Sviluppo del cromoplasto e caratteristiche organolettiche del frutto in Solanum Lycopersicum

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1 ACCADEMIA DEI GEORGOFILI Il cloroplasto e la ricerca biologica per la produzione di cibo ed energia Sviluppo del cromoplasto e caratteristiche organolettiche del frutto in Solanum Lycopersicum Dr. Matteo Ballottari Università di Verona Dipartimento di Biotecnologie FIRENZE 24 MAGGIO 212

2 Analysis of chloroplast-chromoplast transition by a proteomic and physiologic approach 1 cm 1 cm 1 cm 1 cm

3 During Chloroplast Chromoplast transition, thylakoids membranes are disassembled and substituted by lipid-carotenoids aggregated structure, as Plastoglobules. CHLOROPLAST grana CHROMOPLAST plastoglobules plasto globules chromoplast CIMs

4 Analysis of chloroplast-chromoplast transition by a proteomic and physiologic approach SG MG OR R 1 cm 1 cm 1 cm 1 cm SMALL GREEN MATURE GREEN ORANGE RED 4 DIFFERENT DEVELOPMENTAL STAGE CONDITIONS WERE SELECTED BY FRUIT AGE (dpa), VOLUME AND PIGMENT CONTENT

5 Plastids were purified from different batches of fruit and leaves: SG MG OR R Leaves SG MG OR R BROKEN MEMBRANES PLASTIDS chloroplast chromoplast Almost no contamination (Western blot analysis)

6 Shotgun MudPIT analysis: Multidimensional Protein Identification Tecnology Membrane fraction Solubile fraction Membrane fraction Solubile fraction trypsin trypsin

7 1218 different polypeptides were identified using NCBI and TIGR-EST Solanacee databases. Leaves identified proteins are homologous to 95 Arabidopsis proteins, corresponding to 863 Arabidopsis genes OR R 112 Most of the proteins were differently expressed in the different developmental stages SG MG

8 Cell sub-localization of identified proteins (combining data from Target-P; AT_chloro; Gene Ontology and Chromoplast proteome database) NUMBER OF PROTEINS NON PLASTID 297 PLASTID 565 % PROTEIN QUANTITY Barsan et al J EXP BOT 21 NON PLASTID 28% PLASTID 72%

9 MAP MAN Functional classification of the different proteins identified # PROTEINS IDENTIFIED LEAVES R OR AA METABOLISM BIODEGRADATION OF XENOBIOTICS C1-METABOLISM CELL ORGANISATION CELL WALL Co-FACTOR AND VITAMINE METABOLISM DEVELOPMENT DNA SYNTHESIS -Reduction of light reactions photosynthetic proteins during ripening -Presence of several protein metabolism related polypeptides -Accumulation in OR of enzymes involved in carotenoid biosynthesis -Increased amount of protein with unknown function FERMENTATION GLUCONEOGENESIS GLYCOLYSIS HORMONE METABOLISM LIPID METABOLISM MAJOR CHO METABOLISM METAL HANDLING MINOR CHO METABOLISM MISC MITOCHONDRIAL ELECTRON TRANSPORT N-METABOLISM NOT ASSIGNED NUCLEOTIDE METABOLISM OPP POLYAMINE METABOLISM PROTEIN PHOTOSYNTHESIS REDOX RNA S-ASSIMILATION SECONDARY METABOLISM SIGNALLING STRESS TCA - Increase in stress-related proteins, in particular heat shock proteins TETRAPYRROLE SYNTHESIS TRANSPORT MG SG

10 MAP MAN Functional classification of the different proteins identified SG MG OR R 1 cm 1 cm 1 cm 1 cm -heat shock proteins are accumulated in fruits and in particular in chromoplast : HEAT SHOCK PROTEINS LEAVES SG MG OR R

11 MAP MAN Functional classification of the different proteins identified SG MG OR R 1 cm 1 cm 1 cm 1 cm -Plastid Ribosomal proteins in OR and R are strongly reduced. Indeed in chromoplast transcription is strongly reduced (Marano and Carrillo 1992).3 PLASTIDIAL RIBOSOMAL PROTEINS S 3S.1.5 LEAVES SG MG OR R

12 MAP MAN Functional classification of the different proteins identified SG MG OR R -increase of proteases (serine proteases and Clp proteases) in SG and OR. 1 cm 1 cm 1 cm 1 cm PROTEASES LEAVES SG MG OR R

13 MAP MAN Functional classification of the different proteins identified SG MG OR R 1 cm 1 cm 1 cm 1 cm -Increased amount of glycolytic enzymes in OR and R: GLYCOLYTIC ENZYMES LEAVES SG MG OR R

14 MAP MAN Functional classification of the different proteins identified SG MG OR R 1 cm 1 cm 1 cm 1 cm - Nitrogen assimilation: GOGAT GS Nitrite reductase

15 MAP MAN Functional classification of the different proteins identified SG MG OR R 1 cm 1 cm 1 cm 1 cm - Sulfur assimilation: ATP sulfurylase, sulfite reductase and cysteine synthase present until OR Sulfite reductase Cysteine synthase ATP sulfurylase

16 ANALYSIS OF PHOTOSYNTHETIC APPARATUS DURING CHLOROPLAST CHROMOPLAST TRANSITION Being mature fruits mainly heterotrophic tissues, Calvin cycle enzyme in chromoplast may prevent lost of biomass by partial re-fixation of CO2 produced by glycolysis and respiration Imported sugars RESPIRATION GLYCOLYSIS CO 2 ATP + REDUCING POWER BIOSYNTHETIC PATHWAY Calvin cycle Triose-P WHAT IS THE FUNCTION OF RUBISCO AND CALVIN CYCLE ENZYMES IN FRUITS AND IN CHROMOPLASTS?

17 ANKNOWLEDGEMENTS University of Verona Prof. Roberto Bassi Dr.ssa Alessandra Scala Dr.ssa Michela Ronzani CNR, Portici Dr.Pasquale Termolino, Dr.Francesco Didato Dr. Maria Cammereri Dr.ssa Silvana Grandillo ENEA Dr. Giovanni Giuliano Dr. Gaetano Perrotta Dr. Linda Bianco Innovation Center, Turin Prof. Massimo Maffei Dr. Andrea Occhipinti Dr. Simone Bossi PROGETTO FIRB RBLA345SF2

18 THANKS FOR YOUR ATTENTION