Enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana. (C) 2012 Elsevier Masson SAS. All rights reserved.556676Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2008/58035-6

Expression of SofLAC, a new laccase in sugarcane, restores lignin content but not S:G ratio of Arabidopsis lac17 mutant

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Lignin is a complex phenolic heteropolymer deposited in the secondarily thickened walls of specialized plant cells to provide strength for plants to stand upright and hydrophobicity to conducting cells for long-distance water transport. Although essential for plant growth and development, lignin is the major plant cell-wall component responsible for biomass recalcitrance to industrial processing. Peroxidases and laccases are generally thought to be responsible for lignin polymerization, but, given their broad substrate specificities and large gene families, specific isoforms involved in lignification are difficult to identify. This study used a combination of co-expression analysis, tissue/cell-type-specific expression analysis, and genetic complementation to correlate a sugarcane laccase gene, SofLAC, to the lignification process. A co-expression network constructed from 37 cDNA libraries showed that SofLAC was coordinately expressed with several phenylpropanoid biosynthesis genes. Tissue-specific expression analysis by quantitative RT-PCR showed that SofLAC was expressed preferentially in young internodes and that expression levels decrease with stem maturity. Cell-type-specific expression analysis by in situ hybridization demonstrated the localization of SofLAC mRNA in lignifying cell types, mainly in inner and outer portions of sclerenchymatic bundle sheaths. To investigate whether SofLAC is able to oxidize monolignols during lignification, the Arabidopsis lac17 mutant, which has reduced lignin levels, was complemented by expressing SofLAC under the control of the Arabidopsis AtLAC17 promoter. The expression of SofLAC restored the lignin content but not the lignin composition in complemented lac17 mutant lines. Taken together, these results suggest that SofLAC participates in lignification in sugarcane.64617691781Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [BIOEN 08/58035-6]CAPES [201660/2010-5]FAPESP [Ghent University Multidisciplinary Research Partnership 'Biotechnology for a Sustainable Economy'

Tibouchina sellowiana (Cham.) Cogn,: Rooting, anatomy and biochemical analyses in the four seasons

The present work tried to verify the effects of application of different concentration of indolebutyric acid (IBA) in talc In solution, in the rooting of cutting of Tibouchina sellowiana (Cham.) Cogn. collected in the four seasons, as well as its anatomical and biochemical variation. The semiharwood cuttings were treated with 0, 1500 and 3000 mg L(-1) indolebutyric acid (IBA), in powder and alchoolic Solutions. After 65 days in a greenhouse, rooting percentage, length and number of roots, cutting with callus percentage and survival, were evaluated. The use of the IBA was efficient in the induction radicial, being indifferent the form in which it was applied. The most promising stations in the formation of the system radicial were the spring (78,75%) with 3000 mg L(-1) or mg kg(-1) IBA and the summer (63,12 %) with 1500 mg L(-1) or mg kg(-1) IBA. (82,50%) with 3000 mg kg(-1) IBA. Considering the cutting in the base of the stakes, in the moment of the cutting, no possible anatomical barrier or structural difference were detected so that it Should promoted variations in the answers of the rooting uring the four seasons. Biochemical analyses done from the cuttings collected in the four seasons revealed that the largest concentrations of total Sugars were obtained in thewinter (83,2 1 mg g(-1)) and in the autumn (72,79 mg g(-1)). The same stations also presented the largest protein concentrations (4,69 and 3,95 mg g(-1) respectively).184167115917

Lignification in Sugarcane: Biochemical Characterization, Gene Discovery, and Expression Analysis in Two Genotypes Contrasting for Lignin Content

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Sugarcane (Saccharum spp.) is currently one of the most efficient crops in the production of first-generation biofuels. However, the bagasse represents an additional abundant lignocellulosic resource that has the potential to increase the ethanol production per plant. To achieve a more efficient conversion of bagasse into ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Because several studies have shown a negative effect of lignin on saccharification yield, the characterization of lignin biosynthesis, structure, and deposition in sugarcane is an important goal. Here, we present, to our knowledge, the first systematic study of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents. Lignin amount and composition were determined in rind (outer) and pith (inner) tissues throughout stem development. In addition, the phenolic metabolome was analyzed by ultra-highperformance liquid chromatography-mass spectrometry, which allowed the identification of 35 compounds related to the phenylpropanoid pathway and monolignol biosynthesis. Furthermore, the Sugarcane EST Database was extensively surveyed to identify lignin biosynthetic gene homologs, and the expression of all identified genes during stem development was determined by quantitative reverse transcription-polymerase chain reaction. Our data provide, to our knowledge, the first in-depth characterization of lignin biosynthesis in sugarcane and formthe baseline for the rationalmetabolic engineering of sugarcane feedstock for bioenergy purposes.163415391557Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Research Foundation-FlandersGhent University Multidisciplinary Research Partnership 'Biotechnology for a Sustainable Economy' [01MRB510W]European Commission's Directorate General for Research [251130]Hercules program of Ghent University for the Synapt apparatus [AUGE/014]Bijzonder Onderzoeksfonds-Zware Apparatuur of Ghent University for the Fourier transform ion cyclotron resonance mass spectrometer [174PZA05]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2008/58035-6]Ghent University Multidisciplinary Research Partnership 'Biotechnology for a Sustainable Economy' [01MRB510W]European Commission's Directorate General for Research [251130]Hercules program of Ghent University for the Synapt apparatus [AUGE/014]Bijzonder Onderzoeksfonds-Zware Apparatuur of Ghent University for the Fourier transform ion cyclotron resonance mass spectrometer [174PZA05

Germination and initial growth of Campomanesia xanthocarpa O. Berg. (Myrtaceae), in petroleum-contaminated soil and bioremediated soil

In 2000 there was an oil spill at the Getúlio Vargas Refinery (REPAR) in Paraná. Nearly five years after contamination and the use of bioremediation, a study was carried out to identify the effects of the contaminated soil and the bioremediated soil on the germination and initial growth of C. xanthocarpa. The experiment was established with soil from REPAR, with three treatment groups: contaminated soil (C), bioremediated soil (B) and uncontaminated soil (U); with five repetitions of 50 seeds each. There was no significant difference in the percentage of germination and the speed of germination index. The production of total biomass (30 - 60 days) and shoot biomass (60 days) was greater in the bioremediated soil compared to the other treatments. The averages for the root biomass were lower in the contaminated soil than in the bioremediated soil. The shoot length and the total length of the seedling in the contaminated soil and uncontaminated soil were lower than in the bioremediated soil