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Luxurious nitrogen fertilization of two sugarcane genotypes contrasting for lignin composition causes changes in the stem proteome related to carbon, nitrogen and oxidant metabolism but does not alter lignin content Fernanda Salvato, Rashaun Wilson, Juan Pablo Portilla Llerena, Eduardo Kiyota, Karina Lima Reis, Luis Felipe Boaretto, Tiago Santana Balbuena, Ricardo A Azevedo, Jay J Thelen, and Paulo Mazzafera J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00397 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017
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Luxurious nitrogen fertilization of two sugarcane genotypes contrasting for lignin composition causes changes in the stem proteome related to carbon, nitrogen and oxidant metabolism but does not alter lignin content Fernanda Salvato1,3*, Rashaun Wilson2, Juan Pablo Portilla Llerena1, Eduardo Kiyota1; Karina Lima Reis3, Luis Felipe Boaretto3, Tiago S. Balbuena4, Ricardo A. Azevedo3, Jay J. Thelen2, Paulo Mazzafera1,3* 1 Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil 2 Department of Biochemistry, University of Missouri Columbia, MO, USA. 3 Universidade de São Paulo, Escola Superior de Agricultura “ Luiz de Queiroz”, Piracicaba, SP, Brazil. 4 Departamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista "Júlio de Mesquita Filho", Jaboticabal, SP, Brazil.
*Corresponding authors:
[email protected], +55 19 34294148
[email protected] 1 ACS Paragon Plus Environment
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ABSTRACT Sugarcane is an important crop for sugar and biofuel production. Its lignocellulosic biomass represents a promising option as feedstock for second-generation ethanol production. Nitrogen fertilization can affect differently tissues and its biopolymers, including the cell wall polysaccharides and lignin. Lignin content and/or composition are the most important factors associated with biomass recalcitrance to convert cell wall polysaccharides into fermentable sugars.
Thus, it is important to understand the metabolic relationship between nitrogen
fertilization and lignin in this feedstock. In this study, a large-scale proteomic approach based on GeLC-MS/MS was employed to identify and relatively quantify proteins differently accumulated in two contrasting genotypes for lignin composition after excessive nitrogen fertilization. From the approximately 1,000 non-redundant proteins identified, 28 and 177 were differentially accumulated in response to nitrogen from IACSP04-065 and IACSP04-627 lines, respectively. These proteins were associated with several functional categories, including carbon metabolism, amino acid metabolism, protein turnover and oxidative stress. Although nitrogen fertilization has not changed lignin content, phenolic acids and lignin composition was changed in both species but not in the same way. Sucrose and reducing sugars increased in plants of the genotype IACSP04-065 receiving nitrogen. Keywords: Saccharum officinarum, cell wall, lignocellulosic ethanol, lignin, proteomics
2 ACS Paragon Plus Environment
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INTRODUCTION
With the increasing concern over global oil supply added by the urgent need for environment preservation, the search for alternative renewable sources of energy generation became one of the main challenges of the present world economy. The utilization of biofuels is one of the important strategies employed for mitigation of climate changes and improvement of fuel security 1. In this context, sugarcane is a versatile crop that can be used for bioethanol production obtained from the fermentation of sucrose- extracted from crushed stalks2. The juice can also be used to produce sugar. Sugarcane can accumulate up to 60% of dry weight of the mature stem as sucrose 3. Sugarcane is also a promising lignocellulosic feedstock for bioenergy generation, as large amounts of lignocellulosic residues (bagasse, leaves, tops) are generated in the process and are suitable for bioenergy conversion.4
Currently, Brazil is the largest world producer of sugarcane. In 2015/2016 was harvested 666.824 million tons of sugarcane which produced 30.232 billion liters of ethanol and 33.837 million tons of sugar.5 As the lignocellulosic residue (bagasse) is available approximately at a proportion of 138 kg of dried bagasse per ton of processed sugarcane6 it can be estimated that for the 2015/2016 harvest 92 million ton of bagasse was produced. Most of the bagasse is burnt in order to provide energy for the sugarcane mills as well as to be sold in the Brazilian energy market, but it has enormous potential to produce second generation bioethanol or cellulosic ethanol.7,8
The lignocellulosic biomass is mainly composed by polymers of the cell wall, including cellulose, hemicelluloses, and lignin. All these components are arranged in a way that cellulose microfibrils are embedded in a hemicellulose matrix cross-linked with heterogenic lignin polymers.8 Lignin is a key polymer conferring rigidity for upright plant growth, hydrophobicity, and strength of the xylem conduits enabling long distance water transport, and protection against pathogens and abiotic stress.9,10,11 Despite its biological importance in preventing cell wall degradation, it is the major cell wall component responsible for biomass recalcitrance12,13 due to its complex chemical nature and structure.14 The recalcitrance conferred by lignin is the main obstacle to convert cell wall polyssacharides, i.e., mainly cellulose, into fermentable sugars. 3 ACS Paragon Plus Environment
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For this reason, lignin biosynthesis and regulation has been an attractive field for molecular studies applied in sugarcane. Recently, an expression analysis identified 18 transcription factors (TFs) possibly related to cell wall biosynthesis in different ancestral sugarcane genotypes (Saccharum spontaneum and Saccharum officinarum) and a commercial hybrid. The expression analysis also suggested that lignin and bioenergetics-related genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage.15 In a previous study, the evaluation of two commercial hybrid genotypes (Saccharum sp.) differing in lignin content generated a set of 85,151 transcripts being more than 2,000 differently expressed, including many involved in lignin metabolism.16 Two other studies on the genetic control of lignin biosynthesis have also used commercial hybrid genotypes (Saccharum sp.) contrasting for lignin content. Bottcher and co-workers8 evaluated developmental lignin deposition and the phenolic profile in sugarcane stems. They found that rind and pith have different lignin content and composition (Syringyl/Guaiacyl ratio and oligomers composition) and, in addition, they demonstrated the differential expression profile of monolignol biosynthetic genes in these tissues. In the second study, expression profiles of nine transcription factors were shown to be tissue type and developmental stage-dependent.17
Although many studies at the transcriptional level have been conducted in this field, very few proteomic studies have been performed on sugarcane. Protein expression analyses can be directed towards global or targeted analyses complementing transcriptome and metabolome profiling. In sugarcane, proteomics is in its infancy being some studies focused on methodological procedures.7 Amalraj et al.18 established the protein extraction method for 2DE using sugarcane stalk tissues. Later, Calderan-Rodrigues et al.19,20 adapted protein extraction protocols for characterization of sugarcane cell wall proteins. Only few proteomics studies have focused on understanding the physiological responses under abiotic stresses, such as drought stress21,22 and salt stress.23,24
Biotic and abiotic stresses, such as drought, low temperature, UV-radiation, mineral deficiency and pathogen infection can change lignin content as well as its composition in plants.25 Drought and low temperature were shown to alter the expression levels of lignin biosynthetic genes and increase lignin content in sugarcane.26,27 4 ACS Paragon Plus Environment
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Nitrogen fertilization can affect differently tissues and biopolymers (carbohydrates, proteins and lignin). Corn plants fertilized with high levels of N have grain and residues (leaves, stems and reproductive support) affected differently.28 Lignin in corn residues increased almost twice as much as carbohydrates in response to N fertilization, while in the corn grain the response brought minimal benefits in carbohydrate content.28 In an opposite way, studies conducted in woody species such as Eucalyptus and Populus showed that luxurious N fertilization decreased lignin content in wood.29,30 Carbon and nitrogen balance can regulate the expression of genes involved in various aspects of primary metabolism such as glycolysis, carbohydrate biosynthesis and pentose-phosphate pathways.31
For sugarcane, N fertilization recommendation has been based on soil type and crop age, not considering biological fixation and plant or soil nutritional status. For these reasons, sugarcane fertilization is poorly understood. Some studies have reported that increasing N doses in sugarcane resulted in a linear increase of sugar yield and stalk production.32 In the currently study, the goal is to detect differences in the protein profile of sugarcane stems from contrasting genotypes for lignin content. We hypothesize that contrasting genotypes can respond differently to luxurious N fertilization in terms of protein abundance and lignin composition.
EXPERIMENTAL PROCEDURES
Plant Material
Contrasting genotypes of sugarcane (Saccharum spp.) for lignin content were provided by the Sugarcane Center of the Agronomic Institute of Campinas, at Ribeirão Preto, Brazil. Both genotypes, IACSP04-065 (lower lignin content, can reach 4.56%) and IACSP04-627 (higher lignin content, can reach 8.4%) were derived from a breeding program conducted for evaluation of different technological traits, including lignin content. In a previous work, these genotypes showed consistent difference in lignin content only in mature internodes (> 10th internode), although the lignin S/G ratio showed differences from young to mature internodes.8 These genotypes and other from this segregating population have been used in other studies of our group. 8,16,17,26,27 5 ACS Paragon Plus Environment
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The genotypes were grown in vases of 85 L (diameter = 50 cm and height = 50 cm) containing latosol/organic material mixture (1:1) for seven months in the greenhouse under daily irrigation and temperature at 28oC ± 4oC. Four biological replicates composed of three plants each were subjected to nitrogen (N) fertilization using 150 g of urea We used a commercial fertilizer containing 45% of urea. Nitrogen application was split in five doses of 30 g of urea applied in seven days interval. For control, four biological replicates composed of three plants each were used without N fertilization. Sugarcane stalks (5th internode) were collected seven days after the last urea application. The 5th internode was chosen since it corresponds to a transition tissue between young and mature internodes with an active metabolism of lignin.8 In addition, the peak of nitrogen demand of sugarcane plants is during tillering, which happens in the beginning of plant growth. Internodes have their bark removed and immediately frozen in liquid nitrogen. Samples were stored at -80oC.
Soluble lignin oligomers and phenolic compounds analyses by LC-MS/MS
Soluble lignin oligomers extraction and LC-MS analyzes were carried-out as previously described.33 The same extracts were utilized to carry out the phenolic compound assays. Samples were prepared by diluting 5X the original extracts in ethanol 80% (ethanol and water, 4:1, v/v). Quinic, shikimic, cinnamic, chlorogenic and coumaric acids were analyzed as reference compounds in an Acquity CSH(TM) C18 (2.1 x 150 mm; 1.7 µm) column coupled to an Acquity UHPLC-TQD Mass Spectrometer (Waters Corp., Manchester, UK). The column was preequilibrated with 29% acetonitrile (solvent B) and 71% Milli-Q water with 0.1% formic acid (solvent A) and the chromatographic separation was performed using a gradient elution ranging from 29% to 38% of solvent B, in 8 min, flow rate of 0.200 mL·min−1 and column temperature of 30°C. Mass spectrometry data were obtained by Multiple Reaction Monitoring (MRM) method with capillary 3.0 kV and cone 50 V, collision energy 15 eV, ion source temperature 150 °C, desolvation temperature 300 °C and electrospray ionization (ESI) in positive or negative mode, following the MRM transitions: quinic acid – ESI negative mode 191>127, 191>93, 191>85; shikimic acid – ESI negative mode 173>137, 173>93, 173>73; cinnamic acid – ESI negative mode 147>103; chlorogenic acid – ESI negative mode 353>191, 353>85; coumaric acid 6 ACS Paragon Plus Environment
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– ESI positive mode 165>119, 165>91, 165>65. Pure commercial reagents were used as standards and all purchased from Sigma-Aldrich, St Louis, MO. The quantification of the different structures were expressed as means of peak areas considering all replicates and t-test (p
