Towards a critical understanding of the photosystem II repair mechanism and its regulation during stress conditions

Photosystem II (PSII) is vulnerable to high light (HL) illumination resulting in photoinhibition. In addition to photoprotection mechanisms, plants have developed an efficient PSII repair mechanism to save themselves from irreversible damage to PSII under abiotic stresses including HL illumination. The phosphorylation/dephosphorylation cycle along with subsequent degradation of photodamaged D1 protein to be replaced by the insertion of a newly synthesized copy of D1 into the PSII complex, is the core function of the PSII repair cycle. The exact mechanism of this process is still under discussion. We describe the recent progress in identifying the kinases, phosphatases and proteases, and in understanding their involvement in the maintenance of thylakoid structure and the quality control of proteins by PSII repair cycle during photoinhibition.     

Sequence related amplified polymorphism (SRAP) analysis for genetic diversity and micronutrient content among gene pools in mungbean [Vigna radiata (L.) Wilczek]

Vigna radiata (L.) Wilczek, commonly called mungbean is an important pulse crop. Commercial cultivars contain low levels of iron and zinc and it is important to assess genetic variability in the available germplasm for improving micronutrient content in commercial cultivars. The present study was undertaken to study molecular diversity using Sequence-related amplified polymorphism (SRAP) among 21 Vigna radiata genotypes. Twenty nine SRAP primer combinations produced a total of 121 amplified bands which were polymorphic with an average of 4.65 bands per primer. The size of amplified bands ranged from 70 bp to 3,000 bp and 6 out of 29 SRAP primers were most useful in fingerprinting Vigna radiata genotypes under study. The similarity coefficients between different genotypes ranged from 0.45 to 0.96 with an average similarity value of 0.71. At an arbitrary cut-off at 60 % similarity level on a dendrogram, the Vigna radiata accessions were categorized into two major clusters. ML1108 and 2KM115 were found to be genetically similar. SMH99-1A and ML776 showed high iron and zinc content while Satya was poor in iron as well as zinc content. Mapping population involving ML776 and Satya could be used for tagging gene(s) for micronutrient content. The results indicated that SRAP markers were efficient for identification of Vigna radiata genotypes and assessment of the genetic relationships among them.     

Role of highly central residues of P-loop and it's flanking region in preserving the archetypal conformation of Walker A motif of diverse P-loop NTPases

P-loop NTPases represent a large and highly diverse protein family that is involved in variety of cellular functions. Walker A motif forms a typical arched conformation, necessary to accommodate the phosphate moiety of the nucleoside tri (or di-) phosphate in Ploop NTPases. The feature that maintains the ancient architecture of P-loop is unidentified and uncharacterized. Here, using a well established global network parameter, closeness centrality, we identify that Walker A and its flanking regions (N- and C-terminal) have high density of globally connected residue positions. We find that closeness centrality of these residue positions are conserved across common structural core of diverse domains of P-loop NTPase fold. Our results suggest the potential role of globally connected residues in maintaining the local conformation of P-loop.     

Efficient genetic transformation of Withania coagulans (Stocks) Dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture

An efficient and reproducible Agrobacterium-mediated genetic transformation of Withania coagulans was achieved using leaf explants of in vitro multiple shoot culture. The Agrobacterium strain LBA4404 harboring the binary vector pIG121Hm containing β-glucuronidase gene (gusA) under the control of CaMV35S promoter was used in the development of transformation protocol. The optimal conditions for the Agrobacterium-mediated transformation of W. coagulans were found to be the co-cultivation of leaf explants for 20 min to agrobacterial inoculum (O.D. 0.4) followed by 3 days of co-cultivation on medium supplemented with 100 μM acetosyringone. Shoot bud induction as well as differentiation occurred on Murashige and Skoog medium supplemented with 10.0 μM 6-benzylaminopurine, 8.0 μM indole 3-acetic acid, and 50.0 mgl^?1 kanamycin after three consecutive cycles of selection. Elongated shoots were rooted using a two-step procedure involving root induction in a medium containing 2.5 μM indole 3-butyric acid for 1 week and then transferred to hormone free one-half MS basal for 2 weeks. We were successful in achieving 100 % frequency of transient GUS expression with 5 % stable transformation efficiency using optimized conditions. PCR analysis of T₀ transgenic plants showed the presence of gusA and nptII genes confirming the transgenic event. Histochemical GUS expression was observed in the putative transgenic W. coagulans plants. Thin layer chromatography showed the presence of similar type of withanolides in the transgenic and non-transgenic regenerated plants. A. tumefaciens mediated transformation system via leaf explants developed in this study will be useful for pathway manipulation using metabolic engineering for bioactive withanolides in W. coagulans, an important medicinal plant.     

Qualitative and quantitative variations in withanolides and expression of some pathway genes during different stages of morphogenesis in Withania somnifera Dunal

Withania somnifera Dunal is an important and extensively studied medicinal plant; however, there is no report available that relates withanolide content and its profile in relation to the expression of pathway genes during different morphogenic stages. In this study, withanolide A, withaferin A, and withanone, the major withanolides of W. somnifera, were measured in different in vitro stages during organogenesis, viz., shoot to root (direct rhizogenesis)/root to shoot (indirect via callus phase) transition vis-à-vis expression levels of key pathway genes involved in withanolide biosynthetic pathways. The morphogenic transitions were found to be tightly linked to the pattern of accumulation of withanolides. The high expression levels of most of the pathway genes in in vitro shoots in comparison to in vitro root and callus tissues exhibited a direct co-relation with the maximum withanolide content (>2.7 mg/gDW). The biogenesis of withaferin A, a major constituent of the leaves, was however found to be tightly linked to shoots/green tissue. In addition, we were also able to establish an efficient regeneration system from roots for their further utilization in biotechnological applications.     

Evaluation of antifungal potential of Marchantia polymorpha L., Dryopteris filix-mas (L.) Schott and Ephedra foliata Boiss. against phyto fungal pathogens

Methanol and flavonoid extracts (free and bound) of Marchantia polymorpha L., Dryopteris filix-mas (L.) Schott and Ephedra foliata Boiss. were screened against three fungal plant pathogens: Alternaria solani, Fusarium oxysporum and Rhizoctonia solani. The extracts from D. filix-mas and E. foliata showed >80% of mycelial inhibition of A. solani whereas M. polymorpha and D. filix-mas (rhizome) completely inhibited the mycelial growth of R. solani when tested at highest concentration (5 mg/ml). Inhibition of spore germination of fungi (A. solani and F. oxysporum) was observed to be 100% by most of the extracts at 10 mg/ml. Moreover, plant extracts were found effective in increasing seed germination and seed vigour simultaneously thereby decreasing the percentage of pathogen infection. The results of the present study reveal that the plants screened possess the potential to inhibit the crop fungal pathogens and further investigation is required to explore the biologically active constituents of these plants and to use them as natural plant protectants for agriculture.     

Single nucleotide polymorphism (SNP) in growth hormone gene of Jakhrana,a prominent milk goat breed in India

PCR-SSCP patterns on non-degenerative PAGE revealed 6 amplicons in caprine GH exon-4 and 3 alleles A₄, B₄ and C₄ were identified. In exon-5, six SSCP variants revealed three alleles A₅, B₅, and C₅. Out of 54 AA sites of GH-4 coding region, six codons were polymorphic. At codon-6, nucleotide substitution of G/A resulted in to genotypes 6_RR, 6_HH and G/C into genotypes 6_PP, 6_RP. At codon-36, A/G nucleotide substitution resulted in to newer genotypes 36_GG from that of 36_DD in reference Genbank sample. At codon-54, C/T nucleotide substitution caused change of amino acid (AA) from arginine (R) to tryptophan (W) resulted into a new genotype of 54_WW in comparison to 54_RR of Genbank reference sample. In exon-5, out of 67 AA sites 8 codons were polymorphic, but the codons 14 and 60 were preponderant. At codon-14, A/G substitution resulted into 3 genotypes 14_KK, 14_EE and 14_KE with frequency of 0.52, 0.38 and 0.10, respectively. At codon-60, G/C and G/A substitutions resulted in to 3 genotypes 60_GG, 60_RR and 60_GR with frequencies of 0.48, 0.42 and 0.10, respectively. Synonymous mutations as compared to Genbank accession D00476.1 were present at codons 25, 31 and 62 in all the animals of Jakhrana goats. The high genetic variability in GH gene exon-4 and exon-5 may be useful in exploring their associations with milk and growth traits in goat for further genetic improvement.     

Relative Bioavailability of Chlorothiazide from Mucoadhesive Compacts in Pigs

The relative bioavailability of chlorothiazide from mucoadhesive polymeric compacts is compared to commercial oral suspension in pigs. A single-dose randomized study was conducted in 12 healthy pigs that are 9–10 weeks old. After overnight fasting, pigs were divided into two groups of six animals. To the first group, a reference product containing 50 mg of chlorothiazide suspension, and in the second group, test product (mucoadhesive compacts) chlorothiazide (50 mg) was administered with 75 mL of water via gastric tubes. Blood samples were collected between 0 to 24 h using catheters inserted into the jugular vein. Plasma was separated by protein precipitation, and chlorothiazide concentrations were determined using a high-performance liquid chromatography method. The mean T_max and the C_max of chlorothiazide following the administration of oral suspension and mucoadhesive compacts were 0.58 ± 0.20 h and 682.97 ± 415.69 ng/mL and 2.17 ± 0.98 h and 99.42 ± 124.08 ng/mL, respectively. The K_el and T_1/2 of chlorothiazide were found to be 1.06 ± 0.28 h⁻¹ and 0.70 ± 0.21 h from suspension and 0.95 ± 1.11 h⁻¹ and 2.05 ± 1.90 h from the compacts, respectively. The T_max of mucoadhesive compacts were significantly longer (p < 0.05; 2.17 h) than the reference products (0.58 h), whereas the C_max of compacts were significantly lower (99 ng/mL) than the reference product (683 ng/mL; p < 0.05). The area under the curve (AUC) of compacts accounts only 50.15% (404.32 ± 449.93 ng h/mL) of the reference product’s AUC (806.27 ± 395.97 ng h/mL). The relative bioavailability of the compacts was lower than that of the suspension, and this may be due to the narrow window of absorption for chlorothiazide.Key words: bioavailability, chlorothiazide, mucoadhesive compacts, pigs