Degradation Of Cutin Research Articles

Genome Sequencing Highlights the Plant Cell Wall Degrading Capacity of Edible Mushroom Stropharia rugosoannulata

The basidiomycetous edible mushroom Stropharia rugosoannulata has excellent nutrition, medicine, bioremediation, and biocontrol properties. S. rugosoannulata has been widely and easily cultivated using agricultural by-products showing strong lignocellulose degradation capacity. However, the unavailable high-quality genome information has hindered the research on gene function and molecular breeding of S. rugosoannulata. This study provided a high-quality genome assembly and annotation from S. rugosoannulata monokaryotic strain QGU27 based on combined Illumina-Nanopore data. The genome size was about 47.97Mb and consisted of 20 scaffolds, with an N50 of 3.73Mb and a GC content of 47.9%. The repetitive sequences accounted for 17.41% of the genome, mostly long terminal repeats (LTRs). A total of 15,726 coding gene sequences were putatively identified with the BUSCO score of 98.7%. There are 142 genes encoding plant cell wall degrading enzymes (PCWDEs) in the genome, and 52, 39, 30, 11, 8, and 2 genes related to lignin, cellulose, hemicellulose, pectin, chitin, and cutin degradation, respectively. Comparative genomic analysis revealed that S. rugosoannulata is superior in utilizing aldehyde-containing lignins and is possible to utilize algae during the cultivation.

Characterization of recombinant cutinase from Thermobifida cellulosilytica and its application in tomato cutin degradation

Cutinases are desirable biorecycling agents for big molecules because of their great efficiency in generating monomer under mild reaction conditions. In this study, the biochemical characterizations of a thermostable cutinase from Thermobifida cellulosilytica for tomato cutin degradation are presented. The cutinase gene was chosen from the sequence database. The gene was synthesized, cloned, and successfully expressed in the Escherichia coli (E. coli) system. SDS-PAGE analysis revealed that the cutinase had a molecular weight of approximately 29kDa. The recombinant cutinase showed highest activity at an optimum pH of 9.0. The result also showed thermal stability up to 60 °C. The activity of the recombinant cutinase was enhanced by 1 mM NaCl, 5 mM NaCl and 1 mM CaCl2; however, slightly inhibited by 5 mM CaCl2. Cutin from tomato peel was efficiently degraded into free fatty acid monomers. The bonding features between the deteriorated and polymerized cutin were clearly visible in the fourier transformed infrared spectroscopy study. These findings open the possibilities of using cutinases for the generation of free fatty acids from tomato peel. It could be a potential raw material for other industrial applications.

Tissue-specific transcriptome responses to Fusarium head blight and Fusarium root rot.

Fusarium head blight (FHB) and Fusarium root rot (FRR) are important diseases of small-grain cereals caused by Fusarium species. While host response to FHB has been subject to extensive study, very little is known about response to FRR and the transcriptome responses of FHB and FRR have not been thoroughly compared. Brachypodium distachyon (Bd) is an effective model for investigating host responses to both FHB and FRR. In this study the transcriptome response of Bd to F. graminearum (Fg) infection of heads and roots was investigated. An RNA-seq analysis was performed on both Bd FHB and FRR during the early infection. Additionally, an RNA-seq analysis was performed on in vitro samples of Fg for comparison with Fg gene expression in planta. Differential gene expression and gene-list enrichment analyses were used to compare FHB and FRR transcriptome responses in both Bd and Fg. Differential expression of selected genes was confirmed using RT-qPCR. Most genes associated with receptor signalling, cell-wall modification, oxidative stress metabolism, and cytokinin and auxin biosynthesis and signalling genes were generally upregulated in FHB or were downregulated in FRR. In contrast, Bd genes involved in jasmonic acid and ethylene biosynthesis and signalling, and antimicrobial production were similarly differentially expressed in both tissues in response to infection. A transcriptome analysis of predicted Fg effectors with the same infected material revealed elevated expression of core tissue-independent genes including cell-wall degradation enzymes and the gene cluster for DON production but also several tissue-dependent genes including those for aurofusarin production and cutin degradation. This evidence suggests that Fg modulates its transcriptome to different tissues of the same host.

Long-term crop rotation and different tillage practices alter soil organic matter composition and degradation

Conservation agriculture such as conservation tillage and crop rotation can alter soil organic matter (OM) storage and cycling. However, little is known about how these methods alter the molecular-level soil OM composition and turnover. We collected soil samples (0−10 cm) from a 37-year experiment with contrasting diversity crop rotations under conventional tillage and conservation tillage. Soil organic carbon concentrations, molecular-level OM composition and microbial biomass and community structure were measured. Soil organic carbon concentrations were similar between the two tillage practices, likely due to the balance between carbon loss via degradation and carbon gains through incorporation of crop residues in soil. We observed significant changes in soil OM composition between conventional tillage and conservation tillage despite similar soil organic carbon concentrations. Under long-term conservation tillage practice, lower suberin-derived compounds and higher lignin-derived compounds were observed. The degradation of cutin-, lignin-derived compounds and soil OM were lower under conservation tillage among most rotations. The changes in lignin-derived compounds and degradation were microbially driven and likely controlled by substrate utilization. In contrast, the differences in suberin-derived compounds were likely associated with crop residue-induced OM inputs and/or the differential introduction of OM (e.g. root materials) to the upper layer of soil due to conventional tillage vs. conservation tillage managements. These findings reveal that microbial degradation, OM inputs from crop residues and management-induced OM distribution in soil layers may alter agricultural soil OM composition. Additionally, we found that the differences in some specific groups of compounds between conventional tillage and conservation tillage were lower in crop rotations with higher diversity, highlighting the benefits of the interaction between diverse rotations and conservation tillage.

Regulation of the cutinases expressed by Aspergillus nidulans and evaluation of their role in cutin degradation.

Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.

A Middle-Aged Enzyme Still in Its Prime: Recent Advances in the Field of Cutinases

Cutinases are α/β hydrolases, and their role in nature is the degradation of cutin. Such enzymes are usually produced by phytopathogenic microorganisms in order to penetrate their hosts. The first focused studies on cutinases started around 50 years ago. Since then, numerous cutinases have been isolated and characterized, aiming at the elucidation of their structure–function relations. Our deeper understanding of cutinases determines the applications by which they could be utilized; from food processing and detergents, to ester synthesis and polymerizations. However, cutinases are mainly efficient in the degradation of polyesters, a natural function. Therefore, these enzymes have been successfully applied for the biodegradation of plastics, as well as for the delicate superficial hydrolysis of polymeric materials prior to their functionalization. Even though research on this family of enzymes essentially began five decades ago, they are still involved in many reports; novel enzymes are being discovered, and new fields of applications arise, leading to numerous related publications per year. Perhaps the future of cutinases lies in their evolved descendants, such as polyesterases, and particularly PETases. The present article reviews the biochemical and structural characteristics of cutinases and cutinase-like hydrolases, and their applications in the field of bioremediation and biocatalysis.

Two dimensional NMR spectroscopy for molecular characterization of soil organic matter: Application to boreal soils and litter

Organic soils in boreal ecosystems and peatlands represent a huge global carbon pool and their composition strongly affects soil properties. Nevertheless, the characterization of soil organic matter (SOM) molecular composition, which is essential for elucidating soil carbon processes and turnover, is not easily achieved, and further advances in the area are greatly needed. Two dimensional (2D) liquid state 1H–13C nuclear magnetic resonance (NMR) spectroscopy has been used on dimethyl sulfoxide (DMSO) extracts of SOM to achieve molecular level characterization, with signals from many identifiable molecular groups observable. Here we show that a simple and fast sample preparation allows acquisition of 2D 1H–13C NMR spectra from extracts of plant litter and organic layers in boreal ecosystems, with fast data acquisition. Our 2D NMR spectra revealed several differences in the DMSO extracts of different tree litter samples, O-horizons of forest soil, peat-forming moss (Sphagnum) and peat. The results mirror established differences between OM in soils and litter of different forest ecosystems (e.g. between deciduous and coniferous litter) but also provide indications for research to untangle previously conflicting results (e.g. cutin degradation in soil or carbohydrate degradation in peat). Thus, combination of 2D NMR methods can greatly improve analysis of litter composition and SOM composition, thereby facilitating the elucidation of their roles in biogeochemical and ecological processes that are critical for foreseeing feedback mechanisms for SOM turnover as a result of global environmental change.

Molecular composition of soil organic matter with land-use change along a bi-continental mean annual temperature gradient

Soil organic matter (SOM) is critical for maintaining soil fertility and long-term agricultural sustainability. The molecular composition of SOM is likely altered due to global climate and land-use change; but rarely are these two aspects studied in tandem. Here we used molecular-level techniques to examine SOM composition along a bi-continental (from North to South America) mean annual temperature (MAT) gradient from seven native grassland/forest and cultivated/pasture sites. Biomarker methods included solvent extraction, base hydrolysis and cupric (II) oxide oxidation for the analysis of free lipids of plant and microbial origin, ester-bound lipids from cutin and suberin, and lignin-derived phenols, respectively. Solid-state 13C nuclear magnetic resonance (NMR) was used to examine the overall composition of SOM. Soil cultivation was found to increase the amount of microbial-derived compounds at warmer temperatures (up to 17% increase). The cultivated soils were characterized by much lower contributions of plant-derived SOM components compared to the native soils (up to 64% lower at the coldest site). In addition, cultivation caused an increase in lignin and cutin degradation (up to 68 and 15% increase, respectively), and an increase in the amount of suberin-derived inputs (up to 54% increase). Clear differences in the molecular composition of SOM due to soil cultivation were observed in soils of varying mineral composition and were attributed to disturbance, different vegetation inputs, soil aggregate destruction and MAT. A high organic allophanic tropical soil was characterized by its protection of carbohydrates and nitrogen-containing compounds. The conversion of native to cultivated land shows significant shifts in the degradation stage of SOM. In particular, cutin-derived compounds which are believed to be part of the stable SOM pool may undergo enhanced degradation with long-term cultivation and disruption of soil aggregates. On a per year basis, the total amount of cutin decreased only at the two forest sites that were converted to pasture, likely due to cutin degradation or to changes in vegetation and litter quality associated with land-use change. Overall, our study highlights that the implementation of different agricultural management practices enhances the degradation of recalcitrant SOM compounds that may become a source of atmospheric CO2 with increasing land-use and climate change.

Genome sequence of Valsa canker pathogens uncovers a potential adaptation of colonization of woody bark.

Canker caused by ascomycetous Valsa species are among the most destructive diseases of woody plants worldwide. These pathogens are distinct from other pathogens because they only effectively attack tree bark in the field. To unravel the potential adaptation mechanism of bark colonization, we examined the genomes of Valsa mali and Valsa pyri that preferentially infect apple and pear, respectively. We reported the 44.7 and 35.7 Mb genomes of V. mali and V. pyri, respectively. We also identified the potential genomic determinants of wood colonization by comparing them with related cereal pathogens. Both genomes encode a plethora of pathogenicity-related genes involved in plant cell wall degradation and secondary metabolite biosynthesis. In order to adapt to the nutrient limitation and low pH environment in bark, they seem to employ membrane transporters associated with nitrogen uptake and secrete proteases predominantly with acidic pH optima. Remarkably, both Valsa genomes are especially suited for pectin decomposition, but are limited in lignocellulose and cutin degradation. Besides many similarities, the two genomes show distinct variations in many secondary metabolism gene clusters. Our results show a potential adaptation of Valsa canker pathogens to colonize woody bark. Secondary metabolism gene clusters are probably responsible for this host specificity.

Soil warming and nitrogen deposition alter soil organic matter composition at the molecular-level

Rising temperatures and nitrogen (N) deposition, both aspects of global environmental change, are proposed to alter soil organic matter (SOM) biogeochemistry. For example, increased plant productivity and enhanced microbial decomposition of litter and SOM may reduce soil carbon stocks and fertility. To better understand SOM biogeochemical shifts at the molecular-level, we employed an array of biomarker and nuclear magnetic resonance (NMR) techniques to investigate the composition and degradation of SOM components in the forest floor and mineral soil horizons of warmed (5 °C above average soil temperature) and N fertilized (5 g m−2 year−1 N applied in the growing season) plots from the soil warming × nitrogen addition study at the Harvard Forest, MA, USA. Biomarker analyses indicated increased plant-derived inputs into the forest floor under N fertilization. Soil warming promoted the decomposition of plant-derived aliphatic and cyclic compounds in the forest floor. Cutin degradation was observed in the heated forest floor which also exhibited relatively higher microbial activity. Lignin oxidation was also observed but was most pronounced in the mineral horizon of the heated plots. These results suggest that continued soil warming may promote the degradation of lignin- and cuticle-derived SOM. N fertilization also enhanced lignin oxidation but to a lesser extent likely due to a decline in microbial activity. 1H NMR spectra of the mineral soils revealed enrichment of plant-derived alkyl structures and microbial-derived organic matter with both soil warming and N fertilization. Overall, this study shows that the decomposition and accumulation of molecularly distinct SOM components occurs with soil warming and N amendment and may subsequently alter soil biogeochemical cycling.

Carbohydrate-active enzymes in pythium and their role in plant cell wall and storage polysaccharide degradation.

Carbohydrate-active enzymes (CAZymes) are involved in the metabolism of glycoconjugates, oligosaccharides, and polysaccharides and, in the case of plant pathogens, in the degradation of the host cell wall and storage compounds. We performed an in silico analysis of CAZymes predicted from the genomes of seven Pythium species (Py. aphanidermatum, Py. arrhenomanes, Py. irregulare, Py. iwayamai, Py. ultimum var. ultimum, Py. ultimum var. sporangiiferum and Py. vexans) using the “CAZymes Analysis Toolkit” and “Database for Automated Carbohydrate-active Enzyme Annotation” and compared them to previously published oomycete genomes. Growth of Pythium spp. was assessed in a minimal medium containing selected carbon sources that are usually present in plants. The in silico analyses, coupled with our in vitro growth assays, suggest that most of the predicted CAZymes are involved in the metabolism of the oomycete cell wall with starch and sucrose serving as the main carbohydrate sources for growth of these plant pathogens. The genomes of Pythium spp. also encode pectinases and cellulases that facilitate degradation of the plant cell wall and are important in hyphal penetration; however, the species examined in this study lack the requisite genes for the complete saccharification of these carbohydrates for use as a carbon source. Genes encoding for xylan, xyloglucan, (galacto)(gluco)mannan and cutin degradation were absent or infrequent in Pythium spp.. Comparative analyses of predicted CAZymes in oomycetes indicated distinct evolutionary histories. Furthermore, CAZyme gene families among Pythium spp. were not uniformly distributed in the genomes, suggesting independent gene loss events, reflective of the polyphyletic relationships among some of the species.

Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada

The hydrolyzable lipid composition of grassland and forest soils, and their overlying vegetation (i.e., prairie grasses, aspen, and pine) were analyzed by alkaline hydrolysis and gas chromatography–mass spectrometry (GC–MS). Prior to hydrolysis, samples were pre-extracted with organic solvents to remove free lipids. The hydrolysis products from soils and plants consisted of series of aliphatic lipids (alkanoic acids, alkanols, alkanedioic acids, ω-hydroxyalkanoic acids, α-hydroxyalkanoic acids, mid-chain-substituted hydroxy and epoxy acids), benzyls and phenols. Cutin, suberin and plant waxes are believed to be the major sources for the detected lipids. The composition of cutin and suberin monomers in the soils is similar to the patterns observed in the corresponding vegetation reflecting the preservation of plant biomolecules such as cutin and suberin. The long-chain ω-hydroxyalkanoic and α,ω-alkanedioic acids (>C 16) as observed in the grass roots are typical biomarkers for suberin and indicate a major input of root biomass to the soils. Mid-chain-substituted hydroxy and epoxy acids (C 14–C 18) derived from the cutin of leaves were detected in the soils but in low abundance, indicating only a minor input of leaf material. A parameter to estimate the input of leaf vs. root organic matter is developed and applied based on the pattern and distribution of observed cutin and suberin biomarkers. The cutin/suberin ratios decrease from plant material to the mineral horizons likely due to the steady input of fresh root material in the soil horizons rather than the preferential degradation of cutin. The decrease of total hydrolysis products from the overlying vegetation to the soil is consistent with the degradation of hydrolysable biomarkers. Long-chain ω-hydroxyalkanoic and α,ω-alkanedioic acids derived from suberin are observed to decompose without preference of specific constituents. In contrast, mid-chain-substituted hydroxy and epoxy acids appear to be preferentially degraded in soils compared to other cutin and suberin monomers such as ω-hydroxyalkanoic acids. However, the steady input of roots which are rich in ω-hydroxyalkanoic acids and low in mid-chain-substituted acids may in part influence the degradation parameters resulting in an apparent higher rate of degradation.

Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence.

Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate- or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in Vmax. Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.

COMPARATIVE ULTRASTRUCTURAL STUDY OF THE DEGRADATION IN SOIL OF ISOLATED CUTIN FROM ILEX LEAVES AND MALUS FRUIT

SUMMARYCutin was dewaxed and removed from leaves of Ilex aquifolium L. (holly) and fruits of Malus pumila Mill, (apple) by a combination of chemical and enzymic techniques. Discs of cutin were then buried in soil under field conditions, retrieved at intervals and examined using electron microscopy. The ultrastructure of cutin before and after burial was compared with that of cuticle of intact holly leaves and apple fruits. Apple cutin was extensively eroded after being buried for 12 months, but neither apple nor holly cutin was completely degraded even after 2 years in soil. The significance of polysaccharide fibrils embedded in the cutin is discussed in relation to the patterns of cutin degradation which were observed and to the distribution of microorganisms in the decaying cutin.

Cutin Degradation by Plant Pathogenic Fungi

Cutin Degradation by Plant Pathogenic Fungi

Stages during the breakdown of plant cutin by soil microorganisms

1. Soil from cultivated land contains a variety of microorganisms, enabled to decay z. Seven out of ten isolated microorganisms were shown to contain cutinolytic enzymes. 2. The degradation of cutin by Cryptococcus laurentii and Rhodotorula sp. was found to proceed in a threestep pattern, according to the release of free F. A. from the substrate as determined analytically. 3. This pattern was shown to coincide with three stages of oxygen consumption and oxygen release in Warburg assays with Rhodotorula sp. 4. The third stage of cutin degradation, represented by a steady oxidation of continously released free F. A., starts after 42 hours with the Bacterium tested, and after 120 hours with Rhodotorula. Determination of the beginning of the third stage thus provides data for the comparison of cutinolytic activity in different microorganisms. 5. Cutin decomposition proceeds faster, if suitable C- and N-sources are provided with the incubation medium.