modulation of soybean and maize antioxidant activities by caffeic acid and nitric oxide under salt

188 Pages·2013·14.96 MB·English

MODULATION OF SOYBEAN AND MAIZE ANTIOXIDANT
ACTIVITIES BY CAFFEIC ACID A ND NITRIC OXIDE UNDER SALT

STRESS

Ashwil Johan Klein

A thesis submitted in partial fulfilment of the requirements for the degree of Doctor
Philosophiae in the Department of Biotechnology, University of the Western Cape.

Supervisor: Prof. Ndomelele Ndiko Ludidi
Co-Supervisor: Dr. Marshall Keyster

November 2012

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Modulation of soybean and maize antioxidant activities by caffeic acid and
nitric oxide under salt stress

Ashwil Johan Klein

KEYWORDS

Nitric oxide
Salinity
Reactive oxygen species
Cell death
Antioxidant enzymes
Caffeic acid
Lipid peroxidation
Superoxide
Antioxidant gene expression
Salt stress tolerance

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ABSTRACT
Modulation of soybean and maize antioxidant activities by caffeic acid and

nitric oxide under salt stress

A.J Klein

PhD Thesis, Department of Biotechnology, University of the Western Cape

This study explores the roles of exogenously applied nitric oxide, exogenously
applied caffeic acid and salt stress on the antioxidant system in cereal (exemplified
by maize) and legume (using soybean as an example) plants together with their
influence on membrane integrity and cell death.
This study investigates changes in H O content, root lipid peroxidation, root cell
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death and antioxidant enzymatic activity in maize roots in response to exogenously
applied nitric oxide (NO) and salt stress. This part of the study is based on the
partially understood interaction between NO and reactive oxygen species (ROS)
such as H O and the role of antioxidant enzymes in plant salt stress responses. The
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results show that application of salt (NaCl) results in elevated levels of H O and an
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increase in lipid peroxidation, consequently leading to increased cell death. The
study also shows that by regulating the production and detoxification of ROS through
modulation of antioxidant enzymatic activities, NO plays a pivotal role in maize
responses to salt stress. The study argues for NO as a regulator of redox
homeostasis that prevents excessive ROS accumulation during exposure of maize to
salinity stress that would otherwise be deleterious to maize.
This study extends the role of exogenously applied NO to improve salt stress
tolerance in cereals crops (maize) further to its role in enhancing salt stress
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tolerance in legumes. The effect of long-term exposure of soybean to NO and salt
stress on root nodule antioxidant activity was investigated to demonstrate the role of

NO in salt stress tolerance. The results show that ROS scavenging antioxidative

enzymes like SOD, GPX and GR are  differentially regulated in response to
exogenous application of NO and salt stre ss. It remains to be determined if the NO-
induced changes in antioxidant enzyme activity under salt stress are sufficient to
efficiently reduce ROS accumulation in soybean root nodules to levels close to those
of unstressed soybean root nodules.
Furthermore, this study investigates the effect of long-term exposure of soybean to
exogenous caffeic acid (CA) and salt stress, on the basis of the established role of
CA as an antioxidant and the involvement of antioxidant enzymes in plant salt stress
responses. The effect of CA on soybean nodule number, biomass (determined on
the basis of nodule dry weight, root dry weight and shoot dry weight), nodule NO
content, and nodule cyclic guanosine monophosphate (cGMP) content in response
to salt stress was investigated. Additionally, CA-induced changes in nodule ROS
content, cell viability, lipid peroxidation and antioxidant enzyme activity as well as
some genes that encode antioxidant enzymes were investigated in the presence or
absence of salt stress. The study shows that long-term exposure of soybean to salt
stress results in reduced biomass associated with accumulation of ROS, elevated
levels of lipid peroxidation and elevated levels of cell death. However, exogenously
applied CA reversed the negative effects of salt stress on soybean biomass, lipid
peroxidation and cell death. CA reduced the salt stress-induced accumulation of
ROS by mediating changes in root nodule antioxidant enzyme activity and gene
expression. These CA-responsive antioxidant enzymes were found to be superoxide
dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and
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glutathione reductase (GR), which contributed to the scavenging of ROS in soybean
nodules under salt stress.

The work reported in Chapter 2 has been published in a peer-reviewed journal

[Keyster M, Klein A, Ludidi N (2012) Caspase-like enzymatic activity and the

ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by
exogenously applied nitric oxide. Plant Signaling and Behavior 7: 349-360]. My
contribution to the published paper was all the work that is presented in Chapter 2,
whereas the rest of the work in the paper (which is not included in Chapter 2) was
contributed by Dr Marshall Keyster.

November 2012

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AIMS OF THIS STUDY

The study aimed at the following:

i. Investigating the role of exogenously applied NO in antioxidant enzyme-

mediated ROS scavenging in maize under salt stress.
ii. Detection of ROS scavenging maize antioxidant enzyme isoforms whose
activity is mediated by exogenous NO in response to salt stress.
iii. Determining the effect of NO on antioxidant enzymes in soybean root nodules
and how this influences soybean responses to salt stress.
iv. Investigating the role of exogenously applied CA in modulating the antioxidant
system in soybean root nodules during long-term salt stress.

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DECLARATION

I declare that “Modulation of soybean and maize antioxidant activities by caffeic acid
and nitric oxide under salt stress” is my own work, that it has not been submitted for
any degree or examination in any other university, and that all the sources I have
used or quoted have been indicated and acknowledged by complete references.

Ashwil Johan Klein November 2012
Signed ………………………………………………

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ACKNOWLEDGEMENTS

 Firstly, I would like to thank my Heavenly Father for giving me the strength,

guidance and wisdom to pursue my goals in life.
 Secondly, I would like to extend a spe cial thanks to my supervisors Prof. Ndiko
Ludidi and Dr. Marshall Keyster for providing me with the opportunity and

necessary guidance throughout this study.
 I would like to acknowledge my parents (Charmaine and Deon Klein) and in-
laws (Salomo and Sandra Moses), for their endless love, support and patience
through all my years of study until now. All the phone calls, text messages and
always being there with a word of encouragement in the difficult times. Words
cannot express my gratitude.

 “To my fiancée (Maretina Moses) and daughter (Chloé Avril Klein), thank you
for your steadfast patience and support throughout this study. The 2 of you were
my true inspiration and motivation for completing this study. I love you to bits”.

 To my siblings (Ivoidia, Gwendolene, Jacques, Leandra), the Van Wyk family
(John, Yolanda, Candice, Jason) and the rest of the Moses family (Cabral,
Leoni) thank you for always believing in me and your extra word of
encouragement in difficult times.
 Thanks to my friends (Eben B, Kevin, Jarries, Catz, DJ Linas, Randal) and
colleagues especially Dr. Marshall Keyster, Kyle Phillips, Dr. Morne Du Plessis,
Dr. Oom Fani Egbichi, for keeping me sane throughout the writing process with
all the silly talk. If I did not mention you, believe me you are not forgotten.
 The rest of the Plant Biotech Research Group (PBRG) past and present thank
you very much for humor throughout this study.
 Last but not least, this work would not have been possible without financial
support from The University of the Western Cape (UWC) and the National
Research Foundation (NRF, South Africa). Your contribution is highly
appreciated.

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LIST OF ABBREVIATIONS

ANOVA analysis of variance

AO amine oxidase

APX ascorbate peroxidase

AsA ascorbic acid
AtNOA1 Arabidopsis thaliana Nitric Oxide Associated 1
AtNOS1 Arabidopsis thaliana Nitric Oxide Synthase 1
BSA bovine serum albumin
CA caffeic acid
CAT catalase
cGMP cyclic guanosine monophosphate
CYP cysteine protease/ cysteine endopeptidase
CYPs cysteine proteases/ cysteine endopeptidases
DETA Diethylenetriamine
DETA/NO 2,2-(hydroxynitrosohydrazono)bisethanimine
DHAsA dehydroascorbate
DNA deoxyribonucleic acid
DHAR dehydroascorbate reductase
EDTA ethylenediaminetetraacetic acid
GPX glutathione peroxidase
GR glutathione reductase
GSH reduced glutathione
GSSG oxidised glutathione
Lb leghaemoglobin
MDA malondialdehyde
MES 2-(N-Morpholino)ethanesulfonic acid
NADPH nicotinamide adenine dinucleotide phosphate
NBT nitrotetrazolium blue chloride
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NO nitric oxide
NOS nitric oxide synthase

NOX NADPH oxidase

NR Nitrate reductase

OXO oxalate oxidase

PAGE Polyacrylamide gel electrophoresis
PCD programmed cell death
PVP Polyvinylpyrrolidone
RNA ribonucleic acid
ROS reactive oxygen species
SDS sodium dodecyl sulphate
SNP sodium nitroprusside
SOD superoxide dismutase
TBA thiobarbituric acid
TCA trichloroacetic acid
TEMED N,N,N′,N′-Tetramethylethylenediamine
XO Xanthine oxidase
XTT 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxyanilide

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