Somesh Singh

Singlet oxygen- and EXECUTER1-mediated signaling is initiated in grana margins and depends on the protease FtsH2

Significance Singlet oxygen (1O2)- and EXECUTER1 (EX1)-dependent signaling triggers programmed cell death in seedlings and inhibits growth of mature plants of the fluorescent (flu) mutant of Arabidopsis. The EX1 protein has been located in chloroplasts to the grana margins close to where chlorophyll is synthesized and the disassembly of damaged photosystem II (PSII) and reassembly of active PSII take place. With the onset of 1O2-mediated signaling there is a rapid decline of EX1 that depends on the ATP-dependent zinc metalloprotease FtsH. Generation of 1O2 without the decline of EX1 is not sufficient to activate 1O2 signaling. As FtsH cleaves also the D1 reaction center protein of damaged PSII, EX1-dependent signaling seems not only spatially but also functionally linked to the repair of PSII. Formation of singlet oxygen (1O2) has been implicated with damaging photosystem II (PSII) that needs to undergo continuous repair to maintain photosynthetic electron transport. In addition to its damaging effect, 1O2 has also been shown to act as a signal that triggers stress acclimation and an enhanced stress resistance. A signaling role of 1O2 was first documented in the fluorescent (flu) mutant of Arabidopsis. It strictly depends on the chloroplast protein EXECUTER1 (EX1) and happens under nonphotoinhibitory light conditions. Under severe light stress, signaling is initiated independently of EX1 by 1O2 that is thought to be generated at the acceptor side of active PSII within the core of grana stacks. The results of the present study suggest a second source of 1O2 formation in grana margins close to the site of chlorophyll synthesis where EX1 is localized and the disassembly of damaged and reassembly of active PSII take place. The initiation of 1O2 signaling in grana margins depends on EX1 and the ATP-dependent zinc metalloprotease FtsH. As FtsH cleaves also the D1 protein during the disassembly of damaged PSII, EX1- and 1O2-mediated signaling seems to be not only spatially but also functionally associated with the repair of PSII.

Improved method of in vitro regeneration in Leucaena leucocephala - a leguminous pulpwood tree species

Leucaena leucocephala is a fast growing multipurpose legume tree used for forage, leaf manure, paper and pulp. Lignin in Leucaena pulp adversely influences the quality of paper produced. Developing transgenic Leucaena with altered lignin by genetic engineering demands an optimized regeneration system. The present study deals with optimization of regeneration system for L. leucocephala cv. K636. Multiple shoot induction from the cotyledonary nodes of L. leucocephala was studied in response to cytokinins, thidiazuron (TDZ) and N6-benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots. Multiple shoots were induced from cotyledonary nodes at varied frequencies depending on the type and concentration of cytokinin used in the medium. TDZ was found to induce more number of shoots per explant than BA, with a maximum of 7 shoots at an optimum concentration of 0.23 µM. Further increase in TDZ concentration resulted in reduced shoot length and fasciation of the shoots. Liquid pulse treatment of the explants with TDZ did not improve the shoot production further but improved the subsequent rooting of the shoots that regenerated. Regenerated shoots successfully rooted on ½-MS medium supplemented with 0.54 µM α-naphthaleneacetic acid (NAA). Rooted shoots of Leucaena were transferred to coco-peat and hardened plantlets showed ≥ 90 % establishment in the green house.

in Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1).

Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants. Abbreviations Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.

Molecular characterization of farnesyl pyrophosphate synthase from Bacopa monniera by comparative modeling and docking studies

Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens. Abbreviations IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.

Genetic Engineering of Phenylpropanoid Pathway in Leucaena leucocephala

The phenylpropanoid pathway is responsible for the biosynthesis of a variety of products that include lignin, flavonoids and hydroxycinnamic acid conjugates. Many intermediates and end products of this pathway play important role in plants as phytoalexins, antiherbivory compounds, antioxidants, ultra-violet (UV) protectants, pigments and aroma compounds. Lignin has far reaching impacts on agriculture, industry and the environment, making phenylpropanoid metabolism a globally important part of plant biochemistry. The mechanical support provided by lignin prevents lodging, a problem in many agronomically important plants, it also provides a hydrophobic surface, essential for longitudinal water transport, and provides a barrier against pathogens. Finally the many functions of lignin and related products in resistance to biotic and abiotic stresses make the phenylpropanoid pathway vital to the health and survival of plants. Besides its critical role in normal plant health and development, high lignin levels are problematic in the agro-industrial exploitation of various plant species. Lignin is considered an undesirable component in paper manufacture, and has a negative impact on forage crop digestibility. Leucaena leucocephala is one of the most versatile, fast growing commercially important trees for paper and pulp industry in India, contributing 1/4th of the total raw material. Lignin composition, quantity and distribution are known to affect the agro-industrial utilization of plant biomass. High quantity and low Syringyl (S) to Guaiacyl (G) lignin ratio plays a detrimental role in economy and ecology of paper production. Every unit increase in S/G ratio decreases the cost of paper production by two and half times. Moreover chemical processing of pulp for lignin removal releases chlorinated organic compounds in effluent, which are hazardous and a serious threat to the environment. Hence, there is currently intense interest in modifying the content and/or composition of the cell wall structural polymer (lignin) as a means of improving the efficiency of the paper pulping process for forest trees. To engineer plants with agronomically useful lignin related traits, we need to devise strategies that can flexibly and predictably yield reductions in lignin content and/or changes in lignin monomer composition. Our studies have concentrated on attempts to alter the levels of enzymes involved in early as well as late phenylpropanoid pathway, mainly by downregulation or upregulation of the phenylpropanoid pathway genes in transgenic L. leucocephala and tobacco plants. Besides, we are also working on some R2R3 type MYB transcription factors supposed to play important role in lignin biosynthesis and some other genes which are not directly involved in phenylpropanoid pathway, but are important for carrying out polymerization of monolignols (peroxidases) and defense mechanism of plants (β glucosidase: family 1 Glycosyl hydrolase). Major phenylpropanoid pathway genes (C4H, 4-CL, CCoAOMT, CCR, Cald5H and CAD) were isolated using PCR based approach. Their 5’ and 3’ UTR determined by rapid amplification of cDNA race (RACE). We could isolate multiple isoforms of most of the genes in this way well supported by Southern hybridization experiment. All the genes were expressed in E. coli and/or yeast with/without the signal sequence. The over-expressed proteins were purified using suitable methods and were used for raising polyclonal antibodies against them. The most un-conserved region of each gene was cloned in antisense orientation in suitable binary vector and L. leucocephala and tobacco explants were transformed using the antisense construct to down-regulate the targeted gene. In order to increase the S/G ratio of lignin monomers sense construct of the target gene was used. We have also done RNAi based downregulation of Cinnamate 4-Hydroxylase (C4H), a key enzyme of phenylpropanoid pathway and a member of cytochrome P450 family in tobacco. Spatio-temporal expression of each gene was studied in L. leucocephala in different tissues at different periods of their growth. Kinetics study of some of the enzymes has been carried out in our lab. We are also trying to establish the structure function correlation of some enzymes. We have also isolated promoters of some phenylpropanoid pathway genes and have identified R2-R3 type MYB binding domain(s) in them. Two MYB genes have been isolated from L. leucocephala and heterologously expressed in Escherichia coli. Their DNA binding efficiency and role in regulating phenylpropanoid pathway remain to be seen. All the isolated genes shared 70-80% homology with the already reported sequences from other species at nucleotide level and more than 80% identity at amino acid level. Different isoforms of different genes had varying degree of identity between them ranging from 80% to more than 95%. We could easily locate the ribosome binding site in the 5’ UTR and the polyadenylation signal in the 3’ UTR in all the genes. Proteins were expressed in both prokaryotic and eukaryotic system. Some of the genes were difficult to express in BL21 (DE3), primarily because of the translational incompatibility of some of the codons in E. coli and may be partly because of the signal sequence present in most of the proteins. All the genes were found to be actively expressing in lignifying tissues and roots in comparison to leaves albeit a time dependent regular expression pattern could not be drawn in case of every gene. We used three methods of genetic transformation for transferring our gene constructs to L. leucocephala embryo viz: Agrobacterium mediatd, gene gun mediated and gene gun followed by cocultivation with Agrobacterium. The transformation and regeneration efficiency varied with each protocol. The transgenic plants invariably showed stunted vigour and slow growth irrespective of the nature of the gene downregulated or upregulated. Initial screening of the transformants was done on MS medium containing appropriate antibiotic and later confirmation was done using PCR with hptII/nptII, gus specific and CaMV35S promoter specific primers. At least one tobacco plant downregulated for C4H showed rudimentary root system and curled leaves with brown tip. Transformed Leucaena and tobacco plants had reduced lignin content with varying degree in case of every downregulated gene. Histochemical staining of transverse root and stem tissue sections showed reduced lignification as evidenced by immunocytolocalization patterns of the candidate protein under study. Promoters of few lignin biosynthetic pathway genes having R2R3 type MYB-binding signal sequences (AC elements) have been cloned. Two R2R3 type MYB transcription factors from L. leucocephala that have been expressed and purified from E. coli will be used for gel retardation studies with the promoter sequences. Also in vitro synthesized oligonucleotides having the highly conserved MYB-binding motifs will be designed to determine the most probable binding sites of the two MYB proteins. Sense construct for the two MYB genes have been transformed in tobacco and antisense construct have been transformed in Leucaena plants to study the after effects of gene manipulation. In short, we have isolated and characterized several genes belonging to phenylpropanoid pathway and have expressed them in different systems. Transgenics for down-regulation or upregulation of those genes have shown very interesting results.

Enhanced activity of Withania somnifera family-1 glycosyltransferase (UGT73A16) via mutagenesis

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein–ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2–3 fold and 6–7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.