Impacts of sub-ambient and elevated CO2 on above

170 Pages·2017·4.49 MB·English

Impacts of sub-ambient and elevated
CO on above- and below-ground
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microbial interactions with Arabidopsis.

Alex Williams
Department of Animal and Plant Science,
The University of Sheffield
Thesis submitted for the degree of Doctor of Philosophy
Submitted: December 2017

This work is dedicated to Pauline Williams.
The most beautiful life I knew.

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Abstract

Over recent years, an increasing body of evidence has suggested that elevated
atmospheric CO concentrations can alter plant microbial interactions. However,
2
there is limited consensus whether these impacts will be positive or negative for
plants in terms of disease resistance. Accordingly, there is a pressing need to
gain a better understanding of the molecular and physiological mechanisms by
which CO shapes the plant’s ability to interact with its biotic environment, which
2
is essential to predict impacts of future climate scenarios on crop production.
The work described in this thesis has used a range of CO concentrations,
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from past through present to future predicted concentrations, to study the immune
response of the model plant Arabidopsis thaliana to aboveground pathogens and
belowground rhizosphere bacteria. Furthermore, a novel developmental
correction was established, which enables assessing the direct immunological
effects of CO on microbial interactions without bias from age-related resistance
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arising from the stimulatory effects of CO on plant development.
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Changes in disease resistance at elevated CO (eCO ), against the
2 2
necrotrophic fungus Plectosphaerella cucumerina (Pc) and the obligate
biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) were associated with
changes in production and sensitivity of the phytohormones jasmonic acid (JA
and salicylic acid (SA), respectively. However, priming of SA-dependent defence
was not the only mechanisms contributing to eCO -induced resistance against
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Hpa. The increased resistance to Hpa at sub-ambient CO (saCO ) against Hpa
2 2
operated independently of SA signaling and was associated with changes in
cellular redox state and priming of pathogen-inducible intracellular ROS. Based
on the defence phenotypes of knock-down mutants in glycolate oxidase, the
H O -generating enzyme of the photorespiration cycle, and transcriptional
2 2
profiling of the peroxisomal catalase gene CAT2, it is proposed that the enhanced
Hpa resistance at saCO is controlled by photorespiratory ROS.
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Below-ground, the root colonisation of a specialised rhizobacterial strain
Pseudomonas simiae WCS417 was found to be dependent on CO concentration
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and soil-nutritional status, whereas root colonisation by the soil saprophytic strain
Pseudomonas putida KT2440 was largely unaffected by these variables. Hence,
changes in atmospheric CO have a greater impact on specialist rhizosphere
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microbes. Furthermore, the ability of P. simiae WCS417 to promote plant growth
and elicit an induced systemic resistance (ISR) was highly dependent on CO
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and nutritional status of the soil. These results suggest that the effects of
atmospheric CO on rhizosphere microbes depend on the rhizosphere species in
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question and the nutritional status of the soil.
To obtain a more global impression of the impacts of CO on rhizosphere
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interactions, rhizosphere soil was studied for bacterial community diversity and
composition using PCR-based community profiling techniques. This revealed that
CO has a measurable impact on microbial communities in a time-point
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dependent manner, whereby the effects of eCO are more pronounced at earlier
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stages and the effects of saCO are more pronounced at later stages. To study
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the biochemical basis of these CO effects on the rhizosphere effect, a new mass
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spectrometry-based method was developed to study quantitative and qualitative
impacts of CO on non-sterile rhizosphere chemistry. These experiments
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revealed that the diversity of chemical signals greatly increases with rising CO
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concentrations and that saCO and eCO are associated with rhizosphere
, 2 2
enrichment of different classes of chemicals.
Together, the results presented in this thesis provide novel insights into
the mechanisms by which plants have adapted to past CO climates, and the
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potential impacts by which future CO scenarios will affect interactions with
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hostile and beneficial microbes. Further research is required to explore the
combined impacts of eCO and other environmental changes due to global
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climate change, such as elevated temperatures and drought.
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Table of Contents
Abstract ............................................................................................................. v
Abbreviations and nomenclature .................................................................... x
List of tables and figures................................................................................ xii
Chapter 1: General Introduction ...................................................................... 1
1.1 Introduction ................................................................................................ 1
1.2 Shifting global climate. ............................................................................... 2
1.2.1 The greenhouse effect and the importance of CO ............................. 2
2.
1.2.2 Past CO concentrations. .................................................................... 3
2
1.2.3 Current and future CO projections. .................................................... 4
2
1.3 Plant perception, recognition and responses to pathogenic microbes. ...... 6
1.4 Elevated CO effects on plant pathogen interactions. ................................ 8
2
1.4.1 Impacts of eCO on plant physiology. .................................................. 8
2
1.4.2 A meta-analysis of the impacts of eCO on plant disease. .................. 9
2
1.4.3 CO and pre-invasive resistance. ...................................................... 10
2
1.4.4 CO : an enhancer or suppressor of post-invasive defences? ............ 11
2
1.4.5 Host defence responses to sub-ambient CO . .................................. 16
2
1.5 Interactions with below-ground beneficial microbes................................. 18
1.6 Effects of CO on belowground interactions in rhizosphere. .................... 21
2
1.6.1 Effects of CO on rhizosphere chemistry. .......................................... 21
2
1.6.2 Effects of eCO on rhizosphere microbes. ......................................... 23
2
1.6.2 Effects of saCO on rhizosphere microbes. ....................................... 24
2
1.7 Thesis outline. ......................................................................................... 24
Chapter 2: Materials and Methods ................................................................. 27
2.1 Reagents and chemicals. ..................................................................... 27
2.2 Plant cultivation and growth conditions. ................................................ 27
2.3 Experimental set-up of growth system for metabolite collection. .......... 27
2.4 Development measurements and correction. ....................................... 28
2.5 Plant developmental correction (DC). ................................................... 28
2.6 Pathogenicity assays using Hpa and Pc. .............................................. 28
2.7 RT-PCR quantification of pathogen DNA. ............................................ 31
2.8 In situ detection of reactive oxygen species. ........................................ 32
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2.9 Targeted quantification of hormones. ................................................... 32
2.10 Metabolite extraction for untargeted profiling...................................... 33
2.11 Untargeted metabolic profiling by UPLC-qTOF-MSE. ......................... 33
2.12 Data preparation of MS data for statistical analysis. ........................... 34
2.13 Soil inoculation with Pseudomonas simiae WCS417 and Pseudomonas
putida KT2440. ........................................................................................... 38
2.14 Induced systemic resistance assays. ................................................. 39
2.15 Bacterial community profiling by terminal-restriction fragment length
polymorphism analysis. .............................................................................. 39
2.16 Community diversity and composition. ............................................... 40
2.17 Analysis of dissimilarity in T-RFLP patterns between samples. .......... 41
Chapter 3: Mechanisms of glacial-to-future atmospheric CO effects on
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plant immunity (adapted from Williams et al. 2017) ................................... 43
3.1 Abstract ................................................................................................... 43
3.2 Introduction .............................................................................................. 44
3.3 Results..................................................................................................... 46
3.4 Discussion ............................................................................................... 53
3.5 Supporting figures ................................................................................... 57
Chapter 4: Impacts of glacial-to-future atmospheric CO on bacterial
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rhizosphere colonisation in relation to plant growth and systemic
resistance responses. .................................................................................... 67
4.1 Abstract ................................................................................................... 67
4.2 Introduction .............................................................................................. 67
4.3 Results..................................................................................................... 70
4.4 Discussion ............................................................................................... 74
Chapter 5: Impacts of glacial-to-future concentrations of atmospheric CO
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on the bacterial community structure and chemical profile of the
Arabidopsis rhizosphere. ............................................................................... 79
5.1 Abstract ................................................................................................... 79
5.2 Introduction .............................................................................................. 80
5.3 Results..................................................................................................... 82
5.4 Discussion ............................................................................................... 90
Chapter 6: Final Discussion ........................................................................... 99
6.1 Summary of findings ................................................................................ 99
6.1 Interactions between below- and above-ground plant defences ............ 101
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6.2 Impacts of interactions between eCO and other environmental variables
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on plant-microbe interactions. ...................................................................... 104
6.2.1 Elevated Ozone ............................................................................... 104
6.2.2 Elevated temperature ...................................................................... 106
6.2.3 Changing Nutrient availability .......................................................... 110
6.2.4 Temporal considerations and growth-stage effects. ........................ 111
6.3 Outlook .................................................................................................. 113
Reference list ................................................................................................ 115
Appendix 1..................................................................................................... 139
Acknowledgements ...................................................................................... 155

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Abbreviations and nomenclature
ABA Abscisic acid
a- e - saCO Ambient, elevated, sub-ambient CO
2 2
AMF Arbuscular mycorrhizal fungi
ARR Age-related resistance
C Carbon
CFC Chlorofluorocarbon
CFU Colony forming unit
DAB 3,3'-diaminobenzidine
DAMP Damage associated molecular pattern
DC Devlopmental correction
DCFH-DA 2',7'-dichlorofluorescein diacetate
DIMBOA 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one
DNA Deoxyribonucleic acid
dpg Days post germination
dpi Days post inoculation
DW Dry weight
eCO Elevated CO
2 2
ESI Electropray (positive or negative mode)
ET Ethylene
ETI Effector triggered immunity
ETS Effector triggered susceptibility
FACE Free air CO enrichment
2
FAM 6-fluorescein amidite
FDR False discovery rate
FW Fresh weight
GFP Green fluorescent protein
GHG Green-house gas
gs Stomatal conductance
Hpa Hyaloperonospora arabidopsidis
hpi Hours post infection
HR Hypersensitive response
IPCC International panel on climate change
ISR Induced systemic resistance
JA Jasmonic acid
LB Lysogeny broth
LN Leaf number
MS Mass spectrometry
mya Million years ago
N Nitrogen
NAD Nicotinamide adenine
NB-LRR Nucleotide binding lucine rich repeats
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