PhD Research

The Function of Isoprene Emission in Plants New Effects and Old Mechanisms

Christopher M. Harvey

Introduction

2-methyl-1,3-butadiene

C5H8

Boiling point = 34 C

Most basic member of the terpenoid family of compounds

Isoprene emission is present within most plant phylogenies

-Carotene

Limonene

Pinene

Isoprene

Why Study Isoprene?

Global annual production ~ 600 Tg (6*1011 Kg)

Contributes to ozone formation

Global warming may increase emissions

Industrial feedstock rubber, fragrances

Enhances heat tolerance of photosynthesis

BioIsoprene*

Los Angeles Pollution*

* Taken from Wikimedia Commons

MEP Pathway

ISPS

GAP

DXP

MEP

CDP-ME

CDP-MEP

MEcDP

HMBDP

IDP

DMADP

ISP

HigherTerpenoids

CO2

DXS

DXR

CMS

CMK

MCS

HDS

HDR

IDI

NADPH

CTP

ATP

CMP

+

2 Fd

NADPH

Pyr

Banerjee A, et al. (2013) Journal of Biological Chemistry 288: 16926-16936

Environment Influences Isoprene Emission

Figures taken from: Monson RK, et al. (1992) Plant Physiol 98:11751180

Under ideal conditions, 2% of fixed carbon

Under stress conditions, >20% of fixed carbon

Stresses include heat, salt, drought, oxidative

Isoprene emission from velvet beanincreases with illumination

Open circles warm-grownClosed circles cool-grown

Isoprene emission from velvet beanIncreases with illumination

Thermal Protection of Photosynthesis

Figure taken from: Sharkey TD & Singsaas E (1995) Nature 374: 769769

First demonstrated in 1995 in Kudzu, a native emitterPure N2 was used to suppress endogenous isoprene

Exogenous isoprene was added

Has since been demonstrated in both emitting and non-emitting plants

Exogenous isoprene shifts PSII fluorescence increase to higher temperatures in Kudzu

Oxidative Protection of Photosynthesis

Figure taken from: Loreto F, et al. (2001) Plant Physiol 126: 9931000

Photobleaching by 300 ppb O3 is ameliorated by 3 ppm ISP in tobacco

First demonstrated in 2001 in Tobacco and Birch

Demonstrated in Tobacco and Birch, both non-emitters

Has since been demonstrated in both emitting and non-emitting plants

Suggested to be the basis for thermal protection of photosynthesis

The mechanism of photosynthetic protection by isoprene is currently under debate

Isoprene as a Signaling Molecule

Isoprene promotes flowering in barley, oil-seed rape, and Arabidopsis1

Isoprene is sensed by insectsRepels herbivorous caterpillars2

Interferes with recruitment of parasitic wasps to herbivore-infested plants3

Non-emitting Populus x canescens have:Downregulation of stress responsive genes4

Repression of phenylpropanoid biosynthesis4

Fewer photosynthetic proteins and more translational machinery5

1Terry GM, et al. (1995) J Exp Bot 10: 1629-16312Laothawornkitkul J, et al. (2008) PC&E 10: 1410-14153Loivamki M, et al. (2008) PNAS 45: 17430-174354Behnke K, et al. (2010) Plant Molecular Biology 74: 61755Velikova V, et al. (2014) Journal of Proteome Research 4: 20052018

ISPS

GAP

DXP

MEP

CDP-ME

CDP-MEP

MEcDP

HMBDP

IDP

DMADP

ISP

HigherTerpenoids

CO2

DXS

DXR

CMS

CMK

MCS

HDS

HDR

IDI

NADPH

CTP

ATP

CMP

+

2 Fd

NADPH

Pyr

ISPS KO's Have Reduced MEcDP

ISPS KO's Have Reduced MEcDP

Figure taken from: Ghirardo A, et al. (2014) Plant Physiol 165, 3751

Open circles Non-EmittingClosed circles Emitting

MEcDP is a Retrograde Signal

MEcDP controls HPL production1Stress responsive protein

High light & wounding

MecDP is shunted away by glucosylation2

1Xiao Y, et al. (2012) Cell 149(7): 1525-15352Gonzlez-Cabanelas D, et al. (2015) The Plant Journal 82(1): 122-137

Are the changes in non-emitting Populus x canescens due to decreased isoprene or decreased MecDP?

Cataloging Isoprene-Induced Genes

5 week old Arabidopsis thaliana24 hours with/without 20 L L-1 isoprene

23 C and 120 mol m-2 s-1 light

3 replicates

Agilent 4x44 microarrayQuantile normalizationIgnored probe quality flags

Background subtracted gene expression value

Criteria2 Fold Expression Change

Log p-value < 0.05

2-sided, unpaired T-test

No multiple testing correction

Result:95 upregulated genes

72 downregulated genes

#ESGene Ontologies

11.98metal ion binding; cation binding; zinc binding; ion binding

21.29response to organic substance; response to endogenous stimulus; response to hormone stimulus

30.90response to chitin; response to carbohydrate stimulus; transcription factor activity; DNA binding;

40.89ZnF_C2H2; Zinc, C2H2-like; zinc ion binding

50.83Glucose/ribitol dehydrogenase; short-chain dehydrogenase/reductase SDR; NAD(P)-binding domain

60.71Flower, reproductive structure, seed, & fruit development

70.58metal ion transport; cation transport; ion transport

80.48extracellular region; signal peptide; secreted; glycoprotein

90.41intrinsic to membrane; transmembrane; membrane; transport

100.25topological domain-Extracellular; transmembrane region; topological domain-cytoplasmic

110.16chloroplast part; plastid part; chloroplast; plastid

120.12ATP binding; adenyl ribonucleotide binding; phosphorylation

Performed with DAVID Gene Functional Classification Tool: Huang DW, et al. (2007) Genome Biology 8: R183

Upregulated Motifs

#ESGene Ontologies

11.559response to: abiotic stimulus, light stimulus, red or far red light; intracellular signaling cascade

21.170sugar-hydrogen symporter activity; sugar transmembrane transporter activity; integral to membrane

30.897protein serine/threonine kinase activity; enzyme linked receptor protein signaling pathway

40.436phosphoprotein; transmembrane region; vacuole; transport

50.356response to: organic substance, endogenous stimulus, hormone stimulus

60.173transcription regulation; transcription factor activity; nucleus; DNA binding

70.002metal ion, transition metal ion, cation :binding

Downregulated Motifs

Gene Set Enrichment Analysis

Red = increased by isoprene

Blue = decreased by isoprene

Node darkness ~ degree of change

Node diameter ~ genes in ontology

Edge thickness ~ ontology overlap

Graphic created using Enrichment Map. Merico D, et al. (2010) PLoS ONE 11: e13984

Coexpressed Gene Network

IDGeneSymbolDescriptionLogFCp Value

1WRKY40WRKY transcription factor 403.510.185

2BAP1BON association protein 12.180.288

3AT3G02840uncharacterized protein3.920.135

4STZzinc finger protein STZ/ZAT103.890.144

5ERF13ethylene-responsive transcription factor 133.590.088

6BCS1cytochrome BC1 synthesis2.020.055

7CML37calcium-binding protein CML372.630.002

8WRKY46WRKY transcription factor 461.580.018


10DIC2NF-X1-type zinc finger protein NFXL23.890.346

11MYB15myb domain protein 152.920.146

12AT5G51190ethylene-responsive transcription factor ERF1052.640.035

13AT1G61340F-box stress induced 11.070.280

14AT5G52760copper transport family protein1.760.005

15RRTF1ethylene-responsive transcription factor ERF1094.080.158


17ZAT7zinc finger protein ZAT71.670.004

18WRKY18WRKY DNA-binding protein 181.470.442

19AT3G46080C2H2-type zinc finger family protein1.520.008


21AT5G19230GPI-anchored glycoprotein membrane precursor1.410.033

22AT1G20823RING-H2 finger protein ATL801.220.338

Network Function

WRKY and ERF transcription factors mediate stress responses

WRKY/ERF may mediate retrograde signaling

8 of the 22 network genes were upregulated in Multiprotein Bridging Factor 1 overexpressorsAll WRKY/ERF genes, MYB15, CML37

Plants are resistant to osmotic and heat stress

Network involved in abiotic-stress sensing and response

Suzuki N, et al. (2005) Plant Physiol 139: 13131322

Isoprene Induces Phenylpropanoid Biosynthetic Genes

Isoprene Induces PAL and 4CL

PAL and 4CL are generally induced under stress conditionsHigh Light

Cold Stress

Drought and Salt Stress

Conclusions

Isoprene induces stress-responsive genes

Some gene expression changes are inconsistent with ROS quenchingThe gene coexpression network is stress responsive

Phenylpropanoid biosynthesis is upregulated by stress response

Harvey & Sharkey (2015) PC&E 39(6): 1251-1263

Mechanism of Photosynthetic Protection

Photobleaching by 300 ppb O3 is ameliorated by 3 ppm ISP in tobacco2

Exogenous isoprene shifts PSII fluorescence increase to higher temperatures in Kudzu1

Two competing mechanistic theories:Protection by membrane stabilization1

Protection by ROS quenching2

1Sharkey TD & Singsaas E (1995) Nature 374: 7697692Loreto F, et al. (2001) Plant Physiol 126: 9931000

The Causal Dilemna

Oxidation of thylakoid lipids by ROS could disrupt the electron transport chain

Disruption of the electron transport chain leads to ROS production

Velikova V, et al. (2012) Plant Signaling & Behavior 7: 13941

Photosynthetic Protection by Membrane Stabilization

Figure taken from: Sharkey TD (1996) Endeavour 20: 7478

Heat causes:membrane thinning

ion leakage

acyl chain disordering

domain mixing

Isoprene could act:within bulk lipids

within proteins

at lipid:protein boundaries

at protein:protein boundaries

Evidence for a Membrane Mechanism

Isoprene stabilizes LHCII arrays during heat stress1

Isoprene slows ion leakage across thylakoids during heat stress1

Isoprene predicted to decrease the fluidity of acyl-lipid chains2

Velikova V, et al. (2011) Plant Physiol 157: 905916Siwko ME, et al. (2007) BBA - Biomembranes 1768: 198206

20 mol% isoprene causes an increasein lipid acyl chain order equivalent to adecrease in temperature of 10 C

Gramicidin as a Membrane Probe

Gramicidin A dimer2

1 Taken from Lundbaek, et al. (2009) Journal of the Royal Society Interface 44: 373-3952 Taken from Wikimedia Commons. Originally from Lomize A, et al. (1992) Biiorg Khim 18: 182-200

15 amino acid peptide

Val/Ile-Gly-Ala-Leu-Ala-Val-Val-Val-Trp-Leu-Trp-Leu-Trp-Leu-Trp

Residues alternate between D and L stereochemistry

helix

Dimer conducts monovalent cations

Membrane-embedded monomers1

Membrane-embedded dimer1

Gramicidin A Channel Lifetimes

Consistent with increased membrane thickness

Perylene as a Membrane Fluidity Probe

Fluorescent and planar

Time-Correlated-Single-Photon-Counting (TCSPC)Excitation with a vertically polarized laser

Vertically and horizontally polarized emission

Anisotropy decay rate allows determination of membrane fluidity

Effect of Isoprene on Membrane Fluidity

Interpretation: Isoprene does not alter membrane fluidity

Vesicles composed of a series of lipidsDSPC (18:0)

SOPC (18:1)

SLPC (18:2)

30-45 C

SLPC

Physiological Concentration of Isoprene

20 L L-1 isoprene in intercellular airspace

20, 43 mol % used by Siwko et al

Apparatus for Partitioning Measurement

Isoprene Quantification

814 pmole Buffer + Thylakoid

582 pmole Buffer

FIS Trace from 2mL samples

Lipid Quantification

Lipid concentration of DMPC vesicle solutions was controlled

Lipid concentration of thylakoid solutions was determined with GC-FAME

Lipid Species Concentration (nmol * mL-1)

LipidThylakoidLeaf

16:0415315

16:112853

16:3477162

18:0163156

18:14576

18:2160174

18:331641191

sum45512127

Intramembrane Isoprene Concentration

[ISP]measured

DMPC20.53.8

Thylakoid64.47.2

[ISP]calculated

44.0

39.3

Less partitioning into DMPC bilayers

Greater partitioning into thylakoid membranes

Isoprene would need to reach ~1 mole % to affect the dynamics of acyl lipids

Isoprene molecules per million acyl lipids

A Simple Calculation

PTXI = PIPIHI-1 = MI,WMI,WKOW,I = MI,O0.5*ADMPCDP-P,DMPC = VDMPCVDMPC-1 = MDMPC100*MI,OMDMPC-1 = XI,DMPC

Based on the nominal concentration of isoprene in air within leaves of ~20 ppm and the partition constant of isoprene in octanol one would predict an isoprene: lipid ratio of approximately 1:200. - Siwko, et al. (2007)

A Graphical Summary

Conclusions

Isoprene is unlikely to be modifying the acyl lipid phase of the thylakoid membrane

The reduction of gramicidin A channel lifetime is likely due to a protein-isoprene interaction

Harvey CM, et al. (2015) Journal of Bioenergetics and Biomembranes 5: 419-429

Open Questions

How does isoprene modulate gene expression changes?

Is the modulation of gene expression based on protein binding?

Which thylakoid proteins does isoprene bind to?

Acknowledgments

AdvisorDr. Thomas D. Sharkey

Committee MembersDr. Claire VieilleDr. Shelagh Ferguson-MillerDr. David WelikyDr. Gregg Howe

CollaboratorsDr. Gary BlanchardDr. Andreas WeberDr. Nicole LinkaJan Weise

MSU Glassblowing FacilityMSU Research Technology Support Facility

Past and present lab members:Dr. Sean E. WeiseDr. Ziru LiDr. Sarathi WeraduwageDr. Dennis GrayDr. Dilara AllyAparajita BanerjeeAlexandra LantzJon MontgomeryLaura CheaneyFrancisca AnozieAlyssa SchreurGustaf DegenBrittany Salyers

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