Standardization of a screening technique for salinity tOlerance' in groundnut (Arachis hypogaea L.) and pigeonpea (Cajanus cajan L.)

Salinity affects plant growth, development and yield In approximately 100 M ha of arable land worldwide. Besides, various management options available the Introduction of salinity tolerant varieties In such areas could partly ease the Increasing global food demand. Here, six groundnut (ICG (FDRS) 10, ICGS 44, ICGS ICGV 86031, JL 24, and TAG 24) and pigeonpea (ICPL 88039, ICPL 88034, ICPL 87119, ICPL 96058,ICP 7035 and ICPL 366) genotypes were screened by conducting two experiments In soli treated with five different NaCI (mM) concentrations (0, 50, 100, 125, 150) and (0,50, 75, 100, 150) respectively for groundnut and Pigeonpea, under controlled conditions. Salt concentrations of 100-125 mM were found to be critical to screen groundnut genotypes whereas 75 mM NaCI appeared most suited treatmen

Genetic analysis of resistance to late leaf spot in interspecific groundnuts

Late leaf spot (LLS), caused by Phaeoisariopsis personata, is an important foliar fungal disease of groundnut (Arachis hypogaea L.), which causes significant economic losses globally to the crop. Inheritance of resistance to LLS disease was studied in three crosses and their reciprocals involving two resistant interspecific derivatives and a susceptible cultivar to refine strategy for LLS resistance breeding. The traits associated with LLS resistance, measured both in the field and under controlled conditions were studied following generation mean analysis. Results suggested that resistance to LLS is controlled by a combination of both, nuclear and maternal gene effects. Among nuclear gene effects, additive effect controlled majority of the variation. In JL 24 × ICG 11337 cross and its reciprocal only additive effects were important, while in JL 24 × ICG 13919 cross and its reciprocal, both additive and dominance effects contributed to the variation. Among digenic epistatic effects, additive × dominance interactions were significant. Additive–maternal effects were significant in both the crosses, while dominance–maternal effects also contributed to the variation in the crosses between the parents, JL 24 and ICG 13919. Due to significant contribution of additive effects of both nuclear and maternal inheritance to resistance to LLS, the parent, ICG 11337 would be a good donor in breeding programs. It would be worthwhile to use the resistance donor as female parent to tap maternal effects of resistance to LLS. Disease score is the best selection criterion in the field for use in breeding programs because of its high heritability and ease in measurement

Functional genomics of corrinoid starvation in the organohalide-respiring bacterium Dehalobacter restrictus strain PER-K23

De novo corrinoid biosynthesis represents one of the most complicated metabolic pathways in nature. Organohalide-respiring bacteria (OHRB) have developed different strategies to deal with their need of corrinoid, as it is an essential cofactor of reductive dehalogenases, the key enzymes in OHR metabolism. In contrast to Dehalococcoides mccartyi, the genome of Dehalobacter restrictus strain PER-K23 contains a complete set of corrinoid biosynthetic genes, of which cbiH appears to be truncated and therefore non-functional, possibly explaining the corrinoid auxotrophy of this obligate OHRB. Comparative genomics within Dehalobacter spp. revealed that one (operon-2) of the five distinct corrinoid biosynthesis associated operons present in the genome of D. restrictus appeared to be present only in that particular strain, which encodes multiple members of corrinoid transporters and salvaging enzymes. Operon-2 was highly up-regulated upon corrinoid starvation both at the transcriptional (346-fold) and proteomic level (46-fold on average), in line with the presence of an upstream cobalamin riboswitch. Together, these data highlight the importance of this operon in corrinoid homeostasis in D. restrictus and the augmented salvaging strategy this bacterium adopted to cope with the need for this essential cofacto

Corrinoid auxotrophy in the obligate organohalide respiring Dehalobacter restrictus

Background Corrinoids are an essential cofactor of reductive dehalogenases, the key enzymes of the environmental-friendly process of organohalide respiration (OHR). Dehalobacter restrictus strain PER-K23 is an obligate OHR bacterium (OHRB) able to conserve energy with tetrachloroethene, but is unable to de novo synthesize corrinoids (1). Genome analysis of D. restrictus however revealed the presence of the complete corrinoid biosynthesis pathway (2,3). Objectives The aim of the present study is to understand the corrinoid metabolism of D. restrictus at the level of biosynthesis, regulation and transport and to compare it to contrasting situations in other OHR bacteria. Methods Genome analysis was performed with standard bioinformatic tools. Both transcriptomic and proteomic approaches were applied on D. restrictus cells cultivated in media with alternative corrinoid conditions. Gene expression was further addressed using targeted reverse transcription and quantitative PCR. Conclusions Annotation and analysis of genes involved in corrinoid metabolism revealed a 101-bp deletion in the cbiH gene resulting in a shift of the reading frame and leading to a non-functional enzyme. This mutation, which is not present in the genome of other Dehalobacter spp., indicates that cbiH represents a possible checkpoint behind corrinoid auxotrophy. The expression of most corrinoid biosynthetic genes is likely to be controlled by cobalamin riboswitches. Experimental evidence of this latter mechanism is under scrutiny. Comparative ’omics’ analyses of corrinoid-starved cells revealed an increased production of corrinoid transporters and proteins involved in corrinoid salvaging. Taken together, these data suggest that D. restrictus has recently lost its capacity of de novo corrinoid synthesis. References (1) Holliger et al. (1998), Arch Microbiol 169, 313. (2) Kruse et al. (2013), Stand Genomic Sci, submitted. (3) Rupakula et al. (2013), R Soc Phil Trans B, in press

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

Dehalobacter restrictus strain PER-K23 is an obligate organohalide respiring bacterium, which displays extremely narrow metabolic capabilities. It grows only via coupling energy conservation to anaerobic respiration of tetra- and trichloroethene with hydrogen as sole electron donor. Dehalobacter restrictus represents the paradigmatic member of the genus Dehalobacter, which in recent years has turned out to be a major player in the bioremediation of an increasing number of organohalides, both in situ and in laboratory studies. The recent elucidation of the D. restrictus genome revealed a rather elaborate genome with predicted pathways that were not suspected from its restricted metabolism, such as a complete corrinoid biosynthetic pathway, the Wood-Ljungdahl (WL) pathway for CO fixation, abundant transcriptional regulators and several types of hydrogenases. However, one important feature of the genome is the presence of 25 reductive dehalogenase genes, from which so far only one, pceA, has been characterized on genetic and biochemical levels. This study describes a multi-level functional genomics approach on D. restrictus across three different growth phases. A global proteomic analysis allowed consideration of general metabolic pathways relevant to organohalide respiration, whereas the dedicated genomic and transcriptomic analysis focused on the diversity, composition and expression of genes associated with reductive dehalogenase

Genetic analysis of resistance to late leaf spot in interspecific groundnuts

Late leaf spot (LLS), caused by Phaeoisariopsis personata, is an important foliar fungal disease of groundnut (Arachis hypogaea L.), which causes significant economic losses globally to the crop. Inheritance of resistance to LLS disease was studied in three crosses and their reciprocals involving two resistant interspecific derivatives and a susceptible cultivar to refine strategy for LLS resistance breeding. The traits associated with LLS resistance, measured both in the field and under controlled conditions were studied following generation mean analysis. Results suggested that resistance to LLS is controlled by a combination of both, nuclear and maternal gene effects. Among nuclear gene effects, additive effect controlled majority of the variation. In JL 24 × ICG 11337 cross and its reciprocal only additive effects were important, while in JL 24 × ICG 13919 cross and its reciprocal, both additive and dominance effects contributed to the variation. Among digenic epistatic effects, additive × dominance interactions were significant. Additive–maternal effects were significant in both the crosses, while dominance–maternal effects also contributed to the variation in the crosses between the parents, JL 24 and ICG 13919. Due to significant contribution of additive effects of both nuclear and maternal inheritance to resistance to LLS, the parent, ICG 11337 would be a good donor in breeding programs. It would be worthwhile to use the resistance donor as female parent to tap maternal effects of resistance to LLS. Disease score is the best selection criterion in the field for use in breeding programs because of its high heritability and ease in measurement

Proteome analysis of the response of Dehalobacter restrictus to cobalamin starvation

Dehalobacter restrictus strain PER-K23 is an obligate organohalide respiring microorganism known to solely use tetra- or trichloroethene as electron acceptors with H2 as electron donor. The catalytic enzyme, PceA, contains a corrinoid as cofactor, a trait believed to be general for reductive dehalogenases. Genomic studies showed the presence of both anaerobic cobalamin biosynthesis and cobinamide salvaging pathways, however, no growth was found without exogenous cobalamin supply. This observation is most likely due to a deletion in precorrin-3B C17-methyltransferase encoding gene cbiH (Rupakula et al., 2013, Philos Trans R Soc Lond B Biol Sci 368: 1616). In order to determine whether D. restrictus utilizes a salvaging or de novo synthesis strategy to obtain essential corrinoids under partial cobalamin starved conditions, D. restrictus was cultivated in the presence of either 250 (high), 50 (mid) or 10 (low) µg/l of cyanocobalamin. Presence and relative abundance of corrinoid-relevant proteins under the tested growth conditions was studied using shotgun proteomics. A total of 1195 proteins were detected from D. restrictus, corresponding to 42% of 2826 predicted protein coding genes in its genome. We detected CbiJ (precorrin-6x reductase, Dehre_0277), Cbi[ET] (precorrin-6Y methyltransferase, Dehre_2849) and CbiD (cobalamin biosynthesis protein, Dehre_2861) from upper cobalamin biosynthetic pathway, which were not detected in the previously analyzed proteome. However, several proteins belonging to the corrinoid synthesis pathway including CobS (cobalamin 5’-phosphate synthase, Dehre_1613), an alternative CbiJ (precorrin-6x reductase, Dehre_2855), CbiH (precorrin-3B C17-methyltransferase, Dehre_2856) and CbiG (cobalamin biosynthesis protein, Dehre_2858) were still missing from proteome. This suggests an incomplete cobalamin biosynthesis pathway that could explain why D. restrictus is incapable of de novo cobalamin biosynthesis. We identified proteins associated with corrinoid biosynthesis or salvaging pathways translated from five different operons. Proteins encoded by operon 2 showed the highest production levels with on average 45.7 fold up-regulation at low versus high cobalamin concentration, and included proteins predicted as members of corrinoid salvaging pathway and as corrinoid transporters. CbiZ proteins, amidohydrolase required for salvaging the cobalamin precursor cobinamide, showed up-regulation by 80 fold (Dehre_0285) and 58 fold (Dehre_0282) under cobalamin starvation, respectively. Furthermore, proteins encoded in operon 1 were on average 8.4 fold up-regulated between low and high cobalamin conditions, and included an ECF-type cobalt transporter (Dehre_0280), which is involved in the cobalt uptake process, and a precorrin-6x reductase (Dehre_0277). Genomic evidence suggests operon 1 and 2 being unique to D. restrictus in all so far available Dehalobacter strains, suggesting the response of operon 1 and 2 to changing cobalamin concentrations is exclusive to D. restrictus. In conclusion, proteomic data suggest that cobalamin biosynthesis in D. restrictus is non-functional and up-regulation of relevant cobalamin salvaging and transport pathways are the strategy employed to ensure sufficient amounts of corrinoids for growth

All CMOS Integrated 3D-Extended Metal Gate ISFETs for pH and Multi-Ion (Na+, K+, Ca2+) sensing

This paper reports for the first time, smart 3D-Extended-Metal-Gate Ion-Sensitive-Field-Effect-Transistors (3D-EMG-ISFETs), with unique figures of merit: (i) extremely-low-power (down to a record value of 2 pW per sensor under excellent linearity), (ii) all CMOS integrated, (iii) high performance pH and multi-ion (Na+, K+, Ca2+) sensing, and, (iv) uniquely low cross sensitivity experimentally proven. Detailed electrical DC and dynamic characterizations show excellent sensitivities (56.8 mV/pH, -58mV/dec for Na+, -49.5 mV/dec for K+, and -21.9 mV/dec for Ca2+) and high selectivity of each ion sensor against 4 different ions that usually coexist in biofluids, all achieved on same CMOS die. Furthermore, unprecedented results show that the threshold voltage (V-th) variability of such CMOS ISFET is reduced by 78 times. We report a V-th drift rate in liquid conditions of 0.67 mV/h, decreased by one order of magnitude compared to other state of the art CMOS ISFETs. Overall, the reported experimental achievements, supported by SPICE calibrated behavioral model simulations results shown in this paper, are expected to greatly enhance the predictability of high performance multi-analyte ISFETs, which is a big step towards ISFET sensor system mass production