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Isolation and characterization of cysteine-degrading and H_1tn2S-releasing proteins in higher plants [Elektronische Ressource] / von Anja Riemenschneider

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Isolation and characterization of cysteine-degrading and H S-releasing proteins 2in higher plants Der Naturwissenschaftlichen Fakultät der Universität Hannover zur Erlangung des Grades Doktor der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation von Dipl.-Biol. Anja Riemenschneider geboren am 21.03.1978 in Hannover 2006 Referentin: PD Dr. Jutta Papenbrock Koreferent: Prof. Dr. Ahlert Schmidt Tag der Promotion: 30.05.2006 Summary The topic of this work is the characterization of cysteine-degrading and H S-releasing 2enzymes in higher plants. The reaction of two candidates, the OAS-TL isoforms and the cysteine desulfhydrases (CDes) have been observed under different conditions. O-acetyl-L-serine(thiol)-lyase (OAS-TL) is responsible for the formation of the first stable organic sulfur compound in the cysteine biosynthesis. It catalyses the incorporation of sulfide to the β-position of O-acetyl-L-serine (OAS) leading to the formation of cysteine. But OAS-TL is also able to release H S in a side reaction. CDes proteins are mainly responsible for the 2degradation of cysteine to H S, pyruvate and ammonium or to alanine and H S.

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Published 01 January 2006
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Isolation and characterization of
cysteine-degrading
and
H S-releasing proteins 2
in higher plants


Der Naturwissenschaftlichen Fakultät der Universität Hannover
zur Erlangung des Grades
Doktor der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
von
Dipl.-Biol. Anja Riemenschneider

geboren am 21.03.1978
in Hannover



2006
























Referentin: PD Dr. Jutta Papenbrock

Koreferent: Prof. Dr. Ahlert Schmidt

Tag der Promotion: 30.05.2006
Summary
The topic of this work is the characterization of cysteine-degrading and H S-releasing 2
enzymes in higher plants. The reaction of two candidates, the OAS-TL isoforms and the
cysteine desulfhydrases (CDes) have been observed under different conditions. O-acetyl-L-
serine(thiol)-lyase (OAS-TL) is responsible for the formation of the first stable organic sulfur
compound in the cysteine biosynthesis. It catalyses the incorporation of sulfide to the β-
position of O-acetyl-L-serine (OAS) leading to the formation of cysteine. But OAS-TL is also
able to release H S in a side reaction. CDes proteins are mainly responsible for the 2
degradation of cysteine to H S, pyruvate and ammonium or to alanine and H S. 2 2
Fumigation experiments with H S have shown that increased cysteine and glutathione 2
concentrations by using and/or detoxifing atmospheric H S seems to be confirm with 2
enhanced in vivo OAS-TL enzyme activity without significantly higher in vitro enzyme
activity at elevated H S levels. Slight decreases in the OAS concentration in the plants support 2
this theory of detoxification because of syntheses or degrading processes.
Experiments with transgenic OAS-TL antisense potato plants led to the conclusion that OAS-
TL has protected the cysteine homeostasis because OAS-TL was bound in the multi-enzyme
complex to inactivate the reverse reaction. Interestingly, in potato plants the H S-releasing 2
side seemed to be mainly catalysed OAS-TL enzyme activity.
Experiments done in order to clarify the role of H S-releasing enzymes and pathogen attack 2
showed that there seems to be a direct correlation between higher L-cysteine desulfhydrase
enzyme activity and pathogen attacks. Independent from the sulfur status of the plants highest
CDes mRNA accumulation could be observed in stems which would mean that if the
precursor for the degradation to H S is limited, higher sulfur supply would only enhance the 2
possibility of releasing H S. This situation supports the newest discussion where the term 2
sulfur-induced resistance (SIR) should be replaced by sulfur-enhanced defence (SED).
Calculated from a fumigation experiment done with Arabidopsis plants the H S amounts 2
-1potentially released against pathogens could not be higher than 0,5 µl l H S. Otherwise the 2
plants suffer oxidative stress. Innovative experiments with a H S microsensor were done to 2
detect H S concentration in plants in vivo. 2
The identification of D-cysteine desulfhydrases in Arabidopsis and the characterization of the
enzyme activity of one of them were successfully done. The substrates specify might be an
explanation for a purpose separation between D- and L-cysteine degrading CDes.

Keywords: Cysteine desulfhydrases, D- and L-amino acids, microsensor, OAS-TL, SED, SIR
IZusammenfassung
Thema dieser Arbeit ist die Charakterisierung von Cystein-abbauenden und H S-freisetzenden 2
Enzymen in Höheren Pflanzen. Dafür sind zwei Enzyme unter unterschiedlichsten Bedingung
untersucht worden. O-Acetyl-L-Serin(thiol)lyasen katalysieren die Verknüpfung von Sulfid
mit O-Acetyl-L-Serin (OAS), wobei Cystein entsteht. Allerdings können OAS-TLs auch in
einer Seitenreaktion den Abbau von Cystein zu HS katalysieren. Für diesen 2
Degradierungsschritt sind hauptsächlich Cystein-Desulfhydrasen (CDes) zuständig, indem sie
H S, Pyruvat und Ammonium oder Alanin und H S freisetzen. 2 2
H S-Begasungsexperimente mit Arabidopsis thaliana haben gezeigt, dass die ansteigende 2
Cystein- und Glutathion-Konzentration anscheinend mit der Nutzung und/oder der Entgiftung
des atmosphärischen H S durch gesteigerte in vivo OAS-TL Enzymaktivität zustande kommt, 2
ohne dass in vitro erhöhte OAS-TL Enzymaktivität gemessene werden kann. Die tendenzielle
Abnahme von OAS während der Begasung in den Pflanzen unterstützt die Theorie der
Entgiftung durch Synthese- oder degradierende Prozesse.
Experimente mit transgenen OAS-TL Kartoffel-Pflanzen lassen den Schluss zu, dass OAS-TL
für die Erhaltung der Cystein-Homöostase durch die Bindung an den Multi-Enzym-Komplex
Cystein-Synthase verantwortlich ist. Gebunden im Komplex ist es für das OAS-TL Enzym
nicht möglich, eine Rückreaktion zu H S zu katalysieren. Allerdings scheint es so, dass die 2
cytosolische OAS-TL-Isoform maßgeblich an der Freisetzung von H S beteiligt ist. 2
In Experimenten, die unternommen wurden, um die Rolle von H S-freisetzenden Enzymen 2
bei Pathogenbefall zu überprüfen, konnte eine direkte Korrelation von erhöhter L-Cystein
Desulfhydrase Aktivität mit Pathogenbefall gezeigt werden. Unabhängig vom Schwefel-
Status der Pflanzen konnte eine erhöhte Akkumulation von CDes mRNA in Stängeln gezeigt
werden. Dies würde wiederum bedeuten, dass auch wenn das Substrat für eine H S-2
Freisetzung limitiert ist, Schwefelzugabe nur eine Erhöhung der Möglichkeit zur H S-2
Freisetzung bewirkt. Diese Beobachtung spiegelt die derzeitige Diskussion über eine
Begriffsänderung von Schwefel-induzierter Resistenz (sulfur-induced resistence; SIR) zur
Schwefel-erhöhten Abwehr (sulfur-enhanced defence; SED) wider. Auf Grund der Ergebnisse
in einem H S-Begasungs-Versuch, kann spekuliert werden, dass Arabidopsis-Pflanzen nur bis 2
-1zu 0,5 µl l H S gegen Pathogene einsetzen könnten. Höhere H S-Konzentrationen führen zu 2 2
oxidativem Stress. Die Einbeziehung einer HS-Mikroelektrode stellt die innovative 2
Möglichkeit dar, H S Konzentrationen in vivo zu messen. 2
Die Identifikation von D-Cystein-Desulfhydrasen in Arabidopsis und die Charakterisierung
von einem der beiden Enzyme war möglich. Mögliche substratspezifische Aufgabenteilungen
IIzwischen L-und D-Cystein-degradierenden CDes könnte das Vorhandensein von beiden
Enzym-Varianten erklären.

Schlüsselwörter: Desulfhydrasen, D- und L-Aminosäuren, Mikroelekrode, OAS-TL, SED,
SIR

IIIAbbreviation


aa amino acids
ACC1-aminocyclopropane-1-carboxylic acid
Acc. no. accession number
AOA aminooxy acetic acid
At Arabidopsis thaliana
AtStr1 Arabidopsis thaliana sulfurtransferase 1
BCIP 5-bromo-4-chloro-3-indolyl-phosphate
Bn Brassica napus
BSA bovine serum albumine
CAS β-cyano-L-alanine synthase
CDes cysteine desulfhydrase
CDP2-chlor-5-(4-methoyxspiro{1,2-dioxetan-3,2’-
(5’-chlor)tricycle[3.3.1.13,7]decan}-4-yl-1-
phenylphosphate
Cyscysteine
D-CDes D-cysteine desulfhydrase
DIGdigoxigenin
DNAdeoxyribonucleicacid
dNTPsdeoxynucleotidetriphosphates
DTT dithiothreitol
ECenzymeclassification
EDTA ethylenediaminetetraacetic acid
EST expressed sequenced tag
FW fresh weight
GFP green fluorescent protein
GSHglutathione
HShydrogensulfide 2
IPTG isopropyl thio-β-D-galactoside
KCN potassiumcyanide
LCDL-cysteinedesulfhydrase
L-CDesL-cysteine desulfhydrase
IVN nitrogen
NAS N-acetyl-L-serine
NBTnitrobluetetrazolium
OAS O-acetyl-L-serine
OASTL O-acetyl-L-serine(thiol)lyase
OAS-TL O
OD optical density
ORF open reading frame
PCR polymerase chain reaction
PEGpolyethyleneglycol
PLPpyridoxal-5’-phosphate
Rhd rhodanese
RNAribonucleicacid
Ssulfur
SATserineacetyltransferase
SED sulfur-enhancedresistance
SIRsulfur-inducedresistance
St Solanum tuberosum
Wtwildtype

V
The following papers cover part of this thesis:




Bloem E, Riemenschneider A, Volker J, Papenbrock J, Schmidt A, Salac I, Haneklaus S, Schnug E.
2004. Sulphur supply and infection with Pyrenopeziza brassicae influence L-cysteine
desulphydrase activity in Brassica napus L. J Exp Bot 55: 2305-2312. Reprinted by permission of
Oxford University Press (Chapter 5)
Riemenschneider A, Nikiforova V, De Kok LJ, Papenbrock J. 2005. Impact of elevated H S on 2
metabolite levels, activity of enzymes and expression of genes involved in cysteine metabolism.
Plant Physiol Biochem 43: 473-483. Reprinted with permission from Elsevier (Chapter 3)
Riemenschneider A, Wegele R, Schmidt A, Papenbrock J. 2005a. Isolation and characterisation of a
D-cysteine desulfhydrase protein from Arabidopsis thaliana. FEBS J 272, 1291-1304. Reprinted
with permission from Blackwell (Chapter 7)
Riemenschneider A, Riedel K, Hoefgen R, Papenbrock J, Hesse H. 2005b. Impact of reduced O-
acetylserine(thiol)lyase isoform contents on potato (Solanum tuberosum L.) plant metabolism.
Plant Physiol 137: 892-900. Reprinted with permission from ASPB (Chapter 2)
Riemenschneider A, Bonacina E, Schmidt A, Papenbrock J. 2005. Isolation and characterization of a
second D-cysteine desulfhydrase-like protein from Arabidopsis. In: Sulfur Transport and
Assimilation in Plants in the Post Genomic Era. Saito K, De Kok LJ, Stulen I, Hawkesford MJ,
Schnug E, Sirko A, Rennenberg H (Eds.), Backhuys Publishers, Leiden, pp. 103-106. (Chapter 8)
VIContents
Chapter 1................................................................................................................................... 1
General introduction............................................................................................................... 1
Short overview of the sulfur metabolism ........................................................................... 1
The role of O-acetyl-serine(thiol)lyases and cysteine desulfhydrases in H S release ....... 2 2
The role of D-cysteine desulfhydrases in H S release ................................................... 6 2
The aim of the thesis .......................................................................................................... 7
Chapter 2.... 8
Impact of reduced O-acetyl-serine(thiol)lyase isoform contents on potato plant metabolism................. 8
Introduction ........................................................................................................................ 8
Materials and methods ..................................................................................................... 11
Generation of transgenic potato lines........................................................................... 11
Plant cultivation............................................................................................................ 11
RNA extraction and Northern blot analysis ................................................................. 11
SDS-PAGE and Western blotting ................................................................................ 12
Activity staining ........................................................................................................... 12
Determination of enzyme activity 13
Extraction and analysis of soluble thiol compounds.................................................... 13
OAS measurement by GC-MS..................................................................................... 14
Extraction and analysis of soluble amino acids ........................................................... 14
Results .............................................................................................................................. 15
Engineering and screening of plants with reduced OASTL enzyme activity .............. 15
Measurements of OASTL protein content in transgenic potato lines and OASTL
activity in a native gel .................................................................................................. 18
Effect of OASTL antisense inhibition on metabolite levels in source leaves.............. 20
Discussion ........................................................................................................................ 21
Chapter 3................................................................................................................................. 26
Impact of elevated H S on metabolite levels, activity of enzymes and expression of genes 2
involved in cysteine metabolism .......................................................................................... 26
Introduction ...................................................................................................................... 26
Methods............................................................................................................................ 28
Growth, treatment and harvest of plants ...................................................................... 28
RNA extraction and Northern blot analysis ................................................................. 29
SDS-PAGE and Western blot analysis......................................................................... 30
Enzyme activity measurements.................................................................................... 30
Determination of metabolites....................................................................................... 31
Miscellaneous and statistical evaluation 31
Results .............................................................................................................................. 32
Influence of H S fumigation on the phenotype of the Arabidopsis plants................... 32 2
Impact of elevated H S on thiol levels 32 2
Influences of H S on enzyme activity .......................................................................... 32 2
Steady-state protein levels............................................................................................ 34
Northern blot analysis of genes involved in sulfur metabolism................................... 36
Does the fumigation with H S cause oxidative stress in Arabidopsis plants? ............. 37 2
Impact on thiol precursors and other metabolites ........................................................ 38
Discussion ........................................................................................................................ 40
In Arabidopsis the levels of cysteine and glutathione were significantly increased by
fumigation with H S..................................................................................................... 40 2
VIIThe impact of elevated H S levels on activities of cysteine-synthesizing and degrading 2
enzymes was limited .................................................................................................... 41
Expression analysis and indication of oxidative stress ................................................ 43
Effects of H S fumigation on non-sulfur metabolites .................................................. 44 2
Chapter 4................................................................................................................................. 45
H S concentrations in plants detected by an H S microsensor ............................................ 45 2 2
Introduction ...................................................................................................................... 45
Material ............................................................................................................................ 46
Plant material................................................................................................................ 46
H S measurements with the microsensor ..................................................................... 46 2
Results .............................................................................................................................. 47
Discussion ........................................................................................................................ 50
Chapter 5................................................................................................................................. 52
Sulfur supply and infection with Pyrenopeziza brassicae influence L-cysteine
desulfhydrase activity in Brassica napus L.......................................................................... 52
Introduction ...................................................................................................................... 52
Materials and methods ..................................................................................................... 55
Experimental design of the field experiment ............................................................... 55
Chemical analysis......................................................................................................... 56
Measurement of cysteine and glutathione.................................................................... 56
Enzyme activity measurements.................................................................................... 56
Statistical calculations.................................................................................................. 57
Results .............................................................................................................................. 58
Effect of S fertilization on different S fractions and the activity of LCD and OAS-TL
...................................................................................................................................... 58
Effect of N feferent S fr....... 59
Effect of infection with P. brassicae on different S fractions and the activity of LCD
and OAS-TL................................................................................................................. 60
Correlations between the different S fractions and the enzyme activities ................... 60
Discussion ........................................................................................................................ 62
Chapter 6................................................................................................................................. 66
Expression of desulfhydrases and their enzyme activity in Brassica napus........................ 66
Introduction ...................................................................................................................... 66
Methods............................................................................................................................ 67
Growth and harvest of plants ....................................................................................... 67
Cloning procedures ...................................................................................................... 68
RNA extraction and Northern blot analysis ................................................................. 68
Determination of enzyme activity................................................................................ 69
Measurements of thiols and sulfate.............................................................................. 70
Sequence search ........................................................................................................... 70
Results .............................................................................................................................. 70
Enzyme activity in infiltrated Brassica napus plants................................................... 70
Pathogen studies in humid chamber............................................................................. 71
Sequences of desulfhydrases in oilseed rape................................................................ 71
Expression studies........................................................................................................ 73
Thiol concentration and enzyme activity in Brassica napus plants under sulfur
starvation ...................................................................................................................... 77
Discussion ........................................................................................................................ 78
Impact of different pathogens on different lines of winter oilseed rape ...................... 78
Research for desulfhydrases in Brassica napus influenced by sulfur starvation ......... 78
VIII