Cell Wall Digestion Research Articles

Glycoside Hydrolase Genes Are Required for Virulence of Agrobacterium tumefaciens on Bryophyllum daigremontiana and Tomato.

Agrobacterium tumefaciens is a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type IV secretion system where the DNA becomes integrated into the host cell chromosomes. The expression of T-DNA in the plant results in tumor formation. Although the binding of the bacteria to plant surfaces has been studied previously, there is little work on possible interactions of the bacteria with the plant cell wall. Seven of the 48 genes encoding putative glycoside hydrolases (Atu2295, Atu2371, Atu3104, Atu3129, Atu4560, Atu4561, and Atu4665) in the genome of A. tumefaciens C58 were found to play a role in virulence on tomato and Bryophyllum daigremontiana Two of these genes (pglA and pglB; Atu3129 and Atu4560) encode enzymes capable of digesting polygalacturonic acid and, thus, may play a role in the digestion of pectin. One gene (arfA; Atu3104) encodes an arabinosylfuranosidase, which could remove arabinose from the ends of polysaccharide chains. Two genes (bglA and bglB; Atu2295 and Atu4561) encode proteins with β-glycosidase activity and could digest a variety of plant cell wall oligosaccharides and polysaccharides. One gene (xynA; Atu2371) encodes a putative xylanase, which may play a role in the digestion of xylan. Another gene (melA; Atu4665) encodes a protein with α-galactosidase activity and may be involved in the breakdown of arabinogalactans. Limited digestion of the plant cell wall by A. tumefaciens may be involved in tumor formation on tomato and B. daigremontianaIMPORTANCEA. tumefaciens is used in the construction of genetically engineered plants, as it is able to transfer DNA to plant hosts. Knowledge of the mechanisms of DNA transfer and the genes required will aid in the understanding of this process. Manipulation of glycoside hydrolases may increase transformation and widen the host range of the bacterium. A. tumefaciens also causes disease (crown gall tumors) on a variety of plants, including stone fruit trees, grapes, and grafted ornamentals such as roses. It is possible that compounds that inhibit glycoside hydrolases could be used to control crown gall disease caused by A. tumefaciens.

Fodder Production and Livestock Rearing in Relation to Climate Change and Possible Adaptation Measures in Manaslu Conservation Area, Nepal

A study was conducted to find out the production potential, nutrient composition, and the variability of the most commonly available fodder trees along with the varying altitude to help optimize the dry matter requirement during winter lean period. The study was carried out in Lho and Prok Village Development Committee of Manaslu Conservation Area (MCA), located in Gorkha district of Nepal.The study was conducted in two parts: social and biological. Accordingly, a households (HHs) survey was conducted to collect primary data from 70 HHs, focusing to the perception of respondents on impacts of climatic variability to the feeding management. The next part consisted of understanding yield potential and nutrient composition of the four most commonly available fodder trees (M. azedirach, M. alba, F. roxburghii, F. nemoralis), within two altitudes range: (1500-2000 masl and 2000-2500 masl) by using a RCB design in 2*4 factorial combination of treatments, each replicated four times. Fresh yield and dry matter yield of the each fodder trees was significant (P<0.01) between the altitude and within species. Fodder trees yield analysis revealed that the highest dry matter (DM) yield (28 kg/tree) was obtained for F. roxburghii but that remained statistically similar (P>0.05) to the other treatment. On the other hand, most of the parameters: ether extract (EE), acid detergent lignin (ADL), acid detergent fibre (ADF), cell wall digestibility (CWD), relative digestibility (RD), digestible nutrient (TDN), and Calcium (Ca) among the treatments were highly significant (P<0.01).
 Int. J. Appl. Sci. Biotechnol. Vol 7(2): 227-235

Molecular mechanisms of chemotropism and cell fusion in unicellular fungi.

In all eukaryotic phyla, cell fusion is important for many aspects of life, from sexual reproduction to tissue formation. Fungal cells fuse during mating to form the zygote, and during vegetative growth to connect mycelia. Prior to fusion, cells first detect gradients of pheromonal chemoattractants that are released by their partner and polarize growth in their direction. Upon pairing, cells digest their cell wall at the site of contact and merge their plasma membrane. In this Review, I discuss recent work on the chemotropic response of the yeast models Saccharomyces cerevisiae and Schizosaccharomyces pombe, which has led to a novel model of gradient sensing: the cell builds a motile cortical polarized patch, which acts as site of communication where pheromones are released and sensed. Initial patch dynamics serve to correct its position and align it with the gradient from the partner cell. Furthermore, I highlight the transition from cell wall expansion during growth to cell wall digestion, which is imposed by physical and signaling changes owing to hyperpolarization that is induced by cell proximity. To conclude, I discuss mechanisms of membrane fusion, whose characterization remains a major challenge for the future.

Mechanics and Dynamics of Bacterial Cell Lysis

Membrane lysis, or rupture, is a cell death pathway in bacteria frequently caused by cell wall-targeting antibiotics. Although previous studies have clarified the biochemical mechanisms of antibiotic action, a physical understanding of the processes leading to lysis remains lacking. Here, we analyze the dynamics of membrane bulging and lysis in Escherichia coli, in which the formation of an initial, partially subtended spherical bulge (“bulging”) after cell wall digestion occurs on a characteristic timescale of 1 s and the growth of the bulge (“swelling”) occurs on a slower characteristic timescale of 100 s. We show that bulging can be energetically favorable due to the relaxation of the entropic and stretching energies of the inner membrane, cell wall, and outer membrane and that the experimentally observed timescales are consistent with model predictions. We then show that swelling is mediated by the enlargement of wall defects, after which cell lysis is consistent with both the inner and outer membranes exceeding characteristic estimates of the yield areal strains of biological membranes. These results contrast biological membrane physics and the physics of thin, rigid shells. They also have implications for cellular morphogenesis and antibiotic discovery across different species of bacteria.

Relationships between Cell Wall Digestibility and Lignin Content as Influenced by Lignin Type and Analysis Method

Difficulties in measuring the lignin content of forage and biomass crops confounds efforts by plant breeders and molecular biologists to grasp how shifts in lignin chemistry affect fiber digestibility. In this study, relationships between digestibility and lignin content were examined by artificially lignifying Zea mays L. cell walls with bulk polymers composed of p‐hydroxyphenyl‐guaiacyl (HG) and guaiacyl (G) units or with endwise polymers composed of G, guaiacyl‐syringyl (GS), and guaiacyl‐syringyl‐p‐coumaric acid (GSpCA) units. Regressions of in vitro rumen gas production or nonfermented polysaccharides (NFP) vs. lignin content accurately measured by an indirect spectrophotometric method revealed that G‐bulk and especially syringyl (S)‐rich endwise polymers were the least inhibitory to digestion. While providing biased estimates at low lignin contents, the acetyl bromide‐soluble lignin (ABSL) assay correctly ranked how endwise polymers affected the digestibility of highly lignified cell walls, but the method required unique absorption coefficients to assess relationships for bulk polymers. Conversely, the Klason lignin (KL) method provided more accurate estimates at low lignin contents, but varying recovery of KL among lignin types created bias for highly lignified cell walls. Overall, our results indicate that S‐rich lignins are the least inhibitory to digestion, but detecting desirable shifts in lignin chemistry will require the judicious use of several analytical methods.

Recruitment of specific flavonoid B-ring hydroxylases for two independent biosynthesis pathways of flavone-derived metabolites in grasses.

In rice (Oryza sativa), OsF2H and OsFNSII direct flavanones to independent pathways that form soluble flavone C-glycosides and tricin-type metabolites (both soluble and lignin-bound), respectively. Production of soluble tricin metabolites requires CYP75B4 as a chrysoeriol 5'-hydroxylase. Meanwhile, the close homologue CYP75B3 is a canonical flavonoid 3'-hydroxylase (F3'H). However, their precise roles in the biosynthesis of soluble flavone C-glycosides and tricin-lignins in cell walls remain unknown. We examinedCYP75B3 and CYP75B4 expression in vegetative tissues, analyzed extractable flavonoid profiles, cell wall structure and digestibility of their mutants, and investigated catalytic activities of CYP75B4 orthologues in grasses. CYP75B3 and CYP75B4 showed co-expression patterns with OsF2H and OsFNSII, respectively. CYP75B3 is the sole F3'H in flavone C-glycosides biosynthesis, whereas CYP75B4 alone provides sufficient 3',5'-hydroxylation for tricin-lignin deposition. CYP75B4 mutation results in production of apigenin-incorporated lignin and enhancement of cell wall digestibility. Moreover, tricin pathway-specific 3',5'-hydroxylation activities are conserved in sorghum CYP75B97 and switchgrass CYP75B11. CYP75B3 and CYP75B4 represent two different pathway-specific enzymes recruited together with OsF2H and OsFNSII, respectively. Interestingly, the OsF2H-CYP75B3 and OsFNSII-CYP75B4 pairs appear to be conserved in grasses. Finally, manipulation of tricin biosynthesis through CYP75B4 orthologues can be a promising strategy to improve digestibility of grass biomass for biofuel and biomaterial production.

Mutation of 4-coumarate: coenzyme A ligase 1 gene affects lignin biosynthesis and increases the cell wall digestibility in maize brown midrib5 mutants

BackgroundMaize brown midrib (bm) mutants associated with impaired lignin biosynthesis are a potential source for the breed of novel germplasms with improved cell wall digestibility. The spontaneous bm5 mutants had been identified since 2008. However, the gene responsible for the bm5 locus, and the comprehensive effects of bm5 mutation on lignin biosynthesis, soluble phenolics accumulation, and cell wall degradation have yet to be elucidated.ResultsThe bm5 locus was identified to encode a major 4-coumarate: coenzyme A ligase (Zm4CL1) through analyzing MutMap-assisted gene mapping data. Two alleles of Zm4CL1 isolated from bm5 mutants contained two transposons inserted in the first exon and the second intron, respectively, and consequently, the activities of 4CLs in the crude enzyme extracts from bm5 midribs were reduced by 51–62% compared with the wild type. Furthermore, five 4CLs were retrieved from maize genome, and Zm4CL1 was the most highly expressed one in the lignified tissues. Mutation of Zm4CL1 mainly impeded the biosynthesis of guaiacyl (G) lignins and increased the level of soluble feruloyl derivatives without impacting maize growth and development. Moreover, both neutral detergent fiber digestibility and saccharification efficiency of cell walls were significantly elevated in the bm5 mutant.ConclusionsZm4CL1 was identified as the Bm5 gene, since two independent alleles of Zm4CL1 were associated with the same mutant phenotype. Mutation of Zm4CL1 mainly affected G lignin biosynthesis and soluble feruloyl derivatives accumulation in maize lignified tissues. The reduced recalcitrance of the bm5 mutant suggests that Zm4CL1 is an elite target for cell wall engineering, and genetic manipulation of this gene will facilitate the utilization of crop straw and stover that have to be dealt with for environmental protection.

A metagenomics investigation of carbohydrate-active enzymes along the goat and camel intestinal tract.

Studies of the digestive microbiota of ruminant animals most often focus on the bacterial diversity in the rumen or the feces of the animals, but little is known about the diversity and functions of their distal intestine. Here, the bacterial microbiota of the distal intestinal tract of two goats and two camels was investigated by metagenomics techniques. The bacterial taxonomic diversity and carbohydrate-active enzyme profile were estimated for samples taken from the small intestine, the large intestine, and the rectum of each animal. The bacterial diversity and abundance in the small intestine were lower than in the rectal and large intestinal samples. Analysis of the carbohydrate-active enzyme profiles at each site revealed a comparatively low abundance of enzymes targeting xylan and cellulose in all animals examined, similar to what has been reported earlier for sheep and therefore suggesting that plant cell wall digestion probably takes place elsewhere, such as in the rumen.

Progress and Opportunities in the Characterization of Cellulose - An Important Regulator of Cell Wall Growth and Mechanics.

The plant cell wall is a dynamic network of several biopolymers and structural proteins including cellulose, pectin, hemicellulose and lignin. Cellulose is one of the main load bearing components of this complex, heterogeneous structure, and in this way, is an important regulator of cell wall growth and mechanics. Glucan chains of cellulose aggregate via hydrogen bonds and van der Waals forces to form long thread-like crystalline structures called cellulose microfibrils. The shape, size, and crystallinity of these microfibrils are important structural parameters that influence mechanical properties of the cell wall and these parameters are likely important determinants of cell wall digestibility for biofuel conversion. Cellulose–cellulose and cellulose-matrix interactions also contribute to the regulation of the mechanics and growth of the cell wall. As a consequence, much emphasis has been placed on extracting valuable structural details about cell wall components from several techniques, either individually or in combination, including diffraction/scattering, microscopy, and spectroscopy. In this review, we describe efforts to characterize the organization of cellulose in plant cell walls. X-ray scattering reveals the size and orientation of microfibrils; diffraction reveals unit lattice parameters and crystallinity. The presence of different cell wall components, their physical and chemical states, and their alignment and orientation have been identified by Infrared, Raman, Nuclear Magnetic Resonance, and Sum Frequency Generation spectroscopy. Direct visualization of cell wall components, their network-like structure, and interactions between different components has also been made possible through a host of microscopic imaging techniques including scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. This review highlights advantages and limitations of different analytical techniques for characterizing cellulose structure and its interaction with other wall polymers. We also delineate emerging opportunities for future developments of structural characterization tools and multi-modal analyses of cellulose and plant cell walls. Ultimately, elucidation of the structure of plant cell walls across multiple length scales will be imperative for establishing structure-property relationships to link cell wall structure to control of growth and mechanics.

An Automated Protoplast Transformation System.

Efficient plant protoplast production from cell suspension cultures, leaf, and stem tissue allows for single-cell plant biology. Since protoplasts do not have cell walls, they can be readily transformed to enable rapid assessment of regulatory elements, synthetic constructs, gene expression, and more recently genome-editing tools and approaches. Historically, enzymatic cell wall digestion has been both expensive and laborious. Protoplast production, transformation, and analysis of fluorescence have recently been automated using an integrated robotic system. Here we describe its use for bulk protoplast isolation, counting, transformation, and analysis at very low cost for high-throughput experiments.

Cloning and Expression of Lipopolysaccharide Elimination Protein (LEP) in Lactic Acid Bacteria.

Lipopolysaccharide (LPS) is related to human inflammation. Therefore, in the probiotics research field, controlling the mechanisms of LPS neutralization and elimination of inflammation of human intestines are important. This chapter presents a description of the identification of LPS elimination protein (LEP) in lactic acid bacteria (LAB), cloning of its protein, and its expression. First, LEP is extracted from the LAB cell wall digestion fraction using Blue Native PAGE. Then LEP is identified by the elimination activity of LPS on gel pieces. Results show that the LEP is an approx. 200kDa protein part of heat shock protein in lactic acid bacteria. After sequencing amino acids of LEP, LEP cloning is done using a Brevibacillus sp. expression system without a general transformation system but with Gram-negative Escherichia coli having LPS. Results presented in this chapter demonstrate the elimination activity of recombinant LEP.

Bioethanol production from bamboo with alkali-catalyzed liquid hot water pretreatment

Altering recalcitrant structures of bamboo is essential to obtain high yield of bioethanol via bioconversion process. With the goal of improving cell wall digestibility, alkaline liquid hot water was used to pretreat N. affinis. The effects of temperature and alkali dosage on structural alterations were determined by chemical composition, Brunauer Emmett Teller (BET) and gel permeation chromatography (GPC). The relationship between these changes and substrate digestibility was addressed by separate enzymatic hydrolysis and fermentation (SHF). The results indicated that pretreatments partly removed and degraded hemicelluloses and lignin, reducing yields of substrates and molecular weights of carbohydrates. With the change of cell wall structure, specific surface area of materials increased after LHW pretreatment but decreased with further removal of lignin and hemicellulosic fractions. Maximum bioconversion was obtained by pretreatment with 0.5% NaOH aqueous at 170 °C and SHF, yielding 4.8 g/L ethanol.

Simultaneous regulation of F5H in COMT-RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis.

SummaryFerulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

Transcriptome analysis of an incompatible Persea americana-Phytophthora cinnamomi interaction reveals the involvement of SA- and JA-pathways in a successful defense response.

Phytophthora cinnamomi Rands (Pc) is a hemibiotrophic oomycete and the causal agent of Phytophthora root rot (PRR) of the commercially important fruit crop avocado (Persea americana Mill.). Plant defense against pathogens is modulated by phytohormone signaling pathways such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), auxin and abscisic acid. The role of specific signaling pathways induced and regulated during hemibiotroph-plant interactions has been widely debated. Some studies report SA mediated defense while others hypothesize that JA responses restrict the spread of pathogens. This study aimed to identify the role of SA- and JA- associated genes in the defense strategy of a resistant avocado rootstock, Dusa in response to Pc infection. Transcripts associated with SA-mediated defense pathways and lignin biosynthesis were upregulated at 6 hours post-inoculation (hpi). Results suggest that auxin, reactive oxygen species (ROS) and Ca2+ signaling was also important during this early time point, while JA signaling was absent. Both SA and JA defense responses were shown to play a role during defense at 18 hpi. Induction of genes associated with ROS detoxification and cell wall digestion (β-1-3-glucanase) was also observed. Most genes induced at 24 hpi were linked to JA responses. Other processes at play in avocado at 24 hpi include cell wall strengthening, the formation of phenolics and induction of arabinogalactan, a gene linked to Pc zoospore immobility. This study represents the first transcriptome wide analysis of a resistant avocado rootstock treated with SA and JA compared to Pc infection. The results provide evidence of a biphasic defense response against the hemibiotroph, which initially involves SA-mediated gene expression followed by the enrichment of JA-mediated defense from 18 to 24 hpi. Genes and molecular pathways linked to Pc resistance are highlighted and may serve as future targets for manipulation in the development of PRR resistant avocado rootstocks.

Expression of a hyperthermophilic endoglucanase in hybrid poplar modifies the plant cell wall and enhances digestibility

BackgroundExpression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates.ResultsIn this study, transgenic hybrid poplar (Populus alba × grandidentata) was generated to express a hyperthermophilic endoglucanase from Thermotoga neapolitana with an optimal temperature over 100 °C. Functional hyperthermoactive endoglucanase was successfully produced in the transgenic events, and altered phenotypic growth was observed in transgenic lines. Moreover, the line with the highest TnCelB expression in both leaf and developing xylem had reduced lignin content and cellulose crystallinity, resulting in a more digestible cell wall. The activation of TnCelB by a post-harvest heat treatment resulted in enhanced saccharification efficiencies of transgenic poplar lines with moderate TnCelB expression and without alteration of cellulose and lignin when not heat-treated. In planta high-level overexpression of a hyperthermophilic endoglucanase paired with heat treatment following harvest, resulted in biomass that was comparable with wild-type lines that underwent a traditional pretreatment for saccharification.ConclusionsOverexpression of hyperthermophilic endoglucanase in feedstock had impacts on plant growth and cell wall composition, especially when the enzyme was highly expressed. Improved glucan saccharification efficiencies from transgenic lines before and after heat treatment could reduce both the economic and environmental costs associated with ethanol production from lignocellulosic biomass.

Field Assessment of Alfalfa Populations Recurrently Selected for Stem Cell Wall Digestibility

Genetic variability for stem cell wall (CW) digestibility could be exploited to improve rumen‐fermentable energy in alfalfa (Medicago sativa L.) forage. We evaluated in the field the response to recurrent selection for stem CW digestibility in alfalfa. Digestibility was assessed as the concentration of glucose released after enzymatic hydrolysis of fiber (enzyme‐released glucose, ERG). Two initial cultivars, 54V54 and Orca, and populations obtained after successive cycles of divergent selection for stem CW digestibility (D−1, D−2, D+1, and D+2) were established at three field sites in north, central, and south of Québec. Field trials conducted over two growing seasons showed that populations obtained after two selection cycles (D+2) had significantly higher CW digestibility (+20.7 mg ERG g−1 CW) than initial cultivars (average of 13% improvement of digestibility). The D+2 populations did not differ from the initial cultivars with regard to biomass yield, winter survival, and stem water soluble‐carbohydrate concentration. Increases of ERG concentrations were observed in response to each selection cycle, and broad‐sense heritability highlights a moderate control of genetic factors over environmental factors for CW digestibility. Recurrent selection for stem CW digestibility is a valuable approach to increase fermentable energy in alfalfa forage and improve N utilization by ruminants.

Nutritive value of cowpea fodder cultivars and their effect on nutrients intake, digestibility and nitrogen balance in sheep

Three cowpea fodder varieties (BL-1, BL-2 and BL-4) developed by IGFRI were evaluated against check Kohinoor for their nutritional attributes by in vitro and in vivo means. Varieties were grown under identical agronomic practices in Kharif 2016 and animal feeding was started at flower initiation stage. For feeding trial, 20 growing sheep (mean body weight 19.85±0.45 kg) were randomly distributed to 4 varieties (Kohinoor, BL-1, BL-2 and BL- 4) with 5 animals to each. After 3 weeks of feeding, a digestion cum- metabolism trial was conducted to assess the nutrients intake, digestibility and nitrogen balance in sheep. The cowpea fodder varieties CP, NDF and lignin contents differed and values ranged between 22.23–23.41, 50.54–55.10 and 8.48–10.76%, respectively. Protein and carbohydrate fractions of cowpea varieties were similar except the NSC (% DM) and CC (% CHO) which were lower and higher, respectively in BL-2. Cowpea varieties energy values as TDN, DE, NEL, NEM and NEG were identical. In vitro gas and methane production (ml/g DM) was lower from Kohinoor (86.57 and 10.67) and BL-2 (90.79 and 11.40) than BL-1 (104.83 and 13.96) and BL-4 (98.86 and 13.08), respectively. Short chain fatty acids, partition factor and efficiency for microbial production differed significantly amongst the cowpea varieties. Sheep DM, CP and ME intake (% body wt and g/kg W0.75) was comparable on evaluated cowpea varieties. Digestibility of DM, OM and CP in sheep was similar on cowpea varieties; however, NDF and ADF digestibility tended to be lower of BL-4 (63.88 and 61.70%) than BL-2. Sheep nitrogen balance was identical on all the cowpea varieties and ranged between 16.92–18.32 g/d. Cowpea varieties DCP and DE contents were similar and ranged between 18.36– 20.30% and 13.63–14.72 kjoules/g DM, respectively. Results showed that nutritive value of cowpea varieties differed significantly in terms of CP, NDF, NSC%DM, fermentation pattern, and cell wall (NDF and ADF) digestibility.

Pathway-specific genetic pretreatment strategy to improve bioenergy feedstock

The plant secondary wall is composed of a complex mixture of cellulose, hemicellulose (e.g., xylan), and lignin. The transcription factor MYB46 (At5g12870) has been reported as a central regulator of the secondary wall formation. However, constitutive overexpression of MYB46 results in a severe growth penalty, possibly due to ectopic lignification in the parenchymatous and photosynthetic cells. To test this hypothesis, we constitutively overexpressed MYB46 in the genetic background of a T-DNA insertion mutant 4cl1-2 of 4-coumarate-CoA ligase 1 (4CL1), a key lignin biosynthesis gene. As expected, the transgenic Arabidopsis plants 35S::MYB46/4cl1-2 grow normally, similarly to the 4cl1-2 mutant, with elevated expressions of three secondary wall cellulose synthase genes and xylan biosynthesis genes. Consistent with the gene expression changes, the immunohistological staining of stem tissues showed the increased accumulation of both cellulose and xylan contents in the 35S::MYB46/4cl1-2 plants. Further biochemical analyses confirmed that 35S::MYB46/4cl1-2 plants have higher level accumulations of crystalline cellulose and xylan in both leaf and stem tissues than wild-type control plants as well as the 4cl1-2 mutant, up to 1.18- and 1.13-fold respectively, with considerably reduced lignin content (up to 0.40-fold). Subsequent analysis of enzymatic glucose release from cell wall materials revealed that 35S::MYB46/4cl1-2 plants have higher yields up to 1.27-fold increase in both leaf and stem tissues than that of control plants and 4cl1-2 mutant. Our results showed that overexpression of MYB46 with simultaneous reduction of lignin biosynthesis significantly increases both fermentable sugar contents and cell wall digestibility without growth penalty associated with MYB46 overexpression.

Biopolymer - A beginning towards back to nature

Biopolymer is regarded as a polymer which can be biodegradable. Polyhydroxyalkanoates (PHAs) is one of the biopolymer which can be recovered from biomass. PHAs are naturally conserved in the cytoplasm of the bacterial cell during the growth. Bacteria/microbes store their energy from carbon sources in the form of hydrocarbons. Intracellular stored compounds are tightly linked with entire cell resulting difficulty of separation. The work aims to extract PHAs from biomass effectively. Chemical and mechanical separation of PHA can be done from biomass. A pretreatment of cells before chemical and mechanical separation is also effective for separation of PHA and has been carried out. Chemical extraction of PHA includes digestion of cell wall in acidic or alkaline medium and releasing PHA in broth, later sedimentation recovers PHA. In recent work different chemical methods were carried out to extract PHA of medium chain length. In one of these, sodium hypochlorite was used to denature the protein and chloroform was used for extraction of purified PHA. A recovery upto 96.6%, PHA by dried weight of cell, was obtained which is quite high comparing to reported literature. Other chemical disruption by sodium chloride, sodium hydroxide and hydrogen peroxide with and without pretreatment have also been carried out.

Rapid Determination of Resistance to Antibiotic Inhibitors of Protein Synthesis in Staphylococcus aureus Through In Situ Evaluation of DNase Activity.

A rapid assay was designed for the detection of resistant strains of Staphylococcus aureus to antibiotic inhibitors of protein synthesis. The assay was based on the fact that a brief cell wall digestion with lysostaphin resulted in fragmentation of the chromosomal DNA by releasing the characteristic DNase stored in the cell wall. DNase activity was ascertained by visualization of the DNA fragments released from the isolated nucleoids. Lysostaphin-released DNase activity was found to be influenced by ribosomal protein synthesis. Inhibition of protein synthesis resulted in the prevention of lysostaphin-DNase induced DNA fragmentation when susceptible clinical strains were incubated with erythromycin, azithromycin, or doxycycline for 2 hr before enzymatic treatment. However, in nonsusceptible strains where protein synthesis was unsuccessfully inhibited, this suppression of lysostaphin-DNase was not, or only very slightly, evident. This assay was highly efficient, identifying resistance to erythromycin and azithromycin with 88-90.9% sensitivity and 100% specificity and with 100% sensitivity and specificity to gentamicin and doxycycline, within a 2 hr and 45 min period.