1 short title - esalq.usp.br · 1 short title 2 herbivore-triggered electrophysiological reactions...

1

Short title 1

Herbivore-triggered electrophysiological reactions 2

Corresponding author details 3

Dr Matthias Rudi Zimmermann 4

Institute of General Botany and Plant Physiology Friedrich-Schiller-University 5

Dornburgerstraszlige 159 D-07743 Jena Germany 6

Telephone number 0049 3641949234 7

E-MailMatthiasRZimmermannbot1biouni-giessende 8

Article title 9

Herbivore-triggered Electrophysiological Reactions Candidates for Systemic Signals in higher 10

Plants and the Challenge of their Identification 11

Authors 12

Matthias R Zimmermann1 Axel Mithoumlfer2 Torsten Will3 Hubert H Felle4 and Alexandra CU 13

Furch1 14

15

LIST OF AUTHOR CONTRIBUTION 16

Matthias R Zimmermann 17

measurements with substomatal conductance blind piercing surface potentials etc hellip (Fig 18

1+2+4+5+6) analysis and discussion MS editing and conception 19

Axel Mithoumlfer 20

support of caterpillars analysis and discussion MS editing and conception 21

Torsten Will 22

measurements with EPG (Fig 5) analysis and discussion MS editing 23

Hans H Felle 24

measurements with substomatal conductance in H vulgare (Fig 1) analysis and discussion 25

MS editing 26

Plant Physiology Preview Published on February 12 2016 as DOI101104pp1501736

Copyright 2016 by the American Society of Plant Biologists

wwwplantorg on February 18 2016 - Published by wwwplantphysiolorgDownloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved

2

Alexandra CU Furch 27

illustration of plant venation (Fig 3) analysis and discussion MS editing and conception 28

figure preparation 29

Funding information 30

The Deutsche Forschungsgemeinschaft in the frame of FU 9692-1 Fe 21315-1 and 15-2 and 31

The Max Planck Society 32

Affiliations 33 1Institute of General Botany and Plant Physiology Friedrich-Schiller-University 34

Dornburgerstraszlige 159 D-07743 Jena Germany 35 2Department Bioorganic Chemistry Max Planck Institute for Chemical Ecology 36

Hans-Knoumlll-Straszlige 8 D-07745 Jena Germany 37 3Plant Cell Biology Research Group Institute of General Botany Justus-Liebig-University 38

Senckenbergstraszlige 17 D-35390 Gieszligen Germany current address Department 39

Phytopathology Heinrich-Buff-Ring 26-32 D-35392 Gieszligen Germany 40 4retired former address Institute of General Botany Justus-Liebig-University 41

Senckenbergstraszlige 17 D-35390 Gieszligen Germany 42

One sentence summary 43

In plants feeding caterpillars trigger various types of electrophysiological reactions in which the 44

diverse voltage pattern are specific for plant species technical and experimental approaches 45

Present addresses 46

See above 47

Corresponding author with email address 48


MatthiasRZimmermannbot1biouni-giessende 50

51

52


3

ABSTRACT 53

In stressed plants electrophysiological reactions (elRs) are presumed to contribute to 54

long-distance intercellular communication between distant plant parts Because of the focus on 55

abiotic stress-induced elRs in the last decades biotic stress-triggered elRs have been widely 56

ignored It is likely that the challenge to identify the particular elR types ndash action potential (AP) 57

variation potential (VP) and system potential (SP) ndash was responsible for this course of action 58

Thus the present survey focused on insect larva feeding (Spodoptera littoralis Manduca sexta) 59

that triggers distant APs VPs and SPs in monocotyledonous and dicotyledonous plant species 60

(Hordeum vulgare Vicia faba Nicotiana tabacum) APs were detected only after feeding on the 61

stemculm whereas SPs were systemically observed following damage to both stemculm and 62

leaves This was reasoned by the unequal vascular innervation of the plant and a selective 63

electrophysiological connectivity of the plant tissue However striking variations in voltage 64

patterns were detected for each elR type Further analyses (also in Brassica napus Cucurbita 65

maxima) employing complementary electrophysiological approaches in response to different 66

stimuli revealed various reasons for these voltage pattern variations an intrinsic plasticity of elRs 67

a plant-specific signature of elRs a specific influence of the applied (a)biotic trigger the impact of 68

the technical approach andor the experimental set-up As a consequence thereof voltage pattern 69

variations which are not irregular but rather common need to be included in electrophysiological 70

signalling analysis Due to their widespread occurrence systemic propagation and respective 71

triggers elRs should be considered as candidates for long-distance communication in higher 72

plants 73

74

75


4

INTRODUCTION 76

The unimpeded feeding of herbivorous insects on plants has disastrous consequences it 77

causes the loss of plant tissue breaks down tissue integrity negatively impacts physiology and 78

facilitates colonisation by pathogens (van Bel 2003 Hilker and Meiners 2010 Mithoumlfer and 79

Boland 2012) In higher plants several constitutive and induced defence responses against 80

herbivores have been identified however the corresponding initial signals for induced defence 81

responses remain largely unknown (Wu and Baldwin 2010 Mithoumlfer and Boland 2012) Many 82

studies on herbivory-initiated signalling focused on chemical signals such as jasmonates ethylene 83

systemin salicylic acid and NO (Pearce et al 1991 Walling 2000 Kessler et al 2004 Maffei et 84

al 2007 Leitner et al 2009 Wu and Baldwin 2010 Mithoumlfer and Boland 2012) whereas 85

electrophysiological reactions (elRs) are largely disregarded as potential signalling components 86

Three different elR types have been described in higher plants action potential (AP) 87

variation potential (VP) and system potential (SP) (Fig 1 Davies 2004 Davies 2006 Fromm and 88

Lautner 2007 Fromm and Lautner 2012 Zimmermann and Mithoumlfer 2013 Galleacute et al 2014) 89

AP and VP are characteristic depolarisation events of a plasma membrane differing in voltage 90

pattern ionic mechanism and velocity (Stahlberg and Cosgrove 1996 Stahlberg and Cosgrove 91

1997 Davies 2006 Felle and Zimmermann 2007) In contrast SPs are systemically transmitted 92

hyperpolarisation events of a plasma membrane (Zimmermann et al 2009) Most studies trigger 93

elRs by using abiotic stimuli little information is available for the elRs triggered by potential 94

biotic stressors such as herbivores (Zimmermann and Mithoumlfer 2013) Volkov and Haack (1995) 95

described an occurrence of APs in the stem of potato plants (Solanum tuberosum L) as a result of 96

the damage by Colorado beetle larvae (Leptinotarsa decemlineata Say) feeding on young terminal 97

leaflets Maffei and co-workers (2004) presented strong membrane depolarisation events at the 98

biting zone of lima bean leaves (Phaseolus lunatus L) in response to feeding Spodoptera littoralis 99

larvae In both cases the depolarisation event decreased rapidly beyond a distance of 60 mm from 100

the feeding site 101

Recently an interesting report described both negative and positive extracellular voltage 102

changes in local (wounded) and distant leaves of Arabidopsis thaliana (L) Heynh upon S 103

littoralis larvae feeding (Mousavi et al 2013) Unfortunately the voltage changes which were 104

not further specified were named as wound-activated surface potentials (WASPs) Negative 105

WASPs were recorded in the local leaf and directly connected distant leaves (parastichies) 106


5

whereas the same stimulus simultaneously triggered positive WASPs in other distant leaves of the 107

same plant The same group also reported on intracellular recordings of herbivore-induced (Pieris 108

brassicae L) elRs in A thaliana sieve elements of intact neighbouring leaves using a direct current 109

electrical penetration graph with a living aphid as bio-electrode (Salvador-Recatalagrave et al 2014) 110

The negative voltage changes were correlated with the jasmonate pathway due to an increase (up 111

to ~130 fold) of the JASMONATE-ZIM DOMAIN 10 transcript levels (Mousavi et al 2013 112

Salvador-Recatalagrave et al 2014) 113

The rising but still low number of known natural triggers for elRs and the observed 114

inconsistent herbivore-induced voltage patterns enliven the controversy about whether or not elRs 115

might play a role in plant signalling cascades (Zimmermann and Mithoumlfer 2013) In order to 116

clarify this situation the current study presents new results of several herbivore-induced elRs in 117

local and systemic plant parts of dicots (Vicia faba Nicotiana tabacum) and a monocot (Hordeum 118

vulgare) Additionally we provide diverse electrophysiological measurements that were recorded 119

in response to different stimuli 120

RESULTS AND DISCUSSION 121

Herbivore-induced action potentials APs 122

A strong steep and transient extracellular hyperpolarisation (representing intracellular 123

depolarisation see material and methods) event was recorded in V faba and H vulgare when S 124

littoralis larvae fed on their stems (Fig 2A and B lower trace) The timescales and slopes of the 125

recorded elRs were characteristic for APs (Felle and Zimmermann 2007 Zimmermann and Felle 126

2009 Zimmermann and Mithoumlfer 2013) Interestingly the herbivore-induced APs in V faba (Fig 127

2A) and H vulgare (Fig 2B) exhibited pronounced differences in the kinetics of their 128

repolarisation phases The wavelike repolarisation in V faba (Fig 2A) could be distinguished from 129

the biphasic repolarisation event of H vulgare (Fig 2B) indicating a plant-specific response The 130

observed voltage patterns in H vulgare (Fig 2B) were similar to APs elicited with KCl CaCl2 or 131

glutamate (Felle and Zimmermann 2007) In contrast previously described APs in V faba 132

differed considerably from the wavelike repolarisation pattern observed here (Roblin 1985 133

Roblin and Bonnemain 1985 Dziubinska et al 2003 Furch et al 2007 Zimmermann and Felle 134

2009) An analysis with published results of elRs noted additional kinetic differences such as 135


6

longer durations (18 fold) and higher magnitudes (2 to 3 fold) compared to our findings (Volkov 136

and Haack 1995 Maffei et al 2004 Mousavi et al 2013 Salvador-Recatalagrave et al 2014) Thus 137

in various plant-herbivore combinations both a plant-species impact and an impact of the 138

particular trigger to the shape of the APs is suggested 139

Herbivore-induced system potentials SPs 140

Besides APs in stems extracellular depolarisation (=intracellular hyperpolarisation) events 141

were systemically detected in target leafs (t-leafs) of V faba and H vulgare when larvae fed on 142

either stimulus leaf (s-leaf) or the culm (Fig 2C and D Fig 3A) These findings confirm recent 143

results of Mousavi et al (2013) though those results differed in duration (6 to 10 fold) and 144

amplitude (15 to 3 fold) Systemically recorded extracellular depolarisation events SPs were 145

previously described in response to wounding and the application of KCl NaCl MgCl2 CaCl2 or 146

fusicoccin (Zimmermann et al 2009) However compared to herbivory (Fig 2D) 147

CaCl2KCl-induced SPs exhibited different voltage patterns (Fig 2F) indicating the influence of 148

the applied stimuli In accordance with prior results (Zimmermann et al 2009 Mousavi et al 149

2013) a single occurrence of SPs could also be detected (Fig 2C first trace Fig 3A) however 150

most experiments revealed repetitive SPs (Fig 2C and D) These repetitive SPs were interpreted as 151

the consequence of the dynamic larval feeding process and might be confirmed by 152

herbivore-induced multiple hydraulic events in remote areas (Alarcon and Malone 1994) Indeed 153

hydraulic events are generally connected with VPs being potentially contradictory (Zimmermann 154

and Mithoumlfer 2013 Zimmermann et al 2013) However it was found that larvae feeding on the 155

leavesrsquo main vein triggered locally (s = 50 mm) both SPs and VPs (Fig 3B) a combination which 156

was interpreted as the plantrsquos electrophysiological response to the induced change of pressure 157

conditions in the vascular system (Zimmermann et al 2013) 158

A connection between the observed elRs and larval feeding might seem questionable 159

because in some cases elRs were first recorded 75 to 100 min after larvae were placed on the plant 160

(Fig 2C lowest trace Fig 3B) That lag phase can be explained by the caterpillarsrsquo movement and 161

the different feeding behaviour of S littoralis (more greedy) and M sexta (less greedy) Immediate 162

feeding usually followed the application of hungry caterpillars In general since an exact trigger 163

time point cannot be defined for herbivory the critical moment of elRs release cannot be 164

determined The necessary unequal period for recording made it impossible to calculate a velocity 165

for the individual elRs 166


7

Interestingly the close temporal (t = 4-6 min) iterative SP recordings (Fig 2C lower traces) 167

strongly suggest that there is a short or missing refractory period for SPs in contrast to APs where 168

refractory periods are well known and base presumably upon a non-conductive state of Ca2+- 169

release channels (Paszewski and Zawadzki 1976 Fromm and Spanswick 1993 Fromm and 170

Bauer 1994 Wacke et al 2003) 171

172

The plant venation - electrophysiological connectivity for distant plant sections 173

Our results attest to the basal ability of higher plants to release and propagate different elRs 174

(for reviews see Davies 2004 2006 Fromm and Lautner 2007 Fromm and Lautner 2012 175

Zimmermann and Mithoumlfer 2013 Galleacute et al 2014) However it was a striking observation that 176

no herbivore-induced APs could be detected in a distant leaf following larvae feeding confirming 177

previous surveys (Volkov and Haack 1995 Maffei et al 2004 Mousavi et al 2013) Hence the 178

existing results show that AP transmission from leaf-to-leaf does not occur reliably in contrast to 179

SP 180

One reason for this phenomenon might be the unequal innervation of individual plant parts 181

with the vascular system as it offers the most likely longitudinal pathway for elRs The 182

innervation of the whole plant can be illustrated via vascular staining in V faba (Fig 4) The 183

distribution of the blue and red ink demonstrates that each main vascular strand in the stem edges 184

of V faba innervates well-defined plant (Fig 4A-D) and leaf (Fig 4E-H) areas Consequently if a 185

close correlation of elRs propagation and vascular branching is assumed an unequal transmission 186

of elRs would be demanded Such a close relation of vascular anatomy and systemically-recorded 187

elRs was already suggested before (Pickard 1973 Roblin 1985 Roblin and Bonnemain 1985 188

Mousavi et al 2013 Kiep et al 2015) A second reason could be the anatomical higher 189

electrophysiological resistance in the transition zones of the nodes The strength of APs would 190

decrease when the area with the postulated higher electrophysiological resistance is passed and the 191

necessary AP threshold could not be reached The consequence of this would be a loss of the 192

characteristic initial depolarisation phase (all-or-nothing law) Simultaneously the detected SPs 193

(Fig 2C and D) compensate for the loss of the voltage-dependent channel activity which is 194

necessary for APs on their way through the plant body because the subsequent activation of 195

H+-ATPases persists (Zimmermann et al 2009) Therefore the electrophysiological connectivity 196

for SPs seems to be improved in comparison to APs 197


8

A complement measurement of intra- and extracellular voltage of a 198

sub-epidermalmesophyll cell demonstrated that the apoplastic hyperpolarisation is intracellularly 199

mirrored with a lower depolarisation event (Fig 2B) That finding is based on the fact that the 200

electrophysiological resistances of apoplast and symplast differ (Zimmermann and Felle 2009) It 201

may also support a lateral propagation of APs originating from the phloem in addition to the 202

prominent longitudinal pathway (Eschrich et al 1988 Fromm 1991 Fromm and Bauer 1994 203

van Bel 2003 van Bel et al 2011 Salvador-Recatalagrave et al 2014) The lateral ldquopropagationrdquo can 204

also be interpreted as an electrophysiological leakage (= low electrical shield effect) additionally 205

supporting the above-mentioned loss of APs However a fundamental study about the quality of 206

electrophysiological propagation (cable properties) in higher plants as an elementary characteristic 207

for a reliable long-distance signal transduction is unfortunately still missing and needs to be 208

addressed in prospective surveys 209

Insect feeding a two-component process 210

The existence of herbivore-triggered elRs raises the question about the nature of the 211

stimulus The dynamic feeding process of caterpillars implies a series of multiple small bites 212

mechanically wounding the plant tissue and generating an injured surface area that might act as an 213

interface for the chemistry of caterpillar-derived oral secretions and plant tissue (Mithoumlfer and 214

Boland 2008 Mescher and De Moraes 2014) Hence the feeding process can be dissected into a 215

mechanical and a chemical component (Mithoumlfer and Boland 2008 Salvador-Recatalagrave et al 216

2014) 217

It was already shown that various mechanical injuries like pinching in A thaliana (Favre et 218

al 2001) cutting in V faba (Furch et al 2008) and C maxima (Zimmermann et al 2013) 219

triggered elRs near to the site of stimulus (s = 30-90 mm) However we were not able to confirm 220

the presence of elRs in distant t-leafs by using diverse types of leaf damages cutting (razor blade 221

scissors) pricking (needle) picking (forceps) squeezing (tubes) or robotic punching with the 222

so-called ldquoMecWormrdquo Solely a non AP-like extracellular depolarisation event was detected in a 223

t-leaf following stem wounding (Fig 2E) Thus these results suggest the existence of a more 224

complex way of stimulation than simple mechanical wounding as mentioned before (Maffei et al 225

2004) Similar results were obtained when oral secretion of S littoralis was used (= chemical) 226

Oral secretions never systemically triggered any elRs neither when placed on the unwounded plant 227

surface nor on a small wound area These results are in contrast to shown local and systemic 228


9

membrane depolarisation events in response to an application of oral secretion (Maffei et al 2004 229

Maischak et al 2007 Guo et al 2013) Nevertheless the results may support the view of an 230

interplay combining the dynamic mechanical damage (= feeding process) with chemical 231

compounds from feeding larvae to trigger systemically elRs 232

Approaches to explain the observed variability of elRs in higher plants 233

An analysis of prior reports revealed that in higher plants discrepancies in elR characteristics 234

such as variations of voltage kinetics and magnitudes is common (eg Pickard 1973 235

Zimmermann and Mithoumlfer 2013) However that is surprising for APs in particular since the 236

orchestrated interaction of channels and pumps (Felle and Zimmermann 2007 Zimmermann and 237

Mithoumlfer 2013) postulates a similar voltage signature at any time and site Hence those 238

observations are problematic and make an identification of individual elR types complicate Based 239

on our own experiments and data from the literature various explanations for the voltage 240

variations are conceivable all of which will be discussed in more detail in the following 241

(i) Intrinsic plasticity of the elRs An evaluation of numerous CaCl2-induced SPs in V faba 242

and H vulgare showed some regular voltage variations (Fig 5) The common basis is the 243

extracellular depolarisation event accompanied with similar de-repolarisation kinetics or a bit 244

longer lasting repolarisation phase (Fig 5A) a variable initial hyperpolarisation (Fig 5B) a 245

subsequent wave (Fig 5C) a two-kinetics repolarisation phase (Fig 5D) a variable initial and 246

subsequent hyperpolarisation (Fig 5E) andor a double depolarisation phase (Fig 5F) Voltage 247

pattern variations are well known for VPs that correlate with the strength of the local hydraulic 248

pressure change and thus are an intrinsic feature of VPs (Zimmermann and Mithoumlfer 2013) Here 249

although the CaCl2 stimulus strength (concentration and application period) was kept similar 250

variations in voltage patterns were still found justifying the variations of herbivore-induced SPs 251

(Fig 2C and D) Similar de- and repolarisation kinetics as well as a subsequent wave and a 252

hyperpolarisation event were observed for both herbivore- and CaCl2-induced SPs The finding of 253

a two-kinetics depolarisation phase (Fig 5E) supports the hypothesis of a short or even missing 254

refractory period as already mentioned above Like VPs SPs exhibit voltage pattern variations 255

thus making them an intrinsic feature as well 256

(ii) Plant-specific signatures of elRs A proposed plant specificity of an extracellular voltage 257

signature for the various elRs can be reasoned with the physico-chemical features of the apoplast 258

The chemical composition of cell walls differs among plant species (Northcote 1972 Bacic et al 259


10

1988 Sakurai 1998 Sattelmacher 2001 Felle 2001 Burton et al 2010 Wolf et al 2012) and 260

affects the physico-chemical properties of the apoplastic space (eg buffer capacities ionic 261

relations) which in turn influences the detectable voltage kinetics For instance the physiological 262

variability of the apoplast is well illustrated with the lower H+ buffer capacity (027-40 mM H+ 263

pH-1) (Hartung et al 1988 Gollan et al 1992 Oja et al 1999 Sattelmacher 2001 Felle and 264

Zimmermann 2007) in comparison with the symplast (20 to 80 mM H+ pH-1) (Kauss 1987 Oja et 265

al 1999 Felle 2001) Thus lower apoplastic H+ alterations are theoretically needed to reliably 266

measure voltage changes for all other ion species (Kauss 1987 Gollan et al 1992 Granqvist et 267

al 2012) The consequence is a faster detection of electrochemical changes within the apoplastic 268

space accompanied by stronger amplitudes in comparison to corresponding intracellular 269

recordings (Table 1) 270

(iii) Specific influence of the applied (a)biotic trigger Until now elRs have been often 271

triggered with a heat stimulus accompanied by a VP of unpredictable magnitude (Roblin 1985 272

Fromm and Lautner 2007 Furch et al 2007 Fromm and Lautner 2012) Heat-triggered VPs 273

represent the local electrophysiological consequence of an induced hydraulic pressure wave 274

spreading along the xylem vessels The VP magnitude is positively linked to the strength of the 275

hydraulic pressure wave that on the one hand depends on the stimulus intensity and on the other 276

hand the distance between stimulus and recording site (Roblin 1985 Roblin and Bonnemain 277

1985 Stahlberg and Cosgrove 1997 Furch et al 2007 Zimmermann and Mithoumlfer 2013) 278

Hence VPs vary strongly in shape and duration and the contribution of VPs to the entire measured 279

voltage change differs (Furch et al 2007 Furch et al 2009) Therefore it cannot be completely 280

excluded that the repeated mechanical damages of larvae feeding mimics heat-triggered VPs in 281

part Feeding (Fig 3B) damages the vascular system and impacts the vascular pressure conditions 282

as already suggested with respect to several other mechanical damages (Fig 2E Alarcon and 283

Malone 1994 Zimmermann et al 2013 Salvador-Recatalagrave et al 2014) 284

(iv) The technical approach The recorded voltage variations based on the applied technical 285

approaches as well Each technical approach possesses intrinsic characteristics that have to be 286

considered for the studied scientific question and analysis In contrast to extracellular recordings 287

intracellularly measured elRs generate readily comparable voltage signature because of the highly 288

regulated small cytoplasmic volume (cf H+-buffer capacities) and the strong plasma membrane 289

resistance representing a strong electrical shield (Rin = 5-120 MΩ Findlay and Hope 1976 290


11

Stahlberg and Cosgrove 1994 1996 Cheeseman and Pickard 1997 Katicheva et al 2014) In 291

consequence intracellular measurements are influenced to a substantial lower extent by 292

environmental factors and the recorded detection area is more defined than recordings of the 293

extracellular space Simultaneously the low electrical shield of extracellular measurements results 294

in an unknown detection area meaning a higher chance to monitor a conjoined reaction of multiple 295

vascular strands The consequence is an overlap or delay of individual elRs displayed with voltage 296

patterns of differing time courses and variable kinetics (Roblin 1985 Roblin and Bonnemain 297

1985) For instance simultaneous measurements of CaCl2-induced SPs with an electrode placed 298

either sub-stomatal or in an agar block exhibited different kinetics and durations (Fig 6A Table 1) 299

The diversity of voltage patterns can be also observed with two serial-placed electrodes one inside 300

the petiole and the other in the main vein of a C maxima leaf in response to a heat stimulus (Fig 301

6B) Numerous APs were recorded in the petiole and two APs were detected in the main vein The 302

decrease of AP quantity can be deduced from the split of the vascular strands in the transient area 303

of petiole and leaf lamina (Carle and Loy 1996) The main vein exhibits a lower amount of 304

vascular strands than the petiole which is reflected by less APs (Fig 6B) supporting the above 305

mentioned influence of plant venation (Fig 4) 306

A particular aspect of the electrical penetration graph (EPG) technique is the usage of an 307

interconnected aphid that is employed as living bio-electrode (see Material and Methods cf 308

Salvador-Recatalagrave et al 2014) The aphid acts as a variable resistance in an electrical circuit 309

Primary the well-established EPG technique was developed to study the sucking behaviour of 310

aphids (McLean and Kinsey 1964 1965) However well-documented experiences identifying 311

and analysing elRs simultaneously are rare which might explain the hesitation of an elR 312

classification by our colleagues (Salvador-Recatalagrave et al 2014) Explicit differences of blind 313

pierced (Fig 6B) intracellular (Fig 6C) and EPG (Fig 6D) recorded elRs were shown in response 314

to a remote heat stimulus and indicated a longer relay time period when using the EPG technique 315

in comparison to the classic electrophysiological recording set-ups (cf Furch et al 2010) One 316

consequence thereof is a different velocity of the electrical reaction Thus the explicit disparities 317

in time (Fig 6B-D) and the strong decrease of the recorded electrophysiological strength with the 318

increasing distance (Fig 6D) are likely the reason that Salvador-Recatalagrave et al (2014) did not 319

report on any herbivore-induced SPs in the sieve elements Nevertheless the practice of aphid 320

bio-electrodes possesses interesting aspects such as multiple-electrode recordings and 321


12

long-distance observations of electrophysiological responses (Furch et al 2010) The method 322

allows minimum-invasive intracellular measurements but it cannot be excluded that aphid watery 323

saliva is released into the pierced sieve element (Will and van Bel 2006) and affects the reactivity 324

of channels pumps and carriers due to the presence of different effectors (Will et al 2013) 325

(v) The experimental set-up An important aspect for an adequate analysis of elRs is the 326

chosen experimental set-up (Fig 7) The relation between the stimulated location and the recording 327

sites plays a crucial role because the distance the elR type and the quality of the vascular 328

connection influences the propagation These facets can be well demonstrated with the application 329

of a heat stimulus (HF) Despite of the artificial character HF is a useful tool for fundamental 330

electrophysiological studies because of the simple application the reliable release of elRs and the 331

ability to trigger all known elR types Near to the stimulus site all reaction types are superimposed 332

and illustrated by the diffuse and variable voltage patterns known as electropotential wave (Fig 333

7A Furch et al 2007 2009) On its way through the plant body the contribution of VPs decrease 334

rapidly due to their inability of self-propagation and the high electrophysiological resistance of the 335

plant tissue (= cable theory cf Jack et al 1975 Koch 1984 Taylor 2013) The consequence is 336

that the voltage pattern of APs (Fig 7A and E) or SPs (Fig 7D and F) becomes clearer with rising 337

distance confirming partly prior results (Roblin 1985 Roblin and Bonnemain 1985) Therefore 338

the distance can act as a separator of the different elR types It is a common observation that elRs 339

do not equally propagate within the plant (Fig 6B Fig 7C and D) and likely depend on the quality 340

of vascular connection (Fig 4 cf Mousavi et al 2013 Salvador-Recatalagrave et al 2014 Kiep et al 341

2015) Frequently APs get ldquolostrdquo and decreasing sub-threshold hyperpolarisation events are 342

detected (Fig 7B-D) As mentioned above the area of the nodes significantly influenced the 343

propagation and the AP transmission failed (Fig 7C and D) The AP-originated disturbance of the 344

plasma membrane potential activates directly the plasma membrane H+-ATPases for a 345

re-initialisation (Felle and Zimmermann 2007 Zimmermann et al 2009) and in many cases SPs 346

persist (Fig 7D and F) The propagation ability of a pure SP (Fig 7G and F cf Lautner et al 2005) 347

strongly indicates an intercellular electrophysiological coupling of H+-ATPases (Zimmermann et 348

al 2009) but the molecular mechanism has not yet been identified 349

350

CONCLUSION 351


13

Here herbivore-triggered elRs were described for different plant and insect species The 352

results support a general ability of feeding herbivores to trigger elRs both locally and systemically 353

and provide defined elRs as candidates for long-distance signalling However it is a common 354

observation that herbivore feeding provokes various types of elRs (Fig 8) 355

VPs are not able for a self-propagation and therefore can solely be detected near to the 356

wounded plant area The long-distance transmission of APs depends on an appropriate 357

electrophysiological connectivity among the individual plant cells and this is seemingly not given 358

for plant tissue The consequence is a ldquolossrdquo of APs on its way through the plant body Both AP 359

and VP are depolarising events of the plasma membrane inducing directly a stimulation of 360

H+-ATPases to recover the plasma-membrane potential It is a comparative new aspect that the 361

subsequent hyperpolarisation (=SP) is able for a self-propagation (Fig 7F and G) and could explain 362

the high chance of detection in systemic plant parts (Fig 8) The potential information content of 363

SPs is a task for future studies however indications for a natural relevance of SPs are given with 364

the herbivore feeding as a natural stimulus 365

366

367

MATERIALS AND METHODS 368

Plant material 369

Vicia faba cv Witkiem major Hordeum vulgare Nicotiana tabacum Brassica napus and 370

Cucurbita maxima (Gele Reuzen) plants were cultivated in pots in a greenhouse under standard 371

conditions (20-30 degC 60 to 70 relative humidity and a 1410-hour lightdark regime) 372

Supplementary illumination (SONT Agro 400 W Philips Eindhoven The Netherlands) led to an 373

irradiance level of 200 to 250 micromol2 sec-1 at the plant apex Plants were taken in their vegetative 374

phase 17 to 21 days after germination 375

Aphid and larvae cultivation 376

Myzus persicae was reared on 20- to 28-day-old plants of B napus in a 377

controlled-environment at 25degC and a 177 h lightdark regime Larvae of Spodoptera littoralis 378

(Boisd Lepidoptera Noctuidae) were hatched from eggs and reared on an agar-based diet at 379

23ndash25degC with a 168 h lightdark regime (Bergomaz and Boppre 1986) Manduca sexta (L 380


14

Lepidoptera Sphingidae) larvae were hatched from eggs as well cultured in climate chambers 381

(28degC and 168 h lightdark regime) and reared on N attenuata leaves 382

Technical approaches of electrophysiological measurements 383

All extra- and intracellular voltage measurements were carried out on a vibration-stabilized 384

bench with a Faraday cage Electrodes consisted of a microelectrode holder (MEH1SF10 385

MEH3S15 WPI World Precision Instruments Inc Sarasota FL USA) and a glass capillary (tip 386

diameter 1ndash2 microm Hilgenberg GmbH Malsfeld Germany) filled with a 05 M KCl solution 387

Electrodes were connected with a high-impedance amplifier (FD 223 or KS-700 WPI) placed 388

with micromanipulators (model ST 35 Brinkmann Instrumentenbau Mannheim Germany) and 389

optically controlled with a microscope (Leitz Wetzlar) The kinetics was recorded with an 390

analogue pen chart recorder (W+W Recorder Model 314) and noise was reduced with a capacitor 391

(1000 microF 63 V) The reference electrode filled with 05 M KCl was inserted into the soil or 392

placed on a leaf tip inside a bathing solution (Zimmermann et al 2009) Four different technical 393

approaches were applied to monitor elRs 394

(i) ldquosub-stomatal conductancerdquo - For each experiment the capillary tips of two voltage electrodes 395

were simultaneously brought in contact with the apoplast of sub-stomatal cavity or were impaled 396

on subepidermalmesophyll cells via two separate open stomata (Fig 9A) The simultaneous 397

application of two voltage electrodes increased the recording quality due to the simultaneous 398

establishment of a acutecontrolacute electrode and an increase of repetitions For further details see 399

previous studies (Felle and Zimmermann 2007 Zimmermann et al 2009 Felle et al 2000) 400

(ii) ldquoblind piercingrdquo ndash The glass capillary tips were filled with 05 M KCl in 1 (wV) agar and 401

backfilled with 05 M KCl solution (Fig 9B) The gelled agar prevents an uncontrolled outflow of 402

the salt solution into the plant tissue during the piercing process The tips were used to pierce the 403

main vein of a mature leaf or the stem of an intact plant The experiments started after the resting 404

potential settled (approx 5 to 24 h) For technical details see described in Furch et al (2010) and 405

Zimmermann et al (2013) 406

(iii) ldquosurface potentialrdquo ndash Small agar blocks (approx 10 x 5 x 5 mm 1 (wV) 05 M KCl) were 407

fixed on the leaf or stem surface and the glass capillary tip of an electrode was inserted into the 408

blocks (Fig 9B) Agar blocks were set on plant sites with a hydrophobe surface only (the adaxial 409

leaf side of V faba V faba stem and leaves of H vulgare) The hydrophobicity minimizes the 410

tendency of KCl to diffuse between agar block and plant tissue 411


15

(iv) ldquoEPGrdquo - Recordings of EPG were executed according to Will et al (2007) Aphids were 412

placed on the petiole base of a mature leaf of B napus between 60 and 90 mm from the leaf tip 413

(Fig 9B) By carefully burning the leaf tip for 3 s elRs were triggered 414

Stimuli ndash herbivory oral secretions HF CaCl2 KCl and mechanical wounding 415

Herbivore-triggered elRs were induced by the larval feeding of S littoralis and M sexta For 416

the entire experimental time period caterpillars (1-3 individuals third-instar) were placed on the 417

t-leaf an s-leaf or on the stem Subsequent elRs were systemically recorded in a distant t-leaf 418

(distance to s-leaf = 200-300 mm Fig 9A and C) To demonstrate the propagation characteristics 419

of the several elR types plants were further stimulated with HF using a lit match for 3 to 5 s 420

(Furch et al 2007 2008 2009 2010 Zimmermann and Felle 2009) SPs were induced with the 421

application of KCl and CaCl2 to a leaf (Zimmermann et al 2009) The stimulus strength ndash 422

concentration and period ndash is given in the figures Mechanical wounding was executed with razor 423

blades scissors needles forceps tubes or robotic punching (ldquoMecWormrdquo Mithoumlfer et al 2005) 424

Oral secretions were collected from fourth-instar S littoralis larvae by gently squeezing behind the 425

larval head with a forceps inducing an immediate regurgitation (Maffei et al 2004 Guo et al 426

2013) 427

Diverse experimental approaches 428

To study the propagation of elRs diverse experimental approaches were exercised All 429

arrangements are summarized in Fig 9 For each experiment 2 to 3 electrodes were simultaneously 430

used to detect the elRs The electrodes were placed together at one site (see sub-stomatal 431

conductance) or distributed over the plant (see blind piercing surface potential EPG) with 432

differing arrangements on the stem andor the leaves The stimuli were given at the same plant part 433

quite near to the electrodes (local approach) or at another leaf or the stem quite far away of the 434

electrodes (systemic approach) in basipetal as well as acropetal direction to the measuring sites 435

Because of the various combinations the individual experimental approaches are additionally 436

illustrated in the figures for an improved comprehension (Fig 2 6 and 7) 437

Visualization of the plant vascular system 438

To illustrate the unequal innervation of the single plant parts with the vascular system the 439

stem edges of V faba plants were submersed in different commercial coloured ink solutions 440


16

(TG4001 brilliant greenredblack royal blue Pelikanreg Berlin Germany) After 1 to 5 h used 441

inks were resorbed and translocated by the xylem all over the plant The staining of the vascular 442

system was monitored with a digital camera (personal communication AJE van Bel Eschrich 443

1967 Fritz 1973) 444

Convention 445

According to classic intracellular measurements a depolarisation event is defined as a 446

positive voltage change and a hyperpolarisation event as a negative voltage change of a resting 447

potential Similar definitions are applied for an extracellular (apoplastic) voltage change (see also 448

Zimmermann et al 2009) Since apoplastic voltage can be influenced by a variety of several 449

parameters and unlike a membrane potential event is not clearly defined no absolute values are 450

given just the polarity together with relative voltage 451

452

ACKNOWLEDGMENTS 453

The authors thank Nicolas Hans-Rudolf Ruoss for technical assistance concerning the 454

experiment of visualization of the vascular system and Aart JE van Bel in whose laboratory the 455

EPG experiments were conducted We thank E Wheeler Boston for editorial assistance Thomas 456

Burks for the linguistic help and Ralf Oelmuumlller for helpful discussion 457

458

459


17

Literature Cited 460

Alarcon JJ Malone M (1994) Substantial hydraulic signals are triggered by leaf-biting insects in tomato J 461 Exp Bot 45 953-957 462

Bacic ANTONY Harris PJ Stone BA (1988) Structure and function of plant cell walls Biochem Plants 14 463 297-371 464

Bergomaz R Boppre M (1986) A simple instant diet for rearing arctiidae and other moths J 465 Lepidopteristsrsquo Soc 40 131-137 466

Boari F Malone M (1993) Wound-induced hydraulic signals Survey of occurrence in a range of species J 467 Exp Bot 44 741-746 468

Burton RA Gidley MJ Fincher GB (2010) Heterogeneity in the chemistry structure and function of plant 469 cell walls Nat Chem Biol 6 724-732 470

Carle RB Loy JB (1996) Morphology and anatomy of the fused vein trait in Cucurbita pepo L J Am Soc 471 Hortic Sci 121 6-12 472

Cheeseman JM Pickard BG (1997) Electrical characteristics of cells from leaves of Lycopersicon Can J 473 Bot 55 497-510 474

Davies E (2004) New functions for electrical signals in plants New Phytol 161 607-610 475

Davies E (2006) Electrical signals in plants facts and hypotheses In Volkov AG eds Plant 476 Electrophysiology Theory and Methods Springer Berlin Heidelberg pp 407-422 477

Dziubinska H Filek M Koscielniak J Trebacz K (2003) Variation and action potentials evoked by thermal 478 stimuli accompany enhancement of ethylene emission in distant non-stimulated leaves of Vicia faba 479 minor seedlings J Plant Physiol 160 1203-1210 480

Eschrich W Fromm J Evert RF (1988) Transmission of electric signals in sieve tubes of zucchini plants 481 Bot Acta 101 327-331 482

Eschrich W (1967) Bidirektionelle Translokation in Siebroumlhren Planta 73 37-49 483

Favre P Greppin H Agosti RD (2001) Repetitive action potentials induced in Arabidopsis thaliana leaves 484 by wounding and potassium chloride application Plant Physiol 39 961-969 485

Felle HH (2001) pH signal and messenger in plant cells Plant Biol 3 577-591 486

Felle HH Hanstein S Steinmeyer R Hedrich R (2000) Dynamics of ionic activities in the apoplast of the 487 sub-stomatal cavity of intact Vicia faba leaves during stomatal closure evoked by ABA and darkness 488 Plant J 24 297-304 489

Felle HH Zimmermann MR (2007) Systemic signalling in barley through action potentials Planta 226 490 203-214 491

Findlay GP Hope AB (1976) Electrical properties of plant cells methods and findings In Luumlttge U Pitman 492 MG eds Transport in Plants II Part A Cells Springer Berlin Heidelberg pp 53-92 493

Fritz E (1973) Microautoradiographic investigations on bidirectional translocation in the phloem of Vicia 494 faba Planta 112 169-179 495


18

Fromm J (1991) Control of phloem unloading by action potentials in Mimosa Physiol Plant 83 529-533 496

Fromm J Bauer T (1994) Action potentials in maize sieve tubes change phloem translocation J Exp Bot 497 45 463-469 498

Fromm J Lautner S (2007) Electrical signals and their physiological significance in plants Plant Cell 499 Environ 30 249-257 500

Fromm J Lautner S (2012) Generation transmission and physiological effects of electrical signals in 501 plants In Volkov AG eds Plant Electrophysiology Signaling and Responses Springer Berlin Heidelberg 502 pp 207-232 503

Fromm J Spanswick R (1993) Characteristics of action potentials in willow (Salix viminalis L) J Exp Bot 504 44 1119-1125 505

Furch ACU Hafke JB Schulz A van Bel AJE (2007) Ca2+-mediated remote control of reversible sieve tube 506 occlusion in Vicia faba J Exp Bot 61 3697-3708 507

Furch ACU Hafke JB van Bel AJE (2008) Plant-and stimulus-specific variations in remote-controlled 508 sieve-tube occlusion Plant Signal Behav 3 858-861 509

Furch ACU van Bel AJ Fricker MD Felle HH Fuchs M Hafke JB (2009) Sieve element Ca2+ channels as 510 relay stations between remote stimuli and sieve tube occlusion in Vicia faba Plant Cell 21 2118-2132 511

Furch ACU Zimmermann MR Will T Hafke JB van Bel AJE (2010) Remote-controlled stop of phloem 512 mass flow by biphasic occlusion in Cucurbita maxima J Exp Bot 61 3697-3708 513

Galleacute A Lautner S Flexas J Fromm J (2014) Environmental stimuli and physiological responses The 514 current view on electrical signalling Environ Exp Bot 114 15-21 515

Gollan T Schurr U Schulze ED (1992) Stomatal response to drying soil in relation to changes in the xylem 516 sap composition of Helianthus annuus I The concentration of cations anions amino acids in and pH of 517 the xylem sap Plant Cell Environ 15 551-559 518

Granqvist E Wysham D Hazledine S Kozlowski W Sun J Charpentier M et al (2012) Buffering capacity 519 explains signal variation in symbiotic calcium oscillations Plant Physiol 160 2300-2310 520

Guo H Wielsch N Hafke JB Svatoš A Mithoumlfer A Boland W (2013) A porin-like protein from oral 521 secretions of Spodoptera littoralis larvae induces defense-related early events in plant leaves Insect 522 Biochem Mol Biol 43 849-858 523

Hafke JB Ehlers K Foumlller J Houmlll SR Becker S van Bel AJE (2013) Involvement of the sieve element 524 cytoskeleton in electrical responses to cold shocks Plant Physiol 162 707-719 525

Hartung W Radin JW Hendrix DL (1988) Abscisic acid movement into the apoplastic solution of 526 water-stressed cotton leaves Role of apoplastic pH Plant Physiol 86 908-913 527

Hilker M Meiners T (2010) How do plants ldquonoticerdquo attack by herbivorous arthropods Biol Rev 85 528 267-280 529

Jack JJB Noble D Tsien RW (1975) Electric current flow in excitable cells Clarendon Press Oxford pp 530 225-260 531

Kauss H (1987) Some aspects of calcium-dependent regulation in plant metabolism Annu Rev Plant 532 Physiol 38 47-72 533


19

Katicheva L Sukhov V Akinchits E Vodeneev V (2014) Ionic nature of burn-induced variation potential in 534 wheat leaves Plant Cell Physiol 55 1511-1519 535

Kessler A Halitschke R Baldwin IT (2004) Silencing the jasmonate cascade induced plant defenses and 536 insect populations Science 305 665-668 537

Kiep V Vadassery J Lattke J Maaszlig JP Boland W Peiter E Mithoumlfer A (2015) Systemic cytosolic Ca2+ 538 elevation is activated upon wounding and herbivory in Arabidopsis New Phytol doi 101111nph13493 539

Koch C (1984) Cable theory in neurons with active linearized membranes Biol Cybernetics 50 15-33 540

Lautner S Grams EET Matyssek R Fromm J (2005) Characteristics of electrical signals in poplar and 541 responses in photosynthesis Plant Physiol 139 2200-2209 542

Leitner M Vandelle E Gaupels F Bellin D Delledonne M (2009) Nitric oxide signalling in plant defence 543 Curr Opin Plant Biol 12 451-458 544

Maffei M Bossi S Spiteller D Mithoumlfer A Boland W (2004) Effects of feeding Spodoptera littoralis on 545 lima bean leaves I Membrane potentials intracellular calcium variations oral secretions and 546 regurgitate components Plant Physiol 134 1752-1762 547

Maffei ME Mithoumlfer A Boland W (2007) Before gene expression Early events in plant-herbivore 548 interactions Trends Plant Sci 12 310-316 549

Maischak H Grigoriev PA Vogel H Boland W Mithoumlfer A (2007) Oral secretions from herbivorous 550 lepidopteran larvae exhibit ion channel-forming activities FEBS Letters 581 898-904 551

McLean DL Kinsey MG (1964) A technique for electronically recording aphid feeding and salivation 552 Nature 202 1358-1359 553

McLean DL Kinsey MG (1965) Identification of electrically recorded curve patterns associated with aphid 554 salivation and ingestion Nature 205 1130-1131 555

Mescher MC De Moraes CM (2014) The role of plant sensory perception in plantndashanimal interactions J 556 Exp Bot doi 101093jxberu414 557

Mithoumlfer A Boland W (2008) Recognition of herbivory-associated molecular patterns Plant Physiol 146 558 825-831 559

Mithoumlfer A Boland W (2012) Plant defense against herbivores Chemical aspects Annu Rev Plant Biol 560 63 431-450 561

Mithoumlfer A Wanner G Boland W (2005) Effects of feeding Spodoptera littoralis on lima bean leaves 562 Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related 563 volatile emission Plant Physiol 137 1160-1168 564

Mousavi SAR Chauvin A Pascaud F Kellenberger S Farmer EE (2013) Glutamate Receptor-like genes 565 mediate leaf-to-leaf wound signaling Nature 500 422-426 566

Northcote DH (1972) Chemistry of the plant cell wall Annu Rev Plant Physiol 23 113-132 567

Oja V Savchenko G Jakob B Heber U (1999) pH and buffer capacities of apoplastic and cytoplasmatic 568 cell compartments in leaves Planta 209 239-249 569

Paszewski A Zawadzki T (1976) Action potentials in Lupinus angustifolius L shoots III Determination of 570


20

the refractory periods J Exp Bot 27 369-374 571

Pearce G Strydom D Johnson S Ryan CA (1991) A polypeptide from tomato leaves induces 572 wound-inducible proteinase inhibitor proteins Science 253 895-897 573

Pickard BG (1973) Action potentials in higher plants Bot Rev 39 172-201 574

Roblin G (1985) Analysis of the variation potential induced by wounding in plants Plant Cell Physiol 26 575 455-461 576

Roblin G Bonnemain JL (1985) Propagation in Vicia faba stem of a potential variation induced by 577 wounding Plant Cell Physiol 26 1273-1283 578

Sakurai N (1998) Dynamic function and regulation of apoplast in the plant body J Plant Res 111 133-148 579

Salvador‐Recatalagrave V Tjallingii WF Farmer EE (2014) Real‐time in vivo intracellular recordings of 580 caterpillar‐induced depolarization waves in sieve elements using aphid electrodes New Phytol 203 581 674ndash684 582

Sattelmacher B (2001) The apoplast and its significance for plant mineral nutrition New Phytol 149 583 167-192 584

Stahlberg R Cosgrove DJ (1992) Rapid alterations in growth rate and electrical potentials upon stem 585 excision in pea seedlings Planta 187 523-531 586

Stahlberg R Cosgrove DJ (1994) Comparison of electric and growth responses to excision in cucumber 587 and pea seedlings I Short-distance effects are a result of wounding Plant Cell Environ 17 1143-1151 588

Stahlberg R Cosgrove DJ (1996) Induction and ionic basis of slow wave potentials in seedlings of Pisum 589 sativum L Planta 200 416-425 590

Stahlberg R Cosgrove DJ (1997) The propagation of slow wave potentials in pea epicotyls Plant Physiol 591 113 209-217 592

Taylor RE (2013) Cable theory Phys Tech Biol Res 6 219-262 593

van Bel AJE (2003) The phloem a miracle of ingenuity Plant Cell Environ 26 125-149 594

van Bel AJE Knoblauch M Furch ACU Hafke JB (2011) (Questions)n on phloem biology 1 595 Electropotential waves Ca2+ fluxes and cellular cascades along the propagation pathway Plant Sci 181 596 210-21 597

Volkov AG Haack RA (1995) Insect-induced bioeletrochemical signals in potato plants 598 Bioelectrochemistry and Bioenergetics 37 55-60 599

Wacke M Thiel G Huumltt MT (2003) Ca2+ dynamics during membrane excitation of green alga Chara 600 model simulations and experimental data J Membr Biol 191(3) 179-192 601

Walling LL (2000) The myriad plant responses to herbivores J Plant Growth Reg 19 195-216 602

Will T van Bel AJE (2006) Physical and chemical interactions between aphids and plants J Exp Bot 57 603 729-737 604

Will T Tjallingii WF Thoumlnnessen A van Bel AJE (2007) Molecular sabotage of plant defense by aphid 605 saliva PNAS 104 10536-10541 606


21

Will T Furch ACU Zimmermann MR (2013) How phloem-feeding insects face the challenge of 607 phloem-located defenses Front Plant Sci 4 336 608

Wolf S Heacutematy K Houmlfte H (2012) Growth control and cell wall signaling in plants Annu Rev Plant Biol 609 63 381-407 610

Wu J Baldwin IT (2010) New insights into plant responses to the attack from insect herbivores Annu 611 Rev Gen 44 1-24 612

Zimmermann MR Maischak H Mithoumlfer A Boland W Felle HH (2009) System potentials a novel 613 electrical long-distance apoplastic signal in plants induced by wounding Plant Physiol 149 1593-1600 614

Zimmermann MR Felle HH (2009) Dissection of heat-induced systemic signals superiority of ion fluxes 615 to voltage changes in substomatal cavities Planta 229 539-547 616

Zimmermann MR Hafke JB van Bel AJE Furch ACU (2013) Interaction of xylem and phloem during 617 exudation and wound occlusion in Cucurbita maxima Plant Cell Environ 36 237-247 618

Zimmermann MR Mithoumlfer A (2013) Electrical long-distance signaling in plants In Baluška F eds 619 Long-Distance Systemic Signaling and Communication in Plants Springer Berlin Heidelberg pp 291-308 620

621

622

623

624


22

Table 1 ndash Characteristics of dissimilarly recorded system potentials in higher plants 625

extra = extracellular (=apoplastic) recording intra = intracellular recording nd = not determined plusmn = standard deviation 626

stimulus specimen experimental set-up

technical approach location distance

(mm) amplitude

(mV) duration

(s) velocity

(cm min-1) n

Spodoptera littoralis

Vicia faba leaf-to-leaf substomatal

conductance extra 250 plusmn51 1148 plusmn50 343 plusmn172 nd 13

Hordeum vulgare nd 81 plusmn40 201 plusmn78 nd 6

CaCl2 (50mM ~600s)


conductance extra 313 plusmn48 2221 plusmn554 3286 plusmn1289 645 plusmn201 15

Hordeum vulgare 466 plusmn74 2838 plusmn895 1803 plusmn595 588 plusmn15 37

Heatflame

Vicia faba

leaf-to-leaf

substomatal conductance

extra

424 plusmn76 1808 plusmn415 4396 plusmn1920 498 plusmn158 13

Vicia faba blind piercing 278 plusmn67 1133 plusmn375 5868 plusmn1267 223 plusmn075 12 Cucurbita maxima blind piercing 377 plusmn108 1672 plusmn89 6148 plusmn1836 281 plusmn106 10

Diverse Vicia faba Hordeum vulgare

leaf-to-leaf stem-to-leaf


intra 476 plusmn159 -786 plusmn399 2126 plusmn1163 544 plusmn204 21

extra 486 plusmn145 2095 plusmn102 2351 plusmn1246 627 plusmn21 23

627

628

w

ww

plantorg on F

ebruary 18 2016 - Published by

ww

wplantphysiolorg

Dow

nloaded from

Copyright copy

2016 Am

erican Society of P

lant Biologists A

ll rights reserved

23

629

630

FIGURE LEGENDS 631

632

Figure 1 Extracellular recordings of an action potential (AP) variation potential (VP) and system 633

potential (SP) 634

APs and VPs are depolarisations whereas SPs are hyperpolarisations of plasma membranes 635

The depolarisation of APs and VPs is extracellularly recorded with a negative voltage shift and the 636

SP hyperpolarisation is measured with a positive voltage shift 637

t = time U = voltage +- = voltage direction 638

639

Figure 2 Diverse herbivory-triggered electrophysiological reactions in distant leaves of Vicia faba 640

(A C E) and Hordeum vulgare (B D F) 641

All measurements were carried out using the sub-stomatal technique Intracellular 642

measurements were executed in spongy mesophyll cells Larvae of Spodoptera littoralis were 643

allowed to feed on a stimulus leaf or the stemculm of V faba and H vulgare Larvae were left on 644

the plant for the whole period of the experiment With the exception of the intracellular recording 645

(EM) the voltage and temporal scale are valid for all extracellular traces The initiation of larval 646

feeding experiments is depicted with a continuous vertical line 647

(A and B) Following herbivore damage of the stemculm action potentials were 648

systemically (s = 200-250 mm) detected extracellularly (Eapo) in V faba and H vulgare and 649

intracellularly (Em) in H vulgare (C and D) System potentials were recorded after larvae were fed 650

leaf tissue or the stemculm in V faba and H vulgare (s = 200-300 mm) (E) Mechanical damage 651

of the stem rapidly provoked (t = ~10-15 s) a depolarisation event in a distant leaf The distance is 652

illustrated with a vertical bar (F) Examples of typical systemic recordings of system potentials are 653

given in response to CaCl2 and KCl for H vulgare The stimulus period is illustrated with a grey 654

box Each trace shows an independent experiment +- = voltage direction 655

656

Figure 3 Manduca sexta feeding triggered electrophysiological reactions in Vicia faba and 657

Nicotiana tabacum 658


24

All measurements were carried out using the sub-stomatal technique Larvae of M sexta 659

were allowed to feed on V faba or N tabacum plants Larvae were left on the plant for the whole 660

period of the experiment (A) When M sexta larvae fed they induced a system potential (SP) in a 661

distant leaf of a V faba plant (B) Feeding on the vascular systemmain vein of the local leaf (s = 662

50 mm) remotely triggered a wavelike voltage change in N tabacum +- = voltage direction VP = 663

variation potential 664

665

Figure 4 The venation of Vicia faba 666

The vascular branching of V faba is demonstrated with different inks (A) After a cut of the 667

complete stem at the plant basis each single edge (= orthostichy) is individually submerged into an 668

ink solution (B-H) During 30 to 180 min the staining of the single orthostichies can be observed 669

and shows that the leaves are differently innervated with the vascular strands of the four 670

orthostichies 671

672

Figure 5 Common extracellular voltage variations of CaCl2-induced system potentials (SPs) in 673

higher plants 674

All measurements were carried out using the sub-stomatal technique CaCl2 solution (10-50 675

mM) was applied at a cut leaf The subsequent voltage reaction was systemically recorded at 676

another leaf The depolarisation event is marked with an asterisk (A) In most cases SPs are 677

characterized with similar de-repolarisation kinetics or a little longer repolarisation phase In 678

addition voltage variations were commonly observed ndash (B) a variable initial hyperpolarisation 679

(C) a subsequent voltage wave (D) a two-kinetics repolarisation phase (E) a variable initial and 680

subsequent hyperpolarisation andor (F) a subsequent depolarisation The voltage variations are 681

marked with a black arrow -+ = direction of voltage change 682

683

Figure 6 Influence of the various technical approaches for monitoring of electrophysiological 684

reactions in higher plants 685

(A) The combined application of two different technical approaches ndash sub-stomatal 686

conductance (upper trace) and surface potential (lower trace) ndash after stimulation with CaCl2 (50 687

mM) at the stem The different kinetics and durations indicate the impact of the applied technique 688

on the recording The grey box illustrates the stimulus period (B) Two blindly pierced electrodes 689


25

(E1 petiole and E2 main vein of a mature leaf) served differing voltage patterns in response to a 690

heat stimulus (HF) of a distant leaf (s = 280-340 mm) Each single peak represents one or more 691

overlaying APs (C) The tips of two glass capillaries were blindly pierced into the main vein of a 692

leaf The simultaneous intra- (upper trace) and extracellular (lower trace) voltage change in a 693

distant leaf tip is shown in response to HF (s = 295 mm) The stimulus time point is indicated with 694

a straight line (D) Two electrical penetration graphs of different aphids (s = 30 and 60 mm) are 695

shown after stimulation of a leaf tip with HF At the very beginning of the experiment three 696

calibration pulses (50 mV) were given The stimulus period is illustrated with a grey box or a 697

continuous line and all distances are shown in the vertical bars +- = direction of voltage change 698

Em = membrane (intracellular) potential Eapo = apoplastic voltage 699

700

Figure 7 Influence of the experimental set-up to the recorded electrophysiological reaction (elR) 701

types 702

Diverse exemplary extracellular recordings of action potentials (AP) variation potentials 703

(VP) and system potentials (SP) are shown with several experiments in Vicia faba plants by using 704

ldquoagarrdquo electrodes (A-D) and blind piercing approaches (E-G) The experimental set-up is 705

schematically illustrated for each single experiment and the specific distances between stimulus 706

and the various recording sites are outlined with the vertical bars The scale bars for voltage and 707

time period are valid for all recordings Agar blocks are indicated with grey bars and the heat 708

stimulus (HF) area is marked with a grey circle (A) The heat-triggered hyperpolarisation events 709

differ with increasing distance and are most obvious in the systemic leaf (E3) (B) Characteristics 710

of an AP can be also observed with agar electrodes ndash (i) an initial lower kinetic and (ii) the point of 711

breakthrough (see black arrow) (C and D) The uneven propagation of elRs can be observed with 712

electrodes being simultaneously located on the stem (E1) and different pinnas of the same leaf (E2 713

E3) The hyperpolarisation events in the stem disappeared almost completely and can be replaced 714

by a depolarisation event (E) The unknown contribution of VPs (marked with an asterisk) is 715

shown with blindly pierced electrodes into vascular strands The serial located electrodes show the 716

separation of AP and VP with increasing distance (E2) (F) If the mandatory voltage threshold for 717

an AP is not passed an unspecific hyperpolarisation event is detected (E1) and disappears rapidly 718

(E2) while the SP remains (G) The propagation of the pure SP can be also observed with a serial 719

arrangement of electrodes +- = direction of voltage change E1-3 = electrode 1 to 3 720


26

721

Figure 8 Proposed mechanistic model of electrophysiological reactions in higher plants 722

The model illustrates the suggested connections among the single types of 723

electrophysiological reactions and delivers explanations for the common observed voltage pattern 724

variations of electrophysiological reactions in higher plants AP = action potential VP = variation 725

potential SP = system potential 726

727

Figure 9 Experimental and technical set-up of electrophysiological recordings 728

(A) Larvae of Spodoptera littoralis or Manduca sexta were placed on the target leaf 729

(t-leaf) a stimulus leaf (s-leaf) or on the stem with variable distances from the t-leaf The 730

herbivore-induced plant electrophysiological reactions were recorded with two electrodes (see 731

cross-section) The capillary tips of two electrodes were simultaneously inserted via open stomata 732

and brought into contact with the apoplast of the sub-stomatal cavity (SSC) for extracellular 733

measurements or impaled on surrounding parenchyma cells (PCs) for intracellular recordings 734

(Felle et al 2000 Felle and Zimmermann 2007 Zimmermann et al 2009) Typical feeding 735

damage of leaves (20 to 60) after 300 s are shown at the lower inset (B) Voltage changes can be 736

also monitored via the plant surface (surface potential) using small agar blocks or the tip of a glass 737

capillary can be inserted into the plant tissue enabling additionally intracellular recordings (blind 738

piercing) An approach to examine the vascular system is the application of aphids sucking 739

specifically of the phloem sieve elements (SE) Aphids are connected with a small drop of 740

silverglue and a goldwire to an amplifier (C) Illustrations of the technical and experimental set-up 741

are given EC = epidermal cell CC = companion cell OS = oral secretions 742

743


27

744











Parsed CitationsAlarcon JJ Malone M (1994) Substantial hydraulic signals are triggered by leaf-biting insects in tomato J Exp Bot 45 953-957

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bacic ANTONY Harris PJ Stone BA (1988) Structure and function of plant cell walls Biochem Plants 14 297-371Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Bergomaz R Boppre M (1986) A simple instant diet for rearing arctiidae and other moths J Lepidopterists Soc 40 131-137Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Boari F Malone M (1993) Wound-induced hydraulic signals Survey of occurrence in a range of species J Exp Bot 44 741-746Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Burton RA Gidley MJ Fincher GB (2010) Heterogeneity in the chemistry structure and function of plant cell walls Nat Chem Biol6 724-732


Carle RB Loy JB (1996) Morphology and anatomy of the fused vein trait in Cucurbita pepo L J Am Soc Hortic Sci 121 6-12Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cheeseman JM Pickard BG (1997) Electrical characteristics of cells from leaves of Lycopersicon Can J Bot 55 497-510Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Davies E (2004) New functions for electrical signals in plants New Phytol 161 607-610Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Davies E (2006) Electrical signals in plants facts and hypotheses In Volkov AG eds Plant Electrophysiology Theory and MethodsSpringer Berlin Heidelberg pp 407-422


Dziubinska H Filek M Koscielniak J Trebacz K (2003) Variation and action potentials evoked by thermal stimuli accompanyenhancement of ethylene emission in distant non-stimulated leaves of Vicia faba minor seedlings J Plant Physiol 160 1203-1210


Eschrich W Fromm J Evert RF (1988) Transmission of electric signals in sieve tubes of zucchini plants Bot Acta 101 327-331Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

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Alexandra CU Furch 27

illustration of plant venation (Fig 3) analysis and discussion MS editing and conception 28

figure preparation 29

Funding information 30

The Deutsche Forschungsgemeinschaft in the frame of FU 9692-1 Fe 21315-1 and 15-2 and 31

The Max Planck Society 32

Affiliations 33 1Institute of General Botany and Plant Physiology Friedrich-Schiller-University 34

Dornburgerstraszlige 159 D-07743 Jena Germany 35 2Department Bioorganic Chemistry Max Planck Institute for Chemical Ecology 36

Hans-Knoumlll-Straszlige 8 D-07745 Jena Germany 37 3Plant Cell Biology Research Group Institute of General Botany Justus-Liebig-University 38

Senckenbergstraszlige 17 D-35390 Gieszligen Germany current address Department 39

Phytopathology Heinrich-Buff-Ring 26-32 D-35392 Gieszligen Germany 40 4retired former address Institute of General Botany Justus-Liebig-University 41

Senckenbergstraszlige 17 D-35390 Gieszligen Germany 42

One sentence summary 43

In plants feeding caterpillars trigger various types of electrophysiological reactions in which the 44

diverse voltage pattern are specific for plant species technical and experimental approaches 45

Present addresses 46

See above 47

Corresponding author with email address 48


MatthiasRZimmermannbot1biouni-giessende 50

51

52


3

ABSTRACT 53




















plants 73

74

75


4

INTRODUCTION 76
































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172








SP 180



















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2014) 217













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350

CONCLUSION 351


13















366

367


Plant material 369













14

































15
















2013) 427















16




1967 Fritz 1973) 444

Convention 445







452

ACKNOWLEDGMENTS 453





458

459


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21








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potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


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744


















































































































































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ABSTRACT 53




















plants 73

74

75


4

INTRODUCTION 76
































5































6

































7






172








SP 180



















8




















2014) 217













9

































10

































11

































12





























350

CONCLUSION 351


13















366

367


Plant material 369













14

































15
















2013) 427















16




1967 Fritz 1973) 444

Convention 445







452

ACKNOWLEDGMENTS 453





458

459


17





















18




















19





















20






















21








621

622

623

624


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(mm) amplitude

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extra








627

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nloaded from

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erican Society of P

lant Biologists A

ll rights reserved

23

629

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FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


27

744


















































































































































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INTRODUCTION 76
































5































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7






172








SP 180



















8




















2014) 217













9

































10

































11

































12





























350

CONCLUSION 351


13















366

367


Plant material 369













14

































15
















2013) 427















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1967 Fritz 1973) 444

Convention 445







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ACKNOWLEDGMENTS 453





458

459


17





















18




















19





















20






















21








621

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ll rights reserved

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629

630

FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


27

744


















































































































































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SP 180



















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2014) 217













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Plant material 369













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2013) 427















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ACKNOWLEDGMENTS 453





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20






















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629

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FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


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744


















































































































































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2014) 217













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2013) 427















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458

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20






















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ll rights reserved

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629

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FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







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orthostichies 671

672


higher plants 674









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25











700


types 702




















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721






727
















743


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2014) 217













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CONCLUSION 351


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Plant material 369













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2013) 427















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458

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20






















21








621

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624


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23

629

630

FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


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2014) 217













9

































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11

































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350

CONCLUSION 351


13















366

367


Plant material 369













14

































15
















2013) 427















16




1967 Fritz 1973) 444

Convention 445







452

ACKNOWLEDGMENTS 453





458

459


17





















18




















19





















20






















21








621

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(mm) amplitude

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23

629

630

FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


27

744


















































































































































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9

































10

































11

































12





























350

CONCLUSION 351


13















366

367


Plant material 369













14

































15
















2013) 427















16




1967 Fritz 1973) 444

Convention 445







452

ACKNOWLEDGMENTS 453





458

459


17





















18




















19





















20






















21








621

622

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624


22





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lant Biologists A

ll rights reserved

23

629

630

FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


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744


















































































































































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Figure 2

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10

































11

































12





























350

CONCLUSION 351


13















366

367


Plant material 369













14

































15
















2013) 427















16




1967 Fritz 1973) 444

Convention 445







452

ACKNOWLEDGMENTS 453





458

459


17





















18




















19





















20






















21








621

622

623

624


22





(mm) amplitude

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erican Society of P

lant Biologists A

ll rights reserved

23

629

630

FIGURE LEGENDS 631

632


potential (SP) 634





639

















656




24







665






orthostichies 671

672


higher plants 674









683








25











700


types 702




















26

721






727
















743


27

744


















































































































































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2013) 427















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orthostichies 671

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higher plants 674









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orthostichies 671

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types 702




















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orthostichies 671

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higher plants 674









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orthostichies 671

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1 short title - esalq.usp.br · 1 short title 2 herbivore-triggered electrophysiological reactions...

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