The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl2 and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second-generation ethanol production.

BackgroundSugarcane has been used as the main crop for ethanol production for more than 40 years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics.ResultsA predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33 % vs 44 %). About 19 % of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75 % more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins.ConclusionsThe results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane cell wall proteome, and provide target proteins that could be used in future research to facilitate 2G ethanol production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0677-0) contains supplementary material, which is available to authorized users.

Plant cell walls mostly comprise polysaccharides and proteins. The composition of monocots’ primary cell walls differs from that of dicots walls with respect to the type of hemicelluloses, the reduction of pectin abundance and the presence of aromatic molecules. Cell wall proteins (CWPs) differ among plant species, and their distribution within functional classes varies according to cell types, organs, developmental stages and/or environmental conditions. In this review, we go deeper into the findings of cell wall proteomics in monocot species and make a comparative analysis of the CWPs identified, considering their predicted functions, the organs analyzed, the plant developmental stage and their possible use as targets for biofuel production. Arabidopsis thaliana CWPs were considered as a reference to allow comparisons among different monocots, i.e., Brachypodium distachyon, Saccharum spp. and Oryza sativa. Altogether, 1159 CWPs have been acknowledged, and specificities and similarities are discussed. In particular, a search for A. thaliana homologs of CWPs identified so far in monocots allows the definition of monocot CWPs characteristics. Finally, the analysis of monocot CWPs appears to be a powerful tool for identifying candidate proteins of interest for tailoring cell walls to increase biomass yield of transformation for second-generation biofuels production.

By characterizing the cell wall proteomes of different sugarcane organs (leaves and stems) at two developmental stages (young vs mature/apical vs basal), it is possible to address unique characteristics in each of them. Four-month-old leaves show a higher proportion of oxido-reductases and proteins related to lipid metabolism (LM), besides a lower proportion of proteins acting on polysaccharides, in comparison to 4-month-old internodes. It is possible to note that sugarcane leaves and young stems have the highest LM rate than all species, which is assumed to be linked to cuticle formation. The data generated enrich the number of cell wall proteins (CWPs) identified in sugarcane, reaching 277. To our knowledge, sugarcane has now the second higher coverage of monocot CWP in plants.Plant biomass is one of the most abundant renewable resources [1] and it has been assumed that plants devote more than 10% of their genome to the biogenesis of cell walls. [2] During plant growth and development, plant cell walls are continuously modified by enzyme action [3] which are important players in the remodeling of cell wall components. The identification of cell wall proteins (CWPs) has been performed in various plant organs mainly in model plants such as Arabidopsis thaliana and Brachypodium distachyon, but also in crops. [4,5] These data could also be used as a source of information to identify possible targets for plant engineering to produce tailored-cell walls more easily amenable to saccharification and production of ethanol of second generation (E2G). [6] In this work, we have established the CWP atlas of leaves and stems of 4-month-old sugarcane plants. DOI: 10.1002/pmic.201700129The aim was to identify genes encoding proteins of interest to produce tailored cell walls allowing more efficient production of E2G from sugarcane residues. Pooling samples from leaves and stems internodes at different developmental stages from sugarcane variety SP80-3280 were used. The samples were named according the classification on the work of Kuijper, 1915 [cited in [7] ] in which growth in length of the internodes and of the leaves are completed at stages 4 and 0 respectively (see Figure 1). Basal internodes (BI) referring to 5-7 internodes, apical internodes (AI) referring to 1-3 internodes, mature leaves (ML) referring to 1-3 leaves, and young leaves (YL) referring to -3, -2, and -1 leaves. Plants were acclimated in a greenhouse at controlled temperature (26°C) with daily irrigation. Internodes and leaves were collected after 4 months of growth and their proteins were extracted.For the extraction of CWPs a nondestructive method was used, based on [8] and adapted by [9] . Three biological replicates were performed. It is important to mention that the infiltration protocol mostly recovers weakly-bound and labile proteins, and it was chosen because of the already proven high efficiency in recovering sugarcane CWPs compared to destructive techniques. [9] Protein identification was performed by MS subsequently and protein digest were f...

Glycoside hydrolases (GHs) are enzymes that are able to rearrange the plant cell wall polysaccharides, being developmental-and stress-regulated. Such proteins are used in enzymatic cocktails for biomass hydrolysis in the second-generation ethanol (E2G) production. In this chapter, we investigate GHs identified in plant cell wall proteomes by predicting their functions through alignment with homologous plant and microorganism sequences and identification of functional domains. Up to now, 49 cell wall GHs were identified in sugarcane and 114 in Brachypodium distachyon. We could point at candidate proteins that could be targeted to lower biomass recalcitrance. We more specifically addressed several GHs with predicted cellulase, hemicellulase, and pectinase activities, such as β-xylosidase, α and β-galactosidase, α-N-arabinofuranosidases, and glucan β-glucosidases. These enzymes are among the most used in enzymatic cocktails to deconstruct plant cell walls. As an example, the fungi arabinofuranosidases belonging to the GH51 family, which were also identified in sugarcane and B. distachyon, have already been associated to the degradation of hemicellulosic and pectic polysaccharides, through a peculiar mechanism, probably more efficient than other GH families. Future research will benefit from the information available here to design plant varieties with self-disassembly capacity, making the E2G more cost-effective through the use of more efficient enzymes.

Sugarcane (Saccharum spp.), a C4 grass, has a peculiar feature: it accumulates, gradient-wise, large amounts of carbon (C) as sucrose in its culms through a complex pathway. Apart from being a sustainable crop concerning C efficiency and bioenergetic yield per hectare, sugarcane is used as feedstock for producing ethanol, sugar, high-value compounds, and products (e.g., polymers and succinate), and bioelectricity, earning the title of the world’s leading biomass crop. Commercial cultivars, hybrids bearing high levels of polyploidy, and aneuploidy, are selected from a large number of crosses among suitable parental genotypes followed by the cloning of superior individuals among the progeny. Traditionally, these classical breeding strategies have been favoring the selection of cultivars with high sucrose content and resistance to environmental stresses. A current paradigm change in sugarcane breeding programs aims to alter the balance of C partitioning as a means to provide more plasticity in the sustainable use of this biomass for metabolic engineering and green chemistry. The recently available sugarcane genetic assemblies powered by data science provide exciting perspectives to increase biomass, as the current sugarcane yield is roughly 20% of its predicted potential. Nowadays, several molecular phenotyping tools can be applied to meet the predicted sugarcane C potential, mainly targeting two competing pathways: sucrose production/storage and biomass accumulation. Here we discuss how molecular phenotyping can be a powerful tool to assist breeding programs and which strategies could be adopted depending on the desired final products. We also tackle the advances in genetic markers and mapping as well as how functional genomics and genetic transformation might be able to improve yield and saccharification rates. Finally, we review how “omics” advances are promising to speed up plant breeding and reach the unexplored potential of sugarcane in terms of sucrose and biomass production.

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.

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