floral and extrafloral nectar – the same or not the same? theme i/heil floral... · day 1:...

Floral and Extrafloral Nectar –the Same or not the Same?

Martin Heil

General Botany, University of Duisburg-Essen, Germany

&

CINVESTAV, Irapuato, Gto, México

Martin HeilExtrafloral nectar (EFN)

Introduction

-- Ralf KrRalf Krüügerger

-- Juan Carlos Silva Juan Carlos Silva BuenoBueno

-- Dr. Christian KostDr. Christian Kost

-- Prof. Dr. Wilhelm BolandProf. Dr. Wilhelm Boland

-- MPG, DFGMPG, DFG


Introduction

Extrafloral nectar


Extrafloral nectaries on the leaves of passionflower

(© R. F. Mizell)

Introduction

Extrafloral nectar

An ant using the EFN of elderberry in north Florida (© R. F. Mizell)


Introduction

Extrafloral nectar

Barrel cactus EFN(© Barry Sullender)


Introduction

Extrafloral nectar

Pseudomyrmex ants consuming extrafloral nectar (EFN) of Acacia collinsii


Lima bean(Phaseolus lunatus)

Induction of EFN

extrafloral nectaries

© C. Kost

not involved in pollination

taxonomically widespread(> 200 families of ferns, gymnosperms, angiosperms)

containing water, carbohydrates, amino acids, proteins and ???

attractive to predatory arthropods

involved in indirect defence

EFN

Introduction


Introduction

Indirect defence


Introduction

VOCsvolatile organic compounds

VOC-mediateddirect or indirectdefence

JAjasmonic acid Indirect defence


Introduction


Octadecanoid signallingcascade

JA involved in

- wound response (synthesisof nicotine, PI‘s, VOCs, EFN)

- tendril coiling

- suppression of root growth

- senescence

- flower development


Induction of VOCs

1: cis-Hexenylacetate, 2: cis-ß-Ocimene, 3: trans-ß-Ocimene, 4: C11 Homoterpene, 5: C10H14, 6: MeSA, 7: C10H16O, 8: unid.STD: Standard [1-bromodecane], 9, 10: unid., 11: cis-Jasmone, 12: Caryophyllene, 13: TMTT [4,8,12-trimethyltrideca,1,3,7,11-tetraene], 14: MeJA

5 6 7 8 9 10 11 12 13 14 15 16Time [min]

100

75

50

25

0

Rel

ativ

e A

bund

ance

1

43 7STD

9

514

21

8

10

12

13 14

14

3

45

STD

13 14

11

C

JA

6

13

x

100

75

50

25

0

x

Martin HeilExtrafloral nectar (EFN)Introduction

Martin HeilExtrafloral nectar (EFN)Introduction


Martin HeilExtrafloral nectar (EFN)EFN as induced defence

subcluster1

(control)

subcluster2

(JA)

Subcluster 1

(control)

Subcluster 2

(JA)

Heil, J Ecology 92 (2004), 527-536


Heil, J Ecology 92 (2004), 527-536

EFN as induced defence

subcluster1

(control)

subcluster2

(JA)

Subcluster 1

(control)

Subcluster 2

(JA)


controlinduced

Leaves Inflorescences

15

10

5

0

EFN

[mg

g -1

24 h

-1]

Heil, J Ecology 92 (2004), 527-536



400

200

0EFN

[µg

g -1

DM

24

h-1]

control damage

spray control JA



400

200

0EFN

[µg

g -1

DM

24

h-1]

control damage

spray control JA

400

200

0

EFN

[µg

g -1

DM

24

h-1]

control damage+Phe

damage dam+Phe+JA


subcluster1

(control)

subcluster2

(JA)

Subcluster 1

(control)

Subcluster 2

(JA)

Indirect defence in the field


Heil, J Ecology 92 (2004), 527-536

C I

1313N =

ic

Boh

nen/

Clu

ster

150

100

50

0

6

C JA

1313N =

ic

Spr

ossw

achs

tum

[cm

/12

Tage

] 70

60

50

40

30

20

10

0

C JA

Heil, J Ecology 92 (2004), 527-536

shoo

tgro

wth

[cm

12

d-1]

bean

spe

r clu

ster

herbivory

growth

fruits

C JA

Heil, J Ecology 92 (2004), 527-536

subcluster1

(control)

subcluster2

(JA)

Subcluster 1

(control)

Subcluster 2

(JA)



Induction of EFN and VOCs via JA

benefits Lima bean under natural conditions

but:

which factor is the effective one????

BOTH!?!?


Heil, J Ecology 92 (2004), 527-536


plant-plant communication

Plant – plant communication????


V- V+ V- V+

Day 1: volatiles


Heil & Kost, Ecol Lett 9 (2006), 813-817


Rel

ativ

e A

bund

ance

100

50

100

4 6 8 10 12 14

Time (min)

50

(a)

(b)

IS

IS

1

1

2

2

3

3

4

4

5

5

6

6

7

7

8

8

9

9

1, (Z)-3-hexenyl acetate; 2, (E,Z)-β-ocimene; 3, (R)-(-)-linalool; 4, DMNT; 5, C10H14; 6, methyl salicylate; 7, (Z)-jasmone; 8, β-caryophyllene; 9, TMTT; IS, internal standard (1-bromodecane).

paste

herbivory

Artificial volatile paste

Kost & Heil, Basic Appl Ecol 6 (2005), 237-248



V- V+ V- V+

Day 1: volatiles




V- V+ V- V+

Day 1: volatiles Day 2: mechanical damage

M- M- M+ M+




Treatment

EFN

sec

retio

n[µ

g g-

124

h-1 ]

V- M- V+ M- V- M+ V+ M+

250

200

150

100

50

0

(V+M-) + (V-M+)

Priming effect(182 %)

a

b

c

b

V- M- V+ M- V- M+ V+ M+

Day 1: volatiles Day 2: mechanicaldamage

+

V-M- V+M- V-M+ V+M+

Day 1 VOCs day 2 mech. damage

=> VOCs prime EFN for augmented responseto mechanical damage N = 8

But…. The ecologicalrelevance….??




C VOCs

Livi

ngtip

s[%

]

100

80

60

** *

26 31 5 10 15Aug Sep

Cerotomaruficornis

Leaf

num

ber 40

30

20

10

* *

26 31 5 10 15Aug Sep dead shoot tip

Her

bivo

ry [%

]


**

15

10

526 31 5 10 15

Aug Sep

n = 18

Heil & Silva Bueno, PNAS 104 (2007) 5467-5472


Lima bean

Herbivore damage induces EFN by JA

VOCs induce EFN

VOCs serve in plant-plant signalling

EFN is an inducible indirect defence

Martin HeilExtrafloral nectar (EFN)Summary I – EFN as indirect defence


Extrafloral nectar

Pseudomyrmex ants consuming extrafloral nectar (EFN) of Acacia collinsii

Acacia: symbiotic mutualisms

Is EFN secretion byAcacia inducible??


Food body

EFN



Acacia hindsii: inhabited ant-free




Acacia farnesiana

750

500

250

0

control spray holes JAcontrol

EFN

[µg

g-1 D

M 2

4h-1

]




Acacia cornigera

control spray holes JAcontrol

4000

3000

2000

1000

0

EFN

[µg

g-1 D

M 2

4h-1

]



- `ant-loving´

- non-symbiotic ants

attracted from the vicinity

- forage on plant and

defend reliable food sources

facultative interaction

non-myrmecophytes



- `ant-plant´

- specialised, symbiotic ants

housed and nourished

by plant host .

- defend plant as „own nest

and only food source“

obligate interaction

myrmecophytes

- `ant-loving´

- non-symbiotic ants

attracted from the vicinity

- forage on plant and

defend reliable food sources

facultative interaction

non-myrmecophytes



400

200

0

2000

1000

1500

500

A. cochliacantha400

200

0 A. farnesiana400

200

0

50

0

A. macracantha

A. pennatula300

100

0

Non-myrmecophytes

a a b b

cont

rol

spra

y co

nt.

dam

age JA

a a b b

a a b b

a a b b

EFN

[µg

g -1

DM

24

h-1]

1000

500

A. chiapensis

A. cornigera

A. globulifera

A. hindsii

Myrmecophytes

a a a a

cont

rol

spra

y co

nt.

dam

age JA

a a a b

a a a a

a a a a

a

Heil et al, Nature 430 (2004), 205-208



Mimosa tenuiflora

Acacia chiapensis

Acacia cochliacantha

Acacia collinsii

Acacia cornigera

Acacia farnesiana

Piptadenia flava

Acacia hindsii

Prosopis julifloraLeucaena leucocephala

Acacia macracantha

Acacia pennatula10 steps

cp sequence data phylogeny

60

100

100

68

88

57

100

inducible

constitutive

Heil et al, Nature 430 (2004), 205-208

400

200

0

2000

1000

1500

500

1000

500

A. chiapensis

A. cornigera

A. globulifera

A. hindsii

Myrmecophytes

a a a a

cont

rol

spra

y co

nt.

dam

age JA

a a a b

a a a a

a a a a


Acacia: symbiotic mutualisms%

of a

nts

non-myrm.

myrm.

0

5

10

15

20

Glucos

e Fruc

tose

Sucros

eSug

ar-Mix

A. coc

h.A. fa

rn.L.

leuc.

A. coll

.A. h

ind.

Wate

r

non-myrmecophytes

myrmeco-phytes

Cafeteria experiment: generalist ants



Glu

co

se

Fr

uc

to

se

Su

cr

os

e

Su

ga

r-

Mix

A.

c

oc

hlia

ca

nt

ha

A.

f

ar

ne

sia

na

Le

uc

ae

na

A.

c

ollin

sii

A.

h

ind

sii

Wa

te

r

0

1 0

2 020

10

0

20

10

0

Glu

cose

Fruc

tose

Suc

rose

Sug

ar m

ix

A. f

arn.

Leuc

aena

A. c

orni

gera

A. c

hiap

ensi

s

Wat

er

Glu

cose

Fruc

tose

Suc

rose

Suga

r m

ix

A. c

och.

A. f

arn.

Leuc

aena

A. c

ollin

sii

A. h

inds

ii

Wat

er

A. Generalistic antscoast

Isthmus

B. Specialized ants

30

20

10

0

P. ferrugineus

Glu

cose

Fruc

tose

Suc

rose

Suga

r m

ix

A. c

och.

A. f

arn.

Leuc

aena

A. c

ollin

sii

A. h

inds

ii

Wat

er

Glu

cose

Fruc

tose

Suc

rose

Sug

ar m

ix

A. c

och.

A. f

arn.

Leuc

aena

A. c

ollin

sii

A. h

inds

ii

Wat

er

20

10

0

P. mixtecus% o

f ant

s

Heil et al., Figure 1

Glu

co

se

Fr

uc

to

se

Su

cr

os

e

Su

ga

r-

Mix

A.

c

oc

hlia

ca

nt

ha

A.

f

ar

ne

sia

na

Le

uc

ae

na

A.

c

ollin

sii

A.

h

ind

sii

Wa

te

r

0

1 0

2 020

10

0

20

10

0

Glu

cose

Fruc

tose

Suc

rose

Sug

ar m

ix

A. f

arn.

Leuc

aena

A. c

orni

gera

A. c

hiap

ensi

s

Wat

er

Glu

cose

Fruc

tose

Suc

rose

Suga

r m

ix

A. c

och.

A. f

arn.

Leuc

aena

A. c

ollin

sii

A. h

inds

ii

Wat

er


Isthmus

B. Specialized ants

30

20

10

0

P. ferrugineus

Glu

cose

Fruc

tose

Suc

rose

Suga

r m

ix

A. c

och.

A. f

arn.

Leuc

aena

A. c

ollin

sii

A. h

inds

ii

Wat

er

Glu

cose

Fruc

tose

Suc

rose

Sug

ar m

ix

A. c

och.

A. f

arn.

Leuc

aena

A. c

ollin

sii

A. h

inds

ii

Wat

er

20

10

0

P. mixtecus% o

f ant

s


myrmeco-phytes

non-myrme-cophytes

(a) generalist ants (b) specialised antssite 1

site 2

Heil et al, Science 308 (2005), 560-563




A. cochliacantha

L. leucocephala

A. farnesiana

A. collinsii

A. hindsii

A. cornigera

A. pennatula A. chiapensis

GG

F

GG

S

non-myrmecophytes myrmecophytesF F




Invertase (EC 3.2.1.26) :

sucrose => glucose + fructose

quantification as glucose released


Kinetik assay: Heil, M., Büchler, R. & Boland, M. (2005) Quantification of invertaseactivity in ants under field conditions. J. Chem. Ecol. 31, 431-437



Atta

spe

c.

Cam

pono

tus

spec

. B

Ceph

alot

essp

ec.

Cam

pono

tus

spec

. C

Crem

atog

aste

r spe

c.

Cam

pono

tus

spec

. A

P. g

raci

lis

P. fe

rrugi

neus

P. m

ixtec

us

60

40

20

0

a,b

cb,c

cc,d d

e e

generalisticspecia-lized

n = 6 4 4 4 5 6 5 8 6

a

Inve

rtase

activ

ity

[ng

Glu

cose

min

-1]

non-symbiotic

P. fe

rrug

ineu

s

P. m

ixte

cus




A. cochliacantha

L. leucocephala

A. farnesiana

A. collinsii

A. hindsii

A. cornigera

A. pennatula A. chiapensis

GG

F

GG

S

non-myrmecophytes myrmecophytesF F

Inve

rtase

0.5

1.5

Inve

rtase

0.5

1.5

Inve

rtase

0.5

1.5

Inve

rtase

0.5

1.5

Inve

rtase

0.5

1.5

Inve

rtase

0.5

1.5In

verta

se

0.5

1.5

Inve

rtase

0.5

1.5



spots identification (Q-tof Ultima, nano-LC, De novo, MS-BLAST w/highest score)1 and 9: endo-1,3-β-glucosidase or β-1,3-glucanase 8: PE-PGRS family protein, 15: Thaumatin or osmotin like, 18: pathogenesis-related PR-1,21, 23: blight-associated p12

A. collinsii


Martin HeilExtrafloral nectar (EFN)Summary

- EFN is generally inducible, yet secretion scheme can be adapted

- EFN secretion depends on JA

- EFN secretion depends on consumption

- EFN contains mono- and disaccharides, amino acids, and proteins

Floral and extrafloral nectar – the same or not the same????

- Floral nectar inducible by JA (Poster by Radhika Venkatesan)

- Floral nectar secretion dependson consumptionGill, F.B. [1988] Biotropica, Pyke, G.H. [1991] Nature

- Floral nectar contains carbo-hydrates, amino acids, and proteins, the proteins serveprotection from bacteriaCarter, C. & Thornburg, R.W. [2004]Trends Plant Sci.

http://upload.wikimedia.org/wikipedia/commons/4/4a/Tobacco_blossom_1580.jpg


- EFN is generally inducible, yet secretion scheme can be adapted

- EFN secretion depends on JA

- EFN secretion depends on consumption

- EFN contains mono- and disaccharides, amino acids, and proteins

- Floral nectar inducible by JA (Poster by Radhika Venkatesan)

- Floral nectar secretion dependson consumptionGill, F.B. [1988] Biotropica, Pyke, G.H. [1991] Nature

- Floral nectar contains carbo-hydrates, amino acids, and proteins, the proteins serveprotection from bacteriaCarter, C. & Thornburg, R.W. [2004]Trends Plant Sci.



VOCs and flower odours are chemically very similar with many shared compounds

Questions:

- What else do EFN and floral nectar have in common?

- How is the secretion of nectar regulated?

- How do plants avoid induction of EFN by flower odours?

- Ecological interactions among visitors of flower nectar and EFN?

- Evolutionary history?



EFN and floral nectar have much in common, and communication among scientists working on pollination

and on indirect defence will improve future research.

Thank you for your attention!!!




Isthmus

B. Specialized ants

30

20

10

0

P. ferrugineus

P. mixtecus

A. collinsii A. hindsii


15

10

5

0

20

10

0


9

6

3

0A. cornigera A. chiapensis



Isthmus

B. Specialized ants

30

20

10

0

P. ferrugineus

P. mixtecus



15

10

5

0

20

10

0


9

6

3

0A. cornigera A. chiapensis


+ sucrose

% o

f ant

s



Martin HeilDefensive ant-plant interactions

Invertase (EC 3.2.1.26) :

sucrose => glucose + fructose

quantification as glucose released

HK Kit:Glucose + ATP hexokinase Glucose-6-phosphate + ADP

G6P + NAD G6P-dehydrogenase NADH + 6-Phosphogluconate

Extract + Kit => equilibrium ...

+ sucrose ..... new glucose released; slope = invertase activityHeil et al, J Chem Ecol 31 (2005), 431-437

Martin HeilDefensive ant-plant interactions

Ant 3 atta 4 gracilis

± SE

Invertase [ n units µl -1 ] 0 96 192 384 576

± SE

0.8

0.6

0.4

0.2

0

0 5 10 15 20 25 30 35 40 45

Abso

rptio

n

t [min]

Glu

cose

[ng

min

-1]

2000

1000

00 96 192 384 576

A

Invertase [n units µl-1]Invertase [n units µl-1]

t [min]0 30 60 90 120 150

+ sucrose

+ sucrose

B 0.6

0.4

0.2

0

Abso

rptio

n

Ant numberand species

3 Atta mexicana4 P. gracilis

Heil et al, J Chem Ecol 31 (2005), 431-437

Heil & Silva Bueno, Plant Signal Behav 2 (2007) in press

within-plant communication by VOCs

O O

HI-VOCs

E

R

O

COOH

JA

VV

VV VV

VOCs can serve as a cue to elicit EFN secretion in

exactly those parts of another - or

even the same –plant, where resistance

actually is required: in the spatially (yet not necessarily

anatomically) neighboring parts


within-plant communication by VOCs

A

B

C

D

5 10 15

STD118

5

4

18

17

20 23

STD

5 10 15

x

6

711

x

5 10 15

STD11

8

5

41

618

17 x

STD

5 10 15

11

18

17

45

6

8

20x

STD

x

5 10 15

STD

5 10 15



A B C D

Induction

by beetles

artificially

200

100

0

Leaves 4/5

b

a a a

Ap Bp Cp Dp

EFN

sec

retio

n[µ

g g-1

24h-1

]

Ap Bp Cp Dp

200

100

0EFN

sec

retio

n[µ

g g-1

24h-1

] Leaves 1-3

a

a

b b


n = 5

Martin HeilExtrafloral nectar (EFN)within-plant communication by VOCs

Leaves 4,5 Leaves 1-350

25

0Vola

tile

amou

nt[%

gTG

-1]

Vola

tile

amou

nt[%

gTG

-1]

Vola

tile

amou

nt[%

gTG

-1]

Vola

tile

amou

nt[%

gTG

-1]

50

25

0

50

25

0

50

25

0

50

25

0

50

25

0

50

25

0

1 3 5 7 9 11 13 15 17 19 21 23 25

1 3 5 7 9 11 13 15 17 19 21 23 25

1 3 5 7 9 11 13 15 17 19 21 23 25

1 3 5 7 9 11 13 15 17 19 21 23 251 3 5 7 9 11 13 15 17 19 21 23 25

1 3 5 7 9 11 13 15 17 19 21 23 25 1 3 5 7 9 11 13 15 17 19 21 23 25Compound No. Compound No.

Treatment

Ap

Bp

Cp

Dp

125 325 118 240

118 287

134 387Induction

by beetles

artificially

1. cis-Hexenyl acetate*2. 2-Ethylhexanol* (contam.)3. cis-ß-Ocimene*4. trans-ß-Ocimene5. Linalool*6. Nonanal*7. 2-ethenyl-Cyclohexan 8. C11 Homoterpene* [4,8-

dimethyl-1,3,7-nonatriene]9. cis-3-Hexen-1-yl-butyrat*10. MeSA*11. Decanal*12. unidientified13. Unidentified14. cis-3-Hexenylhexanoate*15. unidentified16. cis-Jasmone*17. ß-Caryophyllene18. trans-Geranylacetone*19. cis-3-Hexen-1-ol-Benzoate20. TMTT [4,8,12-

trimethyltrideca,1,3,7,11-tetraene] *

21. MeJA *22. Unidientified23. 5,9,13-Trimethyl- 4,8,12-

Tetradecatrienal24. Stearyl acetate25. Palmitinic acid-isopropylester


Martin HeilPriming of extrafloral nectar (EFN)

floral and extrafloral nectar – the same or not the same? theme i/heil floral... · day 1:...

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