Pragmatic Applications and Universality of DNA Barcoding for Substantial Organisms at Species Level: A Review to Explore a Way Forward

DNA barcodes are regarded as hereditary succession codes that serve as a recognition marker to address several queries relating to the identification, classification, community ecology, and evolution of certain functional traits in organisms. The mitochondrial cytochrome c oxidase 1 (CO1) gene as a DNA barcode is highly efficient for discriminating vertebrate and invertebrate animal species. Similarly, different specific markers are used for other organisms, including ribulose bisphosphate carboxylase (rbcL), maturase kinase (matK), transfer RNA-H and photosystem II D1-ApbsArabidopsis thaliana (trnH-psbA), and internal transcribed spacer (ITS) for plant species; 16S ribosomal RNA (16S rRNA), elongation factor Tu gene (Tuf gene), and chaperonin for bacterial strains; and nuclear ITS for fungal strains. Nevertheless, the taxon coverage of reference sequences is far from complete for genus or species-level identification. Applying the next-generation sequencing approach to the parallel acquisition of DNA barcode sequences could greatly expand the potential for library preparation or accurate identification in biodiversity research. Overall, this review articulates on the DNA barcoding technology as applied to different organisms, its universality, applicability, and innovative approach to handling DNA-based species identification.

Diversity And Distribution Of CO2-Fixing Microbial Community Along Elevation Gradients In Meadow Soils On The Tibetan Plateau

Soil CO2-fixing microbes play a significant role in CO2-fixation in the terrestrial ecosystems, particularly in the Tibetan Plateau. To understand carbon sequestration by soil CO2-fixing microbes and the carbon cycling in alpine meadow soils, microbial diversity and their driving environmental factors were explored along an elevation gradient from 3900m to 5100m, on both east and west slopes of Mila Mountain region on the Tibetan Plateau. The CO2-fixing microbial communities were characterized by high-throughput sequencing targeting the cbbL gene，encoding the large subunit for the CO2-fixing protein ribulose 1, 5-bisphosphate carboxylase/oxygenase. The overall OTU abundance is concentrated at an altitude between 4300m~4900m. The species richness and distribution uniformity on the east slope is better than those on the west slope. In terms of microbial community composition, Proteobacteria is dominant, and the most abundant genera are Cupriavidus, Rhodobacter, Sulfurifustis and Thiobacillus. The CO2-fixing microbial community structure dramatically shifted along the elevation. It was jointly driven by vegetation coverage, soil moisture content, and soil organic carbon and soil particle size, and most environmental factors are positively correlated. Our results are helpful to understand the variation in soil microbial community and its role in soil carbon cycling along elevation gradients.

Melatonin Promotes the Chilling Tolerance of Cucumber Seedlings by Regulating Antioxidant System and Relieving Photoinhibition

Chilling adversely affects the photosynthesis of thermophilic plants, which further leads to a decline in growth and yield. The role of melatonin (MT) in the stress response of plants has been investigated, while the mechanisms by which MT regulates the chilling tolerance of chilling-sensitive cucumber remain unclear. This study demonstrated that MT positively regulated the chilling tolerance of cucumber seedlings and that 1.0 μmol⋅L–1 was the optimum concentration, of which the chilling injury index, electrolyte leakage (EL), and malondialdehyde (MDA) were the lowest, while growth was the highest among all treatments. MT triggered the activity and expression of antioxidant enzymes, which in turn decreased hydrogen peroxide (H2O2) and superoxide anion (O2⋅–) accumulation caused by chilling stress. Meanwhile, MT attenuated the chilling-induced decrease, in the net photosynthetic rate (Pn) and promoted photoprotection for both photosystem II (PSII) and photosystem I (PSI), regarding the higher maximum quantum efficiency of PSII (Fv/Fm), actual photochemical efficiency (ΦPSII), the content of active P700 (ΔI/I0), and photosynthetic electron transport. The proteome analysis and western blot data revealed that MT upregulated the protein levels of PSI reaction center subunits (PsaD, PsaE, PsaF, PsaH, and PsaN), PSII-associated protein PsbA (D1), and ribulose-1,5-bisphosphate carboxylase or oxygenase large subunit (RBCL) and Rubisco activase (RCA). These results suggest that MT enhances the chilling tolerance of cucumber through the activation of antioxidant enzymes and the induction of key PSI-, PSII-related and carbon assimilation genes, which finally alleviates damage to the photosynthetic apparatus and decreases oxidative damage to cucumber seedlings under chilling stress.

Using Transcript Levels of Nitrate Transporter 2 as Molecular Indicators to Estimate the Potentials of Nitrate Transport in Symbiodinium, Cladocopium, and Durusdinium of the Fluted Giant Clam, Tridacna squamosa

Giant clams are important ecosystem engineers of coral reefs because they harbor large quantities of phototrophic Symbiodiniaceae dinoflagellates of mainly genera Symbiodinium, Cladocopium, and Durusdinium. The coccoid dinoflagellates donate photosynthate and amino acids to the clam host, which in return needs to supply inorganic carbon and nitrogen to them. The host can conduct light-enhanced absorption of nitrate (NO3–), which can only be metabolized by the symbionts. This study aimed to clone nitrate transporter 2 (NRT2) from the symbionts of the fluted giant clam, Tridacna squamosa. Here, we report three major sequences of NRT2 derived from Symbiodinium (Symb-NRT2), Cladocopium (Clad-NRT2) and Durusdinium (Duru-NRT2). Phenogramic analysis and molecular characterization confirmed that these three sequences were NRT2s derived from dinoflagellates. Immunofluorescence microscopy localized NRT2 at the plasma membrane and cytoplasmic vesicles of the symbiotic dinoflagellates, indicating that it could partake in the uptake and transport of NO3–. Therefore, the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 could be used as molecular indicators to estimate the potential of NO3– transport in five organs of 13 T. squamosa individuals. The transcript levels of form II ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcII) of Symbiodinium (Symb-rbcII), Cladocopium (Clad-rbcII) and Durusdinium (Duru-rbcII) were also determined in order to calculate the transcript ratios of Symb-NRT2/Symb-rbcII, Clad-NRT2/Clad-rbcII, and Duru-NRT2/Duru-rbcII. These ratios expressed the potentials of NO3– transport with reference to the phototrophic potentials in a certain genus of coccoid dinoflagellate independent of its quantity. Results obtained indicate that Symbiodinium generally had a higher potential of NO3– transport than Cladocopium and Durusdinium at the genus level. Furthermore, some phylotypes (species) of Symbiodinium, particularly those in the colorful outer mantle, had very high Symb-NRT2/Symb-rbcII ratio (7–13), indicating that they specialized in NO3– uptake and nitrogen metabolism. Overall, our results indicate for the first time that different phylotypes of Symbiodiniaceae dinoflagellates could have dissimilar abilities to absorb and assimilate NO3–, alluding to their functional diversity at the genus and species levels.

Characterization of Ribulose-1,5-Bisphosphate Carboxylase-Oxygenase Activase (Rca) Genes in Durum Wheat

Durum wheat is one of the most widely cultivated cereal crop in the Mediterranean area. Its production has been triggered by drought and rising temperature, both affecting the photosynthetic machinery. Rubisco is one of the most important enzymes in plants. Despite its major role in the control of carbon cycle it has a very low efficiency, which is restored by the action of Ribulose-1,5-bisphosphate carboxylase/oxygenase activase (Rca), a protein belonging to the AAA+ family. The main objective of our work was to isolate and characterize Rca genes in durum wheat and determine their phylogeny with other main crops and model species. Besides a genetic and physical position of Rca1 gene was allowed in a RIL mapping population previously developed. In silico analysis, performed in order to understand whether Rca1 gene was differentially expressed under stress condition, highlighted that homoeologous Rca1 genes have different expression levels especially after infections by Zymoseptoria, powdrey mildew and fusarium. A deeper knowledge of Rca genes structures as well as a better understanding of their physiological role in durum wheat might be of greater importance in panning future modern breeding programs to improve crop yield in adverse environmental condition.

Role of Microalgae in Global CO2 Sequestration: Physiological Mechanism, Recent Development, Challenges, and Future Prospective

The rising concentration of global atmospheric carbon dioxide (CO2) has severely affected our planet’s homeostasis. Efforts are being made worldwide to curb carbon dioxide emissions, but there is still no strategy or technology available to date that is widely accepted. Two basic strategies are employed for reducing CO2 emissions, viz. (i) a decrease in fossil fuel use, and increased use of renewable energy sources; and (ii) carbon sequestration by various biological, chemical, or physical methods. This review has explored microalgae’s role in carbon sequestration, the physiological apparatus, with special emphasis on the carbon concentration mechanism (CCM). A CCM is a specialized mechanism of microalgae. In this process, a sub-cellular organelle known as pyrenoid, containing a high concentration of Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), helps in the fixation of CO2. One type of carbon concentration mechanism in Chlamydomonas reinhardtii and the association of pyrenoid tubules with thylakoids membrane is represented through a typical graphical model. Various environmental factors influencing carbon sequestration in microalgae and associated techno-economic challenges are analyzed critically.

Transcriptome-Wide Gene Expression Plasticity in Stipa grandis in Response to Grazing Intensity Differences

Organisms have evolved effective and distinct adaptive strategies to survive. Stipa grandis is a representative species for studying the grazing effect on typical steppe plants in the Inner Mongolia Plateau. Although phenotypic (morphological and physiological) variations in S. grandis in response to long-term grazing have been identified, the molecular mechanisms underlying adaptations and plastic responses remain largely unknown. Here, we performed a transcriptomic analysis to investigate changes in gene expression of S. grandis under four different grazing intensities. As a result, a total of 2357 differentially expressed genes (DEGs) were identified among the tested grazing intensities, suggesting long-term grazing resulted in gene expression plasticity that affected diverse biological processes and metabolic pathways in S. grandis. DEGs were identified in RNA-Seq and qRT-PCR analyses that indicated the modulation of the Calvin–Benson cycle and photorespiration metabolic pathways. The key gene expression profiles encoding various proteins (e.g., ribulose-1,5-bisphosphate carboxylase/oxygenase, fructose-1,6-bisphosphate aldolase, glycolate oxidase, etc.) involved in these pathways suggest that they may synergistically respond to grazing to increase the resilience and stress tolerance of S. grandis. Our findings provide scientific clues for improving grassland use and protection and identifying important questions to address in future transcriptome studies.

The perspectives for DNA barcoding of Rhaponticum carthamoides (Willd.) Iljin using rbcL gene sequence

The methods of biological species identification using nucleotide sequences of short genome regions (DNA barcoding) are actively developed. The universal DNA barcode for plants remains to be discovered, and one of the leading candidates is the plastid gene of the large subunit of ribulose-bisphosphate carboxylase gene (rbcL). In our study, we estimated the part of rbcL gene as a possible marker for molecular identification of Rhaponticum carthamoides (Willd.) Iljin. Due to its officinal properties, the species is susceptible to uncontrolled and illegal harvesting from natural populations. Today, the species needs to be protected and therefore is included into the Red Data Books of the Russian Federation and certain regions. The study was carried out using plants from the natural populations sampled from the Khamar-Daban Ridge (South Siberia) and considering now as Rh. carthamoides var. chamarense (Peschkova) O S Zhirova. It was shown that rbcL gene can be used to identify Rh. carthamoides at least from the populations of the Khamar-Daban Ridge using a fragment of the maximum length or its 3’ region. Apparently, the 5’ region of the gene (rbcLa) most often used as DNA barcode for plants may be of lesser importance for Rh. carthamoides. The rbcL gene sequences can be also used for the development of approaches for Rh. carthamoides identification in the medicinal preparations and products containing dried tissues to prevent their falsification and illegal harvesting of this species. The combination of rbcL gene with additional markers seems to be highly desirable to create effective DNA barcodes for Rhaponticum species.

Nostoc talks back: Temporal patterns of differential gene expression during establishment of the Anthoceros-Nostoc symbiosis

Endosymbiotic association between hornworts and dinitrogen-fixing cyanobacteria form when the plant is limited for combined nitrogen (N). We generated RNA-Seq data to examine the temporal gene expression patterns during culture of N-starved Anthoceros punctatus in the absence and presence of the symbiotically competent cyanobacterium Nostoc punctiforme. Symbiotic nitrogenase activity commenced within 5 days of coculture reaching a maximal by 14 days. In symbiont-free gametophytes, chlorophyll content, chlorophyll fluorescence characteristics and transcription of genes encoding light harvesting and reaction center proteins, as well as the small subunit of ribulose-bisphosphate-carboxylase/oxygenase, were downregulated. The downregulation was complemented in a temporal pattern corresponding to the N. punctiforme provision of N2-derived ammonium. The impairment and complementation of photosynthesis was the most distinctive response of A. punctatus to N-starvation. Increases in transcription of ammonium and nitrate transporters and their N. punctiforme-dependent complementation was also observed. The temporal patterns of differential gene expression indicated N. punctiforme transmits signals to A. punctatus both prior to, and after its provision of fixed N. This is the only known temporal transcriptomic study during establishment of a symbiotic nitrogen-fixing association in this monophyletic evolutionary lineage of land plants.

Cys-SH based quantitative redox proteomics of salt induced response in sugar beet monosomic addition line M14

Background Salt stress is a major abiotic stress that limits plant growth, development and productivity. Studying the molecular mechanisms of salt stress tolerance may help to enhance crop productivity. Sugar beet monosomic addition line M14 exhibits tolerance to salt stress. Results In this work, the changes in the BvM14 proteome and redox proteome induced by salt stress were analyzed using a multiplex iodoTMTRAQ double labeling quantitative proteomics approach. A total of 80 proteins were differentially expressed under salt stress. Interestingly, A total of 48 redoxed peptides were identified for 42 potential redox-regulated proteins showed differential redox change under salt stress. A large proportion of the redox proteins were involved in photosynthesis, ROS homeostasis and other pathways. For example, ribulose bisphosphate carboxylase/oxygenase activase changed in its redox state after salt treatments. In addition, three redox proteins involved in regulation of ROS homeostasis were also changed in redox states. Transcription levels of eighteen differential proteins and redox proteins were profiled. (The proteomics data generated in this study have been submitted to the ProteomeXchange and can be accessed via username: [email protected], password: q9YNM1Pe and proteomeXchange# PXD027550.) Conclusions The results showed involvement of protein redox modifications in BvM14 salt stress response and revealed the short-term salt responsive mechanisms. The knowledge may inform marker-based breeding effort of sugar beet and other crops for stress resilience and high yield.