Study on the leaf proteome of sugarcane cultivars and genotypes

Author(s)

Eduardo Shigueo Kitano

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2017-06-13

Examining board members

Solange Maria de Toledo Serrano; Carlos Alberto Labate; Milton Yutaka Nishiyama Junior; Graziella Eliza Ronsein

• Advisor: Solange Maria de Toledo Serrano

Abstract

Sugarcane, one of the major crops worldwide, is an old energy source for humans and, more recently, has been used as a source for ethanol production. In sugarcane molecular studies, its complex genetic background and the lack of a complete genome sequencing hinder the analysis of proteomes of different tissues. Recently, in a Brazilian research study focused on the transcriptomes of different sugarcane tissues, 17,563 full-length transcripts were sequenced and covered approximately 53% of the predicted sugarcane genome. However, it is known that mRNA levels are not always directly correlated with protein expression levels. Therefore, the study of the sugarcane proteome is important to identify proteins that are actually expressed, complementing the genome studies, and provides predictive information on the proteome composition of different cultivars. However, studies on sugarcane leaves are rare. In this study, with the aim of characterizing the proteome of sugarcane +1 leaves, we selected two ancestral species (Saccharum officinarum [S. off] and S. spontaneum[S. sp]), and a hybrid cultivar (SP80-3280 [SP]), which differ in sucrose production and pathogen resistance. The analysis of physiological parameters (photosynthesis rate, stomatal conductance, transpiration and efficiency in water use) of 12 month-old +1 leaves revealed that in S. off and SP there is a better relationship between the number of H2O molecules lost by transpiration and the number of CO2 molecules assimilated upon photosynthesis than in S. sp. The same parameters were evaluated in +1 leaves of SP upon 4, 8, 11 and 13 months of growth and revealed the role of stomata in the maintenance of leaf water potential, acting in the modulation of the transpiration rate in response to environmental changes, however without inducing drastic changes in the photosynthesis rate, during the period evaluated in this study. In order to obtain proteins in optimal conditions for proteomic analysis, we carried out different protein extraction methods and evaluated the yield and electrophoretic profiles of extracted proteins, and selected a protocol using a combination of phenol and sodium dodecyl sulfate to carry out this study. To characterize and compare the +1 leaf proteomes of S. off, S. sp and SP we used Shotgun/bottom-up, GeLC-MS/MS and Multidimensional Protein Identification Technology (MudPIT) (off-line and \"on-line\") as proteomic approaches, and the software MaxQuant and Perseus as bioinformatic tools for data analysis. The combined results of these approaches allowed for the identification of 5,825 protein groups in +1 leaves of S. off, S. sp and SP, and the Gene Ontology enrichment analysis highlighted the photosynthetic nature of these proteomes, along with high representativeness of organeles involved in the processes of photosynthesis and carbon metabolism. The comparative quantitative analysis of 1,060 protein groups identified using the Shotgun/bottom-up approach revealed that 120 proteins were differentially expressed in +1 leaves of S. off, S. sp and SP, and of these, those that showed at least a 4-fold change are related to biological processes involved in both abiotic and biotic stress (response to other organism, response to cadmium ion, defense response to fungus, and defense to oxidative stress). For the analysis of the ontogenetic variation in the proteome of +1 leaves of SP (of 4, 8, 11 and 13 months old plants) the Shotgun/bottom-up proteomic approach was employed and resulted in the identification of a total of 5,780 peptides corresponding to 1,615 unique protein groups. Sixty five proteins were identified as differentially abundant between the samples and a hierarchical cluster analysis revealed that the 13 months-old leaves showed the most distinct profile, while the others clustered together. The characterization of the phosphoproteomes of +1 leaves of S. off, S. sp and SP using Immobilized Metal Affinity Chromatography (IMAC) as an enrichment method for phosphopeptides, and LC-MS/MS analysis, resulted in the identification of 633 unique phosphopeptides in 515 proteins. The characterization of the phosphoproteome of +1 leaves of SP (of 4, 8, 11 and 13 months old plants) using IMAC and Hydroxy Acid-Modified Metal Oxide Chromatography (HAMMOC) as enrichment methods for phosphopeptides, and LC-MS/MS analysis, resulted in the identification of 558 unique phosphopeptides in 413 proteins. In both analyses various phosphorylated kinases were identified, and the overall Gene Ontology enrichment analysis showed as most enriched terms nucleus and cytoplasm, as cellular components, and regulation of transcription, response to abiotic stress and glycolysis in biological process. This is the first study to characterize the whole proteome of leaves from sugarcane cultivars and genotypes in Brazil, and its results constitute a significant effort to use different proteomic approaches to advance the knowledge on the complex physiological processes modulating sugarcane growth and performance (AU)

• FAPESP's process: 11/11308-0 - Proteomic analysis of leaves of sugarcane cultivars and genotypes
• Grantee: Eduardo Shigueo Kitano
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)