Arabidopsis ERG28 Tethers the Sterol C4-Demethylation Complex to Prevent Accumulation of a Biosynthetic Intermediate That Interferes with Polar Auxin Transport

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development.     

A Phase 1b Randomized,Controlled, Double-Blinded Dosage-Escalation Trial to Evaluate the Safety,Reactogenicity and Immunogenicity of an Adenovirus Type 35 Based Circumsporozoite Malaria Vaccine in Burkinabe Healthy Adults 18 to 45 Years of Age

Background

Ad35.CS.01 is a pre-erythrocytic malaria candidate vaccine. It is a codon optimized nucleotide sequence representing the P. falciparum circumsporozoite (CS) surface antigen inserted in a replication deficient Adenovirus 35 backbone. A Phase 1a trial has been conducted in the USA in naïve adults and showed that the vaccine was safe. The aim of this study is to assess the safety and immunogenicity of ascending dosages in sub Saharan Africa.

Methods

A double blind, randomized, controlled, dose escalation, phase Ib trial was conducted in a rural area of Balonghin, the Saponé health district (Burkina Faso). Forty-eight healthy adults aged 18-45 years were randomized into 4 cohorts of 12 to receive three vaccine doses (day 0, 28 and 84) of 10⁹, 10¹⁰, 5X10¹⁰, 10¹¹ vp of Ad35.CS.01 or normal saline by intra muscular injection. Subjects were monitored carefully during the 14 days following each vaccination for non serious adverse events. Severe and serious adverse events were collected throughout the participant study duration (12 months from the first vaccination). Humoral and cellular immune responses were measured on study days 0, 28, 56, 84, 112 and 140.

Results

Of the forty-eight subjects enrolled, forty-four (91.7%) received all three scheduled vaccine doses. Local reactions, all of mild severity, occurred in thirteen (27.1%) subjects. Severe (grade 3) laboratory abnormalities occurred in five (10.4%) subjects. One serious adverse event was reported and attributed to infection judged unrelated to vaccine. The vaccine induced both antibody titers and CD8 T cells producing IFNγ and TNFα with specificity to CS while eliciting modest neutralizing antibody responses against Ad35.

Conclusion

Study vaccine Ad35.CS.01 at four different dose levels was well-tolerated and modestly immunogenic in this population. These results suggest that Ad35.CS.01 should be further investigated for preliminary efficacy in human challenge models and as part of heterologous prime-boost vaccination strategies.

Trial Registration

ClinicalTrials.gov NCT01018459 http://clinicaltrials.gov/ct2/show/NCT01018459     

Nodulation of certain legumes of the genus Crotalaria by the new species Methylobacterium

We studied a collection of 126 rhizobial isolates from eight species of Crotalaria (C. comosa, C. glaucoides, C. goreensis, C. hyssopifolia, C. lathyroides, C. perrottetii, C. podocarpa, and C. retusa) growing in Senegal. Nodulation and nitrogen-fixation tests on nine Crotalaria species revealed two specificity groups within the genus Crotalaria. Group I consists of plants solely nodulated by very specific fast-growing strains. Group II plants are nodulated by slow-growing strains similar to promiscuous Bradyrhizobium spp. strains already reported to nodulate many tropical legumes. SDS-PAGE studies showed that slow-growing strains grouped with Bradyrhizobium while fast-growing strains constituted a homogeneous group distinct from all known rhizobia. Amplified ribosomal DNA restriction analysis (ARDRA) of 10 representative strains of this group using four restriction enzymes showed a single pattern for each enzyme confirming the high homogeneity of group I. The 16S rDNA sequence analysis revealed that this specific group belonged to the genus Methylobacterium, thus constituting a new branch of nodulating bacteria.     

In situ calibration and [H+] sensitivity of the fluorescent Na+ indicator SBFI

Despite the popularity of Na+-binding benzofuran isophthalate (SBFI) to measure intracellular free Na+ concentrations ([Na+](i)), the in situ calibration techniques described to date do not favor the straightforward determination of all of the constants required by the standard equation (Grynkiewicz G, Poenie M, and Tsien RY. J Biol Chem 260: 3440-3450, 1985) to convert the ratiometric signal into [Na+]. We describe a simple method in which SBFI ratio values obtained during a "full" in situ calibration are fit by a three-parameter hyperbolic equation; the apparent dissociation constant (K(d)) of SBFI for Na+ can then be resolved by means of a three-parameter hyperbolic decay equation. We also developed and tested a "one-point" technique for calibrating SBFI ratios in which the ratio value obtained in a neuron at the end of an experiment during exposure to gramicidin D and 10 mM Na+ is used as a normalization factor for ratios obtained during the experiment; each normalized ratio is converted to [Na+](i) using a modification of the standard equation and parameters obtained from a full calibration. Finally, we extended the characterization of the pH dependence of SBFI in situ. Although the K(d) of SBFI for Na+ was relatively insensitive to changes in pH in the range 6.8-7.8, acidification resulted in an apparent decrease, and alkalinization in an apparent increase, in [Na+](i) values. The magnitudes of the apparent changes in [Na+](i) varied with absolute [Na+](i), and a method was developed for correcting [Na+](i) values measured with SBFI for changes in intracellular pH.     

Spotted in the News: Using Media Reports to Examine Leopard Distribution,Depredation, and Management Practices outside Protected Areas in Southern India

There is increasing evidence of large carnivore presence outside protected areas, globally. Although this spells conservation success through population recoveries, it makes carnivore persistence in human-use landscapes tenuous. The widespread distribution of leopards in certain regions of India typifies this problem. We obtained information on leopard-human interactions at a regional scale in Karnataka State, India, based on systematic surveys of local media reports. We applied an innovative occupancy modelling approach to map their distribution patterns and identify hotspots of livestock/human depredation. We also evaluated management responses like removals of ‘problem’ leopards through capture and translocations. Leopards occupied around 84,000 km² or 47% of the State’s geographic area, outside designated national parks and wildlife sanctuaries. Their presence was facilitated by extent of vegetative cover- including irrigated croplands, rocky escarpments, and prey base in the form of feral and free-ranging dogs. Higher probabilities of livestock/human attacks by leopards were associated with similar ecological features as well as with capture/removals of leopards. Of the 56 cases of leopard removals reported, 91% did not involve human attacks, but followed livestock predation or only leopard sightings. The lack of knowledge on leopard ecology in human-use areas has resulted in unscientific interventions, which could aggravate the problem rather than mitigating it. Our results establish the presence of resident, breeding leopards in human-use areas. We therefore propose a shift in management focus, from current reactive practices like removal and translocation of leopards, to proactive measures that ensure safety of human lives and livelihoods.     

Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene β-cyclase (LCY-B), which converts lycopene into β-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of κ-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to β-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of β-carotene and κ-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of β-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.Later steps of carotenoid biosynthesis involve the formation of diverse cyclic carotenoids. For example, β-carotene, the vitamin A precursor, is synthesized de novo by photosynthetic organisms, limited nonphototrophic bacteria and fungi, and also by aphids (Moran and Jarvik, 2010) according to a multistep pathway that ends with the cyclization of lycopene by lycopene β-cyclase (LCY-B). Similarly, in pepper (Capsicum annuum) chromoplasts, antheraxanthin and violaxanthin are converted into the κ-cyclic carotenoids capsanthin and capsorubin, respectively, by capsanthin-capsorubin synthase (CCS). In both cases, the proposed mechanism involves a concerted protic attack and stabilization of a transient carbocation without any net redox change (Camara, 1980; Bouvier et al., 1994; Britton, 1998). Several cDNAs for LCY-B have been cloned from bacteria (Misawa et al., 1990; Cunningham et al., 1994; Armstrong, 1997; Cunningham and Gantt, 2001), fungi (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001), and plants (Hugueney et al., 1995; Ronen et al., 2000) using functional complementation. Information available from primary structures suggest that the cyclization of lycopene is catalyzed by holomeric proteins in photosynthetic organisms (Cunningham et al., 1994; Maresca et al., 2007), by holomeric (Misawa et al., 1990) or heteromeric (Krubasik and Sandmann, 2000; Viveiros et al., 2000) proteins in nonphotosynthetic bacteria, and by holomeric, bifunctional proteins in fungi that combine the activities of phytoene synthase and lycopene cyclase (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001). This structural diversity of LCY-Bs coupled to a lack of significant amino acid sequence identity between the lycopene cyclases from bacteria, fungi, and plants hinder our understanding of the catalytic mechanism of LCY-Bs and CCS. In addition, the N terminus of plant LCY-B and CCS contains an amino sequence motif characteristic of a polypeptide predicted to adopt a Rossmann fold (Rossmann et al., 1974) and suggests the binding of an as yet unknown dinucleotide prosthetic ligand. It has been shown using recombinant bacterial enzyme that the cyclization of lycopene into β-carotene strictly requires NADPH but proceeds without any net redox change (Schnurr et al., 1996; Hornero-Mendez and Britton, 2002). Under the same conditions, FAD alone could not sustain bacterial LCY-B activity (Schnurr et al., 1996). Much less is known about the dinucleotide requirements of plant carotenoid cyclases, which are highly conserved within plants but are extremely divergent in nonplant organisms. Previously, a crucial acidic domain for lycopene cyclase activity was identified using an affinity-labeling strategy followed by site-directed mutagenesis (Bouvier et al., 1997) in the absence of any crystal structures. This so-called 293-FLEET-297 motif of LCY-B and CCS contained two tandem Glu-295-Glu-296 residues that were essential for LCY-B- and κ-cyclase activities (Bouvier et al., 1997). However, it still remains unclear how the protic mechanism is compatible with the requirement of dinucleotide cofactors.To further explore the mechanism of plant carotenoid cyclases, we first choose pepper CCS as a prototypic enzyme because it displays a strong identity (52%) to pepper LCY-B, and we have shown previously that CCS could also catalyze the cyclization of lycopene into β-carotene (up to 25% of activity compared with LCY-B; Hugueney et al., 1995). Herein, we have shown that monomeric CCS purified to homogeneity from plant chromoplasts or recombinant CCS purified from Escherichia coli-transformed cells are typical flavoproteins containing one noncovalently bound FAD. We also observed that CCS-bound FAD is required for enzyme activity in the presence of NADPH, which functions as a reductant of FAD. During this process, no hydrogen is transferred to β-carotene or κ-cyclic carotenoids. In addition to this cofactor requirement, we also show from extensive site-directed mutagenesis using pepper CCS and LCY-B and Erwinia herbicola LCY-B (Mialoundama, 2009) that Glu-295 of pepper CCS and LCY-B plays a key role in the formation of β-carotene and κ-cyclic carotenoids, and we demonstrate that a similar role is played in structurally divergent bacterial LCY-Bs by Glu-196. These characteristics suggest that plant CCS and LCY-Bs are mechanistically similar to non-metal-assisted terpene cyclases, such as squalene:hopene cyclase and oxidosqualene cyclase, and additionally represent a new subfamily of flavoproteins like isopentenyl diphosphate isomerase type II, which catalyze carotenoid cyclization without any net redox modification of the substrate.     

Arbuscular mycorrhizal symbiosis can counterbalance the negative influence of the exotic tree species Eucalyptus camaldulensis on the structure and functioning of soil microbial communities in a sahelian soil

The hypothesis of the present study was that bacterial communities would differentiate under Eucalyptus camaldulensis and that an enhancement of arbuscular mycorrhizal (AM) density would minimize this exotic plant species effect. Treatments consisted of control plants, preplanting fertilizer application and AM inoculation. After 4 months of culture in autoclaved soil, E. camaldulensis seedlings were either harvested for growth measurement or transferred into containers filled with the same soil but not sterilized. Other containers were kept without E. camaldulensis seedlings. After 12 months, effects of fertilizer amendment and AM inoculation were measured on the growth of Eucalyptus seedlings and on soil microbial communities. The results clearly show that this plant species significantly modified the soil bacterial community. Both community structure (assessed by denaturing gradient gel electrophoresis profiles) and function (assessed by substrate-induced respiration responses including soil catabolic evenness) were significantly affected. Such changes in the bacterial structure and function were accompanied by disturbances in the composition of the herbaceous plant species layer. These results highlight the role of AM symbiosis in the processes involved in soil bio-functioning and plant coexistence and in afforestation programmes with exotic tree species that target preservation of native plant diversity.