Molecular Cloning and Characterization of γ-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694

γ-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydrolytic activity than transfer activity, as compared with other γ-glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard γ-glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a γ-glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.     

Somatic embryogenesis and plant regeneration from cotyledonary explants of green gram [Vigna radiata (L.) Wilezek.]—A recalcitrant grain legume

Summary This study reports a protocol for plant regeneration from cultured explants of green gram [Vigna radiata (L.) Wilezek] via somatic embryogenesis. Somatic embryos were induced from nature cotyledons of var., TAP-7 and Pusa Baisaki when cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid singly or in combination with 2.22–8.88 μM N ⁶-benzylaminopurine (BAP) or 2.32–9.38 μM kinetin. The type and concentration of auxin and plant genotype influenced the frequency of somatic embryogenesis. NAA was the most effective auxin for somatic embryo induction. The well-developed, cotyledonary shaped embryos of var. TAP-7 germinated into plantlets at a frequency of 56.6% on MS medium supplemented with 1.88 μM abscisic acid and 6.66 μM BAP. Regenerated plants were transferred to soil and grown to maturity with 90% survival. The protocol described here offers a good potential for genetic improvement using gene transfer techniques and the production of synthetic seeds of V. radiata.     

Purification and Characterization of γ-Hexachlorocyclohexane (γ-HCH) Dehydrochlorinase (LinA) from Pseudomonas paucimobilis

The linA gene from Pseudomonas paucimobilis was highly expressed in Escherichia coli, and the linA product (LinA), named γ-HCH dehydrochlorinase, was purified to homogeneity. LinA released three chloride ions per one molecule of γ-HCH. Degradation assay of halogenated compounds by purified LinA showed that the substrate specificity of LinA is very narrow.     

Effects of Exogenous γ-Aminobutyric Acid (GABA) on Photosynthesis and Antioxidant System in Pepper (Capsicum annuum L.) Seedlings Under Low Light Stress

The effects of γ-aminobutyric acid (GABA) treatment on parameters of photosynthesis and antioxidant defense system were measured in pepper (Capsicum annuum L.) leaves under low-light (LL) stress. Seedlings exposed to LL stress showed increased chlorophyll content as well as decreased net photosynthetic rate (P _n), stomatal conductance (g _s), maximum quantum yield of PSII (F _v/F _m), actual PSII photochemical efficiency (Φ_PSII), electron transport rates and photochemical quenching coefficient (q _p). However, almost all the photosynthetic parameters above were enhanced markedly in seedlings treated with GABA under LL stress. Moreover, LL stress increased malondialdehyde (MDA) content, superoxide anion radical (O₂ ^·?) and hydrogen peroxide (H₂O₂) production. GABA-treated, LL-stressed seedlings exhibited lower MDA, O₂ ^·? and H₂O₂ production, and showed an activated antioxidant defense system, including increased activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, ascorbate and glutathione. Moreover, seedlings subjected to LL stress showed increased endogenous GABA levels, and the level was further improved by application of exogenous GABA. These results suggest that GABA mitigates the LL-induced stress via regulating the antioxidant defense system and maintaining a high level of photochemical efficiency in pepper seedlings.

     

Molecular and chemical profiling of ‘sweet flag’ (Acorus calamus L.) germplasm from India

In the present study, molecular (DAMD and ISSR) and chemical (α and β-asarone contents) markers were used to characterize the A. calamus genotypes procured from different parts of India. The cumulative analysis carried out for both DAMD and ISSR markers revealed 24.71 % polymorphism across all genotypes of A. calamus. The clustering patterns of the genotypes in the UPGMA tree showed that the genotypes are diverse, and did not show any specific correlation with their geographical provenances, reflecting the low level of genetic diversity and a high genetic differentiation among the genotypes from the same localities. All the 27 genotypes of A. calamus were also analyzed for α and β-asarone contents, and percentage of essential oil. The genotype (Ac13) from Kullu (Himachal Pradesh) showed maximum (9.5 %) percentage of oil, whereas corresponding minimum (2.8 %) was obtained from the genotypes from Pangthang (Sikkim). Similarly, the highest α and β-asarone contents (16.82 % and 92.12 %) were obtained from genotypes from Renuka (Himachal Pradesh) and Udhampur (Jammu & Kashmir), while lowest α and β-asarone contents (0.83 % and 65.96 %) resulted from Auranwa (Uttar Pradesh) and Pangthang (Sikkim) genotypes, respectively. A. calamus harbours tremendous economic value, and it is therefore, important to identify the genotypes with low α and β-asarone contents for its commercial utilization. Further, this study will help in evaluation and documentation of a large number of diverse genotypes for their value traits.     

Molecular cloning and biochemical characterization of α- and β-tubulin from potato plants (Solanum tuberosum L.)

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) α-tubulin and β-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of α- and β-tubulin were 2.0–3.5 mg l^?1 and 1.3–3.0 mg l^?1 of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant α- and β-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.     

Combined Proteomic and Molecular Approaches for Cloning and Characterization of Copper–Zinc Superoxide dismutase (Cu, Zn-SOD2) from Garlic (Allium sativum)

Superoxide dismutases (SODs; EC 1.15.1.1) are key enzymes in the cells protection against oxidant agents. Thus, SODs play a major role in the protection of aerobic organisms against oxygen-mediated damages. Three SOD isoforms were previously identified by zymogram staining from Allium sativum bulbs. The purified Cu, Zn-SOD2 shows an antagonist effect to an anticancer drug and alleviate cytotoxicity inside tumor cells lines B16F0 (mouse melanoma cells) and PAE (porcine aortic endothelial cells). To extend the characterization of Allium SODs and their corresponding genes, a proteomic approach was applied involving two-dimensional gel electrophoresis and LC-MS/MS analyses. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 456?bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 152 residues. The deduced amino acid sequence showed high identity (82-87%) with sequences of Cu, Zn-SODs from other plant species. Molecular analysis was achieved by a protein 3D structural model.     

Source–Sink Adjustment: A Mechanistic Understanding of the Timing and Severity of Drought Stress on Photosynthesis and Grain Yields of Two Contrasting Oat (Avena sativa L.) Genotypes

Journal of Plant Growth Regulation - There is limited knowledge about the effects of the severity and timing of drought stress on oat (Avena sativa) yield and the critical stages at which water...     

Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.)

Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC–MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future.     

Inhibition of Abscisic Acid 8′-Hydroxylase Affects Dehydration Tolerance and Root Formation in Cuttings of Grapes (Vitis labrusca L. × Vitis vinifera L. cv. Kyoho) Under Drought Stress Conditions

The effects of an inhibitor (Abz-E3M) of abscisic acid (ABA) 8′-hydroxylase, which is a primary enzyme of ABA catabolism, on dehydration tolerance and root formation in grape cuttings under drought conditions were investigated. Cuttings of ‘Kyoho’ grape (Vitis labrusca L. × Vitis vinifera L.) were sprayed with 100 μM of Abz-E3M and subjected to water deficit conditions at the stage when their first leaves fully expanded. The physiological and morphological changes in the leaves and basal portions of the cuttings were determined. In Abz-E3M-treated leaves, lower ABA metabolite and higher ABA and indole-3-acetic acid (IAA) concentrations were observed. Compared to the untreated control leaves, higher water potential was significantly maintained in Abz-E3M-treated leaves. Abz-E3M applications resulted in lower proline accumulation and 2,2-diphenyl-2-picrylhydrazyl radical scavenging activity in the leaves and led to enhanced dehydration tolerance. In addition, the percentage of rooted cuttings was significantly increased by Abz-E3M application. In the basal portion of Abz-E3M-treated cuttings, endogenous IAA concentrations and the gene expressions of VvARF6 and VvARF8, which are positive regulators of adventitious root formation, were significantly increased. Moreover, the expression levels of the negative regulator, VvARF17, were significantly lower. These results suggested that the inhibition of ABA 8′-hydroxylase enhanced dehydration tolerance and adventitious rooting and may be an effective strategy for achieving drought stress tolerance in grape cuttings.

     

Examination of Antioxidative System’s Responses in the Different Phases of Drought Stress and During Recovery in Desert Plant <Emphasis Type="Italic">Reaumuria soongorica</Emphasis> (Pall.) Maxim

The aim of this study was to test the protective roles of superoxide dismutases (SODs), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) against oxidative damage and their activities in different phases of the dry down process in Reaumuria soongorica (Pall.) Maxim. leaves. Drought stress was imposed during 100 consecutive days and rewatering after 16, 72, and 100 days. The concentration of hydrogen peroxide (H₂O₂), malondialdehyde, and SODs activities were elevated significantly with progressing drought stress. POD and CAT activities increased markedly in the early phase of drought and decreased significantly with further drought stress continuation, and POD activity was unable to recover after rewatering. Ascorbate, reduced glutathione, APX, and GR activities declined in the initial stages of drought process, elevated significantly with further increasing water deficit progression and recovered after rewatering. These results indicate that: (1) iron SODs-removing superoxide anion is very effective during the whole drought stress; (2) CAT scavenges H₂O₂ in the early phase of drought and enzymes of ascorbate–glutathione cycle scavenge H₂O₂ in further increasing drought stress; and (3) POD does not contribute to protect against oxidative damage caused by H₂O₂ under drought stress.     

Discerning of Rice Landraces (Oryza sativa L.) for Morpho-physiological,Antioxidant Enzyme Activity,and Molecular Markers’ Responses to Induced Salt Stress at the Seedling Stage

Salinity has been identified as key abiotic stress factor limiting rice production in many countries around the globe, including Bangladesh. In the present study, we examined the effects of salt-induced toxicity on growth of rice landraces for screening salt-tolerant genotypes by assessing morpho-physiological, biochemical, and molecular responses. Screening of 28 rice genotypes at seedling stage was performed at 12 dS m⁻¹ salinity level in hydroponic media. Most of the rice genotypes showed an apparent reduction in growth traits, while a fewer showed less reduction under salinity stress. Euclidean clustering and heatmap based on morpho-physiological parameters dissected rice genotypes into three major clusters, viz., susceptible, moderately tolerant, and tolerant. Results of cluster analysis revealed Nonabokra, Hogla, Ghunsi, Holdegotal, Nonabokra, and Kanchon as salt-tolerant rice genotypes. These genotypes also were grouped using three microsatellite markers, viz., RM493, RM3412b, and RM140 that were closely linked to saltol QTL showed Hogla, Ghunsi, Holdegotal, Nonabokra, Kanchon, BINA dhan-8, and BINA dhan-10 as salt-tolerant genotypes considering genetic similarity in dendrogram. The positive relationships of Na⁺/K⁺ ratio with hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), and antioxidant enzymes’ activity in the tolerant rice genotypes indicated their importance for improving salinity tolerance. The salt-tolerant landraces showed lower Na⁺/K⁺ ratio, high proline accumulation, lower H₂O₂ accumulation and MDA content, and higher catalase and ascorbate peroxidase activities. Higher antioxidant enzymes’ activity and lower H₂O₂ accumulation in tolerant genotypes indicate their abilities to fight against oxidative stress. Based on all morpho-physiological clustering, biochemical response, and molecular dendrogram, Nonabokra, Hogla, Ghunsi, Holdegotal, and Kanchon were identified as the salt-tolerant landraces. Therefore, these identified salt-tolerant landraces could be useful to improve breeding program for the development of salt-tolerant high-yielding rice cultivars in future.

     

Characterization of fertility restoration in alloplasmic lines derived from hybridization of self-fertilized offspring of barley-wheat (Hordeum vulgare L. × Triticum aestivum L.) amphiploid with common wheat varieties Saratovskaya 29 and Pyrotrix 28

The problems of fertility restoration in the progeny of barley-wheat hybrids (H. vulgare × T. aestivum) are explained by incompatibility between the cytoplasm of cultivated barley and the nuclear genome of common wheat. Appropriate models for studying these problems are alloplasmic lines that combine the cytoplasm of barley and the nuclear genome of wheat. In this work, the differences of fertility restoration in alloplasmic common wheat lines (H. vulgare)-T. aestivum were studied depending on the influence of wheat varieties Saratovskaya 29 (Sar29) and Pyrotrix 28 (Pyr28) used to produce these lines. The alloplasmic lines were created using hybrids between the 48-chromosome offspring (Amph₁) of the barley-wheat amphiploid H. vulgare (ya-319) × T. aestivum (Sar29) and these wheat varieties. Backcrossing of the Amph₁ (2n = 48) × Sar29 hybrid with the wheat variety Sar29 resulted in the complete sterility in the (H. vulgare)-Sar29 line, which suggests the incompatibility of the nuclear genome of the common wheat variety Sar29 with the cytoplasm of H. vulgare. Crossing of Amph₁ (2n = 48) with Pyr28 resulted in the restoration of self-fertility in the hybrid with 2n = 44. In the alloplasmic lines (2n = 42) formed based on plants of the self-fertilized generations of this hybrid, the barley chromosomes were eliminated, and recombination between the nuclear genomes of the parental wheat varieties Sar29 and Pyr28 took place. Alloplasmic recombinant lines (H. vulgare)-T. aestivum with different levels of fertility were isolated. As was shown by the SSR analysis, differences in the fertility between these lines are determined by differences in the content of the genetic material from the wheat varieties Sar29 and Pyr28. The complete restoration of fertility in these alloplasmic recombinant lines is accompanied by the formation of a nuclear genome in which the genetic material of Pyr28 significantly prevails. The conclusion is made that the common wheat variety Pyrotrix 28 is a carrier of a gene (or genes), which determines the restoration of common wheat fertility on the cytoplasm of cultivated barley.     

Molecular weight and subunit composition of GABAA-ergic compound-sensitive Cl−, HCO 3 − -Stimulated Mg2+ ATPase from the plasma membrane of carp brain (Cyprinus carpio L.)

Molecular weight and subunit composition of Cl^?, HCO ₃ ^? , and picrotoxin-stimulated Mg²⁺-ATPase solubilized with sodium deoxycholate from the plasma membrane fraction of fish brain were studied by gel filtration. These enzymes were eluted from a Sephacryl S-300 column as a single peak and corresponded to a ～300 kDa protein with a Stokes radius of 5.4 nm. The enzyme-enriched fraction concentrated and denatured by SDS was eluted from a Sephacryl S-200 column as a single subunit with a molecular weight of ～56 kDa. SDS-PAGE also revealed a single major protein band with a molecular weight of ～56 kDa. It was concluded that the molecular weight and subunit composition of Cl^?, HCO ₃ ^? -stimulated Mg²⁺-ATPase isolated from the plasma membrane of fish brain are similar to those of GABA_A/benzodiazepine receptor complex from fish brain but differ from those of P-type transport ATPases.     

Cloning,Tissue Expression Analysis,and Functional Characterization of Two Δ6-Desaturase Variants of Sea Bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> L.)

Fish are the main source of the n-3 highly unsaturated fatty acids, which are crucial for human health. Their synthesis from C₁₈ precursors is mediated by desaturases and elongases, but the activity of these enzymes has not been conclusively established in marine fish species. This study reports the cloning, tissue expression, and functional characterization of a sea bass (Dicentrarchus labrax L.) Δ6-desaturase and one of its splicing variants. Two cDNAs with open reading frames of 1,346 and 1,354 bp were cloned and named D6D and D6D-V, respectively. Both deduced protein sequences (445 and 387 amino acids, respectively) contained two transmembrane regions and the N-terminal cytochrome b₅ domain with the HPGG motif characteristic of microsomal desaturases. D6D presents three histidine-rich regions, whereas in D6D-V, an insertion of eight nucleotides in the boundaries of exons 10 and 11 modified the third histidine-rich domain and led to insertion of a premature STOP codon, resulting in a shorter predicted protein. Quantitative real-time polymerase chain reaction assay of gene expression showed that D6D was highly expressed in the brain and intestine, and to a lesser extent, in muscle and liver; meanwhile, D6D-V was expressed in all tissues tested, but at level at least 200-fold lower than D6D. Functional analysis in yeast showed that sea bass D6D encodes a fully functional Δ6-desaturase with no residual Δ5-desaturase activity. This desaturase does not exhibit a clear preference for n-3 versus n-6 C₁₈ substrates. Interestingly, D6D-V is a nonfunctional protein, suggesting that the C-terminal end is indispensable for protein activity.