Acetylation of polysaccharides in plant cell wall

Plant cell wall in woody tissues is a complex matrix, which consists of cellulose, matrix polysaccharides and lignin. The matrix polysaccharides are substituted with acetyl group that are hypothesised to play important roles in determining properties of these polysaccharides. The aim of this thesis was to understand the role of O-acetylation in plants and investigate possibilities for improvement of woody lignocellulose for biorefinery applications by reducing wood O-acetylation. To alter acetylation specifically in woody tissues, a promoter from Glycosyl Transferase 43 family (GT43) in Populus was isolated that had a very specific expression in secondary cell wall forming cells. This xylem specific promoter (pGT43B) was more effective in modification of wood acetylation by overexpression and suppression strategies as compared to 35S promoter (Paper I). To reduce xylan acetylation using transgenic approach, acetyl xylan esterase from Aspergillus niger, AnAXE1, was targetted specifically to the cell wall in Arabidopsis (Paper II) and in Populus (Paper III). Plants expressing AnAXE1 grew as well as wild type and had increased acetyl esterase activity. This has led to reduction in cell wall acetyl content and in xylan O-acetylation. Moreover, transgenic Arabidopsis exhibited increased resistance against a biotrophic pathogen Hyaloperonospora arabidopsidis. Both transgenic plants had improved sugar yields in saccharification with different pretreatments and without pretreatment. To reduce acetylation using cisgenic approach, Populus Reduced Wall Acetylation (RWA) gene family was characterised by suppression of the two clades RWA-AB, and RWA-CD (Paper IV). Both clades were shown to be involved in xylan acetylation in the wood. Both clades were therefore suppressed under control of xylem specific promoter pGT43B to improve wood saccharification potential. Transgenic plants had reduced wood acetyl content, normal growth, and increased sugar yield and glucose conversion % in saccharification without pretreatment. Glucose yield was also slightly increased in saccharification after acid pretreatment. These results show that reduction of cell wall acetylation by 10-30% does not alter plant growth and development, but improves yields in lignocellulose saccharification (with and without pretreatment) (Papers II, III and IV). To identify Quantitative Trait Loci (QTLs) related to cell wall acetyl content and other chemical traits in Salix, the mapping population of 463 progenies of S. viminalis and S. schwerinii was analysed by FT-IR and acetyl content assay (Paper V). 28 QTLs were identified for different cell wall chemical traits, which were co-located with several cell wall related genes and gene clusters. These QTLs and genes can be used in the future to improve wood chemical traits in Salix and Populus for biofuel production by breeding

Awareness and Pattern of Tobacco Use among the Medical Students of Government Medical College

Introduction: Nearly 6 million people die due to tobacco every year and this figure will increase to 8 million tobacco-attributed deaths per year by 2030 with 80% of them occurring in developing countries. Objective: To study the awareness and pattern of tobacco use among the undergraduate medical students of Government Medical College, Haldwani. Material and Methods: A Cross-sectional study was conducted among 303 medical students using a predesigned and pretested, semi-structured self-administered anonymous questionnaire. Data was analyzed by using SPSS v 16. Results: Among 303 participants, 44(14.5%) were smokers.  Majority of the students (97.73%) were more than 20 years of age at the time of initiation of tobacco use. Cigarette smoking was most common form (79.55%) of abuse. Male students were using tobacco significantly higher than that of females (?2=36.68). The effect of parental tobacco use on tobacco consumption habits of the users were significantly higher than non-tobacco users (?2=180.75). The tobacco consumption was significantly increased among the senior students as compared to that among the juniors (?2=15.29). Awareness about harmful effects of tobacco abuse was very high (90.76%) and mainly they got knowledge from media. 84.16% students support ban on tobacco use in public places. Conclusion: We can conclude that though the awareness among medical students regarding harmful effects of tobacco use was very high but they got this knowledge mainly from the electronic media, so it is necessary to introduce teaching on tobacco dependence and cessation early in the courses of the medical colleges

Optical observations of the bright long duration peculiar GRB 021004 afterglow

The CCD magnitudes in Johnson $B,V$ and Cousins $R$ and $I$ photometric passbands are determined for the bright long duration GRB 021004 afterglow from 2002 October 4 to 16 starting $\sim$ 3 hours after the $\gamma-$ray burst. Light curves of the afterglow emission in $B$,$V$,$R$ and $I$ passbands are obtained by combining these measurements with other published data. The earliest optical emission appears to originate in a revese shock. Flux decay of the afterglow shows a very uncommon variation relative to other well-observed GRBs. Rapid light variations, especially during early times ($\Delta t < 2$ days) is superposed on an underlying broken power law decay typical of a jetted afterglow. The flux decay constants at early and late times derived from least square fits to the light curve are $0.99\pm0.05$ and $2.0\pm0.2$ respectively, with a jet break at around 7 day. Comparison with a standard fireball model indicates a total extinction of $E(B-V)=0.20$ mag in the direction of the burst. Our low-resolution spectra corrected for this extinction provide a spectral slope $\beta = 0.6\pm0.02$. This value and the flux decay constants agree well with the electron energy index $p\sim 2.27$ used in the model. The derived jet opening angle of about $7^{\circ}$ implies a total emitted gamma-ray energy $E_{\gamma} = 3.5\times10^{50}$ erg at a cosmological distance of about 20 Gpc. Multiwavelength observations indicate association of this GRB with a star forming region, supporting the case for collapsar origin of long duration GRBs.Comment: 19 pages, 3 figures, BASI, 31, 1

Hybrid Aspen Expressing a Carbohydrate Esterase Family 5 Acetyl Xylan Esterase under Control of a Wood-Specific Promoter Shows Improved Saccharification

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.Peer reviewe

SN 2011hw: Helium-Rich Circumstellar Gas and the Luminous Blue Variable to Wolf-Rayet Transition in Supernova Progenitors

We present optical photometry and spectroscopy of the peculiar Type IIn/Ibn supernova SN2011hw. Its light curve exhibits a slower decline rate than normal SNeIbc, with a peak absolute magnitude of -19.5 (unfiltered) and a secondary peak of -18.3 mag (R). Spectra of SN2011hw are unusual compared to normal SN types, most closely resembling the spectra of SNeIbn. We center our analysis on comparing SN 2011hw to the well-studied TypeIbn SN2006jc. While the two SNe have many important similarities, the differences are quite telling: compared to SN2006jc, SN2011hw has weaker HeI and CaII lines and relatively stronger H lines, its light curve has a higher luminosity and slower decline rate, and emission lines associated with the progenitor's CSM are narrower. One can reproduce the unusual continuum shape of SN2011hw with equal contributions of a 6000K blackbody and a spectrum of SN2006jc. We attribute this emission component and many other differences between the two SNe to extra opacity from a small amount of additional H in SN2011hw, analogous to the small H mass that makes SNeIIb differ from SNeIb. Slower speeds in the CSM and elevated H content suggest a connection between the progenitor of SN2011hw and the class of Ofpe/WN9 stars, which have been associated with LBVs in their hot quiescent phases between outbursts, and are H-poor - but not H-free like classical Wolf-Rayet (WR) stars. We conclude that the similarities and differences between SN2011hw and SN2006jc can be largely understood if their progenitors exploded at different points in the transitional evolution from an LBV to a WR star.Comment: 11 pages, 7 figures, submitted to MNRA

Morpho-biochemical characterization of a RIL population for seed parameters and identification of candidate genes regulating seed size trait in lentil (Lens culinaris Medik.)

The seed size and shape in lentil (Lens culinaris Medik.) are important quality traits as these influences the milled grain yield, cooking time, and market class of the grains. Linkage analysis was done for seed size in a RIL (F5:6) population derived by crossing L830 (20.9 g/1000 seeds) with L4602 (42.13 g/1000 seeds) which consisted of 188 lines (15.0 to 40.5 g/1000 seeds). Parental polymorphism survey using 394 SSRs identified 31 polymorphic primers, which were used for the bulked segregant analysis (BSA). Marker PBALC449 differentiated the parents and small seed size bulk only, whereas large seeded bulk or the individual plants constituting the large-seeded bulk could not be differentiated. Single plant analysis identified only six recombinant and 13 heterozygotes, of 93 small-seeded RILs (<24.0 g/1000 seed). This clearly showed that the small seed size trait is very strongly regulated by the locus near PBLAC449; whereas, large seed size trait seems governed by more than one locus. The PCR amplified products from the PBLAC449 marker (149bp from L4602 and 131bp from L830) were cloned, sequenced and BLAST searched using the lentil reference genome and was found amplified from chromosome 03. Afterward, the nearby region on chromosome 3 was searched, and a few candidate genes like ubiquitin carboxyl-terminal hydrolase, E3 ubiquitin ligase, TIFY-like protein, and hexosyltransferase having a role in seed size determination were identified. Validation study in another RIL mapping population which is differing for seed size, showed a number of SNPs and InDels among these genes when studied using whole genome resequencing (WGRS) approach. Biochemical parameters like cellulose, lignin, and xylose content showed no significant differences between parents and the extreme RILs, at maturity. Various seed morphological traits like area, length, width, compactness, volume, perimeter, etc., when measured using VideometerLab 4.0 showed significant differences for the parents and RILs. The results have ultimately helped in better understanding the region regulating the seed size trait in genomically less explored crops like lentils

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.Peer reviewe

Fomento y transversalidad de la lengua inglesa en el Grado de Historia: propuesta interdisciplinar en torno a los conceptos de resiliencia y precariedad

Memoria ID-0099. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2017-2018