LCIB functions as a carbonic anhydrase: evidence from yeast and Arabidopsis carbonic anhydrase knockout mutants.

Chlamydomonas reinhardtii evolved a CO2-concentrating mechanism (CCM) because of the limited CO2 in its natural environment. One critical component of the C. reinhardtii CCM is the limiting CO2 inducible B (LCIB) protein. LCIB is required for acclimation to air levels of CO2. C. reinhardtii cells with a mutated LCIB protein have an 'air-dier' phenotype when grown in low CO2 conditions, meaning they die in air levels of CO2 but can grow in high and very low CO2 conditions. The LCIB protein functions together with its close homolog in C. reinhardtii, limiting CO2 inducible C protein (LCIC), in a hexameric LCIB-LCIC complex. LCIB has been proposed to act as a vectoral carbonic anhydrase (CA) that helps to recapture CO2 that would otherwise leak out of the chloroplast. Although both LCIB and LCIC are structurally similar to ßCAs, their CA activity has not been demonstrated to date. We provide evidence that LCIB is an active CA using a Saccharomyces cerevisiae CA knockout mutant (∆NCE103) and an Arabidopsis thaliana ßCA5 knockout mutant (ßca5). We show that different truncated versions of the LCIB protein complement ∆NCE103, while the full length LCIB protein complements ßca5 plants, so that both the yeast and plant mutants can grow in low CO2 conditions.

The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

Bacteria and Archaea diversity within the hot springs of Lake Magadi and Little Magadi in Kenya.

BACKGROUND: Lake Magadi and little Magadi are hypersaline, alkaline lakes situated in the southern part of Kenyan Rift Valley. Solutes are supplied mainly by a series of alkaline hot springs with temperatures as high as 86 °C. Previous culture-dependent and culture-independent studies have revealed diverse groups of microorganisms thriving under these conditions. Previous culture independent studies were based on the analysis of 16S rDNA but were done on less saline lakes. For the first time, this study combined illumina sequencing and analysis of amplicons of both total community rDNA and 16S rRNA cDNA to determine the diversity and community structure of bacteria and archaea within 3 hot springs of L. Magadi and little Magadi. METHODS: Water, wet sediments and microbial mats were collected from springs in the main lake at a temperature of 45.1 °C and from Little Magadi "Nasikie eng'ida" (temperature of 81 °C and 83.6 °C). Total community DNA and RNA were extracted from samples using phenol-chloroform and Trizol RNA extraction protocols respectively. The 16S rRNA gene variable region (V4 - V7) of the extracted DNA and RNA were amplified and library construction performed following Illumina sequencing protocol. Sequences were analyzed done using QIIME while calculation of Bray-Curtis dissimilarities between datasets, hierarchical clustering, Non Metric Dimensional Scaling (NMDS) redundancy analysis (RDA) and diversity indices were carried out using the R programming language and the Vegan package. RESULTS: Three thousand four hundred twenty-six and one thousand nine hundred thirteen OTUs were recovered from 16S rDNA and 16S rRNA cDNA respectively. Uncultured diversity accounted for 89.35 % 16S rDNA and 87.61 % 16S rRNA cDNA reads. The most abundant phyla in both the 16S rDNA and 16S rRNA cDNA datasets included: Proteobacteria (8.33-50 %), Firmicutes 3.52-28.92 %, Bacteroidetes (3.45-26.44 %), Actinobacteria (0.98-28.57 %) and Euryarchaeota (3.55-34.48 %) in all samples. NMDS analyses of taxonomic composition clustered the taxa into three groups according to sample types (i.e. wet sediments, mats and water samples) with evident overlap of clusters between wet sediments and microbial mats from the three sample types in both DNA and cDNA datasets. The hot spring (45.1 °C) contained less diverse populations compared to those in Little Magadi (81-83 °C). CONCLUSION: There were significant differences in microbial community structure at 95 % level of confidence for both total diversity (P value, 0.009) based on 16S rDNA analysis and active microbial diversity (P value, 0.01) based on 16S rRNA cDNA analysis, within the three hot springs. Differences in microbial composition and structure were observed as a function of sample type and temperature, with wet sediments harboring the highest diversity.

BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes.

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication.

Endoreplication controls cell fate maintenance.

Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level.

A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae.

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur−Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.

Screening for antifolate and artemisinin resistance in Plasmodium falciparum dried-blood spots from three hospitals of Eritrea.

Background: Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea's population resides in the malaria-endemic western lowlands of the country. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with P. falciparum resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea. Methods: Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800) and PfK13 (PF3D7_1343700) partial gene regions. Results: Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant Pfdhfr (S108N, C59R, N51I) haplotype, the double-mutant Pfdhfr (N51I, S108N) haplotype, the single-mutant Pfdhfr (K540E) haplotype, and the mixed-mutant Pfdhfr-Pfdhps (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous Pfdhfr V45A mutation in four isolates, and a synonymous Pfdhps R449R in one isolate. Conclusions: The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.

Origin, Genetic Diversity, and Population Structure of Rabbits (Oryctolagus cuniculus) in Kenya.

To evaluate the origin, genetic diversity, and population structure of domesticated rabbits in Kenya, a 263-base pair region of mtDNA D-loop region of 111 rabbits sampled from Kakamega, Vihiga, and Bungoma counties in the western region, Laikipia and Nyandarua counties in the central region, and Kitui, Machakos, and Makueni in the eastern region of the country were analyzed. The average haplotype (0.40702) and nucleotide (0.01494) diversities observed were low, indicating low genetic diversity of domesticated rabbits in Kenya. This study resolved 5 unique haplotypes in the mtDNA D-loop region. A population genetic structure distinguishing Europe grouping and domesticated rabbits in Kenya was obtained on incorporating 32 known haplotypes. Domesticated rabbits in Kenya clustered together with rabbits from other geographic regions, suggesting common origin. The results suggested that the Kenyan domesticated rabbits may have originated from Europe. Integration of exotic breeds into breeding programmes could have contributed to the low genetic diversity. These results provide useful information for breeding and conservation decisions by the relevant stakeholders in the agriculture industry in Kenya.

Constitutive Expressor Of Pathogenesis-related Genes5 affects cell wall biogenesis and trichome development.

BACKGROUND: The Arabidopsis thaliana CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene has been previously implicated in disease resistance, cell proliferation, cell death, and sugar sensing, and encodes a putative membrane protein of unknown biochemical function. Trichome development is also affected in cpr5 plants, which have leaf trichomes that are reduced in size and branch number. RESULTS: In the work presented here, the role of CPR5 in trichome development was examined. Trichomes on cpr5 mutants had reduced birefringence, suggesting a difference in cell wall structure between cpr5 and wild-type trichomes. Consistent with this, leaf cell walls of cpr5 plants contained significantly less paracrystalline cellulose and had an altered wall carbohydrate composition. We also found that the effects of cpr5 on trichome size and endoreplication of trichome nuclear DNA were epistatic to the effects of mutations in triptychon (try) or overexpression of GLABRA3, indicating that these trichome developmental regulators are dependant on CPR5 function for their effects on trichome expansion and endoreplication. CONCLUSION: Our results suggest that CPR5 is unlikely to be a specific regulator of pathogen response pathways or senescence, but rather functions either in cell wall biogenesis or in multiple cell signaling or transcription response pathways.

Development stage, storage temperature and storage duration influence phytonutrient content in cowpea (Vigna unguiculata L. Walp.).

Cowpea (Vigna unguiculata) plays an important role in sustainable food security and livelihood improvement in Sub-Saharan Africa (SSA). The crop is rich in phytonutrients and minerals, which are key in solving malnutrition and hunger crisis, a major challenge in SSA. However, physiological status, storage temperature and duration affect phytonutrient levels and postharvest life of the leafy vegetable. Despite the significant importance of cowpeas, the maturity and postharvest storage effects on quality of the leafy vegetable remains unrevealed. The aim of this study was to analyze the dynamics of phytonutrients in cowpea leaves during development under field conditions in Kenya and in storage. The total carbohydrates (glucose, fructose, sucrose and starch) were highest at 90 d after planting (105.9 ± 2.5 g kg-1) compared to 30, 60 and 120 d. The total Phenolics (Gallic acid equivalents) increased gradually with age up to 12.0 ± 0.2 g kg-1 by 120 d. Catechin equivalent flavonoids, trolox equivalent antioxidants (TEA) and chlorophyll were highest in concentrations at 60 d after planting with 8.0 ± 0.5 g kg-1, 26.19 ± 0.5 g kg-1 and 5.7 ± 0.4 g kg-1, respectively. Quercetin equivalent flavonoids and total carotenoids did not show significant changes with age, while mineral concentration dynamics were specific for each element. Storage of cowpea leaves at room temperature (50-55 % relative humidity) led to a stronger decline of phytonutrients after 4 d, but mostly they remained stable at cold storage (5 °C). Results of this study highlight the importance of developmental stage at harvest, storage conditions and duration for the optimal availability of phytonutrients in freshly consumed leaves and for postharvest management strategies.

SIAMESE cooperates with the CDH1-like protein CCS52A1 to establish endoreplication in Arabidopsis thaliana trichomes.

Endoreplication, also known as endoreduplication, is a phyogenetically widespread modified version of the cell cycle in which DNA replication is not followed by cell division. The SIAMESE (SIM) gene of Arabidopsis thaliana encodes the founding member of a novel class of plant-specific cyclin-dependent kinase (CDK) inhibitors and is a key regulator of endoreplication during the development of trichomes (shoot epidermal hairs). Here, we have identified mutations in the CCS52A1 gene as genetic modifiers of the multicellular trichome phenotype of sim mutants. Loss-of-function ccs52A1 mutations dramatically enhance the multicellularity of sim mutants trichomes in double mutants, whereas overexpression of CCS52A1 completely suppresses the sim mutant phenotype. CCS52A1 encodes a CDH1/FZR-like protein, a class of proteins that function as activators of the anaphase-promoting complex. Unicellular ccs52A1 trichomes become multicellular upon overexpression of B-type cyclin, consistent with repression of the accumulation of mitotic cyclins in the developing trichome by CCS52A1. As these M-phase-specific cyclins are known to accumulate in sim mutant trichomes, our data suggest that CCS52A1 and SIM cooperate in repressing accumulation of mitotic cyclins to establish the trichome endocycle. Comparison with endoreplication pathways in Drosophila and mammals indicates that while these organisms all use similar components to initiate endoreplication, the components are deployed differently in each organism.