rbcS Gene Research Articles

Expression of carbohydrate-related genes underlying 3,5,6-TPA-induced fruitlet abscission in citrus

In citrus, the synthetic auxin 3,5,6-trichloro-2-pyridyloxyacetic acid (3,5,6-TPA), applied as a foliar spray at a concentration of 15 mg l− 1 during physiological fruitlet abscission, caused additional fruitlet drop and reduced the number of fruits reaching maturity. The effect was much more pronounced at full physiological abscission than after. In this study, this thinning effect was successfully exploited for the first time in sour orange trees grown in an urban environment, reducing harvesting costs by up to almost 40%. This effect is mediated by the leaves, which alter their photosynthetic activity. Our results show a reduction of carbon fixation and sucrose synthesis in the leaf, by 3,5,6-TPA repression of the RbcS, SUS1 and SUSA genes, its transport to the fruit, as shown by the reduced expression of the sucrose transporter genes SUT3 and SUT4, and its hydrolysis in the fruit, mainly by repression of the SUS1 gene expression. Genes involved in auxin homeostasis in the fruit, TRN2 and PIN1, were also repressed. The coordinated repression of all these genes is consistent with the decrease in the fruit cell division rate, as shown by the repression of CYCA1-1 gene, leading to the production of ethylene, which ultimately induces fruitlet abscission.

Microbial mechanisms for CO2 and CH4 emissions in Robinia pseudoacacia forests along a North–South transect in the Loess Plateau

Forest soil microbes play a crucial role in regulating atmospheric-soil carbon fluxes. Environmental heterogeneity across forest types and regions may lead to differences in soil CO2 and CH4 emissions. However, the microbial mechanisms underlying these emission variations are currently unclear. In this study, we measured CO2 and CH4 emissions of Robinia pseudoacacia forests along a north–south transect in the Loess Plateau. Using metagenomic sequencing, we investigated the structural and functional profiles of soil carbon cycling microbial communities. Results indicated that the forest CO2 emissions of Robinia pseudoacacia was significantly higher in the north region than in the south region, while the CH4 emission was oppositely. This is mainly attributed to changes in gene abundance driven by soil pH and moisture in participating carbon degradation and methane oxidation processes across different forest regions. The gene differences in carbon fixation processes between regions primarily stem from the Calvin cycle, where the abundance of rbcL, rbcS, and prkB genes dominates microbial carbon fixation in forest soils. Random forest models revealed key genes involved in predicting forest soil CO2 emissions, including SGA1 and amyA for starch decomposition, TYR for lignin decomposition, chitinase for chitin decomposition, and pectinesterase for pectin decomposition. Microbial functional characterization revealed that interregional differences in CH4 emissions during methane metabolism may originate from methane oxidation processes, and the associated gene abundances (glyA, ppc, and pmoB) were key genes for predicting CH4 emissions from forest soils. Our results provide new insights into the microbial mechanisms of CO2 and CH4 emissions from forest soils, which will be crucial for accurate prediction of the forest soil carbon cycle in the future.

Genome-Wide Analysis and Expression Profiling of Soybean RbcS Family in Response to Plant Hormones and Functional Identification of GmRbcS8 in Soybean Mosaic Virus.

Rubisco small subunit (RbcS), a core component with crucial effects on the structure and kinetic properties of the Rubisco enzyme, plays an important role in response to plant growth, development, and various stresses. Although Rbcs genes have been characterized in many plants, their muti-functions in soybeans remain elusive. In this study, a total of 11 GmRbcS genes were identified and subsequently divided into three subgroups based on a phylogenetic relationship. The evolutionary analysis revealed that whole-genome duplication has a profound effect on GmRbcSs. The cis-acting elements responsive to plant hormones, development, and stress-related were widely found in the promoter region. Expression patterns based on the RT-qPCR assay exhibited that GmRbcS genes are expressed in multiple tissues, and notably Glyma.19G046600 (GmRbcS8) exhibited the highest expression level compared to other members, especially in leaves. Moreover, differential expressions of GmRbcS genes were found to be significantly regulated by exogenous plant hormones, demonstrating their potential functions in diverse biology processes. Finally, the function of GmRbcS8 in enhancing soybean resistance to soybean mosaic virus (SMV) was further determined through the virus-induced gene silencing (VIGS) assay. All these findings establish a strong basis for further elucidating the biological functions of RbcS genes in soybeans.

Molecular evolution of the rbcS multiple gene family in Oryza punctata

AbstractRubisco is assembled from large subunits (encoded by chloroplast gene rbcL) and small subunits (encoded by the nuclear rbcS multigene family), which are involved in the processes of carbon dioxide fixation in the Calvin cycle of photosynthesis. Although Rubisco has been studied in many plants, the evolutionary divergences among the different rbcS genes are still largely unknown. Here, using a rice closely related wild species, Oryza punctata Kotschy ex Steud, we investigated the differential properties of the rbcS genes in the species. We identified five rbcS genes (OprbcS1 through OprbcS5), OprbcS1 showed a different evolutionary pattern from the remaining four genes in terms of chromosome location, gene structure, and sequence homology. Phylogenetic analysis revealed that plant rbcS1 and other non‐rbcS1 genes originated from a common ancient duplication event that occurred at least in seed plants ancestor. RbcS1 was then retained in a few plant lineages, including Oryza, whereas non‐rbcS1 was mainly amplified in angiosperms. OprbcS1, OprbcS2–OprbcS4, and OprbcS5 were prominently expressed in stems and seeds, young leaves, and mature leaves, respectively. The yeast two‐hybrid assay detected a significant decrease in the interaction between OprbcS1 and OprbcL compared to the other four pairs of proteins (OprbcS2–OprbcS5 and OprbcL). We propose that OprbcS1 might be assigned a divergent function that was predominantly specific to nonphotosynthetic organs, whereas OprbcS2–OprbcS5, having different affinity in the assembly process of Rubisco, might be subfunctionalized in photosynthetic organs. This study not only deepens our understanding of the fine assembly of Rubisco, but also sheds some light on future de novo domestication of wild rice.

Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.

Physiological responses of marine Chlorella sp. exposed to environmental levels of triphenyltin.

Triphenyltin (TPT) is a herbicide and antifouling agent that has been widely used. After TPT flows into water bodies, it will cause toxic effects on marine life. We evaluated the effect of environmental concentration level (0, 10, 100, and 200ng/L) on the cell density, antioxidant capability, and photosynthesis-related genes in the marine Chlorella sp. The results showed that 10 and 100ng/L TPT can promote the growth of marine Chlorella sp., 200ng/L TPT can inhibit the growth of marine Chlorella sp., and the TPT toxicity was accumulative. The chlorophyll composition changed. The content of chlorophyll a in 100ng/L and 200ng/L groups was significantly higher than that in the control group (p < 0.05) in 13days. The content of chlorophyll b in the 100ng/L and 200ng/L groups in 1day and 13days was significantly different from that in the control group (p < 0.05). The content of total chlorophyll in the 100ng/L and 200ng/L groups in 13days was higher than that in the control group (p < 0.05). The 200ng/L group began to suffer oxidative damage on the 12th day, and the pigment protein complex responded to oxidative damage through self-feedback regulation. On the 18th day, chld, cao, psy, rbcS, and rbcL genes were downregulated, and psbA gene was upregulated in the 10ng/L and 100ng/L groups, which may be a feedback regulation of self-oxidative damage. This paper analyzed toxicity of environmental levels of TPT to marine Chlorella sp., which provided new data support for the comprehensive evaluation of its marine ecological toxicity.

Temperature-induced changes in Arabidopsis Rubisco activity and isoform expression.

In many plant species, expression of the nuclear encoded Rubisco small subunit (SSu) varies with environmental changes, but the functional role of any changes in expression remains unclear. In this study, we investigated the impact of differential expression of Rubisco SSu isoforms on carbon assimilation in Arabidopsis. Using plants grown at contrasting temperatures (10 °C and 30 °C), we confirm the previously reported temperature response of the four RbcS genes and extend this to protein expression, finding that warm-grown plants produce Rubisco containing ~65% SSu-B and cold-grown plants produce Rubisco with ~65% SSu-A as a proportion of the total pool of subunits. We find that these changes in isoform concentration are associated with kinetic changes to Rubisco in vitro: warm-grown plants produce a Rubisco having greater CO2 affinity (i.e. higher SC/O and lower KC) but lower kcatCO2 at warm measurement temperatures. Although warm-grown plants produce 38% less Rubisco than cold-grown plants on a leaf area basis, warm-grown plants can maintain similar rates of photosynthesis to cold-grown plants at ambient CO2 and 30 °C, indicating that the carboxylation capacity of warm-grown Rubisco is enhanced at warmer measurement temperatures, and is able to compensate for the lower Rubisco content in warm-grown plants. This association between SSu isoform expression and maintenance of Rubisco activity at high temperature suggests that SSu isoform expression could impact the temperature response of C3 photosynthesis.

Transcriptome Analysis of Air Space-Type Variegation Formation in Trifolium pratense.

Air space-type variegation is the most diverse among the species of known variegated leaf plants and is caused by conspicuous intercellular spaces between the epidermal and palisade cells and among the palisade cells at non-green areas. Trifolium pratense, a species in Fabaceae with V-shaped air space-type variegation, was selected to explore the application potential of variegated leaf plants and accumulate basic data on the molecular regulatory mechanism and evolutionary history of leaf variegation. We performed comparative transcriptome analysis on young and adult leaflets of variegated and green plants and identified 43 candidate genes related to air space-type variegation formation. Most of the genes were related to cell-wall structure modification (CESA, CSL, EXP, FLA, PG, PGIP, PLL, PME, RGP, SKS, and XTH family genes), followed by photosynthesis (LHCB subfamily, RBCS, GOX, and AGT family genes), redox (2OG and GSH family genes), and nitrogen metabolism (NodGS family genes). Other genes were related to photooxidation, protein interaction, and protease degradation systems. The downregulated expression of light-responsive LHCB subfamily genes and the upregulated expression of the genes involved in cell-wall structure modification were important conditions for air space-type variegation formation in T. pratense. The upregulated expression of the ubiquitin-protein ligase enzyme (E3)-related genes in the protease degradation systems were conducive to air space-type variegation formation. Because these family genes are necessary for plant growth and development, the mechanism of the leaf variegation formation in T. pratense might be a widely existing regulation in air space-type variegation in nature.

Short-term exposure to silver nano-particles alters the physiology and induces stress-related gene expression in Nelumbo nucifera

Lotus (Nelumbo nucifera) was used as model plant in this study to explore its physiology and molecular response upon short-term exposure to silver nano-particles (AgNPs). Accumulation patterns demonstrated a potential uptake of AgNPs by roots and transport to the leaves as a likely key translocation route in lotus. AgNPs exposure was negatively correlated with lotus growth, including germination rate and petiole length in a concentration-dependent manner. Synthesis of chloroplast pigments in lotus leaves was enhanced by low AgNPs concentration, but were inhibited at high concentration. Hydrogen peroxide (H2O2) was detected in lotus leaves after AgNPs treatment. Proline accumulation in lotus leaves was induced with the increase in AgNPs concentration and exposure time. Antioxidant enzyme activities of superoxide dismutase (SOD), peroxidase (POD) as well as catalase (CAT) were enhanced after the first day of AgNPs exposure, but declined with increased exposure time, indicating a time-dependent toxicity of AgNPs. In addition, real-time PCR revealed that two detoxification-related genes, GSH1 and GST, could be activated on the first day of AgNPs exposure, but down-regulated with prolonged AgNPs treatment. Photosynthesis-related RbcS gene was up-regulated, however, no obvious difference in the expression of RbcL was observed after the first day of AgNPs exposure. Moreover, WRKY70a and WRKY70b transcription factors exhibited similar expression patterns, with the highest induction after a 5 mg/L AgNPs exposure on the first day, which decreased with prolonged exposure time. This study provides useful references for further evaluation of the toxic mechanism of AgNPs and their bio-effects on aquatic plants and ecosystems.

Comprehensive toxic effects of povidone iodine on microalgae Chlorella pyrenoidosa under different concentrations

Povidone iodine (PVP-I) is widely used as an efficient antiseptic disinfectant, which can kill various pernicious microbe. PVP-I also exhibits certain toxic effects to the environment. To study the comprehensive toxic effects of PVP-I on microalgae, different concentrations including 0, 7, 14, 28, 56 and 112 mg/L were set. The effects of PVP-I on the growth performance, microstructure, photosynthesis, antioxidant levels and photosynthesis associated genes expressions of microalgae were investigated. The results showed that PVP-I had significant effects on the growth and physiological functions of C. pyrenoidosa. Under the observation of scanning electron microscope, PVP-I of 56 and 112 mg/L could destroy the cytoderm of C. pyrenoidosa directly. Under the PVP-I exposure, the activity of SOD and CAT fluctuated up and down, while MDA content continued to rise. The relative gene expression of PsbA gene was significantly up-regulated in high concentration of 112 mg/L PVP-I. The relative gene expression of rbcL and rbcS gene were upregulated in 28 mg/L PVP-I. The results of present study showed that PVP-I could induce the production of oxidative stress response in C. pyrenoidosa, inhibit the expression of genes related to photosynthesis of C. pyrenoidosa and affect the photosynthesis of C. pyrenoidosa. The present study will provide a reference for the safe application of PVP-I.

Low Light Alters the Photosynthesis Process in Cigar Tobacco via Modulation of the Chlorophyll Content, Chlorophyll Fluorescence, and Gene Expression

Shading or low light (LL) conditions are a key and necessary cultivation technique in cigar wrapper tobacco production. However, the effect of low light on the photosynthesis in cigar tobacco is not clear. Therefore, this study is designed to know the photosynthesis of cigar tobacco under different light intensities (T200, T100, and T50 μmol m−2 s−1). The results reveal that under low light, T50 especially improved the light interception and increased carbon utilization, as witnessed by a higher specific leaf area and lower specific leaf weight. Low light intensity caused better light interception and carbon utilization in cigar tobacco leaves, and thus thinner leaves are more able to use low light efficiently. The chlorophyll content is related to the photosynthesis process; thus, LL affected the photosynthesis process by lowering the chlorophyll content. Similarly, LL also altered the photosynthetic efficiency by lowering the QY_Lss, qP_Lss, and Rfd_Lss. Additionally, higher expression of Lhcb4.2, Lhcb6, PsbA, PsbB, and PsbD under low light, especially T50, shows that the PSII and antenna proteins complex efficiently utilized the absorbed energy for photosynthesis. Finally, the lower photosynthesis, particularly in T50, is attributed to the downregulation of genes related to NADPH production (petH) and the rubisco enzyme synthesis-related gene (rbcs) for CO2 fixation in the Calvin cycle. Overall, the results show that the photosynthesis is decreased under LL intensities which might be related to lower chlorophyll content and downregulation of petH and rbcs genes.

An Antisense Circular RNA Regulates Expression of RuBisCO Small Subunit Genes in Arabidopsis.

Circular RNA (circRNA) is a novel class of endogenous long non-coding RNA (lncRNA) and participates in diverse physiological process in plants. From the dataset obtained by high-throughput RNA sequencing, we identified a circRNA encoded by the sense strand of the exon regions spanning two RuBisCO small subunit genes, RBCS2B and RBCS3B, in Arabidopsis thaliana. We further applied the single specific primer-polymerase chain reaction (PCR) and Sanger sequencing techniques to verify this circRNA and named it ag-circRBCS (antisense and across genic-circular RNA RBCS). Using quantitative real-time PCR (qRT-PCR), we found that ag-circRBCS shares a similar rhythmic expression pattern with other RBCS genes. The expression level of ag-circRBCS is 10–40 times lower than the expression levels of RBCS genes in the photosynthetic organs in Arabidopsis, whereas the Arabidopsis root lacked ag-circRBCS expression. Furthermore, we used the delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) to deliver in vitro synthesized ag-circRBCS into Arabidopsis seedlings. Our results indicate that ag-circRBCS could significantly depress the expression of RBCS. Given that ag-circRBCS was expressed at low concentration in vivo, we suggest that ag-circRBCS may represent a fine-tuning mechanism to regulating the expression of RBCS genes and protein content in Arabidopsis.

Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein

Pigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

Algal Lysis by Sagittula stellata for the Production of Intracellular Valuables.

The purpose of this study was to examine the efficacy of the algicidal bacterium Sagittula stellata on the cell lysis of Nannochloropsis oceanica, a microalga found in the marine environment, in order to extract intracellular valuables. Algicidal bacteria are capable of lysing algal cell walls while keeping lipids and proteins intact yet separated. We obtained these microbes from locations with consistent algae blooms and found that the bacterium Sagittula stellata displayed significant algicidal properties toward Nannochloropsis oceanica, achieving an algicidal rate of 80.1%. We detected a decrease of 66.2% in in vivo fluorescence intensity in algae cultures, obtained a recoverable crude lipid content of 23.3% and a polyunsaturated fatty acid (PUFA) ratio of 29.0% of bacteria-treated algae, and observed the lysis of the cell membrane and the structure of the nucleus of algae. We also identified the inhibited transcription of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcS) gene and proliferating cell nuclear antigen (PCNA)-related genes and the upregulated heat shock protein (hsp) gene in algal cells during bacterial exposure. Our results indicate that Sagittula stellata effectively lysed microalgae cells, allowing the recovery of intracellular valuables. The algicidal method of Sagittula stellata on Nannochloropsis oceanica cells was confirmed to be a direct attack (or predation), followed by an indirect attack through the secretion of extracellular algicidal compounds. This study provides an important framework for the broad application of algicidal microorganisms in algal cell disruption and the production of intracellular valuables.

CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum.

Engineering the small subunit of the key CO2-fixing enzyme Rubisco (SSU, encoded by rbcS) in plants currently poses a significant challenge, as many plants have polyploid genomes and SSUs are encoded by large multigene families. Here, we used CRISPR-Cas9-mediated genome editing approach to simultaneously knock-out multiple rbcS homologs in the model tetraploid crop tobacco (Nicotiana tabacum cv. Petit Havana). The three rbcS homologs rbcS_S1a, rbcS_S1b and rbcS_T1 account for at least 80% of total rbcS expression in tobacco. In this study, two multiplexing guide RNAs (gRNAs) were designed to target homologous regions in these three genes. We generated tobacco mutant lines with indel mutations in all three genes, including one line with a 670 bp deletion in rbcS-T1. The Rubisco content of three selected mutant lines in the T1 generation was reduced by ca. 93% and mutant plants accumulated only 10% of the total biomass of wild-type plants. As a second goal, we developed a proof-of-principle approach to simultaneously introduce a non-native rbcS gene while generating the triple SSU knockout by co-transformation into a wild-type tobacco background. Our results show that CRISPR-Cas9 is a viable tool for the targeted mutagenesis of rbcS families in polyploid species and will contribute to efforts aimed at improving photosynthetic efficiency through expression of superior non-native Rubisco enzymes in plants.

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the βA-βB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.

Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.

The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

Coevolution in Hybrid Genomes: Nuclear-Encoded Rubisco Small Subunits and Their Plastid-Targeting Translocons Accompanying Sequential Allopolyploidy Events in Triticum.

The Triticum/Aegilops complex includes hybrid species resulting from homoploid hybrid speciation and allopolyploid speciation. Sequential allotetra- and allohexaploidy events presumably result in two challenges for the hybrids, which involve 1) cytonuclear stoichiometric disruptions caused by combining two diverged nuclear genomes with the maternal inheritance of the cytoplasmic organellar donor; and 2) incompatibility of chimeric protein complexes with diverged subunits from nuclear and cytoplasmic genomes. Here, we describe coevolution of nuclear rbcS genes encoding the small subunits of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) and nuclear genes encoding plastid translocons, which mediate recognition and translocation of nuclear-encoded proteins into plastids, in allopolyploid wheat species. We demonstrate that intergenomic paternal-to-maternal gene conversion specifically occurred in the genic region of the homoeologous rbcS3 gene from the D-genome progenitor of wheat (abbreviated as rbcS3D) such that it encodes a maternal-like or B-subgenome-like SSU3D transit peptide in allohexaploid wheat but not in allotetraploid wheat. Divergent and limited interaction between SSU3D and the D-subgenomic TOC90D translocon subunit is implicated to underpin SSU3D targeting into the chloroplast of hexaploid wheat. This implicates early selection favoring individuals harboring optimal maternal-like organellar SSU3D targeting in hexaploid wheat. These data represent a novel dimension of cytonuclear evolution mediated by organellar targeting and transportation of nuclear proteins.

WHIRLY1 maintains leaf photosynthetic capacity in tomato by regulating the expression of RbcS1 under chilling stress.

Rubisco, which consists of eight large subunits (RBCLs) and eight small subunits (RBCSs), is a major photosynthetic enzyme that is sensitive to chilling stress. However, it is largely unclear how plants maintain high Rubisco content under low temperature conditions. Here, we report that tomato WHIRLY1 (SlWHY1) positively regulates the Rubisco level under chilling stress by directly binding to the promoter region of SlRbcS1, resulting in the activation of SlRbcS1 expression. SlRbcS1-overexpressing lines had higher Rubisco contents and were more resistant to chilling stress compared with the wild type. Quantitative real-time PCR analyses showed that, among the five RbcS genes, only SlRbcS1 expression is up-regulated by chilling treatment. These results indicate that SlWHIRLY1 specifically enhances the levels of SlRbcS1 and confers tolerance to chilling stress. The amino acid sequence of SlRBCS1 shows 92.67% identity with those of another two RBCS proteins and three residues are specifically found in SlRBCS1. However, mutation of these residues to alanine in SlRBCS1 does not influence its function during cold adaptation. Thus, we conclude that high levels of Rubisco, but not the specific residues in SlRBCS1, play important roles in tolerance to chilling stress in tomato.

Designing a new generation of expression toolkits for engineering of green microalgae; robust production of human interleukin-2.

Introduction: Attributable to some critical features especially the similarity of the protein synthesis machinery between humans and microalgae, these microorganisms can be utilized for the expression of many recombinant proteins. However, low and unstable gene expression levels prevent the further development of microalgae biotechnology towards protein production. Methods: Here, we designed a novel "Gained Agrobacterium -2A plasmid for microalgae expression" (named GAME plasmid) for the production of the human interleukin-2 using three model microalgae, including Chlamydomonas reinhardtii, Chlorella vulgaris , and Dunaliella salina . The GAME plasmid harbors a native chimeric hsp70/Int-1/rbcS2 promoter, the microalgae specific Kozak sequence, a novel hybrid 2A peptide, and Int-1 and Int-3 of the rbcS2 gene in its expression cassette. Results: The obtained data confirmed that the GAME plasmid can transform the microalgae with high transformation frequency. Molecular and proteomic analyses revealed the stable and robust production of the hIL-2 by the GAME plasmid in the microalgae. According to the densimetric analysis, the microalgae can accumulate the produced protein about 0.94% of the total soluble protein content. The ELISA data confirmed that the produced hIL-2 possesses the same conformation pattern with the acceptable biological activity found naturally in humans. Conclusion: Most therapeutic proteins need post-translational modifications for their correct conformation, biological function, and half-life. Accordingly, microalgae could be considered as a cost-effective and more powerful platform for the production of a wide range of recombinant proteins such as antibodies, enzymes, hormones, and vaccines.