Carboxylase Large Subunit Research Articles

Purification, crystallization, and X-ray crystallographic analysis of ribulose bisphosphate carboxylase large subunit from Hydrogenovibrio marinus

Purification, crystallization, and X-ray crystallographic analysis of ribulose bisphosphate carboxylase large subunit from <i>Hydrogenovibrio marinus</i>

Potential application of DNA barcoding technology for identification of medicinal plant species traded in Nairobi County, Kenya

There exists a long history of herbal products use but recently there are concerns on authenticity and safety of some these products. Majority of the herbal products traded in Kenyan markets are processed plant parts, which have been dried or in powder form and are extremely difficult to identify taxonomically. This study was carried out in selected markets in Nairobi County, the capital city of Kenya to assess authenticity of herbal products traded in the region using deoxyribonucleic acid (DNA) barcoding technology by applying internal transcribed spacer (ITS) and ribulose -1,5-bisphosphate carboxylase Large subunit (rbcL) markers. One hundred and twelve herbal products were purchased from practicing herbalists and analyzed using ITS and rbcL DNA barcoding markers. DNA was extracted from acquired samples, amplified and sequences generated. BLAST search with edited generated sequences against the NCBI database was done to recover the expected species. Our findings showed that 35% of the herbal products were wrongly labeled and contained different species compared to those listed on the labels. Out of 92 samples with positive sequences, 58 matched their expected species after the BLAST search. Among the authentic species identified are O. lanceolata, M. oleifera, W. ugandensis, O. europaea, Acacia species, and C. spinarum. However, 32 species did not match the expected species as listed on labels and were concluded to have been substituted. Among them were R. prinoides, T. abyssinica, U. massaica and A. indica, which were substituted with their close relatives, while R. tridentate, Z. usambarense and Prunus africana were substituted with unrelated species with no reported medical benefits. ITS marker had remarkable identification efficiency at 98% to species level compared to rbcL at 89%. We recommend further studies to establish at what stage substitution takes place, as well as utilization of DNA barcoding technology for medicinal plants authentication and verification

Guilt by Association: DNA Barcoding-Based Identification of Potential Plant Hosts of Phytoplasmas from Their Insect Carriers.

Phytoplasmas are small phloem-restricted and insect-transmissible bacteria that infect many plant species, including important crops and ornamental plants, causing severe economic losses. Our previous studies screened phytoplasmas in hundreds of leafhoppers collected from natural habitats worldwide and identified multiple genetically different phytoplasmas in seven leafhopper species (potential insect vectors). As an initial step toward determining the impact of these phytoplasmas on the ecosystem, ribulose 1,5-biphosphate carboxylase large subunit (rbcL), a commonly used plant DNA barcoding marker, was employed to identify the plant species that the phytoplasma-harboring leafhoppers feed on. The DNA of 17 individual leafhoppers was PCR amplified using universal rbcL primers. PCR products were cloned, and five clones per amplicon were randomly chosen for Sanger sequencing. Moreover, Illumina high-throughput sequencing on selected PCR products was conducted and confirmed no missing targets in Sanger sequencing. The nucleotide BLAST results revealed 14 plant species, including six well-known plant hosts of phytoplasmas such as tomato, alfalfa, and maize. The remaining species have not been documented as phytoplasma hosts, expanding our knowledge of potential plant hosts. Notably, the DNA of tomato and maize (apparently cultivated in well-managed croplands) was detected in some phytoplasma-harboring leafhopper species sampled in non-crop lands, suggesting the spillover/spillback risk of phytoplasma strains between crop and non-crop areas. Furthermore, our results indicate that barcoding (or metabarcoding) is a valuable tool to study the three-way interactions among phytoplasmas, plant hosts, and vectors. The findings contribute to a better understanding of phytoplasma host range, host shift, and disease epidemiology.

Enhanced genome-wide association reveals the role of YABBY11-NGATHA-LIKE1 in leaf serration development of Populus.

Leaf margins are complex plant morphological features that contribute to leaf shape diversity, which affects plant structure, yield, and adaptation. Although several leaf margin regulators have been identified to date, the genetic basis of their natural variation has not been fully elucidated. In this study, we profiled two distinct leaf morphology types (serrated and smooth) using the persistent homology mathematical framework (PHMF) in two poplar species (Populus tomentosa and Populus simonii, respectively). A combined genome-wide association study (GWAS) and expression quantitative trait nucleotide (eQTN) mapping were applied to create a leaf morphology control module using data from P. tomentosa and P. simonii populations. Natural variation in leaf margins was associated with YABBY11 (YAB11) transcript abundance in poplar. In P. tomentosa, PtoYAB11 carries a premature stop codon (PtoYAB11PSC), resulting in the loss of its positive regulation of NGATHA-LIKE1 (PtoNGAL-1) and RIBULOSE BISPHOSPHATE CARBOXYLASE LARGE SUBUNIT (PtoRBCL). Overexpression of PtoYAB11PSC promoted serrated leaf margins, enlarged leaves, enhanced photosynthesis, and increased biomass. Overexpression of PsiYAB11 in P. tomentosa promoted smooth leaf margins, higher stomatal density, and greater light damage repair ability. In poplar, YAB11-NGAL1 is sensitive to environmental conditions, acts as a positive regulator of leaf margin serration, and may also link environmental signaling to leaf morphological plasticity.

Proteomics and photosynthetic apparatus response to vermicompost attenuation of salinity stress Vicia faba leaves

Crop production and growth are severely affected by salt stress. Nevertheless, the bio-fertilizer vermicompost (VC) can be participated as a potent inhibitor of salinity on plant growth and crop production by regulating photosynthetic efficiency. We investigated the effect of VC on photosynthetic performance of salt-stressed broad bean (Vicia faba L. Aspani cultivar). Seeds were grown in soil mixture; clay and sand in ratio 1:2 by volume with five different volumetric ratios of VC; 0, 2.5, 5, 10 and 15% irrigated with either water and/or 200 mM NaCl. Leaf area, Na and K contents, chlorophylls, photosystem II efficiency, Rubisco content, soluble sugars, chloroplasts’ organization and proteomics were analyzed. The imposed stress decrease leaf area, chlorophyll contents, maximum quantum efficiency (Fv/Fm), Rubisco content, increase soluble sugars and damage chloroplasts organization. Salinity upregulated glucose-1-phosphate adenylyl transferase, ribulose bisphosphate carboxylase large subunit and chloroplastic peptidyl-prolyl cis–trans isomerase. The increased leaf area, chlorophyll a, b and carotenoids, maximum quantum efficiency of photosystem II, Rubisco content, improving the degeneration of thylakoid lamellae and lessening plastoglobuli number in thylakoid membranes are the major benefits attained with vermicompost treatments under salt stress.Analysis of proteomic revealed that VC upregulated chloroplastic ferredoxin–NADP reductase, plastocyanin, polyphenol oxidase, peptidyl-prolyl cis–trans isomerase, alpha-glucan phosphorylase H isozyme and maturase expression under salt stress. The results suggest that VC controls protein expression at the level of transcriptional and translational which may conserve photosynthetic components and prevent salt-induced harmful effects in broad bean plants.

DNA BARCODING OF THE EGYPTIAN MEDICINAL PLANT Astragalus spinosus

Medicinal plants have served as the basis of alternative medicine and have become the primary source for developing new medications. Astragalus spinosus (Forssk.) Muschl. belongs to family Fabaceae and due to its active constituent’s composition, it is used traditionally to treat various ailments. Additionally, various Astragalus-based dietary supplements were sold for supporting immunity and cardiovascular system. Therefore, authentication of the medicinal plant material is important and vital to provide an extensive database with DNA data for easy identification, adulteration detection and conservation of genetic resources. In the current study, three partial chloroplast coding genes were examined in A.spinosus; ribulose 1,5biphosphate carboxylase large subunit (rbcL), RNA polymerase (rpoC1) and maturase K (matK). When compared to the National Center for Biotechnology Information (NCBI) database, the results showed that rbcL was successful in identifying and discriminating A.spinosus at the genus and species levels. The, rpoC1 and matK genes revealed a high level of identity with Astragalus species, indicating that they are capable of identifying A.spinosus at the genus level. Phylogenetic trees were established based on the expected amino acids sequence of each coding gene, demonstrating the extent of relatedness with recorded plant species in the NCBI database.

The biphasic responses of nanomaterial fullerene on stomatal movement, water status, chlorophyll a fluorescence transient, radical scavenging system and aquaporin-related gene expression in Zea mays under cobalt stress

Nanomaterial fullerene (FLN) has different responses called the hormesis effect against stress conditions. The favorable/adverse impacts of hormesis on crop quality and productivity are under development in agrotechnology. In this study, the effect of FLN administration (100-250-500mg L−1 for FLN1-2-3, respectively) on growth, water management, gas exchange, chlorophyll fluorescence kinetics and cobalt (Co)-induced oxidative stress in Zea mays was investigated. The negative alterations in relative growth rate (RGR), water status (relative water content, osmotic potential and proline content) and gas exchange/stomatal regulation were removed by FLNs. FLNs were shown to protect photosynthetic apparatus and preserve the photochemistry of photosystems (PSI–PSII) in photosynthesis, chlorophyll fluorescence transients and energy flux damaged under Co stress. The maize leaves exposed to Co stress exhibited a high accumulation of hydrogen peroxide (H2O2) due to insufficient scavenging activity, which was confirmed by reactive oxygen species (ROS)-specific fluorescence visualization in guard cells. FLN regulated the gene expression of ribulose-1,5-bisphosphate carboxylase large subunit (rbcL), nodulin 26-like intrinsic protein1-1 (NIP1-1) and tonoplast intrinsic protein2-1 (TIP2-1) under stress. After stress exposure, FLNs successfully eliminated H2O2 content produced by superoxide dismutase (SOD) activity of catalase (CAT) and peroxidase (POX). The ascorbate (AsA) regeneration was achieved in all FLN applications together with Co stress through the elevated monodehydroascorbate reductase (MDHAR, under all FLNs) and dehydroascorbate reductase (DHAR, only FLN1). However, dose-dependent FLNs (FLN1-2) provided the induced pool of glutathione (GSH) and GSH redox state. Hydroponically applied FLNs removed the restrictions on metabolism and biological process induced by lipid peroxidation (TBARS content) and excessive ROS production. Considering all data, the modulation of treatment practices in terms of FLN concentrations and forms of its application will provide a unique platform for improving agricultural productivity and stress resistance in crops. The current study provided the first findings on the chlorophyll a fluorescence transient and localization of ROS in guard cells of Zea mays exposed to FLN and Co stress.

Enhancement of Biomass Production in Colony-Forming Green Algae, Botryosphaerella sudetica, Under Mixotrophic Cultivation.

In this study, we characterized the potential of colony-forming green algae, Botryosphaerella sudetica KNUA107, isolated from Ulleung Island, South Korea, as a bioresource and analyzed the effects of mixotrophic cultivation on its bioresource production efficiency. Internal transcribed spacer (ITS) (ITS1, 5.8S, and ITS2), ribulose bisphosphate carboxylase large subunit (rbcL), and elongation factor Tu (tufa) regions were used for molecular identification and phylogenetic analysis. B. sudetica KNUA107 had a strong relationship with the green algae of Botryococcus and Botryosphaerella genera, which are colony-forming species, and was also associated with members of the Neochloris genus. To improve biomass productivity, we tested mixotrophic cultivation conditions using several organic carbon sources. Glucose supplementation stimulated B. sudetica KNUA107 growth and reduced the time needed to reach the stationary phase. In addition, the colony size was 1.5–2.0 times larger with glucose than in photoautotrophic cultures, and settleability improved in proportion to colony size. The total lipid content and biomass productivity were also higher in cultures supplemented with glucose. Among the lipid components, saturated fatty acids and monounsaturated fatty acids had the highest proportion. Our study suggests that B. sudetica KNUA107, which has enhanced efficiency in biomass production and lipid components under mixotrophic cultivation, has high potential as a bioresource.

Afrotropical sand fly-host plant relationships in a leishmaniasis endemic area, Kenya.

The bioecology of phlebotomine sand flies is intimately linked to the utilization of environmental resources including plant feeding. However, plant feeding behavior of sand flies remains largely understudied for Afrotropical species. Here, using a combination of biochemical, molecular, and chemical approaches, we decipher specific plant-feeding associations in field-collected sand flies from a dry ecology endemic for leishmaniasis in Kenya. Cold-anthrone test indicative of recent plant feeding showed that fructose positivity rates were similar in both sand fly sexes and between those sampled indoors and outdoors. Analysis of derived sequences of the ribulose-1,5-bisphosphate carboxylase large subunit gene (rbcL) from fructose-positive specimens implicated mainly Acacia plants in the family Fabaceae (73%) as those readily foraged on by both sexes of Phlebotomus and Sergentomyia. Chemical analysis by high performance liquid chromatography detected fructose as the most common sugar in sand flies and leaves of selected plant species in the Fabaceae family. Analysis of similarities (ANOSIM) of the headspace volatile profiles of selected Fabaceae plants identified benzyl alcohol, (Z)-linalool oxide, (E)-β-ocimene, p-cymene, p-cresol, and m-cresol, as discriminating compounds between the plant volatiles. These results indicate selective sand fly plant feeding and suggest that the discriminating volatile organic compounds could be exploited in attractive toxic sugar- and odor- bait technologies control strategies.

Epi-Brassinolide Positively Affects Chlorophyll Content and Dark-Reaction Enzymes of Maize Seedlings

Brassinosteroids participate in many physiological processes in plants; however, their regulatory roles on the activities of the enzymes involved in dark phase of photosynthesis remains elusive. In this study, detached leaves and protoplasts of maize seedlings were treated with epi-brassinolide (EBR) and brassinazole followed by the determination of the contents of chlorophyll (a+b) and soluble sugars, and the activity of dark reaction enzymes and the expression of the relevant genes. The results showed that chlorophyll (a+b) content increased by 7.4% under 0.1 μM EBR treatment for 48 h; furthermore, chlorophyll (a+b) content increased by 34% in detached leaves that were continuously soaked in brassinazole. In addition, the transcription levels of glyceraldehyde-3-phosphate dehydrogenase subunit A (GAPA), cytoplasmic FBPase (cyFBPase), ribulose-1,5-bisphosphate carboxylase small subunit (rbcS), phosphoenolpyruvate carboxylase (PEPC), fructose-1,6-bisphosphatase (FBPase), Rubisco activase β subunit (RCAβ), ribulose-l,5-bisphosphate carboxylase large subunit (rbcL), and glutathione reductase (GR) were 96.8%, 48.4%, 79.3%, 41%, 85.6%, 133.3%, 68.8%, and 119.8% higher, respectively, in 0.1 μM EBR than in the control group. The activity of RCA and Rubisco and soluble sugar content increased by 53.4%, 28.7%, and 35.4% under 0.1 μM EBR. Brassinazole inhibits the expression of these genes. However, the transcription level, protein content, and activity of some dark-reaction enzymes cannot be increased at the same time under EBR treatment. These results indicate that the effect of EBR on dark-reaction is mainly in transcription level.

Molecular Identification and Evaluation of the Genetic Diversity of Dendrobium Species Collected in Southern Vietnam

Dendrobium has been widely used not only as ornamental plants but also as food and medicines. The identification and evaluation of the genetic diversity of Dendrobium species support the conservation of genetic resources of endemic Dendrobium species. Uniquely identifying Dendrobium species used as medicines helps avoid misuse of medicinal herbs. However, it is challenging to identify Dendrobium species morphologically during their immature stage. Based on the DNA barcoding method, it is now possible to efficiently identify species in a shorter time. In this study, the genetic diversity of 76 Dendrobium samples from Southern Vietnam was investigated based on the ITS (Internal transcribed spacer), ITS2, matK (Maturase_K), rbcL (ribulose-bisphosphate carboxylase large subunit) and trnH-psbA (the internal space of the gene coding histidine transfer RNA (trnH) and gene coding protein D1, a polypeptide of the photosystem I reaction center (psaB)) regions. The ITS region was found to have the best identification potential. Nineteen out of 24 Dendrobium species were identified based on phylogenetic tree and Indel information of this region. Among these, seven identified species were used as medicinal herbs. The results of this research contributed to the conservation, propagation, and hybridization of indigenous Dendrobium species in Southern Vietnam.

A DNA barcode survey of marine macroalgae from Bergen (Norway)

ABSTRACTSafeguarding the services provided by marine forests inherently depends on an accurate understanding of macroalgal species diversity. Here, we provide the first DNA barcode survey of marine macroalgae from Norway, with a focus on the Bergen area, and compared our findings to morphological listings for the corresponding location. Specimens were sampled 14–20 April and 3–13 June 2016, and variously sequenced for several genetic markers, including the five prime end of the cytochrome c oxidase subunit I gene (COI-5P), elongation factor tufA, and the ribulose-1, 5-biphosphate carboxylase large subunit gene (rbcL). We generated 655 new barcode records for COI-5P, 11 for tufA, and 50 for rbcL, representing nine species of Chlorophyta, 51 species of Phaeophyceae, and 74 species of Rhodophyta. Sequence data confirmed the presence of 113 morphological species. A further 17 genetic groups indicated the presence of new species for the area, only six of which were linked to formally described species. The remaining four genetic records were uncertain in terms of morphological species assignment and relation to the local flora. We recommend further DNA barcoding surveys in the area, as only a third of the listed morphological species were genetically confirmed.

Development of a next generation DNA sequencing-based multi detection assay for detecting and identifying Leishmania parasites, blood sources, plant meals and intestinal microbiome in phlebotomine sand flies

Leishmaniasis is a disease caused by Leishmania parasites transmitted by phlebotomine sand flies (Diptera: Psychodidae). Human infections with different Leishmania species cause characteristic clinical manifestations; cutaneous or visceral leishmaniasis. Here we describe the development and application of a Miseq Next GenerationSequencing (NGS)-based Multi Detection Assay (MDA) designed to characterize metagenomics parameters pertinent to the sand fly vectors which may affect their vectorial capacity for Leishmania. For this purpose, we developed a MDA by which, DNA fragments were amplified through polymerase chain reactions (PCR) and then sequenced by MiSeq/NGS. PCR amplification was achieved using some published and some new primers designed specifically for identifying Leishmania spp. (ITS1), sand fly spp. (cytochrome oxidase I), vertebrate blood (Cytochrome b), plant DNA ribulose-1,5-bisphosphate carboxylase large subunit gene (rbcL), and prokaryotic micobiome (16 s rRNA). This MDA/NGS analysis was performed on two species of wild-caught sand flies that transmit different Leishmania spp. in two ecologically distinct, but geographically neighboring locations. The results were analyzed to identify, quantitate and correlate the measured parameters in order to assess their putative importance in the transmission dynamics of leishmaniasis.

Amplification and Analysis of Rbcl Gene (Ribulose-1,5-Bisphosphate Carboxylase) of Clove in Ternate Island

DNA Barcoding is recommended as a tool for identifying and confirming species within the taxonomy framework. The rbcL gene is the barcode DNA for plant species. Ternate Island is one of clove plantation center in North Maluku. The diversity and productivity of cloves on the Ternate island known since in earlier times. This study has a purpose to amplify the rbcL gene in clove plants collected from clove plantations of communities in Ternate island. Isolation of total DNA carried out with using ZymoBiomic (Zymo Research DNA Extraction) Kit from leaves tissue, then DNA specimen is amplified based on rbcL gene with forwarding sequence rbcLaF 5′-ATG TCA CCA CAA ACA GAG ACT AAA GC-3′ and reverse sequence is rbcLaR 5′-GTA AAA TCA AGT CCA CCR CG-3′. The results showed that the specimen was successfully amplified with an amplicon size of 600 bp. Furthermore, BLASTN analysis results note that the sequence has a similarity of 99% with Syzygium cumini vc. J.R. Abbott 23676 (FLAS) ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) gene, but phylogenetically the precise position of sample could not found because the limitation of rbcL gene.

DNA barcoding of the marine macroalgae from Nome, Alaska (Northern Bering Sea) reveals many trans-Arctic species

Marine macroalgal species are expected to shift northward with temperature regimes in the wake of climate change. Monitoring ecosystem shifts in the Arctic will depend on accurate and consistent species level datasets from key locations. The Northern Bering Sea is an area with limited sampling, and will be critical to monitoring the potential movement of Pacific species into the Arctic. We therefore sampled the marine flora of Nome, Alaska, and surrounding area, August 12–22 (2017). Specimens were pressed and/or preserved in silica for DNA barcoding procedures. Several genetic markers were sequenced including the 5′ end of the cytochrome c oxidase subunit I gene (COI-5P; n = 551), elongation factor Tu (tufA; n = 41), full length reads of the internal transcribed spacer of the ribosomal cistron (ITS; n = 56), and full or partial reads of the ribulose-1, 5-biphosphate carboxylase large subunit (rbcL or rbcL-3P, respectively; n = 24). These collections represented eight species of green algae, 21 species of brown algae, and 24 species of red algae. Most of the species sampled (66%, n = 35) represented trans-Arctic species, 17% (n = 9) represented species with distributions restricted to the Pacific, 8% (n = 4) represented species restricted to the Arctic, and the remaining 9% (n = 5) represented previously unsampled genetic groups. Our survey revealed that the marine flora of Nome, Alaska, is largely contiguous with the North-west Atlantic. Repeated sampling from Nome and other key locations in the Northern Bering Sea (e.g., St. Lawrence Island) will be critical to further establishing baseline data for monitoring ecosystem shifts in an era of climate change.

Trans‐Arctic speciation of Florideophyceae (Rhodophyta) since the opening of the Bering Strait, with consideration of the “species pump” hypothesis

AbstractAimThe opening of the Bering Strait initiated significant biotic interchange that is postulated to have played a major role in phylogeographical patterns of northern marine flora and fauna. In addition, the “species pump” hypothesis asserts that glaciation events promoted speciation due to repeated isolation of populations over the past 2.6 million years. Here, trans‐Arctic speciation events in red marine macroalgae (Florideophyceae) were assessed using time‐calibrated phylogenies, and the applicability of the “species pump” hypothesis was considered.LocationSpecies records and sequence data for trans‐arctic genera of marine macroalgae were amalgamated and supplemented with sampling from the Northwest Atlantic, Northern Alaska (Beaufort Sea), Norway, and Nome, Alaska (Bering Sea; 2014–2017).MethodsBayesian and maximum likelihood phylogenies were variously built using the 5′ end of the cytochrome c oxidase subunit I gene (COI‐5P), and/or the full‐length nuclear internal transcribed spacer region (ITS), and/or the ribulose‐1 5‐biphosphate carboxylase large subunit gene (rbcL), and nodes were timed using calibrated COI‐5P and rbcL molecular clocks. The final dataset represented approximately 184 species, broadly representing 14 trans‐arctic lineages.ResultsPacific to Atlantic migration and subsequent speciation was inferred in 11 cases, whereas the opposite scenario, atlantic to pacific, was inferred once; only three speciation events appeared to occur during the Pleistocene.Main conclusionsOur results are in agreement with previous studies in that trans‐arctic speciation events postdated the opening of the Bering Strait with a clear pacific to atlantic bias. Evidence for the “species pump” (as applied to trans‐arctic interchange) was lacking given the frequency of trans‐arctic speciation events was not amplified during the Pleistocene.

Molecular characterization and in vitro interaction analysis of Op14-3-3 μ protein from Opuntia ficus-indica: identification of a new client protein from shikimate pathway

In plants, 14-3-3 proteins are important modulators of protein-protein interactions in response to environmental stresses. The aim of the present work was to characterize one Opuntia ficus-indica 14-3-3 and get information about its client proteins. To achieve this goal, O. ficus-indica 14-3-3 cDNA, named as Op14-3-3 μ, was amplified by 3’-RACE methodology. Op14-3-3 μ contains an Open Reading Frame of 786 bp encoding a 261 amino acids protein. Op14-3-3 μ cDNA was cloned into a bacterial expression system and recombinant protein was purified. Differential Scanning Fluorimetry, Dynamic Light Scattering, and Ion Mobility-Mass Spectrometry were used for Op14-3-3 μ protein characterization, and Affinity-Purification-Mass Spectrometry analysis approach was used to obtain information about their potential client proteins. Pyrophosphate-fructose 6-phosphate 1-phosphotransferase, ribulose bisphosphate carboxylase large subunit, and vacuolar-type H+-ATPase were identified. Interestingly chorismate mutase p-prephenate dehydratase was also identified. Op14-3-3 μ down-regulation was observed in Opuntia calluses when they were induced with Jasmonic Acid, while increased accumulation of Op14-3-3 μ protein was observed. The putative interaction of 14-3-3 μ with chorismate mutase, which have not been reported before, suggest that Op14-3-3 μ could be an important regulator of metabolites biosynthesis and responses to stress in Opuntia spp. SignificanceOpuntia species are important crops in arid and semiarid areas worldwide, but despite its relevance, little information about their tolerance mechanism to cope with harsh environmental conditions is reported. 14-3-3 proteins have gained attention due to its participation as protein-protein regulators and have been linked with primary metabolism and hormones responses. Here we present the characterization of the first Opuntia ficus-indica 14-3-3 (Op14-3-3) protein using affinity purification-mass spectrometry (AP-MS) strategy. Op14-3-3 has high homology with other 14-3-3 from Caryophyllales. A novel Op14-3-3 client protein has been identified; the chorismate mutase p-prephenate dehydratase, key enzyme that links the primary with secondary metabolism. The present results open new questions about the Opuntia spp. pathways mechanisms in response to environmental stress and the importance of 14-3-3 proteins in betalains biosynthesis.

Molecular identification of Allium ochotense and Allium microdictyon using multiplex-PCR based on single nucleotide polymorphisms

Allium ochotense and Allium microdictyon are commonly known as ‘Mountain garlic’ and are popular, economically important species in many countries such as Korea, China, and Mongolia. Their leaves are used as culinary side dishes and in traditional medicines. In Korea, these two species are at risk of extinction due to damage to their natural habitat and thus, conservation and breeding programs are needed. However, their identification relies mostly on morphological data, which is limited and until recently, led to classifying these two species under A. victorialis. In the present study, a simple and reliable method of molecular identification was developed to distinguish A. ochotense from A. microdictyon that targets four barcoding regions: the internal transcribed spacer (ITS), the maturase K gene (matK), the chloroplast psbA-trnH intergenic region, and the ribulose-bisphosphate carboxylase large subunit gene (rbcL). Single nucleotide polymorphisms (SNPs) were found in ITS and matK regions, and species-specific primers were designed based solely on the SNP at position 680 of the ITS region that could differentiate A. ochotense from A. microdictyon. Using these primers in amplification refractory mutation system (ARMS)-PCR, A. ochotense, and A. microdictyon could be simultaneously and efficiently distinguished. This study is the first to report a simple, rapid, and efficient method for discriminating A. ochotense and A. microdictyon, indicating the utility of species-specific markers in the development of conservation and breeding programs.

Validation of reference genes for qRT-PCR data normalisation in lentil (Lens culinaris) under leaf developmental stages and abiotic stresses.

Lentil (Lens culinaris) is one of the most important staple food crops of developing countries. Transcriptome based global gene expression profiling followed by validation of expression of important genes through quantitative real time-PCR (qRT-PCR) has achieved significance in recent years. However, there is a severe scarcity of information regarding stable reference genes in lentil, which is mandatory for qRT-PCR data normalisation. Hence, the present study was under-taken to identify the most stable reference gene(s) in lentil. Expression stability of eight candidate genes viz. ribulose 1,5-bisphosphate carboxylase large subunit (Rbcl), ribosomal protein L2 (RPL2), 18S rRNA, tubulin (Tub), elongation factor 1α (EF1α), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), heat shock protein (HSP70), and Maturase (mat K) was evaluated in five varieties of lentil at three different stages of leaf development and abiotic stress conditions using qRT-PCR. The results were analysed using four types of statistical software viz., geNorm, BestKeeper, NormFinder and RefFinder; all softwares identified RPL2 as most stable under abiotic stress conditions and developmental stages followed by Tub and Rbcl; while, HSP70 was identified as least stable. Relative expression of the target genes, defensin and PR4, was evaluated under abiotic stress conditions and data normalisation was done using two stable reference genes, RPL2 and Tub, either alone or in combination and with two least stable genes, HSP70 and 18S. The present work provides a list of potential reference genes in lentil, which will help in selection of appropriate reference gene for qRT-PCR data normalization depending upon the experiment.

Remodeling of chloroplast proteome under salinity affects salt tolerance of Festuca arundinacea.

Acclimation of photosynthetic apparatus to variable environmental conditions is an important component of tolerance to dehydration stresses, including salinity. The present study deals with the research on alterations in chloroplast proteome of the forage grasses. Based on chlorophyll fluorescence parameters, two genotypes of a model grass species-Festuca arundinacea with distinct levels of salinity tolerance: low salt tolerant(LST) and high salt tolerant (HST), were selected. Next, two-dimensional electrophoresis and mass spectrometry were applied under both control and salt stress conditions to identify proteins accumulated differentially between these two genotypes. The physiological analysis revealed that under NaCl treatment the studied plants differed in photosystem II activity, water content, and ion accumulation. The differentially accumulated proteins included ATPase B, ATP synthase, ribulose-1,5-bisphosphate carboxylase large and small subunits, cytochrome b6-f complex iron-sulfur subunit, oxygen-evolving enhancer proteins (OEE), OEE1 and OEE2, plastidic fructose-bisphosphate aldolase (pFBA), and lipocalin. A higher level of lipocalin, potentially involved in prevention of lipid peroxidation under stress, was also observed in the HST genotype. Our physiological and proteomic results performed for the first time on the species of forage grasses clearly showed that chloroplast metabolism adjustment could be a crucial factor in developing salinity tolerance.