AtGCS promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes-mediated transformation.

Agrobacterium rhizogenes-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma-glutamylcysteine synthetase promoter, termed AtGCSpro, with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus, and tomato roots. Using the pAtGCSpro-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p2 × 35Spro-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the pAtGCSpro-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.

Overexpression of γ-glutamylcysteine synthetase gene from Caragana korshinskii decreases stomatal density and enhances drought tolerance.

BACKGROUND: Gamma-glutamylcysteine synthetase (γ-ECS) is a rate-limiting enzyme in glutathione biosynthesis and plays a key role in plant stress responses. In this study, the endogenous expression of the Caragana korshinskii γ-ECS (Ckγ-ECS) gene was induced by PEG 6000-mediated drought stress in the leaves of C. korshinskii. and the Ckγ-ECS overexpressing transgenic Arabidopsis thaliana plants was constructed using the C. korshinskii. isolated γ-ECS. RESULTS: Compared with the wildtype, the Ckγ-ECS overexpressing plants enhanced the γ-ECS activity, reduced the stomatal density and aperture sizes; they also had higher relative water content, lower water loss, and lower malondialdehyde content. At the same time, the mRNA expression of stomatal development-related gene EPF1 was increased and FAMA and STOMAGEN were decreased. Besides, the expression of auxin-relative signaling genes AXR3 and ARF5 were upregulated. CONCLUSIONS: These changes suggest that transgenic Arabidopsis improved drought tolerance, and Ckγ-ECS may act as a negative regulator in stomatal development by regulating the mRNA expression of EPF1 and STOMAGEN through auxin signaling.

Evolutionary history and genetic diversity of apomictic allopolyploids in Hieracium s.str.: morphological versus genomic features.

PREMISE: The origin of allopolyploids is believed to shape their evolutionary potential, ecology, and geographical ranges. Morphologically distinct apomictic types sharing the same parental species belong to the most challenging groups of polyploids. We evaluated the origins and variation of two triploid taxa (Hieracium pallidiflorum, H. picroides) presumably derived from the same diploid parental pair (H. intybaceum, H. prenanthoides). METHODS: We used a suite of approaches ranging from morphological, phylogenetic (three unlinked molecular markers), and cytogenetic analyses (in situ hybridization) to genome size screening and genome skimming. RESULTS: Genotyping proved the expected parentage of all analyzed accessions of H. pallidiflorum and H. picroides and revealed that nearly all of them originated independently. Genome sizes and genome dosage largely corresponded to morphology, whereas the maternal origin of the allopolyploids had no discernable effect. Polyploid accessions of both parental species usually contained genetic material from other species. Given the phylogenetic distance of the parents, their chromosomes appeared only weakly differentiated in genomic in situ hybridization (GISH), as well as in overall comparisons of the repetitive fraction of their genomes. Furthermore, the repeatome of a phylogenetically more closely related species (H. umbellatum) differed significantly more. CONCLUSIONS: We proved (1) multiple origins of hybridogeneous apomicts from the same diploid parental taxa, and (2) allopolyploid origins of polyploid accessions of the parental species. We also showed that the evolutionary dynamics of very fast evolving markers such as satellite DNA or transposable elements does not necessarily follow patterns of speciation.

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity.

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.

Buthionine sulfoximine is a multitarget inhibitor of trypanothione synthesis in Trypanosoma cruzi.

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

Clinical and molecular characterization of 6 children with glutamate-cysteine ligase deficiency causing hemolytic anemia.

Glutathione (gamma-glutamylcysteinylglycine) has diverse functions including free radicals scavenging and modulating many critical cellular processes. Glutathione is synthesized by the consecutive action of the enzymes glutamate-cysteine ligase (GCL) and glutathione synthetase. GCL is composed of a catalytic subunit encoded by the GCLC gene and a regulatory subunit encoded by the GCLM gene. GCL deficiency due to homozygous mutations in GCLC has been reported in 6 individuals from 4 independent families. All presented with hemolytic anemia and 4 had additional neurological manifestations including cognitive impairment, neuropathy, ataxia, and myopathy. In this report, we present additional 6 children from 2 independent consanguineous families with GCL deficiency. All the children presented with neonatal hemolytic anemia. Beyond the neonatal period, they did not have jaundice or hemolysis, but continued to have mild anemia. They all had normal development and neurological examination. The affected children from the first family had the homozygous mutation c.1772G>A (p.S591N) and the second family had the homozygous mutation c.514T>A (p.S172T) in GCLC. GCL deficiency can have a mild non-neurological phenotype or a more severe phenotype with neurological manifestations. GCL deficiency can be an underdiagnosed cause of hemolytic anemia, thus awareness may aid in early diagnosis, appropriate genetic counseling, and management.

Ornithine decarboxylase or gamma-glutamylcysteine synthetase overexpression protects Leishmania (Vianna) guyanensis against antimony.

Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (SbIII)-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in SbIII-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of SbIII in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to SbIII than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to SbIII, and confirms a role of these genes in the SbIII-resistance phenotype.

Whey protein increases muscle weight gain through inhibition of oxidative effects induced by resistance exercise in rats.

Whey protein (WP) is known for its nutritional value and antioxidant properties. The aim of this study was to evaluate whether the antioxidant properties of WP could contribute to muscle weight gain in response to resistance exercise (RE). We hypothesized that WP ingestion could increase muscle weight gain in rats subjected to an RE program, through inhibition of oxidative effects induced by high-intensity RE. Thirty-two male Fischer rats were randomly assigned to control sedentary, control exercised, WP sedentary, and WP exercised groups (n=8/group). The RE consisted of inducing the rats to perform sets of jumps for 8 weeks. Body and muscle weight gains, muscle glutathione content, histopathology, muscle antioxidant enzyme activities, and gene expression were evaluated. Body and muscle weight gains of exercised rats fed WP were higher than those of control exercised rats. Concomitantly, RE induced an increase in phagocyte infiltration, protein oxidation, and down-regulation of glutathione peroxidase and gamma-glutamylcysteine synthetase messenger RNA expression in gastrocnemius muscle (P<.05), effects that were inhibited by WP ingestion. Cytosolic superoxide dismutase and catalase messenger RNA expression were reduced only by RE (P<.05), and muscle glutathione content was increased only by WP (P<.05) with no significant interaction observed (P>.05). These findings suggest that differences in body and muscle weight gain in exercised rats fed control or WP diets were mediated, in part, by the antioxidant properties of WP, and indicate that when associated with RE, WP represents a nutritional aid to support muscle growth.