Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has inherently low catalytic efficiency, making it a key target for photosynthesis and crop improvement. However, introducing mutations to the chloroplast-encoded Rubisco large subunit (rbcL), which contains the enzyme’s catalytic sites, is technically challenging. In this study, we successfully generate a range of chloroplast-genome-edited Arabidopsis thaliana plants targeting rbcL by a targeted base editor, ptpTALECD. The M309I and D397N substitutions in rbcL result in an increased Rubisco catalytic rate (kcat) without any reductions of Rubisco content, thereby enhancing photosynthetic rates and plant growth under both current atmospheric CO2 concentrations (i.e., 381 μmol mol−1) and projected future concentrations (i.e., 549 μmol mol−1). Cryo-electron microscopy structural analysis shows that the M309I and D397N substitutions, although located far from the catalytic site, induce structural alterations in the catalytic (60 s) loops. Our findings highlight the potential of Rubisco engineering to improve plant photosynthesis and growth, and underscore the unique opportunities that chloroplast genome editing offers for enhancing photosynthesis and crop productivity and reducing atmospheric CO2 levels in a non-GMO context.

Quinoa, a resilient crop adapted to marginal environments, offers a promising alternative under water-limited conditions. To gain new insights into its drought tolerance, we combined physiological analyses with quantitative proteomics under progressive drought (1-2weeks) and subsequent recovery (2weeks). Drought stress induced significant reductions in biomass (56%) and relative water content (46%) after two weeks, with leaf osmotic potential decreasing by up to 48%. However, plants exhibited strong recovery in these parameters upon rehydration. Proteomic analysis identified 114 differentially abundant proteins between drought treatments and control. Proteins involved in cell defense and cytoskeleton organization including S-adenosylmethionine synthetase, caffeoyl-CoA O-methyltransferase, actin, and Exocyst complex component EXO84A, showed an increase under short-term drought conditions. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) increased during prolonged drought and further during recovery, while heat shock proteins (HSP90) accumulated under stress and declined after rewatering, supporting protein stability and oxidative defense. Multiple ribulose bisphosphate carboxylase large and small subunits (RuBisCO) showed increased abundance under drought and further increased during recovery, while oxygen-evolving enhancer proteins (OEE1, OEE2) accumulated early (D1W), helping to stabilize PSII under stress. Interestingly, key proteins involved in amino acid metabolism, including ACT domain-containing protein (ACR11), glutamine synthetase, and Ferredoxin-dependent glutamate synthase (Fd-GOGAT), were identified as regulators of nitrogen assimilation and oxidative stress regulation. Betaine aldehyde dehydrogenase was decreased under stress and increased during recovery, reflecting osmoprotectant accumulation. This study, integrating physiological and proteomic data, demonstrates that quinoa deploys immediate protective mechanisms while activating adaptive processes related to stress memory and recovery-associated metabolic changes, highlighting its resilience under water deficit.

Grain chalkiness is attributable to polygenic traits, including inadequate photosynthate supply, imbalanced source-sink dynamics, or restricted biosynthesis of storage materials. Nevertheless, the precise regulatory mechanism of chalkiness formation remains to be elucidated. In this study, a CRISPR/Cas9 gene editing mutant (oss40-2cr) of a novel transcription factor OsS40-2 in rice exhibited a longer and stay-green flag leaf, a lower CO2 assimilation rate, as well as a transparent endosperm, accompanying by augmented grain weight and diminished grain size compared to the japonica rice cultivar Nipponbare (NIP). The complementation line oss40-2com partially restored the oss40-2cr phenotype to its NIP state. The integrative analysis of the transcriptome and the tsCUT&Tag dataset of the flag leaf and grain at varying developmental stages of the oss40-2cr relative to NIP demonstrated that OsS40-2 functions both as an activator and as a repressor for various downstream genes, thereby regulating enzyme activities related to catalysis, kinase, and transferase in the carbohydrate and protein metabolic processes. OsS40-2 directly targeted and activated the transcription of the precursor of the Rubisco large subunit 1 (OspreRBCL1) before pollination and that of the sucrose transporter OsSWEET7d before seed maturation. However, OsS40-2 exerts a contrary effect by repressing the transcription of OsUGT201 and OsGPIT1 gene at the seed development stage. OsS40-2 plays a critical node linking photosynthetic efficiency, grain filling, and seed component metabolism through the maintenance of source-sink homeostasis in a developmentally dependent manner, thereby affecting grain weight and grain quality.

Cytonuclear interactions play a pivotal role in eukaryotic adaptation, particularly in polyploid organisms, which must achieve coordinated expression and functional compatibility between nuclear-encoded and cytoplasmically inherited (e.g., chloroplast- and mitochondrial-encoded) genes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) a hetero-oligomeric enzyme composed of chloroplast-encoded large subunits (rbcL) and nuclear-encoded small subunits (rbcS) provides an ideal model system for investigating the evolutionary dynamics of cytonuclear coadaptation. To elucidate patterns of cytonuclear evolution following polyploidization, we comparatively analysed rbcL and rbcS sequences, expression profiles, and functional interactions across diploid progenitors (Oryza species bearing B and C genomes) and their derived allotetraploids. We performed Sanger sequencing of rbcL and rbcS coding regions across multiple accessions of B- and C-genome diploids and corresponding allotetraploids. Sequence variation was characterized using nucleotide diversity (π), haplotype analysis, and dN/dS ratio estimation to infer selective regimes. Gene conversion events were detected via phylogenetic incongruence and alignment-based methods. Quantitative real-time PCR (qRT-PCR) was employed to assess tissue-specific and developmental expression levels of rbcS paralogs and rbcL. Protein-protein interaction affinities between rbcL and rbcS variants were systematically evaluated using yeast two-hybrid (Y2H) assays under standardized conditions. Amino acid substitutions in rbcL specifically within the structural interface region known to mediate physical contact with rbcS were identified in both B- and C-genome lineages. In contrast, rbcS homologs exhibited exceptionally high sequence conservation across all taxa, consistent with strong purifying selection (ω = dN/dS ≪ 1); notably, no nonsynonymous single-nucleotide polymorphisms (nsSNPs) were observed in rbcS coding sequences of the allotetraploids. Neither SNP distribution nor quantitative expression data revealed consistent parental-biased allelic expression or dosage effects. However, Y2H assays demonstrated significantly stronger interaction intensities between maternally inherited rbcL alleles and paternally inherited rbcS alleles, suggesting preferential functional compatibility across parental origins. While no direct mechanistic linkage was established between cytonuclear coordination and maternal inheritance of rbcS, the pronounced evolutionary constraint on rbcS underscores its critical role in maintaining Rubisco functionality. The observed asymmetry in rbcL-rbcS interaction preferences may reflect nascent coadaptation processes rather than fixed coevolutionary outcomes. Given the relatively recent origin of Oryza allotetraploids (estimated at <0.5 million years), ongoing cytonuclear adjustment potentially mediated by regulatory rewiring, subfunctionalization or epigenetic modulation likely contributes to the stabilization of photosynthetic machinery during early polyploid evolution.

Enzymes are adapted to perform optimally in different thermal regimes that would otherwise alter kinetics and stability. Whether adaptive evolution in the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) also compensates for thermal variation remains uncertain. We examined molecular evolution and modelled the change in folding free energy (ΔΔG, where negative values indicate stabilization) of the rubisco large subunit (RbcL) in four phylogenetically distant plant genera: wood ferns (Dryopteris), sea lavenders (Limonium), pines (Pinus), and viburnums (Viburnum). Using codon evolutionary models in each genus, we observed widespread positive selection and parallel substitution in the catalytic α/β barrel domain. Species with warmer growing seasons had derived amino acids with stronger hydrogen bond contributions to ΔΔG. Protein structure-based modelling showed that the hydrogen bond contribution to stability tracked the growing season temperature of species carrying the derived amino acid. Stronger hydrogen bonds were offset by weaker contributions from hydrophobic solvation interactions, such that total ΔΔG showed no relationship with growing season temperature. In Viburnum, the strength of positive selection differed among biomes, with cold temperate and cloud forest clades showing stronger positive selection. These patterns are consistent with environmental tuning of non-covalent interactions within the enzyme. However, modest effect sizes indicate that other components of the rubisco holoenzyme likely also contribute to its thermal evolution.

Bee microbiota form important symbiotic relationships with their hosts, but microbial communities vary across bee species, sociality, and environment. Comparing the microbiome of bees with different social roles and foraging behaviours may uncover the ways in which microbiota are environmentally acquired and subsequently introduced and spread into the nest environment. Here, we performed metabarcoding of the 16S rRNA, ITS, and ribulose biphosphate carboxylase large (rbcL) regions on mothers, dwarf eldest daughters, and regular daughters in nests of the facultatively social, small carpenter bee, Ceratina calcarata, contrasting bacteria, fungi, and plant associates. We also performed two different sampling types by characterizing the microbiome using whole-guts and whole-bodies. Social role in nest impacted the microbial community composition and mothers were found to demonstrate increased plant diversity compared to their daughters, more specifically in whole-bodies, highlighting the ability to determine plants that bees are visiting during foraging through DNA metabarcoding. We also found that metabarcoding of the whole-body recovered increased fungal and plant diversity compared to whole-guts, suggesting that including microbiota from beyond the gut offers an opportunity to characterize uncommon associates that bees encounter, particularly through plant-pollinator relationships. As the transmission of beneficial symbionts and pathogens between individuals are studied for its impact on bee health, microbial analyses of bees across different environments and levels of sociality provides unique biomonitoring that can indicate the health of the larger bee community.

Background: DNA barcoding is an efficient molecular biology technique that utilizes a short genetic locus with sufficient variability to enable precise organism identification. Typically, regions such as ribulose-bisphosphate carboxylase (rbcL) and maturase K (matK) in plants are widely used due to their balance between interspecific divergence and intraspecific conservation. These standardized genomic fragments are amplified by Polymerase Chain Reaction (PCR) and subsequently sequenced for comparative analysis. Every species has its own characteristic DNA barcode, which can be matched against curated reference libraries, enabling accurate identification even when morphological traits are ambiguous, damaged, or insufficient for taxonomic classification. This approach has been particularly transformative in biodiversity monitoring, ecological studies, and the detection of cryptic or invasive species. Presenting DNA barcodes in a graphical form provides an alternative and powerful way to store and display sequence information as it facilitates cross-comparison. The generated graphical outputs can be incorporated into machine learning algorithms for species recognition in further large-scale ecological and conservation research.Methods: The matK and rbcL genes of the collected five senna species were selected as DNA barcodes to confirm and distinguish these plant samples. An alternative method was also presented to extract the characteristics of the DNA sequences with graphical operation based on Chaos Game Representation (CGR).Results: It was found that the power to discriminate between species was high enough when a two-locus barcode approach was applied with 90% successful amplification using the provided protocol optimization. The similarity/dissimilarity comparison of the collected plant samples was also achieved by the obtained CGR.Conclusion: Grouping of individuals based on genetic relationship was consistent with morphology and taxonomy, particularly when the primers for matK were used, whereas the rbcL barcode was less effective in distinguishing species.Keywords:DNA barcode, matK, rbcL, Senna species, Chaos Game Representation

GmAP1 delays flowering time and confers sensitivity to salt stress in soybean.

Previously, the formation of a stable adduct 2H3BO3·3CsCl (BC) was observed in CsCl(aq) but not in RbCl(aq). The phase equilibria of the H3BO3 + RbCl + CsCl + H2O system were studied to assess the potential for selective separation of Cs from Rb. Solubility isotherms were determined experimentally. Appropriate liquid composition ranges were preselected using modeling with ISLEC’s equilibrium software. Schreinemakers’ wet solid method, XRD, Raman and IR spectra, Roozeboom’s plots, and SEM-EDS were applied to identify the four phase regions H3BO3, 2H3BO3·3(Cs,Rb)Cl (Bss), (Rb,Cs)Cl, and (Cs,Rb)Cl. Notably, in the presence of RbCl, the BC phase incorporates Rb+, forming a substitutional solid solution, Bss, which inhibits selective separation of CsCl. Roozeboom’s plot analysis revealed that RbCl exhibits higher solubility in the Bss phase compared to the (Cs,Rb)Cl solid solution. The separation factor βCs/Rb between the solid Bss phase and the corresponding solution was 2.0, significantly lower than that of (Cs,Rb)Cl (βCs/Rb = 9.5) in the ternary system without H3BO3. This indicates a stronger coordination between CsCl and H3BO3 relative to that of RbCl in the ternary system without H3BO3. The above understanding provides new strategies designing novel B–OH compounds to enhance Cs/Rb separation efficiency in aqueous systems.

Limitations in accurate and efficient species identification techniques at various stages of development and sample conditions are major problems in the conservation of local plants. Conventional morphology-based identification approaches often encounter obstacles due to phenotypic variation and diagnostic character limitations. This study aims to examine the potential of DNA barcoding as a biotechnology innovation in supporting the identification and conservation of local plants. The research method used was a systematic literature study of national and international scientific articles published between 2020 and 2025. Data sources were obtained from the PubMed, Scopus, ScienceDirect, and Google Scholar databases, then analyzed thematically by grouping findings based on the type of genetic marker, identification procedure, and relevance to plant conservation. The results of the study show that DNA barcoding is a species identification technique carried out through the analysis of deoxyribonucleic acid sequences from specific standard marker genes. In plant taxonomy, chloroplast genes commonly used as genetic markers include ribulose bisphosphate carboxylase large chain, maturase K, and internal transcribed spacer. The genetic sequences obtained are then compared with a global database to accurately determine species suitability. This technique has been proven to provide fast and reliable species identification under various sample conditions. With these advantages, DNA barcoding has great potential as a key supporting tool in local plant conservation efforts. Keywords: DNA Barcode, Biotechnology, Plant Conservation

Genome scale engineering has enabled codon compression of the universal genetic code to eliminate seven codons in Escherichia coli, but to allow more radical schemes for codon compression and reassignment to be tested at genome scale, while avoiding significant technical challenges, smaller, simpler genetic systems are needed. Here, we report a recoding scheme for the 205 kb Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine, and serine, all of which have two cognate transfer RNAs (tRNAs), are absent, compressing the genetic code to 51 codons. Several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native sequence without affecting expression of a reporter protein or strain fitness under standard laboratory conditions. The same strategy was successfully used for codon compression of ycf1, encoding a subunit of the chloroplast translocon, psaA and psbA, intron-containing highly expressed genes encoding reaction center subunits of both photosystems, and an 8.5 kb operon encoding essential and nonessential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of Rubisco, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codons were recovered. For all recoded genes, viable homoplasmic lines were obtained, showing the efficacy of our codon compression scheme.

Effect of Nitrosomonas europaea on Chlorella vulgaris in bio-hydrogels for startup of microalgal-bacterial granular sludge: Performance and microscopic mechanism.

Very few true Pseudomonas methylotrophic strains have been described, and in none of them have the pathways for one-carbon (C1) substrate metabolism been elucidated. The genomes of three Pseudomonas strains able to grow on methanol as the sole source of carbon (C) and energy (E) were sequenced and analyzed, and one of the strains was further characterized at the proteomic and physiological level. None of the three strains possesses a classic methanol dehydrogenase enzyme, and they apparently employ generalist type-I alcohol dehydrogenases (ADHs) to catabolize methanol to formaldehyde. In two of the strains' genomes, the only complete route encoded for incorporating methylotrophic carbon is the Calvin-Benson-Bassham (CBB) cycle, while other more typical pathways for C1-carbon assimilation (serine cycle, ribulose monophosphate cycle) appear incomplete. The indispensability of the QedA1 alcohol dehydrogenase and of ribulose bisphosphate carboxylase for growth on methanol was demonstrated by insertion mutagenesis of the qedA1 and cbbL genes in one of the strains. To the author's knowledge, all wild-type methylotrophic Pseudomonadota (i.e., "Gram-negative bacteria") so far described employ a specific dehydrogenase distinctively adapted to using methanol as a substrate (MxaFI, XoxFI, or Mdh2). The methylotrophic Pseudomonas strains described here lack MDH and employ generalist ADHs, thus demoting methanol dehydrogenase (MDH) from the position of a critical enzyme for methanol utilization and expanding the range of enzymes (and genes) that enable methylotrophy in nature. The second remarkable result of this work is the discovery of the utilization of the CBB cycle by a Pseudomonas strain during methylotrophic growth, an absolute novelty for this very relevant bacterial genus.

Legundi (Vitex trifolia) is one of the crucial ethno-pharmacological plants. However, the genetic exploration of these plants in Indonesia remains limited. Moreover, a set of primers can be an initial important step to explore the genetics of V. trifolia as well as to molecularly identify the species of Legundi. This study aimed to develop the two pairs of primers of maturase-K (matK) and ribulose bisphosphate carboxylase large subunit (rbcL) genes. This type of research is exploratory research using a quantitative approach. The research sampling are the leaves of the V. trifolia species, which were collected from Makassar, Indonesia, and the purposive sampling method. The data were obtained through DNA barcoding, and the data were analyzed using bioinformatics analysis. This study found two pairs of developed primers, matK and rbcL, successfully amplified both matK and rbcL target genes of V. trifolia. The newly developed species-specific primers successfully amplified the matK and rbcL genes of V. trifolia, and sequence analysis revealed high similarity values in BLAST and BOLD databases ranging from 99.2 to 100%, with PCR amplification of the matK marker showing particularly high DNA concentration and specificity for species-level identification. This study supports the genetic exploration and identification of useful ethno-pharmacy plants, V. trifolia. The conclusion of the study shows that two primer pairs of maturase-K (matK) and ribulose bisphosphate carboxylase large subunit (rbcL) genes have been developed from V. trifolia plants. This study supports the genetic exploration and identification of ethnopharmaceutical plants of V. trifolia and the role of bioinformatics tools in molecular studies of medicinal plants.

Spirogyra are green algae that occur widespread in freshwater habitats across northeastern Thailand, yet its species diversity and chemical characteristics remain poorly understood. This study presents a novel integrated approach combining morphological identification, rbcL-based molecular analysis, and chemical profiling (LC-MS and FTIR) to comprehensively characterize Thai Spirogyra species. Six Spirogyra samples were collected from different water resources in Sakon Nakhon province and identified using both morphological characteristics and ribulose-bisphosphate carboxylase (rbcL) gene sequences. The sequence data of the rbcL gene was analyzed by using the database of the National Center for Biotechnology Information (NCBI). The results found that 6 Spirogyra samples were able to be identified as 3 Spirogyra submaxima and 1 sample each of S. fluviatilis, S. maxima, and S. chungkingensis. All Spirogyra samples were extracted with ethanol, yielding 6 crude ethanol extracts (SPE1 - SPE6). The chemical compositions were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS). LC-MS analysis of the 6 crude ethanol extracts revealed a total of 60 compounds, of which thirty were consistently detected across all Spirogyra samples, while the remaining compounds were specific to individual extracts. In addition, the FTIR profiles of SPE1-SPE6 were examined using the ATR-FTIR technique. The FTIR spectra indicated similar functional groups across samples, with absorption bands attributed to hydroxyl groups, CH₂ and CH₃ stretching vibrations, and carbonyl functionalities. These findings demonstrate that integrating morphological, genetic, and chemical analyses provides a more reliable framework for distinguishing Spirogyra species and reveals locality-specific chemical variation that may be valuable for future biochemical and taxonomic research. HIGHLIGHTS Molecular identification of 6 Spirogyra species was performed using the rbcL marker. Morphological characteristics of 6 Spirogyra specimens were described and compared. Phylogenetic relationships among 6 Spirogyra samples were constructed using Maximum Likelihood and Neighbor-Joining methods based on the rbcL gene locus. Comparative analysis of the chemical compositions of 6 Spirogyra species collected from different water resources was conducted. GRAPHICAL ABSTRACT

Spontaneous reshaping of microalgae carbon metabolism under organophosphate esters stress: Insights into adaptive strategies for wastewater treatment.

Photosynthetic bacteria (PSB) are protein-rich and a high-quality producer of microbial proteins. In this study, PSB were cultivated using self-fermented kitchen waste fermentation broth under controlled light intensity and light cycle conditions. Biomass and protein concentrations were measured daily, and microbial community composition and functional succession were analyzed using high-throughput sequencing and bioinformatic tools to investigate the effects of photoperiod on PSB growth and protein synthesis. The results showed that PSB had the highest biomass and protein production of 1356.5mg/L and 564.3mg/L at 4000lx and 24h light/0h dark, respectively. Organic pollutant removal was also the highest, with 89.7% chemical oxygen demand (COD) removal and 65.8% ammonia nitrogen removal. Microbiological analysis indicated that the selected light intensity and light/dark cycles were highly favorable for PSB growth. Under these conditions, the dominance of Rhodopseudomonas was further strengthened. During the cultivation process, PSB adjusted its metabolic pathway and shifted its metabolic focus from carbon metabolism to nitrogen metabolism. In addition, the activities of ribulose bisphosphate carboxylase (Rubisco), a key enzyme for photosynthesis, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase (SDH), key enzymes of the tricarboxylic acid (TCA) cycle, were enhanced in PSB. These findings provide an important reference for an in-depth understanding of the growth characteristics, metabolic responses, and protein biosynthesis of PSB in the treatment of kitchen waste fermentation broth.

Jasmonates regulate metabolic cascade towards Eicosapentaenoic acid and Docosahexaenoic acid biosynthesisin filamentous microalga Tribonema minus.

Differential expression of RubisCO and PEPC during the salinity stress recovery of facultative CAM plants.

The morphological traits of Vaccinium uliginosum L., including plant height, leaf area, and fruit weight, have changed significantly across an elevational gradient in the Changbai Mountains. To elucidate the molecular mechanisms underlying these morphological variations, RNA-Seq technology was employed to identify differentially expressed genes (DEGs), key metabolic pathways, and associated biological functions of V. uliginosum at seven elevations in the Changbai Mountains. A total of 1190 DEGs significantly associated with morphological variations were identified. These genes are mainly involved in lipid synthesis, carbohydrate metabolism, energy metabolism, and signal transduction. Redundancy analysis (RDA) revealed that fatty acyl-ACP thioesterase B (FATB) and ribulose-bisphosphate carboxylase small subunit (cbbS) exhibited a significant association with morphological variation. Integrated analysis indicated that high-altitude plants likely enhance lipid synthesis and cell wall stability while also inhibiting photosynthesis and carbohydrate metabolism. The regulatory mechanisms underlying hormone signal transduction may be relatively complex, as evidenced by the enhanced activity of gibberellin and reduced biological effects of auxin, abscisic acid, and ethylene. This study is the first to provide transcriptomic evidence elucidating the genetic basis of altitudinal morphological adaptation in V. uliginosum, integrating phenotypic traits with gene expression profiles across an elevational gradient.