Strategies Shaping the Transcription of Carbohydrate-Active Enzyme Genes in Aspergillus nidulans

Understanding the coordinated regulation of the hundreds of carbohydrate-active enzyme (CAZyme) genes occurring in the genomes of fungi has great practical importance. We recorded genome-wide transcriptional changes of Aspergillus nidulans cultivated on glucose, lactose, or arabinogalactan, as well as under carbon-starved conditions. We determined both carbon-stress-specific changes (weak or no carbon source vs. glucose) and carbon-source-specific changes (one type of culture vs. all other cultures). Many CAZyme genes showed carbon-stress-specific and/or carbon-source-specific upregulation on arabinogalactan (138 and 62 genes, respectively). Besides galactosidase and arabinan-degrading enzyme genes, enrichment of cellulolytic, pectinolytic, mannan, and xylan-degrading enzyme genes was observed. Fewer upregulated genes, 81 and 107 carbon stress specific, and 6 and 16 carbon source specific, were found on lactose and in carbon-starved cultures, respectively. They were enriched only in galactosidase and xylosidase genes on lactose and rhamnogalacturonanase genes in both cultures. Some CAZyme genes (29 genes) showed carbon-source-specific upregulation on glucose, and they were enriched in β-1,4-glucanase genes. The behavioral ecological background of these characteristics was evaluated to comprehensively organize our knowledge on CAZyme production, which can lead to developing new strategies to produce enzymes for plant cell wall saccharification.

Dietary Fiber in Plant Cell Walls—The Healthy Carbohydrates

Dietary fiber (DF) is one of the major classes of nutrients for humans. It is widely distributed in the edible parts of natural plants, with the cell wall being the main DF-containing structure. The DF content varies significantly in different plant species and organs, and the processing procedure can have a dramatic effect on the DF composition of plant-based foods. Given the considerable nutritional value of DF, a deeper understanding of DF in food plants, including its composition and biosynthesis, is fundamental to the establishment of a daily intake reference of DF and is also critical to molecular breeding programs for modifying DF content. In the past decades, plant cell wall biology has seen dramatic progress, and such knowledge is of great potential to be translated into DF-related food science research and may provide future research directions for improving the health benefits of food crops. In this review, to spark interdisciplinary discussions between food science researchers and plant cell wall biologists, we focus on a specific category of DF—cell wall carbohydrates. We first summarize the content and composition of carbohydrate DF in various plant-based foods, and then discuss the structure and biosynthesis mechanism of each carbohydrate DF category, in particular the respective biosynthetic enzymes. Health impacts of DF are highlighted, and finally, future directions of DF research are also briefly outlined.

Identification and Fine Mapping of the Recessive Gene BK-5, Which Affects Cell Wall Biosynthesis and Plant Brittleness in Maize

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant–pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.

Comparative Proteome Analysis of the Penultimate Internodes of Barley Genotypes Differing in Stem Reserve Remobilization Under Drought Stress

Barley yield relies more on stem reserves under stress conditions at the grain filling stage. At terminal drought stresses, the remobilization of reserved assimilates from stem to seed contributes a major role in yield. To understand the molecular mechanism of stem reserve utilization during drought stress, a comparative proteome and physiological analyses were performed on the penultimate internodes of three genotypes of barley Yousef (tolerant), Morocco (susceptible), and PBYT17 (semi-tolerant) under drought stress at 21 and 28 days after anthesis (DAA). Under water stress and well-watered conditions Yousef showed significantly higher RWC, grain yield, and stem reserve remobilization capacity than susceptible and semi-tolerant genotypes. The proteome analysis led to the identification of 1580 differentially abundant proteins (DAPs), of which 759 and 821 proteins were differentially expressed at 21 and 28 DAA, respectively. Tolerant genotype in response to drought stress increased the abundance of several plant cell wall polysaccharide degradation proteins and protein kinases associated with posttranslational-associated, which might accelerate remobilization process for seed biomass formation compared to susceptible one under drought stress. However, the susceptible genotype increased the abundance of proteins involved in RNA metabolism and transcriptional changes to save energy sources for the growth and survival during drought stress. These findings suggest that barley might response to water stress by efficiently remobilize assimilates from stem to grain through specific remobilization processes.

Assessing Comparative Microbiome Performance in Plant Cell Wall Deconstruction Using Multi-omics-Informed Network Analysis

Plant cell walls are interwoven structures recalcitrant to degradation. Both native and adapted microbiomes are particularly effective at plant cell wall deconstruction. Studying these deconstructive microbiomes provides an opportunity to assess microbiome performance and relate it to specific microbial populations and enzymes. To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from time-dependent gene expression identified key deconstructive groups within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmanimonadetes populations. Functional analysis of gene expression demonstrated that the microbiomes proceeded through successional stages that are linked to enzymes that deconstruct plant cell wall polymers. This combination of network and functional analysis highlighted the importance of cellulose-active Actinobacteria in differentiating the performance of these microbiomes.

Comparative genome analysis of plant ascomycete fungal pathogens with different lifestyles reveals distinctive virulence strategies

Background Pathogens have evolved diverse lifestyles and adopted pivotal new roles in both natural ecosystems and human environments. However, the molecular mechanisms underlying their adaptation to new lifestyles are obscure. Comparative genomics was adopted to determine distinct strategies of plant ascomycete fungal pathogens with different lifestyles and to elucidate their distinctive virulence strategies. Results We found that plant ascomycete biotrophs exhibited lower gene gain and loss events and loss of CAZyme-encoding genes involved in plant cell wall degradation and biosynthesis gene clusters for the production of secondary metabolites in the genome. Comparison with the candidate effectome detected distinctive variations between plant biotrophic pathogens and other groups (including human, necrotrophic and hemibiotrophic pathogens). The results revealed the biotroph-specific and lifestyle-conserved candidate effector families. These data have been configured in web-based genome browser applications for public display (http://lab.malab.cn/soft/PFPG). This resource allows researchers to profile the genome, proteome, secretome and effectome of plant fungal pathogens. Conclusions Our findings demonstrated different genome evolution strategies of plant fungal pathogens with different lifestyles and explored their lifestyle-conserved and specific candidate effectors. It will provide a new basis for discovering the novel effectors and their pathogenic mechanisms.

A unified view of LCRs across species

Low complexity regions (LCRs) in proteins play a major role in the higher order assemblies of organisms, such as the nucleolus and extracellular matrix. Despite recent focus on how certain features affect the function of LCRs in intracellular higher order assemblies, the relationships between LCRs within proteins, captured by their type and copy number, has yet to be systematically studied. Furthermore, we still lack a unified view of how the sequences, features, relationships and functions of LCRs relate to each other. Here, we developed a systematic and comprehensive approach using dotplot matrices and dimensionality reduction to define LCR relationships proteome-wide and to create a map of LCR sequences capable of integrating any LCR features. As a proof of concept of the importance of LCR relationships, we demonstrate the biological significance of LCR copy number for higher order assembly of the nucleolar protein RPA43 both in vitro and in vivo. Using the LCR map, we revealed the boundaries and connections between regions of sequence space occupied by LCRs, and that LCRs of certain higher order assemblies populated specific regions of sequence space. The integration of LCR relationships and the LCR map provided a unified view of LCRs which uncovered the distribution, distinguishing features, and conserved prevalence of glutamic acid-rich LCRs among nucleolar proteins. When applied across multiple species, this approach highlights how differential occupancy of certain regions of LCR sequence space corresponds to the conservation and emergence of higher order assemblies, such as the plant cell wall or metazoan extracellular matrix. Additionally, we identified previously undescribed regions of LCR sequence space, including a teleost-specific threonine/histidine-rich cluster which exhibits signatures of higher order assemblies. By providing this unified view of LCRs, our approach enables discovery of how LCRs encode higher order assemblies of organisms.

Genomic insights into the phylogeny and biomass-degrading enzymes of rumen ciliates

Understanding the biodiversity and genetics of the gut microbiome has important implications for host physiology. One underexplored and elusive group is ciliated protozoa, which play crucial roles in regulating gut microbial interactions. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies for all major abundant clades. With these genomes, we firstly resolved the taxonomic and phylogenetic framework that reclassified them into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Via extensive horizontal gene transfer and gene family expansion, rumen ciliates possess a broad array of enzymes to synergistically degrade plant and microbial carbohydrates. In particular, ~80% of the degrading enzymes in Diplodiniinae and Ophryoscolecinae act on plant cell wall, and the high activities of their cellulase, xylanase and lysozyme reflect the potential of ciliate enzymes for biomass-conversion. Additionally, the new ciliate dataset greatly facilitated the rumen metagenomic analyses by allowing ~12% of reads to be classified.

Delivery of DNA into Human Cells by Functionalized Lignin Nanoparticles

Lignin is an aromatic plant cell wall polymer that is generated in large quantities as a low-value by-product by the pulp and paper industry and by biorefineries that produce renewable fuels and chemicals from plant biomass. Lignin structure varies among plant species and as a function of the method used for its extraction from plant biomass. We first explored the impact of this variation on the physico-chemical properties of lignin nanoparticles (LNPs) produced via a solvent exchange procedure and then examined whether LNPs produced from industrial sources of lignin could be used as delivery vehicles for DNA. Spherical LNPs were formed from birch and wheat BioLignin™ and from poplar thioglycolic acid lignin after dissolving the lignin in tetrahydrofuran (THF) and dialyzing it against water. Dynamic light scattering indicated that the diameter of these LNPs was dependent on the initial concentration of the lignin, while electrophoretic light scattering indicated that the LNPs had a negative zeta potential, which became less negative as the diameter increased. The dynamics of LNP formation as a function of the initial lignin concentration varied as a function of the source of the lignin, as did the absolute value of the zeta potential. After coating the LNPs with cationic poly-l-lysine, an electrophoretic mobility shift assay indicated that DNA could adsorb to LNPs. Upon transfection of human A549 lung carcinoma basal epithelial cells with functionalized LNPs carrying plasmid DNA encoding the enhanced green fluorescent protein (eGFP), green foci were observed under the microscope, and the presence of eGFP in the transfected cells was confirmed by ELISA. The low cytotoxicity of these LNPs and the ability to tailor diameter and zeta potential make these LNPs of interest for future gene therapy applications.