Cell Wall Digestion Research Articles

Precise control of water stress in the field reveals different response thresholds for forage yield and digestibility of maize hybrids.

With dwindling global freshwater supplies and increasing water stress, agriculture is coming under increasing pressure to reduce water use. Plant breeding requires high analytical capabilities. For this reason, near-infrared spectroscopy (NIRS) has been used to develop prediction equations for whole-plant samples, particularly for predicting dry matter digestibility, which has a major impact on the energy value of forage maize hybrids and is required for inclusion in the official French catalogue. Although the historical NIRS equations have long been used routinely in seed company breeding programmes, they do not predict all variables with the same accuracy. In addition, little is known about how accurate their predictions are under different water stress-environments. Here, we examined the effects of water stress and stress intensity on agronomic, biochemical, and NIRS predictive values in a set of 13 modern S0-S1 forage maize hybrids under four different environmental conditions resulting from the combination of a northern and southern location and two monitored water stress levels in the south. First, we compared the reliability of NIRS predictions for basic forage quality traits obtained using the historical NIRS predictive equations and the new equations we recently developed. We found that NIRS predicted values were affected to varying degrees by environmental conditions. We also showed that forage yield gradually decreased as a function of water stress, whereas both dry matter and cell wall digestibilities increased regardless of the intensity of water stress, with variability among the tested varieties decreasing under the most stressed conditions. By combining forage yield and dry matter digestibility, we were able to quantify digestible yield and identify varieties with different strategies for coping with water stress, raising the exciting possibility that important potential selection targets still exist. Finally, from a farmer's perspective, we were able to show that late silage harvest has no effect on dry matter digestibility and that moderate water stress does not necessarily result in a loss of digestible yield.

Adaptability of regression algorithms to protein Plant behavior

The behavior of components of protein plant is of vital importance for animals that consume them in their diet. The objective of this research is to evaluate regression algorithms, to determine the behavior of the expressions that best adapt to the procedures of a traditional laboratory and to estimate the chemical components of protein plants, in this sense the MULAN library of java has been used, that contain automatic learning algorithms capable of adapting to dissimilar problems. Three data set were created for each species treated in this study; each of these include the main elements to be evaluate in each experiment, these are delimits by: secondary metabolites, cell wall components and digestibility element for training files one, two and three, respectively; subsequently, they were evaluated through learning supervised and cross-validation of each to determine the best fit by aRMSE (Average Root Mean Square Error). The learning results were compare with previous experiments, where there was a learning variant that contained in a single dataset all the components to be evaluates in a single prediction. The result of the comparison shows that the lazy algorithms based on instances have a better learning behavior than the others evaluate.

Pectins, hemicellulose and lignocellulose profiles vary in leaves among different aromatic Vanilla species (Orchidaceae)

The alcohol-insoluble residue in Vanilla dressleri, V. odorata, V. planifolia and V. pompona mature leaves was fractionated into water-, oxalate-, HCl- and NaOH-soluble pectins, hemicellulose and lignocellulose. Neutral sugars and uronic acids of each fraction were determined by high-performance anion exchange chromatography-pulsed amperometric detection. V. dressleri differentiated from the other three species based on the higher amounts of the HCl-soluble pectin fraction and the amount of glucose exceeding 5–7 times the values found in the NaOH-soluble pectin fraction in the other species, as well as a cellulose/hemicellulose ratio of ca. 2/3. Analogously, V. planifolia could also be distinguished by its higher cellulose/hemicellulose ratio of ca. 7. In addition to taxonomical implications, these results might be instrumental for defining adequate conditions for cell wall digestion aiming at protoplast isolation and appropriate media composition for cell wall regeneration during protoplast culture, which has been elusive in the Orchidaceae.

Forages and Pastures Symposium: revisiting mechanisms, methods, and models for altering forage cell wall utilization for ruminants.

Several ruminant animals rely almost exclusively on the complex polysaccharide matrix from the plant cell wall (CW) as their primary energy source via volatile fatty acids produced through ruminal and some hindgut fermentation processes. The CW contains different types and proportions of polysaccharides, proteins, phenolic compounds, and minerals in their macromolecular structure that influence the rate and extent of fiber digestion and selective retention of particulate matter due to its physical characteristics (buoyancy and comminuting) in the reticulorumen. The biosynthetic formation of the CW dictates possible manipulation mechanisms (targeted plant and microbes selection) and processing methods (physical, chemical, microbial, and enzymatic treatments and the use of genetically engineered bacteria) to increase its digestibility, leading to better utilization of the CW by the ruminant animal and hopefully lower the contribution of ruminants' greenhouse gas emissions. Early studies on lignin biosynthesis have led to more advanced studies focusing on replacing traditional monolignols with homopolymers that are easier to deconstruct or degrade. Concurrently, laboratory methods must be developed, evaluated, and modified to accurately reflect the digestibility and nutritive value of CW brought about by modern manipulation mechanisms or processing methods. However, the laboratory methods must also be reliable, precise, feasible, trivial, easy to implement, and cost-effective, but at the same time environmentally friendly and aware. For instance, although the acid detergent lignin has been demonstrated to behave uniformly as a nutritional entity, its chemical determination and association with carbohydrates still lack consensus. Spectroscopy (near-infrared and Raman) and in vitro gas production techniques have been adopted to assess plant chemical composition and nutritive value, but an incomplete understanding of the impacts caused by disrupting the CW for sample processing still exists. Different variations of multicompartmental and time- and age-dependent mathematical models have been proposed to determine the ruminal rates of degradation and passage of fiber. However, low-quality and incomplete data due to inconsistent marker results used to determine passage rates and transit time of fiber in the gastrointestinal tract have hindered advancements and adoptions of the next generation of computer models to understand ruminal fiber degradation.

Expansion Microscopy of Plant Cells (PlantExM).

Expansion microscopy (ExM) achieves super-resolution imaging without the need for sophisticated super-resolution microscopy hardware through a combination of physical and optical magnification. Samples are fixed, stained, and embedded in a swellable gel. Following cross-linking of fluorophores to the gel matrix, the components of the sample are digested away and the gel expanded in water. Labeled objects which are too close to be resolved by diffraction-limited microscopy are moved far enough apart that these can now be resolved as individual objects on a standard confocal. Originally developed for animal cells and tissues, ExM for plants requires the additional consideration of cell wall digestion. Super-resolution can be limited in plants due to the size of cells, light scattering of tissues, and variations in refractive index. By removing the components which cause these limitations, ExM opens up the possibility of super-resolution at depth within plant tissues for the first time. Here we describe our method for PlantExM which is optimized for cytoskeleton resolution, which, when also coupled with compatible optical super-resolution technologies, can produce images of the plant cytoskeleton in unprecedented detail.

Solid-state fermentation alters the fate of red kidney bean protein during buccal and gastrointestinal digestion: Relationship with cotyledon cell wall integrity

The relationship between legume cotyledon cell wall and macromolecular nutrient digestibility has attracted increased attention. In this study, the effect of solid-state fermentation by Rhizopus oligosporus RT-3 on the digestibility of red kidney bean protein and its relationship with cotyledon cell integrity were investigated. Buccal digestion and gastrointestinal digestion were performed to compare the fate of protein between unfermented (F0) and fermented samples. Results showed a remarkable disruption in cotyledon cell integrity at the late fermentation period, and it was accompanied by a possible migration/degradation of protein matrix. Buccal and gastrointestinal digestion barely affected cell wall integrity at F0 but notably disintegrated cell morphology at 29 h of fermentation (F29). As this fermentation time, gastrointestinal digestion resulted in higher contents of soluble proteins, peptides, and free amino acids by 1.4-, 1.8-, and 2.5-fold, respectively. Therefore, solid-state fermentation facilitated the structural breakdown of cotyledon cell walls, thereby further improving protein digestibility.

Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion

Secretory vesicle clusters transported on actin filaments by myosin V motors for local secretion underlie various cellular processes, such as neurotransmitter release at neuronal synapses,1 hyphal steering in filamentous fungi,2,3 and local cell wall digestion preceding the fusion of yeast gametes.4 During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes.5,6,7 The position and coalescence of the vesicle focus are controlled by local signaling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis,6,8,9,10 but the mechanisms of focusing have been elusive. Here, we show that the regulatory N terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation invivo and forms dense assemblies that exclude ribosomes. Fus1 lacking its IDR fails to concentrate in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, the replacement of Fus1 IDR with a heterologous low-complexity region that forms molecular condensates fully restores Fus1 focusing and function. By contrast, the replacement of Fus1 IDR with a domain that forms more stable oligomers restores focusing but poorly supports cell fusion, suggesting that condensation is tuned to yield a selectively permeable structure. We propose that condensation of actin structures by an IDR may be a general mechanism for actin network organization and the selective local concentration of secretory vesicles.

200 Forage Cell Wall Organization, Complexes, and Components

Abstract It has been nearly 31 years since the International Symposium on Forage Cell Wall Structure and Digestibility was held in Madison, WI. The text resulting from that symposium, first published in 1993, has been an invaluable resource for animal and plant scientists alike but, unfortunately, has yet to be updated. Symposium organizers recognized that there was "a critical lack of information on the chemistry of the cell walls of forage plants and especially how this chemistry relates to digestibility and absorption of the materials and how the digestibility can be improved through plant breeding". They proceeded to organize an excellent symposium that covered nearly every aspect of that broad topic. Whereas "fiber" (plant cell wall) always has been a mainstay in ruminant animal feeding, its importance in the areas of human and non-ruminant animal nutrition has now been realized as indicated by the extensive scientific literature published on the topic since the time of the symposium, and by the fact that dietary fiber is now considered an "essential nutrient" for the human. In this presentation, the components of the plant cell wall will be discussed and their organization within the plant defined. Their relationship to the gut microbiota will be highlighted as will those factors critical in determining accurate fiber concentrations in forages and fibrous ingredients using the latest fiber methodologies. Rather than being viewed simply as "roughage" or as "filler" or as an "anti-nutritional factor", cell walls now have a prominent role in all of animal and human nutrition, so understanding their characteristics at all levels becomes all the more important.

Effect of daily or intermittent addition of fenugreek (Trigonella foenum graecum L.) seeds to concentrate on intake, digestion, and growth performance of Barbarine lamb

An 82-day experiment was carried out to determine the response of growing lambs to the intermittent administration of fenugreek (FG) seeds (Trigonella foenum graecum L.) into concentrate. Twenty-Four 3-month-old Barbarine lambs (initial body weight 16.6 ± 0.42 kg) were divided into four equal groups (n = 6). All lambs received 600 g alfalfa pellets and 250 g concentrate containing or not 12% ground FG seeds. Control concentrate (CC) was composed (as fed) of barley (200 g), faba bean (40 g) and mineral and vitamin supplement (MVS, 14 g). The experimental concentrate (CE) contained barley (200 g), FG seeds (30 g) and MVS (14 g). The two concentrates have the same crude protein content. The control group (FG-0) received CC while groups FG-D, FG-2D and FG-4D received CE daily, or in alternation with concentrate CC at 2 or 4-day intervals (FG-2D or FG-4D, respectively). The feeding trial was followed by a 5-day in vivo digestibility trial. The frequency of distribution of FG-containing concentrate (CE) had no effect (P > 0.05) on diet intake and diet apparent digestibility of dry matter, organic matter, crude protein and cell wall (NDFom), nitrogen balance and the concentrations of urea, total protein and glucose in sheep plasma. The intermittent supply of FG seeds in concentrate tended to increase (P = 0.087)the average daily gain in FG-2D and FG-4D lambs and the microbial nitrogen synthesis (P = 0.052)with the FG administration. However, it decreased (P < 0.001) ruminal protozoa count by 33%, 44% and 66% in the FG-D, FG-2D and FG-4D lambs, respectively compared to FG-0 group. Similarly, the intermittent administration of FG seeds decreased ruminal ammonia nitrogen (NH3-N) concentrations (P < 0.001) by 28%, 31% and 35% in the FG-D, FG-2D and FG-4D, respectively. The antiprotozoal activity of FG seeds could have been induced by secondary compounds like saponins. This activity was more efficient when the frequency of incorporation of FG seeds into the concentrate is wider. It is concluded that the intermittent administration of 120 g/kg to concentrate (DM basis), had an antiprotozoal effect but failed to improve diet digestibility and growth performance of Barbarine lambs. Therefore, there is a need to emphasize further investigation to identify the active components responsible for the antiprotozoal activity of FG seeds.

CRISPR/Cas9 suppression of OsAT10, a rice BAHD acyltransferase, reduces p-coumaric acid incorporation into arabinoxylan without increasing saccharification.

Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important roles in crosslinking within cell wall arabinoxylans (AX) and between AX and lignin in grass cell walls. The addition of hydroxycinnamates to AX, is mediated by the Mitchell clade of BAHD acyl-coenzyme A-utilizing transferases. Overexpression of OsAT10 (a Mitchell clade BAHD acyl transferase) in rice, has previously been shown to increase p-CA content in AX in leaves and stems, leading to increased cell wall digestibility, potentially associated with a concomitant decrease in FA content. To investigate the physiological role of OsAT10 we established CRISPR/Cas9 rice knock-out mutants devoid of OsAT10. Our analysis of hydroxycinnamic acid content in wild type plants revealed that AX associated p-CA is found almost exclusively in rice husks, with very little found in other tissues. Mutant plants were essentially devoid of ester-linked p-CA associated with AX, indicating that OsAT10 represents the major enzyme responsible for the addition of p-CA to arabinoxylan in rice plants. We found no change in the digestibility of rice husk lacking AX-associated p-CA, suggesting that the changes in digestibility seen in OsAT10 overexpressing plants were solely due to compensatory decreases in AX-associated FA.

First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.

Partial enzymatic cell wall disruption of Oocystis sp. for simultaneous cultivation and extraction

A green extraction process is proposed to reduce energy and petroleum solvent’s consumption while maintaining environmental safety and quality that allows multiple product recovery. The goal of the study was to generate a highly permeable cell, through the use of crude fungal enzymes, that could permit simultaneous cultivation and extraction. A novel pretreatment method is developed which combines partial enzymatic digestion of cell walls of microalgae, while maintaining their viability, thus enabling further product recovery from the same biomass and has not been reported in literature yet. Partial digestion of the cell wall was accomplished through regulation of the contact time of crude enzymes with the microalgal cells leading to a highly permeable cell that is viable and actively growing. Experimentally this was achieved through controlling the time of incubation of microalgal cells with the crude enzymes. The effects of enzymatic treatment using crude fungal enzymes from Trichoderma reesei (cellulase 10.98 ± 0.17 U/ml and amylase 20.28 ± 0.13 U/ml) were investigated on the structural properties of marine microalga Oocystis sp. Results showed over 2-fold increase in protein efflux; 41% increase in released sugars and 24% decrease in cell count after 1 h of enzyme treatment at 33% (v/v) enzyme concentration. Controlled hydrolysis of algal cells could be achieved through optimizing the contact time (1 h) of enzymes with microalgal cells which retained their viability. This was validated through FTIR, SEM, XRD and Flow Cytometric analysis. The carotenoid content increased by approximately 9% and lipid content rose by nearly 6.2%.

Down-regulation of PvSAMS impairs S-adenosyl-L-methionine and lignin biosynthesis, and improves cell wall digestibility in switchgrass.

S-adenosyl- l-methionine (SAM) is the methyl donor involved in the biosynthesis of guaiacyl (G) and syringyl (S) lignins in vascular plants. SAM is synthesized from methionine through the catalysis of the enzyme S-adenosylmethionine synthase (SAMS). However, the detailed function of SAMS in lignin biosynthesis has not been widely investigated in plants, particularly in monocot species. In this study, we identified PvSAMS genes from switchgrass (Panicum virgatum L.), an important dual-purpose fodder and biofuel crop, and generated numerous transgenic switchgrass lines through PvSAMS RNA interference technology. Down-regulation of PvSAMS reduced the contents of SAM, G-lignins, and S-lignins in the transgenic switchgrass. The methionine and glucoside derivatives of caffeoyl alcohol were found to accumulate in the transgenic plants. Moreover, down-regulation of PvSAMS in switchgrass resulted in brownish stems associated with reduced lignin content and improved cell wall digestibility. Furthermore, transcriptomic analysis revealed that most sulfur deficiency-responsive genes were differentially expressed in the transgenic switchgrass, leading to a significant increase in total sulfur content; thus implying an important role of SAMS in the methionine cycle, lignin biosynthesis, and sulfur assimilation. Taken together, our results suggest that SAMS is a valuable target in lignin manipulation, and that manipulation of PvSAMS can simultaneously regulate the biosynthesis of SAM and methylated monolignols in switchgrass.

The release and catabolism of ferulic acid in plant cell wall by rumen microbes: A review.

Ferulic acid (FA) is one of the most abundant hydroxycinnamic acids in the plant world, especially in the cell wall of grain bran, in comparison with forage and crop residues. Previous studies noted that FA was mainly linked with arabinoxylans and lignin in plant cell walls in ester and ether covalent forms. After forages were ingested by ruminant animals or encountered rumen microbial fermentation in vitro, these cross-linkages form physical and chemical barriers to protect cell-wall carbohydrates from microbial attack and enzymatic hydrolysis. Additionally, increasing studies noted that FA presented some toxic effect on microbial growth in the rumen. In recent decades, many studies have addressed the relationships of ester and/or ether-linked FA with rumen nutrient digestibility, and there is still some controversy whether these linkages could be used as a predicator of forage digestibility in ruminants. The authors in this review summarized the possible relationships between ester and/or ether-linked FA and fiber digestion in ruminants. Rumen microbes, especially bacteria and fungi, were found capable of breaking down the ester linkages within plant cell walls by secreting feruloyl and p-coumaroyl esterase, resulting in the release of free FA and improvement of cell wall digestibility. The increasing evidence noted that these esterases secreted by rumen microbes presented synergistic effects with xylanase and cellulase to effectively hydrolyze forage cell walls. Some released FA were absorbed through the rumen wall directly and entered into blood circulation and presented antioxidant effects on host animals. The others were partially catabolized into volatile fatty acids by rumen microbes, and the possible catabolic pathways discussed. To better understand plant cell wall degradation in the rumen, the metabolic fate of FA along with lignin decomposition mechanisms are needed to be explored via future microbial isolation and incubation studies with aims to maximize dietary fiber intake and enhance fiber digestion in ruminant animals.

Bacterial-Assisted Extraction of Bioactive Compounds from Cauliflower

The market for nutraceutical molecules is growing at an impressive pace in all Western countries. A convenient source of bioactive compounds is found in vegetable waste products, and their re-use for the recovery of healthy biomolecules would increase the sustainability of the food production system. However, safe, cheap, and sustainable technologies should be applied for the recovery of these beneficial molecules, avoiding the use of toxic organic solvents or expensive equipment. The soil bacterium Bacillus subtilis is naturally endowed with several enzymes targeting complex vegetable polymers. In this work, a raw bacterial culture supernatant was used to assist in the extraction of bioactives using isothermal pressurization cycles. Besides a wild-type Bacillus subtilis strain, a new strain showing increased secretion of cellulases and xylanases, pivotal enzymes for the digestion of the plant cell wall, was also used. Results indicate that the recovery of compounds correlates with the amount of cellulolytic enzymes applied, demonstrating that the pretreatment with non-purified culture broth effectively promotes the release of bioactives from the vegetable matrix. Therefore, this approach is a valid and sustainable procedure for the recovery of bioactive compounds from food waste.

Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure

The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.

Alfalfa biotypes with putative enhanced cell wall digestibility and effects on performance of growing beef steers.

Three alfalfa biotypes were chosen based on the presumption that they would be sources of alfalfa herbage that differed in lignin concentration and therefore cell wall digestibility. The hypothesis was that a lesser lignin concentration would result in greater alfalfa neutral detergent fiber (NDF) digestibility and greater beef steer growth performance. The three alfalfa biotypes were HarvXtra (Forage Genetics International), Hi-Gest 360 (Alforex Seeds), and a control alfalfa, LegenDairy XHD (Winfield Solutions LLC). High-moisture wrapped bales were prepared from second-harvest, d 30 crops. Digestibility of NDF was determined using in vitro incubations and a steer digestibility trial. Alfalfa baleage and trace mineral salt were fed to Angus steers (300 kg initial body weight, 4 pens/treatment) in an 83-day growing-phase trial. Alfalfa acid detergent lignin concentrations were 75.6, 71.8, and 63.0 g/kg dry matter (P = 0.34) for LegenDairy, Hi-Gest and HarvXtra, respectively. Based on in vitro total-tract NDF digestibility coefficients, HarvXtra tended (P ≥ 0.09) to have the highest NDF digestibility. Alfalfa biotype affected in vivo apparent total tract digestibility of NDF (P < 0.001) and there was a trend for an effect on acid detergent fiber digestibility (P = 0.051). Hi-Gest and HarvXtra had similar in vivo apparent NDF digestibilities, which were greater than for LegenDairy (P < 0.05). There was no alfalfa biotype effect on daily alfalfa dry matter intake (DMI; P = 0.51) or average daily gain (P = 0.25) by growing steers. The absence of an effect by the novel alfalfa biotypes on DMI by growing steers suggests that the compositional and digestibility differences of the novel alfalfa biotypes compared to LegenDairy were not sufficient to alleviate the limitation of physical fill (if evident) on DMI. If more disparity in cell wall composition and NDF digestibility were to exist between control and reduced-lignin biotypes, then perhaps an advantage in cattle growth performance for a reduced-lignin alfalfa biotype would be detectable.

Seasonal Differences in Structural and Genetic Control of Digestibility in Perennial Ryegrass.

Perennial ryegrass is an important forage crop in dairy farming, either for grazing or haying purposes. To further optimise the forage use, this study focused on understanding forage digestibility in the two most important cuts of perennial ryegrass, the spring cut at heading and the autumn cut. In a highly diverse collection of 592 Lolium perenne genotypes, the organic matter digestibility (OMD) and underlying traits such as cell wall digestibility (NDFD) and cell wall components (cellulose, hemicellulose, and lignin) were investigated for 2 years. A high genotype × season interaction was found for OMD and NDFD, indicating differences in genetic control of these forage quality traits in spring versus autumn. OMD could be explained by both the quantity of cell wall content (NDF) and the quality of the cell wall content (NDFD). The variability in NDFD in spring was mainly explained by differences in hemicellulose. A 1% increase of the hemicellulose content in the cell wall (HC.NDF) resulted in an increase of 0.81% of NDFD. In autumn, it was mainly explained by the lignin content in the cell wall (ADL.NDF). A 0.1% decrease of ADL.NDF resulted in an increase of 0.41% of NDFD. The seasonal traits were highly heritable and showed a higher variation in autumn versus spring, indicating the potential to select for forage quality in the autumn cut. In a candidate gene association mapping approach, in which 503 genes involved in cell wall biogenesis, plant architecture, and phytohormone biosynthesis and signalling, identified significant quantitative trait loci (QTLs) which could explain from 29 to 52% of the phenotypic variance in the forage quality traits OMD and NDFD, with small effects of each marker taken individually (ranging from 1 to 7%). No identical QTLs were identified between seasons, but within a season, some QTLs were in common between digestibility traits and cell wall composition traits confirming the importance of hemicellulose concentration for spring digestibility and lignin concentration in NDF for autumn digestibility.

Methyltransferase as Antibiotics Against Foodborne Pathogens: An In Silico Approach for Exploring Enzyme as Enzymobiotics.

The development of resistance in microbes against antibiotics and limited choice for the use of chemical preservatives in food lead the urgent need to search for an alternative to antibiotics. The enzymes are catalytic proteins that catalyze digestion of bacterial cell walls and protein requirements for the survival of the cell. To study methyltransferase as antibiotics against foodborne pathogen, the methyltransferase enzyme sequence was modeled and its interactions were analyzed against a membrane protein of the gram-positive and gram-negative bacteria through in silico protein–protein interactions. The methyltransferase interaction with cellular protein was found to be maximum, due to the maximum PatchDock Score (15808), which was followed by colicin (12864) and amoxicillin (4122). The modeled protein has found to be interact more significantly to inhibit the indicator bacteria than the tested antibiotics and antimicrobial colicin protein. Thus, model enzyme methyltransferase could be used as enzymobiotics. Moreover, peptide sequences similar to this enzyme sequence need to be designed and evaluated against the microbial pathogen.

Protoplast Isolation and Transfection in the Single-Cell C4 Species Bienertia sinuspersici.

We have developed an optimized protocol for isolating protoplasts from chlorenchyma cells of the single-cell C4 species Bienertia sinuspersici. The isolated protoplasts maintained the integrity of the unique single-cell C4 intracellular compartmentation of organelles as observed in chlorenchyma cells after cell wall digestion. Approximately over 80% of isolated protoplasts expressed the fusion reporter gene following the polyethylene glycol-mediated transfection procedures. Overall, fluorescent protein fusion tagged with various intraorganellular sorting signals validated the potential use of the transient gene expression system in subcellular localization and organelle dynamics studies.