Molecular analysis of relationship between plants and environment by a proteomic approach BIOS Research Doctorate School in BIOmolecular Science Universitá degli Studi di Pisa

Universitá degli Studi di Pisa

Research Doctorate School in BIOmolecular Science

BIOS

Course: MOLECULAR BIOTECHNOLOGIES

Molecular analysis of relationship between plants and

environment by a proteomic approach

Author: Francesca Fanucchi Advisor: Prof. Amedeo Alpi

Co-advisors:Prof. Pierdomenico Perata Dott. Massimo Alessio

Zeus non concesse più ai legni la forza del fuoco indefesso

per gli uomini mortali. Ma il prode figlio di Iapeto lo

ingannò rubando il bagliore del fuoco in una ferula cava.

[Esiodo, Teogonia vv. 563-569]. Come Zeus se ne accorse,

ordinò ad Efesto di inchiodare il corpo di quello sul monte

Caucaso. E Prometeo, inchiodato, vi rimase legato per una

serie di lunghi anni; ed ogni giorno un’aquila, volando su di

lui gli rodeva i lobi del fegato, che ricrescevano durante la

notte. Dunque Prometeo, per aver rubato il fuoco, pagava

questa pena, finché Eracle colpì con frecce sul Caucaso

l’aquila che rodeva il fegato di Prometeo e lo liberò.

[Apollodoro, Bibliotheca, I, 7, 1]

INDEX

1 INTRODUCTION

1.1 Freezing stress response in higher plants 3

1.2 Transcriptional regulation 9

1.3 Post-transcriptional regulation 12

1.4 mRNA processing and export from the nucleus 13

1.5 Post-translational regulation 15

1.6 Proteomics of freezing stress response in

Arabidopsis thaliana 16

1.7 Aim of the study 20

2 MATERIALS AND METHODS

2.1 Plant materials and growth conditions 22

2.1.1 Plants age and cold acclimation treatment 22 2.1.2 Freezing temperature and time of exposition to each

Temperature 22

2.2 Freezing tolerance studie

s

2.2.1 Electrolyte leakage 23

2.2.2 Photosynthetic pigments 24 2.3 Analysis on 20-days-old plants 25

2.3.1 Cold acclimation treatment 25

2.3.2 Freezing tolerance studies 25

2.3.3 Electrolyte leakage 26

2.3.4 Photosynthetic pigments 26

2.4 Protein extraction from Arabidopsis plant material 26

2.4.1 Evaluation of protein extracts concentration 27

2.4.2 Two dimensional electrophoresis (2DE) analysis:

Isoelectric focusing (IEF) and SDS PAGE 28

2.4.3 Staining of 2D gel 30

2.4.3.a Silver staining 30

2.4.3.b Vorum - Silver staining 31

2.4.3.c Coomassie staining 32

2.4.4 Computer analysis 33 2.5 Protein identification 33

2.5.1 Trypsin in-gel digestion 33

2.5.2 Mass spectrometry and database search 34

3 RESULTS

3.1 Screening conditions set-up 38

3.2 Experiments to establish the best growth conditions for

freezing tolerance studies 38

3.3 Freezing tolerance studies on 20-days-old plants 45

3.4 Proteomic analysis 51

3.4.1 Spot identification by mass spectrometry analysis 55

4 DISCUSSION

4.1 Freezing Tolerance Studies 71

4.2 Proteomic analysis 74

4.2.1.a Photosynthetic apparatus 77

• Ribulose bisphosphate carboxylase/oxygenase

large chain and Ribulose bisphosphate

carboxylase/oxygenase activase 77

• OEE2 (oxygen-evolving enhancer protein 2-1)

and OEE1 (oxygen-evolving enhancer protein 1-1 ) 80

• Coproporphyrinogen III oxidase 81

4.2.1.b Antioxidant enzime 82

• Peroxiredoxin-IIB 82

4.2.1.c Sugar transport 83

• EDL18 (Sugar transporter ERD6 protein) 83

4.2.1.d Sugar epimerase family protein 86

• Uncharacterized protein At2g37660 86

4.2.1.e RNA-binding protein and signal transduction 87

• Glycine-rich RNA-binding protein (GRP-7) 87

• ROC1 Putative ribonucleoprotein At2g37220

and ROC2, 29 kDa ribonucleoprotein 88

4.2.1.f Transport and signal transduction 89

• AtRAB7-Ras related protein Rab7 and

Ras related protein ARA-3 (Small GTPase superfamily) 89

• WBC24. Probable white-brown complex

homolog protein 24 – FRAGMENT 90

• Drl43. Probable disease resistance protein – FRAGMENT 91

• Uncharacterized protein SCRL2 92

4.2.1.g Protein biogenesis 92

• Mitochondrial ribosomal protein S2 and

Probable nucleolar protein NOP5-2– FRAGMENT 92

4.3 Conclusion and prospective 93