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.

Microtubule-associated IQD9 guides cellulose synthase velocity to shape seed mucilage

Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes is guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis (SCE). Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles for cell wall polysaccharide biosynthesis and cortical microtubule (MT) organization. Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. Double mutant analyses revealed that their closest paralogs (IQD10 and KLCR2, respectively) are not required for mucilage biosynthesis. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. Similar to the previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein, IQD9 is required to maintain the velocity of cellulose synthases. Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in guiding the distribution of cell wall polysaccharides. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.

Machine learning prediction of novel pectinolytic enzymes in Aspergillus niger through integrating heterogeneous (post-) genomics data

Pectinolytic enzymes are a variety of enzymes involved in breaking down pectin, a complex and abundant plant cell-wall polysaccharide. In nature, pectinolytic enzymes play an essential role in allowing bacteria and fungi to depolymerize and utilize pectin. In addition, pectinases have been widely applied in various industries, such as the food, wine, textile, paper and pulp industries. Due to their important biological function and increasing industrial potential, discovery of novel pectinolytic enzymes has received global interest. However, traditional enzyme characterization relies heavily on biochemical experiments, which are time consuming, laborious and expensive. To accelerate identification of novel pectinolytic enzymes, an automatic approach is needed. We developed a machine learning (ML) approach for predicting pectinases in the industrial workhorse fungus, Aspergillus niger. The prediction integrated a diverse range of features, including evolutionary profile, gene expression, transcriptional regulation and biochemical characteristics. Results on both the training and the independent testing dataset showed that our method achieved over 90 % accuracy, and recalled over 60 % of pectinolytic genes. Application of the ML model on the A. niger genome led to the identification of 83 pectinases, covering both previously described pectinases and novel pectinases that do not belong to any known pectinolytic enzyme family. Our study demonstrated the tremendous potential of ML in discovery of new industrial enzymes through integrating heterogeneous (post-) genomimcs data.

Biochemical and Cytological Interactions Between Callose Synthase and Microtubules in the Tobacco Pollen Tube

Callose is a cell wall polysaccharide involved in several fundamental biological processes, ranging from plant development to response to abiotic and biotic stresses. To understand how callose deposition is regulated, it is important to know how its synthesizing enzyme, i.e., callose synthase, is regulated and if it interacts with vesicular-cytoskeletal system of plant cells. Actin filaments are thought to determine the long-range distribution of callose synthase through transport vesicles. Unlike other enzymes (such as cellulose synthase) that synthesize cell wall polysaccharides, the spatial and biochemical relationships between callose synthase and microtubules are poorly understood. Some experimental evidence already support the association between callose synthase and tubulin, however, despite its importance in maintaining plant integrity, knowledge about regulation of callose biosynthesis is still limited. Here we investigated the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in a model system, pollen tube, where callose is an essential cell wall component. Native 2-D electrophoresis and isolation of the callose synthase complex confirmed that callose synthase is associated with tubulin and can interface with cortical microtubules. In contrast, actin and sucrose synthase (which supplies UDP-glucose to callose synthase) are not permanently associated with callose synthase. Immunogold labeling showed strong colocalization of the enzyme and microtubules; this association is occasionally mediated by vesicles. The association between callose synthase and vesicles was also demonstrated by co-distribution between the enzyme and Rab11b; in addition, the not homogeneous distribution of callose synthase in cell membranes is also shown by analysis of membrane microdomains.

Modification of physical and functional characteristics of chitin extracted from microwave-treated Nelumbo nucifera rhizome flour

Chitin, a cell wall polysaccharide, extracted from Nelumbo nucifera rhizome (NNR), was subjected to microwave treatment to modify its physical and functional characteristics. The NNR flour was irradiated at different levels of the microwave treatment period (1, 2, 3, 4, and 5 min). Chitin was extracted from the native and microwave-treated samples and analyzed for physical and functional characteristics. The microwave treatment resulted in some variations in the extract yield, structure, morphology, and composition of chitin that were directly correlated with its functional properties. Regression analysis of the data showed a significant ( p < 0.05) time-dependent linear decrease in extract yield, polynomial decrease in water-holding and swelling capacities, an exponential increase in oil holding, and an exponential decrease in iron-binding capacity of chitin extracted from microwave-treated flour. These variations in the studied functional properties may be due to microwave-induced hydrolytic degradation of chitin, structural rearrangements, and exposure of some lipophilic functional groups on the surface of chitin. The data would be a valuable contribution to the literature regarding microwave-induced modification in physical and functional characteristics of chitin present in N. nucifera rhizome and other plant-based biomaterials of industrial importance.

Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus luchuensis

The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3/1,4-glucosidic linkages.

Liberation of eicosapentaenoic acid and degradation of the major cell wall polysaccharide porphyran by fermentation of nori, the dried thalli of Pyropia yezoensis, with koji

In this study, dried sheets of nori, shredded and processed thalli of the red alga Pyropia yezoensis, were fermented with either barley, rice or soybean koji. High-performance liquid chromatographic analyses of the lipid extracts of the fermented products indicated that the fermentation of nori with all kinds of tested koji released free fatty acids, including the eicosapentaenoic acid, from ester lipids. We found that approximately half of the eicosapentaenoic acid in nori had been released as the free fatty acid at up to 4 weeks of fermentation at 30 °C and more than 65% at 8 to12 weeks in the fermented products with barley and rice koji. We also demonstrated the degradation of porphyran, a major cell wall polysaccharide of nori, by gel chromatography on Sephacryl S-300 HR of hot water extracts of the fermented products of nori with barley koji. Approximately two-third of porphyran had been degraded to porphyran oligosaccharides up to 6 weeks of fermentation. Fermentation of nori with koji may bring out the potential health-promoting functions of nori.

Comparison of Cell Wall Polysaccharide Composition and Structure Between Strains of Sporothrix schenckii and Sporothrix brasiliensis

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa. By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-β-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-β-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.