Studies of Gamma-Hydroxybutyrate and Gamma-Aminobutyrate Metabolism in Apple and ...

173 Pages·2013·3.76 MB·English

Studies of Gamma-Hydroxybutyrate and Gamma-Aminobutyrate Metabolism
in Apple and Arabidopsis

by

Greta Z. Chiu

A Thesis
presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Master of Science
in
Plant Agriculture

Guelph, Ontario, Canada

© Greta Z. Chiu, October, 2013
ABSTRACT

Studies of Gamma-Hydroxybutyrate and Gamma-Aminobutyrate Metabolism
in Apple and Arabidopsis

Greta Z. Chiu Advisors:
University of Guelph, 2013 Barry J. Shelp
Gale G. Bozzo

γ-Hydroxybutyrate (GHB) is an intermediate of γ-aminobutyrate (GABA) catabolism in
plants subjected to abiotic stress and its formation is catalyzed by two NADPH-dependent
glyoxylate/succinic semialdehyde reductases (GLYRs). GABA and/or GHB accumulation in
‘Honeycrisp’ and ‘Empire’ apple fruit stored under controlled atmosphere (CA) conditions (i.e.,
low temperature, low O , elevated CO ) in the presence or absence of the ethylene-antagonist 1-
2 2
methylcyclopropene, coincided with the onset of physiological injury, suggesting an association
with cellular disruption. Salinity and chilling stresses differentially influenced the expression of
GABA pathway genes and the levels of GHB among various GABA pathway mutants of
Arabidopsis. Furthermore, the occurrence of GHB in glyr1/glyr2 double knockout mutants
indicates the presence of an additional pathway for GHB production. Evidence for GHB
oxidation was not detectable in cell-free leaf extracts, suggesting the existence of a novel enzyme
for GHB turnover.
ACKNOWLEDGEMENTS
Many thanks to my advisors, Dr. Barry Shelp and Dr. Gale Bozzo, for their unwavering
guidance and for sharing their expertise in experimental design and data interpretation over the
past few years. This work would not exist without them. A note of thanks goes out to Dr.
Jennifer DeEll for her valuable insights, especially in postharvest apple disorders, and for serving
as a member of my advisory committee.
Special thanks to Dr. Adel Zarei who was responsible for the gene expression analysis
presented here, in addition to the creation of the glyr1/glyr2 double mutants, and for his help in
developing and carrying out the Arabidopsis experiments. Thanks also to Dr. Gordon Hoover for
his amino acid analyses in apple fruit that are presented here, his instruction in HPLC-use and
amino acid integration, his aid in adapting the amino acid analysis protocol for Arabidopsis, and
his patience in helping me locate apple samples.
The GHB quantitation would not have been possible without the guidance of Dr. Armen
Charchoglyan, who not only showed me how to use two GC-MS machines, but frequently shared
his expertise in alleviating my chromatography and detection troubles. Thanks to Dr. Dyanne
Brewer for her advice and consistent support in my analytical problems also, and for allowing
me to make the AAC a second home where I was frequently found preparing samples past
midnight.
Thanks to Dr. Stephen Bowley for providing suggestions on the statistical analysis of the
‘Honeycrisp’ experiment. Thanks also to Dr. Behrouz Ehsani-Mogghadam for conducting
maturity analyses in ‘Honeycrisp’. I am also very grateful to my labmates, fellow graduate
students, and certain roommates for their advice and support during rough times, and for
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tolerating my frequent ‘sighs’ in the office. Last but not least, many thanks to my family and
friends for their unwavering support and for persuading me to get in touch with the outside world
once in a while.
Funding for this project was provided by the Apple Growers of Ontario, MITACS, Rohm
and Haas, and the Natural Sciences and Engineering Research Council of Canada (NSERC). I
am very grateful for the NSERC CGS-M, the Ontario Graduate Scholarship, as well as other
University awards that I received during this time for making my life as a graduate student just a
little bit easier.

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Table of Contents
ABSTRACT .................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................... iii
LIST OF FIGURES ..................................................................................................................... viii
LIST OF APPENDICES ................................................................................................................. x
LIST OF ABBREVIATIONS ........................................................................................................ xi
Chapter 1. Literature Review .......................................................................................................... 1
1.1. Introduction .......................................................................................................................... 1
1.2. GHB Production from GABA .............................................................................................. 2
1.2.1. GABA Production ......................................................................................................... 4
1.2.1.1. Glutamate decarboxylase ........................................................................................ 4
1.2.1.2. Polyamines .............................................................................................................. 6
1.2.2. GABA Catabolism ......................................................................................................... 7
1.2.2.1. GABA transaminase ............................................................................................... 7
1.2.2.2. Succinic semialdehyde dehydrogenase ................................................................... 9
1.2.2.3. Glyoxylate reductase (Succinic semialdehyde reductase) .................................... 10
1.3. GHB Catabolism ................................................................................................................ 13
1.3.1. Plants ........................................................................................................................... 13
1.3.2. Mammalian systems .................................................................................................... 15
1.3.3. Bacteria and yeast ........................................................................................................ 16
1.4. Postharvest Metabolic Processes in Fruit ........................................................................... 18
1.4.1 Effects of CA conditions on GABA ............................................................................. 18
1.4.2. Physiological storage disorders in pears ...................................................................... 19
1.4.3. Physiological storage disorders in apples .................................................................... 21
1.4.3.1. Preharvest factors .................................................................................................. 21
1.4.3.2. Postharvest factors ................................................................................................ 22
1.4.3.2.1. Temperature ................................................................................................... 22
1.4.3.2.2. CA conditions ................................................................................................. 24
1.4.3.2.3. 1-Methylcyclopropene .................................................................................... 25
1.5. Concluding Remarks, Hypotheses and Objectives ............................................................ 27
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1.5.1. Hypothesis 1: GABA accumulation is associated with physiological injury in stored
‘Honeycrisp’ fruit treated with 1-MCP. ................................................................................ 27
1.5.2. Hypothesis 2: GHB accumulation is associated with physiological injury in stored
apples. .................................................................................................................................... 27
1.5.3. Hypothesis 3: A novel enzyme is responsible for the conversion of GHB to SSA in
plants. ..................................................................................................................................... 28
1.5.4. Hypothesis 4: Metabolite and gene responses of the GABA pathway are stress-
specific. .................................................................................................................................. 28
1.5.5. Hypothesis 5: SSA is the sole source of GHB in plants subjected to abiotic stress. ... 29
Chapter 2. Materials and Methods ................................................................................................ 31
2.1. Postharvest Storage of ‘Honeycrisp’ Apples ..................................................................... 31
2.1.1. Apple material ............................................................................................................. 31
2.1.2. Fruit maturity evaluations ............................................................................................ 31
2.1.3. Postharvest treatment ................................................................................................... 32
2.1.4. Evaluation of physiological storage disorders ............................................................. 34
2.2. Response of Arabidopsis thaliana to Salt or Chilling Stress ............................................. 35
2.2.1. Plant and chemical materials ....................................................................................... 35
2.2.2. Cultivation and stress conditions ................................................................................. 37
2.3. Extraction and Analysis of Amino Acids by HPLC .......................................................... 39
2.4. Extraction and Analysis of GHB and GABA by GC-MS/MS ........................................... 40
2.5. Analysis of Gene Expression in Arabidopsis during Chilling or Salinity Stress ............... 43
2.6. Statistical Analysis ............................................................................................................. 44
Chapter 3. Results ......................................................................................................................... 46
3.1. ‘Honeycrisp’ 2010 Storage Trials ...................................................................................... 46
3.1.1. Effects of chilling, CO , and 1-MCP on the incidence of storage disorders ............... 46
2
3.2. ‘Empire’ 2009 Storage Trials ............................................................................................. 55
3.2.1. Effects of chilling and CO on GHB and amino acid levels ........................................ 55
2
3.3. Metabolite and Expression Analysis of the GABA Pathway in Arabidopsis Mutants
Subjected to Abiotic Stress ....................................................................................................... 59
3.3.1. Phenotypic differences in response to exogenous SSA and GHB application ............ 59
3.3.2 Effects of high salinity on plant growth and levels of gene expression, GHB, and
amino acids ............................................................................................................................ 66
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3.3.3 Effect of chilling on levels of GHB and amino acids ................................................... 71
Chapter 4. Discussion ................................................................................................................... 76
4.1. GABA accumulation is associated with flesh browning in ‘Honeycrisp’ apple fruit during
CA storage ................................................................................................................................. 76
4.2. GHB accumulates in ‘Empire’ apple fruit during CA storage ........................................... 84
4.3. SSA and GHB have negative effects on growth and development in Arabidopsis ........... 86
4.4. GABA pathway genes and metabolites are differentially expressed during the early stress
response to salinity and cold ..................................................................................................... 90
4.5. GHB is produced via multiple pathways............................................................................ 94
Chapter 5. Conclusions ............................................................................................................... 100
LIST OF REFERENCES ............................................................................................................ 102
APPENDICES ............................................................................................................................ 124

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LIST OF FIGURES
Figure 1.1. Pathway for the metabolism of glutamate to GHB...................................................... 3
Figure 1.2. Known GHB degradation pathways in non-plant organisms .................................... 17
Figure 1.3. Common apple storage disorders .............................................................................. 23
Figure 2.1. Design of 2010 ‘Honeycrisp’ storage trial ................................................................ 33
Figure 2.2. Design of 2009 ‘Empire’ storage trial ....................................................................... 41
Figure 3.1. Effect of 1-MCP and CA on the incidence of flesh browning during storage at 20 C
and 3 C ............................................................................................................................ 48
Figure 3.2. Effect of 1-MCP and CA on the relative amino acid composition of ‘Honeycrisp’
fruit during storage at 20 C.. ........................................................................................... 51
Figure 3.3. Effect of 1-MCP and CA on the relative concentrations of metabolites of
‘Honeycrisp’ fruit during storage at 3 C. ........................................................................ 53
Figure 3.4. Effects of low temperature and elevated CO on amino acid and GHB levels in
2
‘Empire’ fruit. ................................................................................................................... 57
Figure 3.5. Effects of SSA on WT, glyr1/glyr2-RNAi, glyr1/glyr2, and GLYR1 Ox lines of
Arabidopsis grown on solid media at 6 °C for 27 d .......................................................... 60
Figure 3.6. Effects of 2mM SSA on WT, glyr1/glyr2-RNAi, glyr1/glyr2, and GLYR1 Ox lines of
Arabidopsis grown on solid media for 21 d at 6 °C and observed at higher magnification
........................................................................................................................................... 62
Figure 3.7. Effects of SSA on WT, glyr1/glyr2, glyr1, and glyr2 lines of Arabidopsis grown on
solid media at 6 °C for 27 d. ............................................................................................. 63
Figure 3.8. Effects of GHB on WT, glyr1/glyr2-RNAi, glyr2/glyr2, and GLYR1 Ox lines of
Arabidopsis grown on solid media at 6 °C for 27 d .......................................................... 64
Figure 3.9. Effects of GHB on WT, glyr1/glyr2, glyr1, and glyr2 lines of Arabidopsis grown on
solid media at 6 °C for 27 d. ............................................................................................. 65
Figure 3.10. Effects of 0, 50, 100, and 150 mM NaCl on WT, GLYR1 Ox, gaba-t, glyr1/glyr2,
and GABA-T/GLYR1 Ox lines of Arabidopsis grown in liquid culture for 21 d............... 67
viii
Figure 3.11. The impact of 150 mM NaCl on the expression of GABA pathway genes in WT,
gaba-t, GABA-T Ox, glyr1/glyr2, and GLYR1 Ox lines of 18-day-old Arabidopsis plants
grown in liquid culture. ..................................................................................................... 69
Figure 3.12. The impact of 150 mM NaCl on the amino acid and GHB levels in WT, gaba-t,
GABA-T Ox, glyr1/glyr2, and GLYR1 Ox lines of 18-day-old Arabidopsis plants grown in
liquid culture. .................................................................................................................... 70
Figure 3.13. The impact of chilling on the expression of GABA pathway genes in WT,
glyr1/glyr2, and GLYR1 Ox lines of 18-day-old Arabidopsis grown in liquid culture..... 73
Figure 3.14. The impact of chilling on the amino acid and GHB levels in WT, glyr1/glyr2, and
GLYR1 Ox lines of 18-day-old Arabidopsis grown in liquid culture. .............................. 74
Figure 4.1. Schematic representation of metabolic changes in 1-MCP-treated ‘Honeycrisp’
apples in response to elevated CO storage at 3 C and their proposed relationship with
2
flesh browning disorders. .................................................................................................. 81
Figure 4.2. A possible alternative pathway for GHB production from phospholipids ................ 99
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LIST OF APPENDICES

Appendix A. Materials and Methods .................................................................................... 1244
A.1. Analysis of gene expression in Arabidopsis .............................................................. 1244
A.2. Creating standards and standard curves for GHB analysis ........................................ 1255
A.3. GHB enzymatic assay in Arabidopsis ....................................................................... 1299
Appendix B. Supplementary Results .................................................................................... 1322
Appendix C. ANOVA Tables ............................................................................................... 1333
C.1. 2010 ‘Honeycrisp’ Experiment .................................................................................. 1333
C.1.1. ANOVA tables for treatments at 20 °C ............................................................... 1333
C.1.2. ANOVA tables for treatments at 3 °C ................................................................. 1433
C.1.3. Correlation tables for treatments at 3 °C ........................................................... 14355
C.2. 2009 ‘Empire’ Experiment ........................................................................................ 1556

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