expressions of rbcL (at both heading and grain-filling stages), the expression of rbcS (at heading stage), and the carboxylation activity of Rubisco (at grain-filling stage). Anatomical structure analysis of the chloroplasts showed SHW with longer chloroplasts and more chloroplast grana and grana lamella. In the present study, HT, OT, and Chinese Spring-rye disomic addition lines with rye chromosomes displayed greater photosynthetic capability than BC and SHW, and could be applied in breeding programs to improve the photosynthetic efficiency of wheat.