Influence of floral visitation on nectar-sugar composition and nectary surface changes in Eucalyptus

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Influence of floral visitation on nectar-sugar composition

and nectary surface changes in Eucalyptus

Ar Davis

To cite this version:

Original

article

Influence of floral visitation

on

_nectar-sugar

composition

and

_nectary

surface

_changes

in

_Eucalyptus

AR

Davis

Department

of Biology,

112 Science Place, University

of

Saskatchewan,

Saskatoon, SK, Canada S7N 5E2

(Received 23

_July

1996;

accepted

29

_January

1997)

Summary —

Floral nectaries and their

_production

of

_major

nectar

_{carbohydrates}

were studied in three

species

of

_Eucalyptus

in Australia. In E

_cosmophylla,

E _grandis and E

_{pulverulenta,}

the _nectary is located on the inner surface of the

_hypanthium,

below the stamen filaments.

_Nectary

_surfaces pos-sessed hundreds of modified stomata that were

_solitary,

distributed

_{uniformly, asynchronous}

in

development,

and served as exits for nectar flow. Nectar

_yields

per

bagged

flower were _greatest in E

_cosmophylla

and least in E

_{grandis, correlating}

with flower size but not nectary stomatal

_density.

The nectar of

_{E pulverulenta}

was _{sucrose-rich,} but hexose-rich for the others. Few

_changes

in

nec-tar

_{carbohydrate composition}

were detected between flowers whether

_protected

or

_continually

exposed

to visitors

_{(eg, honeybees),}

_{and whether young} or _old,

_indicating

an overall _constancy in

com-position

for the

_{long period}

of nectar

_{availability.}

Eucalyptus

/

_honeybee

/ modified stomata / nectar

carbohydrate

/ nectary surface

INTRODUCTION

Floral nectar from

_Eucalyptus

trees

_provides

Australia’s most

_important

source of

_honey

production

(Brimblecombe,

1946;

Clem-son,

1985),

allowing

the

_country’s

_average

honey

yields

per bee

colony

to _{rank among}

the

_highest

in the world

_(Anonymous,

1985,

1987).

Today,

their

potential

for

_honey

and timber

_production,

as well as their

orna-mental

value,

has fostered cultivation of

eucalypts

in many other countries

_(Pellett,

1923; Lovell, 1926; Vansell, 1941;

Barbier,

1951;

Lupo

and

Eisikowitch,

1990).

Chem-ical

_analyses

of Australian

_honeys

derived from individual

_eucalypt

_species

are avail-able

_{(Chandler et al, 1974). However,}

little research has been devoted to

_Eucalyptus

nectar,

_particularly

within Australia

_(Bond

and Brown,

1979;

Moncur and

Boland,

1989).

Although

the

_{overwhelming majority}

of

species

secrete floral nectar of

_relatively

constant

_composition

(Percival, 1961), some

changes

may occur. The

_carbohydrate

(≤

24

_h)

flowers

(Freeman, 1986;

Petanidou

et

_{al, 1996),}

flowers of intermediate

_(2-4

days)

duration

_(Loper

et

_{al, 1976),}

or

long-lived

_(5-8

_days)

flowers

_(Christ

and

Schnepf,

1988;

Kronestedt-Robards et

al,

1989).

An increased amino acid

concen-tration is common in nectar of

_aging

flowers

(Gottsberger

et

_{al, 1990;}

Petanidou et

_al,

1996).

Additionally,

the

_composition

of

flo-ral nectar _{may be altered}

_by

animal visits

(Corbet, 1978; Willmer, 1980;

Freeman and

Wilken, 1987).

Flowers of

_{eucalypt species}

are

_generally

_{long-lived, lasting}

4-18

_days

post-anthesis

(Ashton, 1975;

Hodgson,

1976; Núñez, 1977; Griffin and Hand, 1979;

Moncur and

Boland, 1989;

Lupo

and

Eisikowitch,

1990; Ellis and

_Sedgley,

_1992).

However, the _constancy of

_Eucalyptus

nec-tar

_composition

either

_during

flower

phe-nology,

or

_following

animal

visits,

has not

been examined in Australia.

Nectar escape in most

_Myrtacean

flowers

occurs from the inner surface of the

hypan-thium,

in a

_{region extending}

from the base of the

_staminophore

to _{the upper surface of the}

ovary

(Carr

and

_{Carr, 1987, 1990;}

Beard-sell et al, 1989;

Moncur and

Boland, 1989;

O’Brien et al, 1996).

Structures referred to

as stomates

(Davis,

1968, 1969), stomata

(Beardsell

et

_{al, 1989),}

stomatal-like

(Mon-cur and

_{Boland, 1989),}

modified stomata

(O’Brien

et

_{al, 1996)}

_{and pore cells}

_(Carr

and Carr,

1987, 1990)

now have been

detected on the surfaces of floral nectaries from over 40

_{Myrtacean (mostly}

Eucalyp-tus)

species.

On the _nectary surface of

E stellulata, Davis

_{( 1969)}

_reported

that these

structures remain

_permanently

_open, whereas Carr and Carr

₍₁₉₈₇₎

_postulated

that

_they

are able to _open and

close,

and

may

regulate

nectar flow

_through

them. The

_objectives

of this

_study

were to

deter-mine,

in three

_Eucalyptus

_{spp in}

Australia,

various nectar characteristics

_including

the

constancy

of

_{nectar-carbohydrate}

composi-tion

_following

flower visits and as flowers

age; and to examine the _nectary

surfaces,

to

_investigate

further the mechanism of

nec-tar escape.

MATERIALS AND METHODS Plant material

The trees

_{investigated,}

one _per

_species,

were

growing

as ornamentals at The Australian National

_University

in Canberra. E

_cosmophylla

F Muell (native to South Australia; Penfold and

Willis, 1961) grew between

buildings

of the Department of _Forestry and the Research School of

_Biological

Sciences. Both E

_grandis

Hill (Maiden) (coastline of _Queensland and New South Wales; Penfold and Willis, 1961) and E

pulverulenta

Sims (southeastern NSW; Peters et

al, 1990) were studied near the western border in the Culture Area of the

_Department

of

_Botany.

Of these

_species,

E

_{grandis especially}

_is recog-nized as an

_{important honey plant}

_(Penfold and

Willis, 1961; Clemson, 1985).

Floral stages

E

_cosmophylla

and

_{E pulverulenta typically}

pos-sessed _{three buds per umbel}

_(fig

1 and 7);

_E

gran-dis had seven. Five floral _{stages (I-V;} see

_figs

_2,

5 and ₈₎ were _designated. Flowers entered _stage

I

_following

dehiscence of the bud cover

(oper-culum;

fig

2 _top left), when the stamens

com-menced

_unfolding

from their natal

_position

occu-pying

the

_{hypanthial region.}

The

_protandrous

flowers at _stage II had most stamens still with their filaments bent _inward;

_only

a

_minority

of

innermost stamens still

_occupied

the

_hypanthium.

Continual

unfolding

of innermost stamens

cou-pled

with

_{outstretching}

of the outermost stamens

characterized _stage III. A _strong

_fragrance

was

evident

_by

this _stage. At _{stage IV,} stamens were

extended

_fully

to form an obvious

_ring

encir-cling

the central

_{style. Stage-V}

_{flowers still} pos-sessed all stamens, but in a

_{collapsed ring owing}

to the shrivelled filaments.

To prevent visits

_by

birds

_{(honeyeaters)}

and

large

insects

_(primarily

_Apis

_{mellifera; fig}

₁₎ to

some _{flowers, many inflorescences per} tree were

protected (fig 1)

when anthesis was imminent,

by

enclosure within

_bags

_(mesh size _approx 2.5 mm; see

_fig

I 1 of Davis, 1992) fitted over

operculum

was

_{preceded by}

its

_change

from

green to

_{yellow (fig}

5 _{top left;}

_Hodgson,

1976);

predictors

of anthesis in the other

_species

were

not obvious.

Nectar collection and

analysis

Small numbers of flowers were harvested

peri-odically

from

_May-August

1990, and

immedi-ately

carried into the

_{laboratory nearby.}

In _total, five

_{(occasionally}

four or _six) flowers per _stage (both

exposed

and

_bagged)

_per tree were taken

for nectar collection. Nectar was removed

_using

a

_technique

_(Davis and

_Gunning,

1991)

com-bining

Drummond

_{Microcaps®}

(1-10

μL)

and then

_filter-paper

wicks _(McKenna and

Thom-son, 1988) to _{soak up residual} nectar.

Nectar-solute concentrations were

_{assayed immediately}

by expelling

nectar from a

_capillary

onto

Belling-ham and

_Stanley

refractometers

_(Tunbridge

Wells, UK), then corrected to 20 °C. Concen-trations were

_expressed

as _{g per 100 mL}

_using

the formula of

_Búrquez

and Corbet _{(1991). Wicks}

were allowed to

_air-dry

before storage in clean labeled vials at room _temperature.

Total nectar _{volume per flower} was calcu-lated

_by

summation of filled

_capillaries,

and that of the wicked nectar (< 2

_&mu;L)

estimated from concentration data _{(above) and sugar}

_yields

(below).

Contents of sucrose,

glucose

and fructose in nectar were

_{assayed enzymatically}

(Kronestedt-Robards et al, 1989). All enzymes were of

ana-lytical grade (Boehringer-Mannheim; Sigma).

Briefly, sample

wicks were

_placed

in known vol-umes _{(0.5-1.0 mL)} of distilled water in

dispos-able

_{Eppendorf centrifuge}

tubes and

_agitated

on a Vortex Genie@. After 30 min elution time on

ice, tubes were

_centrifuged

before 5.0-200 _&mu;L

aliquots

were introduced into _separate cuvettes

containing

100 mM Tris-HCl buffer

_(pH

4.5, for

sucrose _{determination;}

_pH

_7.6, for hexoses), 30 mM ATP, 5 mM NADP and 0.175 U

glu-cose-6-phosphate dehydrogenase

(EC 1.1.1.49).

Depending

on _{the assay,}

_{5-&mu;L aliquots}

of inver-tase

_{(&beta;-fructosidase,}

EC _3.2.1.26), hexokinase (EC 2.7.1.1) or

_{phosphoglucose}

isomerase (EC 5.3.1.9) were added to initiate the reaction. Final

volumes were 800-900

_&mu;L

_per cuvette. Reduc-tion of NADP+to NADPH was _measured

spec-trophotometrically (Eppendorf)

at 340 nm

_using

a chart recorder and

_compared

to standards of

freshly-prepared carbohydrate

solutions.

Contents of sucrose,

glucose

and fructose _per

nectar

_sample

were

_expressed

as _percentages.

Means were calculated for each floral _stage and

compared by pooled,

two-tailed t tests _(P = _0.05).

Ratios of

_glucose

to fructose _(G/F), and sucrose

to hexose [S/(G+ F)], were also

_compared

sta-tistically.

Examination of

_nectary

surfaces

Mature, pre-secretory buds and post-secretory

flowers of each

_species

were harvested and

immediately prepared

for

_{low-temperature}

scan-ning

electron

microscopy

(SEM). Sliced buds and flowers were mounted

_nectary-side

_{up with} Tissue Tek&reg;/colloidal

_graphite

_(1:1) on stubs

and

_plunged

into

_liquid

_N

₂

_slush

_{(-230 °C).} The

flash-frozen

_specimens

were then transferred under vacuum into a Hexland 1000A

_CryoTrans

cold _stage attached to a

_Cambridge

Stereoscan 360 SEM. Nectaries were coated with

_gold,

viewed

_directly

at 15 kV and -180 °C, and

pho-tographed

with Ilford SEM film.

RESULTS

Nectar

_volumes,

concentrations and _sugar

quantities

as flowers

_aged

Flower duration was shortest in E

_grandis,

taking

4-6

_days

from anthesis to _stage IV and about 6

_days

more to _stage V. In E

cos-mophylla,

stage

III was reached 4-6

_days

after

_operculum

dehiscence and _stage V 7-9

days

later. In

_{E pulverulenta,}

_stage

III was

reached

_6-10-days,

_stage IV

_12-days,

and

stage V

_{17-days post-anthesis, respectively.}

In all

_species,

nectar was absent in flow-ers with

_{opercula just}

_dehiscing,

but was

collectable

_by

_stage I

_(figs

14-16,

upper

plots)

when flowers of E

_cosmophylla

pos-sessed 20-50 times more than the others.

In that

_species,

nectar secretion

_preceded

anther dehiscence. At _stage

I,

all E

cosmo-phylla

flowers secreted nectar,

compared

to

E pulverulenta

(70%)

and E

_grandis

_(17%).

Thereafter,

nectar was available from _stages

Patterns of

_{nectar-standing}

_crop were

similar for all

_species;

flowers

_exposed

con-tinually

to animal visits contained volumes that were not

_{significantly}

different across

stages II-IV

_(figs

14-16).

By

stage V,

nec-tar

_yields

declined.

In flowers that

_opened

within

_bags

and

were

_continually

inaccessible to

_large

visi-tors,

copious

volumes of nectar

accumu-lated

_(figs

14&mdash;16).

Nectar

_yields

were

great-est at _stage III in E

_{pulverulenta,}

and at IV

in the others. At these

_peak

_levels,

flowers

of E

_{cosmophylla averaged}

4.4 and 8.9 times

more nectar than E

_pulverulenta

and E

_grandis,

_{respectively.}

_Bagged

flowers of

stage V contained less nectar than _{stage IV,}

but this difference was

_{statistically}

signifi-cant

_(P

<

_0.05)

_only

for E

_grandis.

Nectar-sugar

quantities

per floral stage

(figs

14-16, bottom

_{plots) corresponded}

floral _stage within a

_species,

nectar-solute

concentrations were not

_{significantly}

dif-ferent between

_bagged

and

_exposed

flow-ers. In

_bagged

flowers,

average

quantities

of

nectar _sugar were maximal at _stage III

(E pulverulenta)

or IV

_(others),

with E

cos-mophylla secreting

1.2 and 5.6 times more

sugar per flower than E

pulverulenta

and E

_{grandis, respectively.}

Average

nectar-solute concentrations at

the five floral _stages

_ranged

from

16.0-37.3

_g/100

mL

_(exposed)

and

14.1-29.6

_g/100

mL

_(bagged)

in E

cosmo-phylla.

In

_Egrandis,

concentrations varied from 14.8-68.2

_g/100

mL

_(exposed)

and

19.1-41.9 g/100 mL (bagged). In E

pul-verulenta,

concentrations

_ranged

from 17.8-30.6

_g/100

mL

_(exposed)

and

23.3-49.6

_g/100

mL

_(bagged).

_Overall,

nec-tar-solute concentrations were

_usually

Nectar-sugar

composition

in

_exposed

and

protected flowers,

as flowers

_aged

In E

_cosmophylla,

flowers that were

pro-tected from visits

_by

_netting

showed no

sta-tistically-significant

changes

in

_quantity

of

each nectar

_{carbohydrate (glucose,}

_fructose,

sucrose)

as flowers

_{aged (fig}

_14,

middle

plot).

The same held true for

_exposed

flow-ers. In E

_grandis,

the levels of the different sugars remained

highly

consistent for

bagged

and

_exposed

flowers

_(fig

_15).

Sim-ilarly,

no

_{statistically}

_significant

differences

occurred in

_{E pulverulenta,}

whether

_bagged

or

_exposed,

as flowers

_{progressed through}

and E

_grandis,

the sucrose content of nectar

was

_usually

lower than each hexose

_(figs

14 and

_15;

table

_I),

whereas nectar sucrose in

E pulverulenta

(fig

16) averaged

33% over

all

_flowering

_{stages (table I).}

When the

S/(G+F)

ratios were

_compared

between

bagged

and

_exposed

flowers for each floral

stage in all three

_species,

and then overall

(table I),

no

_{statistically significant}

differ-ences were detected.

For all

_species

_{the average} content

_of

glu-cose in nectar exceeded fructose

(figs

14-16).

Significant

differences in the ratio of G/F between

_exposed

and

_bagged

flowers occurred at

_stage

II in E

cosmo-phylla

(fig

14;

P <

_0.05)

and _stage III in E

_{pulverulenta (fig}

16;

P <

_0.01).

When

mean

_composition

data were combined for

all floral _stages

_{(table I),}

nectar of

_bagged

flowers of

_{E pulverulenta}

had a

_{significantly}

greater G/F ratio than that of

_exposed

flow-ers

_(P

<

_0.02).

Nectary

features as flowers

aged

The

_nectary

surface

_spanned

the inner

hypanthium

from the

_style

base to near the

staminophore

and bore modified stomata,

except for a zone of variable width below

the stamens: E

_cosmophylla

_{(250-450 pm),}

E

_grandis

₍₂₅₀

_pm),

E

_{pulverulenta (fig}

_9;

200-250

_&mu;m).

In all

_species,

the modified

stomata were

_{usually solitary}

and distributed

regularly (figs

6,

9 and

10).

Stomatal

fre-quency remained constant in _pre- and post-secretory flowers and

_averaged

348 ± 15

(SE,

n = 7; E

cosmophylla),

299 ± 10

(n

=

_3;

E

_grandis)

and 163 ± 7

_(n

=

_3;

_E

pulveru-lenta)

per

mm

2

of nectary surface.

In all

_species,

modified stomata of

vari-ous

_{developmental}

_stages could be detected

in a

_single

_nectary

_epidermis.

_In

pre-secre-tory

buds could be found immature

_(figs

3 left and

₁₀₎

and

_{maturing (figs}

3

_right

and

11)

stomata, and those with _open

_(figs

_4,

10 and

12)

pores, as well as modified stomata

with occluded _pores

_(figs

10 and

_13).

Sim-ilarly,

this

_asynchrony

in

_development

was

detected in _{post-secretory} nectaries

_(fig

6).

The

_occluding

material of

_light

electron

den-sity

on the surface of the

_guard

cells of

E

_{pulverulenta (fig}

₁₃₎

_may

_correspond

to

the small red dots

_apparent

with the naked

eye,

especially

in fresh flowers of

_stages

IV and V.

In E

_cosmophylla

the _nectary surface had

post-secretion. In E

_grandis

and

_{E pulverulenta}

the

_nectary

_{appeared shiny}

_and,

_except for

many small flecks of wax in mature buds of the

former,

lacked wax

_{throughout (figs}

_6,

10-13).

Nectary

color

_changed

from mature bud

to

_stage

III to _{stage V,} as follows: E

cos-mophylla

&mdash;

pale

green to

_bright

_yellow

to

yellow-orange;

E

_grandis

&mdash;

bright

lemon-yellow

to butterscotch

_{(see Clemson, 1985)}

to

_light

butterscotch with mottled black

(fig

5,

bottom

_{right); E pulverulenta&mdash;pale}

green to

_bright

_orange to butterscotch with

red or brown

_{mottling (fig}

_8,

bottom

_right).

DISCUSSION

Floral nectar characteristics

Nectar traits of these

_species

differed in

sev-eral _respects. Both maximal nectar volume and sugar

production

per flower were related

to flower

_size,

_{being highest}

in E

cosmo-phylla

and lowest in E

_grandis.

The

ten-dency

for nectar-solute concentration to

increase

_during

_{stages I-IV may relate} to

exposure of

standing

nectar to the

atmo-sphere, during

the

_{progressive unfolding}

of

the stamens

_during

flower

_phenology.

In

stage

V,

further

_evaporation

of water from

nectar and a diminishment in rate of nectar

volume

_secreted,

_probably

contributed to

the

_higher

concentrations observed.

Nectar-sugar composition

remained

con-stant _per

_species,

in contrast to other

_species

with

_long-lived

flowers

_{(see Introduction).}

Individual sugar levels remained

similar,

as

did the ratios G/F and

_S/(G+F).

Carbohy-drate

_{composition appeared}

most consistent for nectar

_{of E grandis,}

which contained the

lowest sucrose content

_(14%),

and most

vari-able for E

_{pulverulenta,}

in which sucrose

levels were

_highest

_(33%).

In

_California,

Vansell

₍₁₉₄₁₎

_reported

that for E

globu-lus,

the ratio between invert and total _sugars varied over 5

_days

from 1:1.566 to

1:2.192,

but it was not disclosed which floral

_stages

were involved nor whether this

_change

was

statistically significant.

It would be

inter-esting

to determine whether _pre-nectar of

Eucalyptus

flowers travels two _separate routes

_(which

_may

_impact

final nectar

com-position) through

the

_{nectary (Nichol}

and

Hall, 1988),

and if so, what

proportion

of

the nectar travels each route,

particularly

during periods

of differential rates of

secre-tion

_(eg,

_stage I or

_V,

versus III or

_IV).

That

G/F ratios were above

_unity

_(1.2-1.4)

sug-gests that the _{secretory process in these}

euca-lypts

is more

_complicated

than

_simple

inver-sion of sucrose to

_{equal quantities}

of the

hexoses.

For reasons

_unknown,

G/F ratios

occa-sionally

were

_{significantly}

_different,

_though

not in a consistent _pattern. In E

_cosmophylla

(stage

II)

exposed

flowers had a

_higher

G/F

ratio than

_bagged

ones, but in E

pulveru-lenta

_(III)

the reverse was true. In E

cos-mophylla, variability

in _nectar-sugar

com-position

was

_always

_greater from

_exposed

flowers than

_bagged

(see

SE values in

table

_I),

but was

irregular

for the others.

Willmer

₍₁₉₈₀₎

found

_changes

in

amino-acid

_composition

of nectar

_following

vis-its; however,

amino acids were not detected

in

_eucalypt

nectar

_{(Nú&ntilde;ez, 1977).}

Microorganisms

can occur in nectar and

may affect its

composition

(Lüttge,

1961;

Gilliam et

_{al, 1983),}

and

_algae

have been

detected on nectaries of

_eucalypts

_(Carr

and

Carr, 1987).

Although

not

_sought,

uniden-tified

_{green-pigmented algae}

were found

macroscopically

on nectaries of two

_bagged

flowers of E

_grandis,

_during

nectar collec-tion.

However,

the

_carbohydrate

composi-tion of these

_{algal-contaminated}

nectars did

not differ from others of _stage III or IV.

Evidently

the

_algal

_{spores entered the} nectar

of these two flowers

_{by passing through}

the

mesh

airborne,

or on the bodies of

_thrips,

common inhabitants of all flowers studied.

Overall,

the

_S/(G+F)

ratios for nectar of

between 0.1-0.499

(’hexose-rich’;

Baker

and

_{Baker, 1983),}

but were in _{the range} 0.5-0.999

(’sucrose-rich’)

for

_E

pulveru-lenta. These

_{designations generally}

_agree

with observations of floral visitors to these

species.

For

instance,

short-tongued

bees

like A

_mellifera

are

_regular

visitors of taxa

that

_produce

nectar

_having

a wide range of

sucrose/hexose

_ratios,

but

_particularly

those

which are hexose-dominant or -rich

_(Baker

and

_{Baker, 1983).}

_{Similarly, species}

polli-nated

_{principally by honeyeasters}

_(birds)

have hexose-dominant or -rich nectar,

only

(Baker

and

_{Baker, 1983).}

These

nectar-car-bohydrate compositions

are similar to

pre-vious

_analyses

of other

_eucalypts

in Cali-fornia

_{(Vansell, 1941,}

1944; Baker and

Baker, 1990),

Argentina

(Nú&ntilde;ez, 1977)

and Australia

_(Moncur

and

_{Boland, 1989)}

which

ranged

from hexose-dominant to

sucrose-rich. However, here the G/F ratios exceeded

unity. Non-eucalypt

nectars of the

Myr-taceae are hexose-rich or -dominant

(Perci-val, 1961;

Beardsell et

_{al, 1989;}

O’Brien et

al, 1996).

The

_phenomenon

of net

_reabsorption

of

nectar

_{carbohydrates}

is well established in

various taxa

_(Pedersen

et

al, 1958; Shuel,

1961;

Corbet and

_{Delfosse, 1984;}

_Búrquez

and

_{Corbet, 1991)}

and now for

_Eucalyptus.

Interestingly,

no

_changes

in sugar

compo-sition occurred here

_during

the

_period

of

nectar

_{reabsorption.}

Therefore,

net recla-mation of uncollected nectar did not occur

selectively,

for _any of the three

carbohy-drates in

_particular,

but instead as a mixture

(Shuel,

1961; Freeman, 1986).

Flowers of

stage V

_appeared

to resorb nectar at a rate

inversely proportional

to the volumes of

stage IV

_(eg,

_compare E

_cosmophylla

and E

_{grandis, figs}

14 and

₁₅₎

that accumulated

by protection

from visitation. Contactable

nectary-surface

area available for

reabsorp-tion limits the process

(Búrquez

and

Cor-bet,

1991).

How _{pore occlusion influences}

reabsorption

remains to be determined.

Nectary

surfaces

The floral nectaries differed in color and

amounts of surface wax.

_Changes

in color,

such as the

corolla,

affect

foraging

behaviour

(Weiss, 1995).

In the absence of

_prominent

petals, changes

in nectary appearance

during

flower

_phenology

_may

_provide

visual

for-aging

cues to

_eucalypt

visitors

_(O’Brien

et

al,

1996).

However, the

_frequency

of mod-ified stomata on the _nectary did not

_change

during phenology,

nor was it correlated with

nectar volume or

_carbohydrate

_production.

The terms ’modified stomata’

_(Fahn,

1979;

Davis and

_Gunning,

₁₉₉₂₎

are used here to _{represent the pore} structures of

Euca-lyptus

nectaries.

_They

share

_specific

devel-opmental

features

_(eg,

_{breakthrough;}

Carr

and

_{Carr, 1987)}

and likeness with foliar

stomata, but

evidently

are unable to close

their _pores

_by

_guard-cell

movements

_(Davis,

1969). Instead,

modified stomata

_(even

on

buds)

can be found with _pores occluded

(Davis

and

_Gunning,

_1992).

Occlusion

apparently

involves release of

_hydrophobic

material(s)

from the

_guard

cells to seal the

pore. Unknown is whether an occluded pore

completely

restricts the _passage of exudate

from the

_gland,

or _{disallows any re-entry}

during

nectar

_{reabsorption.}

It would _prove

interesting

to determine whether modified

stomata without

_overlying

nectar

_droplets

(see

Beardsell et

_{al, 1989)}

are

_possibly

immature or occluded. Unlike the stamens

and

_style,

the _nectary does not abscind

_(Carr

and Carr,

1990).

Hence, it is

_possible

that occlusion serves to deter

_pathogen

_entry

through

nectary pores to the

_developing

seeds below.

Low-temperature

SEM minimizes

arte-facts

_{(Robards, 1984)}

and here

_provided

no

evidence that nectar flow in

_eucalypts

is

reg-ulated

_by

_{pore aperture} movements

(Carr

and

Carr, 1987).

Rarely

were modified

stomata of the _nectary observed to have

guard

cells almost

_meeting,

or

_touching,

clo-sure could occur

_by

total occlusion.

Inter-estingly, partial

stomatal occlusion

(as

polar

flaps

and

_pseudo-outer

stomatal

_ledges)

also has been detected in leaves of certain

euca-lypts,

where it involves

_deposition

of new

cuticle and wall

_(Carr

and

_{Carr, 1980).}

Even

after nectar secretion had

_ceased,

modified

stomata with _{open pores} were still found.

Furthermore,

pre-secretory buds

_possessed

open pores on their nectaries.

Therefore,

the modified stomata did not control floral

nectar secretion of these

_species,

because

commencement of nectar release did not

coincide with

_synchronous,

initial

_opening

of pores of the modified stomata, nor did

secretion cease as a result of stomatal

clo-sure. _{At any} floral

_stage,

modified stomata

could be found on the _nectary surface in all

of four

_ontogenetic

_stages:

immature,

break-ing through,

open and occluded. This

asyn-chronous _pattern in

_development

of modi-fied stomata, itself sides

_against

stomatal

regulation

of nectar flow.

Indeed,

like the

nectary

(fig

6),

the leaf of E

_grandis

shows

asynchrony

in

_development

between

_nearby

stomata

_(fig

20 of Carr and

_Carr,

_1991).

In

an

_evolutionary

_sense,

_early

land

_plants

bore

stomata before

leaves,

and stomata in the

plant kingdom

possess different

degrees

of

functionality (Ziegler,

1987).

These close

similarities between leaf and _nectary struc-tures in

_Eucalyptus

favor utilization of the

existing terminology,

’modified stomata’

(Fahn,

1979;

Davis and

_Gunning,

1992)

and

’guard

cells’,

instead of

_’pore

cells’

_(Carr

and

Carr, 1987, 1990).

Further studies are

_required

to

investi-gate the

_anatomy

and ultrastructure of

Euca-lyptus

nectaries. A

_{key question}

_(Carr

and

Carr, 1987)

still to be addressed in

euca-lypts

is whether the _nectary

_guard

cells have

plasmodesmata

and are involved in

sym-plastic

transport of nectar. There is

insuffi-cient evidence from Chamelaucium unci-natum, the

_{only Myrtacean species}

whose

nectary modified stomata have been stud-ied

_by

transmission electron

_microscopy

(O’Brien

et

al, 1996).

However, in floral

nectaries of Vicia, the

_{plasmodesmata}

detectable in

_{primary pit-fields}

of

guard-cell walls of immature modified stomata,

were no

_{longer complete}

at stomatal

matu-rity

(Davis

and

_Gunning,

1992).

Further evidence

_against

the modified stomata

mak-ing

a direct contribution as

_part

of a

sym-plastic

secretory

pathway

for nectar in that

species,

is considerable

_(Davis

and

Gun-ning,

1993).

ACKNOWLEDGMENTS

It is a

_pleasure

to thank Dr P Macnicol,

Phy-totron, CSIRO Division of Plant

Industry,

Can-berra, ACT, for his excellent introduction to enzy-matic

_analysis

of

_{carbohydrates.}

BES

_Gunning,

J Jacobson and RW

_{King kindly provided}

labo-ratory space and invertase. The technical exper-tise with

_{low-temperature}

SEM of R

_Heady

and M Ciszewski, Electron

_Microscopy

Unit, RSBS,

also was

_appreciated.

D

_Dyck

assisted

_greatly

with

_preparation

of the

_figures.

A research grant (ANU-1H) from the Australian

_Honey

Research

Council, and a

_{Postgraduate Scholarship}

from NSERC of Canada,

provided

the necessary funds.

Résumé &mdash; Influence de la visite florale

sur la

_{composition glucidique}

du nectar et

sur les modifications de la surface

necta-rifère de

_{l’Eucalyptus.}

Les

_eucalyptus

(Eucalyptus spp)

constituent la source de

nectar floral la

_plus

_importante

_{pour la} pro-duction de miel en Australie. Leur nectar a

fait _pourtant

_l’objet

de _peu de recherches.

Les nectaires floraux et la

_production

des

principaux

sucres du nectar ont été étudiés chez trois

_{espèces d’eucalyptus}

_poussant

à

Canberra,

Australie : E

_{cosmophylla, E}

grandis

et E

_{pulverulenta.}

Chez ces trois

espèces,

le nectaire est situé à la surface interne de

_{l’hypanthium,}

sous le

stamino-phore (fig

9).

La

_microscopie

_{électronique}

à

balayage

à basse

_température

a montré _que

la surface des nectaires

_possédait

des

cos-mophylla)

stomates/mm

2

.

Le

développe-ment des stomates modifiés est

_asynchrone

(figs

6, 10),

chaque

nectaire examiné au

stade

_{présécréteur}

et

_{postsécréteur}

possé-dant à la fois des stomates modifiés

imma-tures

_(figs

3

_gauche,

_{6, 10),}

en cours de

maturation

_(figs

3

_{droite, 11),}

avec un _pore

ouvert

_(figs

_{4, 10, 12)}

ou fermé

_(figs

10,

13).

On n’a pas trouvé de preuve que le sto-mate modifié s’ouvre et se ferme pour

régu-ler la sécrétion nectarifère. Il est en revanche

évident que la fermeture du pore se fait _par

occlusion et non _{par des} mouvements de cellules

_stomatiques.

Grâce à une

_technique

combinée

_{capillaire-mèche,}

le nectar a été

prélevé

à

_cinq

stades floraux

_(I-V)

sur

chaque espèce

(figs

2, 5, 8) :

depuis

les

fleurs fraîchement écloses

_qui

venaient de

perdre

leur

_{operculum jusqu’aux}

fleurs de 12

à 17

_jours

dont les étamines étaient

forte-ment flétries.

_{À chaque}

_stage, le nectar a été

prélevé

sur des fleurs ensachées

_(fig

1)

quand l’operculum

était déhiscent

_(fig

2 en

haut à

_gauche)

et sur des fleurs laissées libres

d’accès aux

visiteurs,

_{principalement}

l’abeille

_{domestique Apis mellifera}

_(figs

1,

7)

et le

_guêpier

_(oiseaux).

Les rendements en

nectar et les

_quantités

de sucre _{par fleur}

ensachée avaient un niveau maximum chez

E

_{cosmophylla (fig}

_14),

intermédiaire chez

E

_{pulverulenta (fig}

₁₆₎

et minimum chez E

grandis (fig

15),

conformément à la taille de la fleur mais pas au nombre de stomates

des nectaires.

_{L’analyse enzymatique}

du

glucose,

du fructose et du saccharose dans les échantillons de nectar a montré

_qu’E

cosmophylla

et E

_grandis

sont riches en

hexose

_(0,1

<

_S/(G

+

_F)

<

0,499 ;

tableau

_I),

tandis que

E pulverulenta

est riche en

sac-charose

_(0,5

<

_S/(G

+

F)

<

_{0,999 ;}

tableau

I).

Le _rapport

_{glucose/fructose}

se situait autour

de

1,2-1,4

pour les trois

(tableau I).

On a

trouvé _peu de

_changements

_{dans la}

compo-sition

_glucidique

du nectar _{selon que les}

fleurs

_{étaient jeunes}

ou

vieilles,

ensachées ou

à l’air libre

_(figs

_14-16),

ce

_{qui indique}

une

constance dans la

_composition

tout au

_long

du processus de secrétion des stages I-IV.

Au stade

V,

une

_{réabsorption}

nette du

nec-tar s’est

_{produite (figs}

_14&mdash;16)

et la constance

dans la

_{composition glucidique}

du nectar

implique

que cette

_{réabsorption}

n’ait lieu

de

_façon

sélective _pour aucun des trois

sucres, mais

plutôt

sous forme d’un

_mélange

complexe.

Il est nécessaire de

_poursuivre

les recherches _pour étudier la fermeture des

stomates modifiés et le rôle

_joué

_par les

pores obturés dans l’écoulement et la

réab-sorption

du nectar.

Apis mellifera

/

_Eucalyptus

/ nectar/

glu-cide / nectaire / stomate

Zusammenfassung &mdash;

Einflu&szlig; des

Blü-tenbeflugs

auf die

_{Zusammensetzung}

der Zucker im Nektar und

_Änderung

der Oberflächenbeschaffenheit der Nekta-rien bei

_Eucalyptus

_ssp

_(Myrtaceae).

Obwohl

_{Eukalyptusbäume}

in Australien die

wichtigste

Quelle

von Blütennektar zur

Honigproduktion

darstellen,

ist

Eukalyp-tusnektar

_bislang

nur

_wenig

untersucht

wor-den. Die floralen Nektarien sowie die Sekre-tion der

_wichtigsten

_{Nektarkohlenhydrate}

wurde bei drei

_{Eukalyptusarten}

in

Canberra,

Australien untersucht. Bei E

_cosmophylla,

E

grandis

und E

_pulverulenta

ist das

Nekta-rium auf der inneren Oberfläche des

Blü-tenbechers unterhalb der

_Staminophore

gele-gen

(Abb 9).

Rasterelektronenaufnahmen bei

_niedrigen

_Temperaturen

_zeigten,

da&szlig; die Oberflächen der Nektarien hunderte von

modifizierten Stomata besitzen. Diese

lie-gen zumeist einzeln und sind relativ

gleich-mä&szlig;ig

verteilt

_{(Abb 6, 9, 10).}

Ihre Dichte

beträgt

163

_{(E pulverulenta)}

bis 348

_(E

cos-mophylla)

pro

mm

2

der Oberfläche. Die

Ent-wicklung

dieser modifizierten Stomata

erfolgte

nicht

_gleichzeitig

_(Abb

_{6, 10).}

Bei allen untersuchten Nektarien kamen diese sowohl vor als auch nach der Sekretion unreif

_(Abb

3

_{links, 6, 10)}

durchbrechend

(Abb

3

_{rechts, 11),}

mit offenem

_{(Abb 4, 10,}

12)

oder

_verstopftem

Porus vor

_{(Abb 10,}

Stomata zur

_Regulierung

des Nektarflusses

öffnen oder schlie&szlig;en. Stattdessen wurden

die Poren durch

_Verstopfung

verschlossen,

offensichtlich nicht durch

_Bewegung

der

Begleitzellen.

Mit einer

Kapillar-Docht-Kombinationstechnik wurde Nektar von

jeder

Art aus fünf Blühstadien

_(I-V)

gesam-melt

_(Abb

2, 5,

8).

Diese Stadien umfassten

frischgeöffnete

Blüten,

die

_gerade

ihren

Knospendeckel

verloren

hatten,

bis zu

meh-rere

_Tage

älteren Blüten mit stark

verwelk-ten

_{Staubgefä&szlig;en.}

Bei

_jedem

Stadium wurde Nektar sowohl von

_Blüten,

die beim

Auf-platzen

des

_{Knospendeckels}

(Abb 2,

oben

links)

eingewickelt

(Abb 1) wurden,

als auch

von

Blüten,

die

ganzzeitig

für

Blütenbesu-cher

_{(zumeist Apis mellifera)}

_{(Abb 1, 7),}

und

_{Honigfresser (Vögel) zugänglich}

waren.

Der

_Nektarertrag

und die

_Zuckermengen

pro

eingewickelter

Blüte waren bei E

cos-meophylla

am

_{grö&szlig;ten}

_{(Abb 14)}

und bei E

grandis

am

_geringsten

_{(Abb 15);}

bei

_E

pul-verulenta

_{(Abb 16)}

_lagen

sie dazwischen. Diese

_{Reihenfolge entsprach}

der

Blüten-grösse,

nicht aber der Anzahl der Stomata auf den Nektarien. Die

_enzymatische

Ana-lyse

der

_Glucose,

der Fructose und der

Saccharose in den

_{Nektarproben zeigte,}

da&szlig;

E

_cosmophylla

und E

_grandis

reich an

ein-fachen Zuckern

(Hexosen)

sind

[0.1

<

S/(G+F)

<

_0.499;

Tabelle

_I],

während

_E

pul-verulenta vor allem das Disaccharid

Saccha-rose enthält

_[0.5

<

_S/(G+F)

<

_0.999;

Tabelle

_I].

Das Verhältnis von Glukose zu

Fruktose

_betrug

bei allen Arten im Mittel

1.2 bis 1.4

_{(Tabelle I).}

Es konnten nur

geringe

Unterschiede der

Nektarzuckerzu-sammensetzung

zwischen

_jungen

und alten oder

_{eingewickelten}

und

_{zugänglichen}

Blü-ten

_{(Abb 14-16)}

_festgestellt

werden,

was

auf eine

_{gleichmä&szlig;ige Zusammensetzung}

während des Sekretionsverlaufes der Sta-dien I-IV hindeutet. Im Stadium V trat eine

Netto-Reabsorption

des Nektars auf

_(Abb

14-16).

Die auch in diesem Stadium

festge-stellte

_{Gleichartigkeit}

der

Nektarzusam-mensetzung lässt darauf

schlie&szlig;en,

da&szlig; diese

Reabsorption

nicht selektiv für einzelne der

drei Zucker

ist,

sondern die _gesamte

Mischung

betrifft. Zur

_Klärung

der

Ver-stopfung

der modifizierten Stomata und zu

der Rolle der

_verstopften

Poren während

des Nektaraustritts und der

_Reabsorption

werden weitere

_{Untersuchungen benötigt.}

Apis mellifera

/

_Eucalyptus

/ Nektar /

Zucker / Stomata / Nektarien

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