Nitric oxide in plants: investigation of synthesis pathways and role in defense against avirulent Pseudomonas [Elektronische Ressource] / vorgelegt von Jagadis Gupta Kapuganti

Bot1047

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

1

Nitric oxide in plants: Investigation of synthesis
pathways and role in defense against avirulent
Pseudomonas






Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg




Vorgelegt von

JAGADIS GUPTA KAPUGANTI

aus

Dharmavaram, VZM (DT)
Andhra Pradesh
INDIEN



Würzburg 2007












2





Eingereicht am:




Mitglieder des Promotionskommission:



Vorsitzender:
Gutachter: Prof. Dr. Werner M. Kaiser
Gutachter: Prof. Dr. Martin J. Müller




Tag des Promotionskolloquiums:

Doktorurkunde ausgehändigt am:














3































To my parents
Gowriswari and Venkata Ramana
















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Contents

Summary ................................................................................................................ 8
Zusammenfassung ............................................................................................... 11

A Introduction ......................................... 14
1. Chemistry of NO ............................................................ 15
2. Atmospheric NO.............................................................. 16
3. Biological sources of NO................................................ 17
3.1 NO in soil ......................... 17
3.2 Sources of NO in plants.................................................................... 17
3.2.1 Non enzymatic synthesis of NO ....................... 17
3.2.2 Nitrate reductase ............................................................................... 18
3.2.3.1. Nitric Oxide Synthase ....... 22
3.2.3.1 NOS in animals ................. 22
3.2.3.2 NOS in plants.................................................................................... 25
3.2.3.3 NOS in context with mitochondria ................... 27
4 NO in plant growth and development ........................... 28
4.1 Nitric Oxide and Hormone ............................................................... 30
5 NO in plant - pathogen interaction 34
6 Methods to detect and measure nitric oxide ................................................. 35
DAF ......................................................................... 35
Electrochemical method .......... 36
6.3 Oxyhaemoglobin assay ............................................................................. 36
6.4 Griess reagent assay ................. 36
6.5 Electron paramagnetic resonance ............................................................ 36
6.6 Arginine-Citrulline assay.......................................... 37
6.7 Chemiluminescence.................................................... 38
Aims of this thesis ................................................................ 39

B Results
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Chapter 1:
1.1 NO emission from roots................................................................................. 40
1.2 Inhibitor studies indicate possible involvement .......... 42
of mitochondrial electron transport
2 NO emission by mitochondria isolated from roots ........................................ 43
2.1 Determination of Km for nitrite dependent NO production...................... 46
2.2NO emission by mitochondria isolated from tobacco suspension cells .... 47
3 NO emission from roots or mitochondria in response
to different oxygen tension ............................................................................. 48
4 NO-scavenging by mitochondria ..... 50
5 NO emission from leaf mitochondria .............................................................. 53
6. Nitrite to NO reduction takes place on mitochondrial
membranes but not in the matrix ................................... 57
Conclusions............................................................................ 60

Chapter 2

1 Nitric Oxide Synthase ....................................................................................... 62
2.NOS activity in crude extracts of roots ........................... 64
3.NOS activity in root mitochondria .................................................................. 65
4.DAF-fluorescence from mitochondria ............................ 67
5.Measurement of commercial recombinant NOS by chemiluminescence .... 71
6.Scavenging of NO by the mitochondria using NOS as a source of NO........ 74
Conclusions ........................................................................................................... 77

Chapter 3:

Role of nitrate and NO in plant/pathogen interaction
Introduction............................................................................................................ 78
1 Cryptogein induced cell death is independe nt
of presence or absence of NO ................... 80
2 Is NR is required for hypersensitive response induced by TMV ............... 81
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3 Is NR required for HR induced by avirulent Pseudomonas syringae ........ 83
4 Is in vivo bacterial growth correlated with lesion formation?.................... 85
5 Is bacterial growth correlated with sugar or
amino acid contents in leaf cells or in the leaf apoplast ............................. 86
6 To what extent is an HR indiced by cryptogein
restricting bacterial growth ........................................................................... 88
Concluding rema rks ............................................................................................ 92
C Discussion
1NO production by root mitochondria .............................................................. 93
2 Scavenging of NO by mitochondria ................................ 94
3 Why do leaf mitochondria not reduce nitrite to NO ..... 96
4 Possible roles of Nitrite-NO reduction by roots and root mitochondria ..... 97
5. NOS or no NO ................................................................................................ 100
6. Is DAF a reliable NO indicator..... 100
7 Do nitrate and NR play a role in the HR ...................................................... 101

D Material and methods ................................................ 103
1 Plant materials ................................................................................................ 103
2. Preparation of root and leaf segments ......................... 104
2.1 Root segments ........................................................................................... 104
2.2 Leaf slices ................................. 104
3 Nicotiana tabacum cell suspension culture s .................................................. 104
4 Mitochondria preparatin ............................................... 106
4.1 Fractionation of mitochondria .............................................................. 106
5 Gas Phase NO measurements ........................................ 107
5.1 Description of the analyzer CLD 770 AL ppt .. 108
5.2 NO gas measurement.......................................................................... 109
6 NO scavenging by mitochondria ... 110
7 DAF- and DAR-4M- fluorescence ................................................................. 111
8 Nitric Oxide synthase (NOS) assay ............................... 111
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9 Nitrate/nitrite trace analysis .......................................................................... 112
10 Nitrite consumption ...................... 112
11 Measurement of NADH and NADPH oxidation ........ 112
12 Growth of plant pathogens and infection ................................................... 113
12.1 Growth and treatment of Pseudomonas strains. ..... 113
12.2 Cryptogein treatment............................................................................... 113
12.3 TMV infection......................... 113
13 Nitrate reductase activity and nitrite content ............................................ 114
14. Sugar and Amino acid analysis .................................. 115
15. Northern Blotting ........................................................ 115
15.1 Total RNA isolation............................................... 115
15.2 Synthesis of c DNA ............................................... 116
15.3 Polymerase Chain Reaction (PCR) ....................................................... 116
15.4 Electrophoresis of RNA ........................................ 118
15.5 Transfer of RNA .................................................... 120
15.6 Radioactive labelling of nucleic
acids and hybridization o