Regulation of Light Energy Distribution between Photosynthetic
Pigment Systems; a Possible Role of Leaf Anatomy
G. H arnischfeger and G. Zenk
Schaper & Brümmer, Salzgitter-Ringelheim and Lehrstuhl für Biochemie der Pflanze, Univer­
sität Göttingen
Z. Naturforsch. 38 c, 6 0 0 -6 0 3 (1983); received April 27, 1983
Photosynthetic Pigments, Energy D istribution, Leaf Anatomy
The complex anatomical structure of an intact leaf results in a distribution o f photosynthetically active energy between photosynthetic pigm ents which is different from that observed in
isolated chloroplasts. The variance is due mainly to scattering at the gas-liquid interface between
cells and intercellular space which tends to increase light absorption by the long wavelength
absorbing pigments through secondary fluorescence. Evidence is given in support of an active use
of this feature by the higher plant to regulate energy flow at the photophysical level of light
absorption.
Introduction
The com plex system o f le af structure has been
viewed so far only as a m eans to achieve an op tim al
balance betw een transpiration, resp iratio n and
d istribution o f nutrients and products in relatio n to
photosynthetic capacity. Little em phasis has been
placed on the logical consequence, th at this balance
is a dynam ic one being in constant need for a d ju st­
ment.
Since photosynthesis is a key factor for th e m a in ­
tenance o f overall m etabolic stability it can be
assum ed, that the chloroplast is the m ain lever for
the pertinent regulatory m echanism s. A variety o f
such systems are known, ranging from those acting
at the pigm ent level (e.g. red istrib u tio n th ro u g h
spillover and dissipation by fluorescence) to those
influencing the
Blackm ann reaction
th ro u g h
m odulation o f gas exchange.
Investigations o f regulatory features at th e p ig ­
m ent level have so far used isolated chloroplasts as
experim ental system and resulted in a coherent
m odel o f interactions betw een several pig m en t sys­
tems [1]. Key p ara m ete r in m any o f these ex p eri­
ments was the m easurem ent o f the fluorescence
energy em itted at 77 °K from the various p igm en t
species m aking up the different pigm ent com plexes.
The interpretation o f these results to the in vivo
situation tacitly assum es th a t the organ izatio n o f
Supported by a grant o f the Deutsche Forschungsgemein­
schaft.
Reprint requests to Prof. Dr. Harnischfeger.
0341-0382/83/0700-0600
$01.30/0
chloroplasts w ithin the living le af and the structural
features o f this plant-organ itself are o f no or only
m inor consequence for the proposed functioning o f
the pigm ent-level regulation. A lthough experim ents
concerning the conform ational change o f plastids in
vivo [2] ap p eared to b ear o u t this conclusion, it
rem ains still a disp u tab le notion.
It seemed, therefore, w arran ted to investigate the
possible p articip atio n o f le a f structure on the energy
distribution o f absorbed light. The results are
reported in the follow ing sections.
Materials and Methods
Leaves o f 6 week old tobacco plants, grown in the
greenhouse, w ere used for the fluorescence m ea­
surem ents w ith o u t fu rth er processing. T h eir ch lo ro ­
phyll content, given per unit area, was determ in ed
according to A m o n [3].
W hole chloroplasts w ere prepared using the
m ethod o f N ak atan i and B arber [4], T he isolation
m edium contained 0.33 m sucrose, 0.2 mM M gC l 2
and 20 mM M ES pH 6.5, the final suspension
m edium 0.33 m sucrose — 0.5 mM TR IC IN E pH 7.5.
Both the tobacco leaves an d the chloroplast sus­
pensions were d ark ad ap ted for 15 min before use.
For the m easurem ents o f leaf fluorescence in
relation to tim e o f day, d ark ad aption was om itted.
The m aterial for co m p arativ e m easurem ents of
various plant species was o b tain ed in the G öttingen
botanical garden.
The fluorescence spectra were determ in ed at
room tem p eratu re using th e instrum ental setup
described earlier [5]. T he le a f was illum inated w ith
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G. Hamischfeger and G. Zenk • Regulation o f Light Energy Distribution
the aid o f one arm o f a double arm fiber optic, the
fluorescence was taken at 360° from the excitation
using the other. C hloroplast suspensions w ere p u t
into a petri-dish. The illum ination and fluorescence
m easurem ents were m ade through its bottom using
the sam e setup.
Excitation was betw een 475 - 500 nm w ith an
intensity o f 1 mW x cm -2. T he fluorescence was
scanned between 6 6 0 -7 6 0 nm. T he resulting spectra
were corrected against filter tailing, m onochrom atoraberrations and photo m u ltip lier sensitivity using
appropriate blanc-sam ples and ca lib ratio n curves.
The standard deviation, determ ined in series experi­
ments using 10 pieces o f individual tobacco leaves,
was 7%.
601
fluorescence (reabsorption o f energy em itted at
690 nm by the chlorophylls fluorescing at 742 nm )
and is not caused by intrath y lak o id al red istrib u tio n
o f energy, attem pts were m ade to m odel this b e­
haviour. The d ata o f Fig. 2 exclude, th at a sim ple
packing o f plastids, i.e. an increase o f secondary
fluorescence by enlarging the pro b ab ility for re a b ­
sorption o f em itted energy, accounts for th e size o f
the phenom enon. T aking the ratio F142/F 69q as a
m easure, dense packing can effect only h a lf o f the
value o f 0.8 observed in vivo.
Results
A com parison o f the fluorescence em ission spec­
trum o f a whole leaf section and th a t o f a chloro­
plast suspension, adjusted to the sam e chlorophyll
content per illum inated area and the sam e th ick ­
ness, shows a considerable am ount o f em ission in
the long wavelength region for the form er w hile its
fluorescence band around 690 nm is appreciab ly
lower (Fig. 1). The area under the curves, a m easure
o f the total am ount o f energy em itted, is a p p ro x i­
mately equal. Obviously, a shift o f em ission from
the pigm ent systems takes place d ue to structural
features o f the leaf itself.
Starting with the hypothesis, th a t this observation
rests basically on the phenom enon o f secondary
/ jq chlorophyll x c m '2
Fig. 2. Influence of chloroplast density per illum inated
area on the ratio of fluorescence emission Fli 7/Ff, at
room temperature. Suspension layer thickness 1.6 mm; the
corresponding ratio of the intact leaf was 0.8 at 51 |ig
chlorophyll x cm-2.
excitation: 475-500 nm
nm emission
Fig. 1. Room temperature fluorescence emission spectrum
of chloroplasts as compared to that of an intact leaf. The
chloroplast suspension was adjusted in chlorophyll content
and thickness of the suspension layer to the values of the
leaf.
g ethyl-cellulose
Fig. 3. Increase o f fluorescence ratio F742/F690 of isolated
chloroplasts through addition of ethylcellulose. C hloro­
phyll concentration was 51 ng/cm 2, suspension layer thick­
ness 1.6 mm. The concentration of ethyl-cellulose is given
per 3 ml total suspension volume.
602
G. Hamischfeger and G. Zenk • Regulation of Light Energy Distribution
e xc ita tio n : 1 7 5 -5 0 0 nm
Fig. 4. Comparison of room tem perature fluo­
rescence emission spectra of an intact tobacco
leaf before (control) and after infiltration with
water.
nm emission
chlorophyll content in/ugx cm-2
Fig. 5. G raphic summary of fluorescence emission ratios
F1A2/Fm at room tem perature of intact leaves belonging to
species of the various families indicated. Between 6 and 15
species of every plant family, all leaves with sim ilar
anatomical features par family, were used. The standard
deviation is given by the respective bars.
A m ajor share o f the observed increase in second­
ary fluorescence m ust th u s be attrib u ted to scatter­
ing on non chloroplast m aterial o f the leaf. T he
experim ents depicted in Fig. 3, m odeling the in vivo
situation by using increasing am ounts o f fibrous
ethylcellulose, indicate clearly, th at th e increase in
em ission ratio is d o m in ated by this feature. S catter­
ing in the leaf ap p ears to be caused largely at the
interphase betw een gas and liquid, nam ely the
b o rd er betw een cells and intercellular space. A
sim ple p ro o f for this reasoning is available.
A bolishing the p hase b o u n d ary by infiltrating the
leaf w ith w ater u n d er gentle evacuation should and
does result in a change o f the fluorescence spectrum
tow ards th at observed w ith isolated plastids
(Fig- 4).
100
5 0 -s
Fig. 6. Variation of the room tem perature
fluorescence ratio FU2/Fm ° f a tobacco leaf in
relation to sun-light intensity and time of day.
Tobacco plant of Göttingen botanical garden;
measurements without dark adaptation.
time of day (MEZ)
G. Hamischfeger and G. Zenk • Regulation of Light Energy Distribution
Table. Fluorescence emission ratios F142/F690 of various
monocotyledons showing the C4-pathway of carbon as­
similation.
Species
Ratio
Saccharum officinale
Sorghum bicolor
Sorghum cernum
Zea mays
0.67
0.58
0.55
0.35
The experim ental results confirm thus the close
relation betw een the em ission ratio and the stru c­
ture o f the leaf.
To strengthen this conclusion, leaves o f 41 plan t
species o f the G öttingen botanical garden, belong­
ing to 5 different fam ilies and taken at random ,
w here checked accordingly. T he presence o f u n i­
form anatom ical features w ithin a fam ily was
assessed before-hand w ith m icroscopic techniques.
Fig. 5 shows the sum m ary o f these m easurem ents
for 4 fam ilies o f dicotyledons.
W ithin the variations depicted, a correlation b e ­
tween the closely related species o f a fam ily and the
em ission ratio seems to be m anifest. T he d a ta o f
C-4 plants w ith th eir bundle sheath structure range
m uch low er (Table) and are in line w ith this
reasoning.
The d ata are the starting point for the hypothesis
that a change in le af structure, presum ably a shift in
the extend o f interface scattering due to stom atal
regulation and leaf-w ater content, m ay influence
photosynthetic m etabolism . T he results o f Fig. 6
support such a feature. T he em ission ratio taken
throughout the day varies in m uch the sam e m an n er
as the photosynthetic activity in the experim ents o f
Lange [6, 7],
[1] W. L. Butler, Ann. Rev. Plant Physiol. 29, 3 4 5 -378,
(1978).
[2] U. Heber, Biochim. Biophys. Acta 180, 3 0 2 -3 1 9
(1969).
[3] D. I. Amon, Plant Physiol. 24,1 - 1 5 (1949).
[4] H. Y. N akatani and J. Barber, Biochim. Biophys. Acta
461,510-512(1977).
[5] G. Hamischfeger and B. Herold, Ber. Dtsch. Bot. Ges.
91,477-486 (1978).
[6] E. D. Schulze, O. L. Lange, and W. Koch, Oecologia
9,317-340(1972).
[7] O. L. Lange, E. D. Schulze, L. Kappen, U. Buschbom,
and M. Evenari, pp. 121-143 in (D. M. Gates, and
603
Discussion
The existence o f a sizeable long w avelength em is­
sion at room te m p eratu re in w hole leaves as co m ­
pared to its very low intensity in isolated ch lo ro ­
plasts reflects prim arily the responsibility o f second­
ary fluorescence for the form er. T he experim ental
findings are congruent w ith d ata by Virgin [8], w ho
used chloroplast soaked glass-wool as experim ental
model. T hey u n d erlin e the fact, th a t the com plexity
o f leaf-structure, esp. the physical p aram eters
scattering, diffraction, refraction d ue to th e exis­
tence o f cell organelles, cell walls and gas-filled
intercellular spaces, has to be included in assessing
the energy d istrib u tio n w ithin the p hotosynthetic
pigm ent system o f higher plants. T he d istortion
o f spectral intensities co m p ared to the isolated
plastids is in the o rd er o f m ag n itu d e o f those
observed by M u rata at 77 °K [9]. It should how ever
be rem em bered, th a t in the latter case an artificial
system (isolated chloroplasts, ad d itio n o f ions, low
tem perature) has been used and th at the in te rp re ta ­
tion o f its causes and its relevance to the in vivo
situation is radically different.
The notion, th a t le af anatom y influences the
energy d istrib u tio n w ithin the p h otosynthetic
apparatus, is by itself n eith er new (e.g. [ 10]) nor
particularly interesting. It gains, how ever, in im ­
portance if a link can be established betw een energy
d istribution and photosynthesis w hich is actively
altered according to m etabolic d em and th ro u g h
subtle changes in le af anatom y. C ircum stantial
evidence has been forw arded in this paper, w hich is
in concordance w ith d ata rep o rted by B jörkm an [11]
and L ichtenthaler [12] on sun- and shade leaves.
However, m ore experim ental w ork is clearly
w arranted to establish this point.
[8]
[9]
[10]
[11]
[12]
R. B. Schmerl eds.), Perspectives of Biophys. Ecol­
ogy, Springer, Berlin 1979.
H. I. Virgin, Physiol. Plant 7 ,5 6 0 -5 7 0 (1959).
N. Murata, Biochim. Biophys. Acta 189, 171-181
(1969).
D. M. Gates, Biophysical Ecology, pp. 215-248,
Springer-Verlag, Berlin 1980.
O. Björkman, Encyclopedia of Plant Physiol. Vol. 12a,
pp. 5 7 - 107, Springer-Verlag, Berlin 1981.
H. K Lichtenthaler, in Photosynthesis G. Akoyuno­
glou ed. Vol. 6, pp. 273-278 Balaban, Philadelphia
1981.