Received for publication October 19, 1989
Accepted March 15, 1990

Plant Physiol. (1990) 93, 1083-1088
0032-0889/90/93/1 083/06/$01 .00/0

Patterns of Chlorophyll Fluorescence Kinetics in Relation to
Growth and Expansion in Cucumber Leaves1
Judith G. Croxdale* and Kenji Omasa
Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 (J.G.C.), and National Institute forEnvironmental Studies, Japan Environment Agency, Onogawa, Tsukuba, Ibaraki 305, Japan (K.O.)
MATERIALS AND METHODSABSTRACT
Photochemical development was studied in developing cucumber (Cucumis sativus L. cv Hokushin) leaves to determine if thespatial pattern coincided with relative growth rates of expanding
leaves, intercalary cell division, or position relative to the vascular
tissue. Both leaf surfaces undergo a series of similar changes inchlorophyll a fluorescence kinetics, but the upper surface more
quickly achieved the characteristic response. Imaging of fluorescence showed an individual developing leaf has four regions
differing in kinetics, but these regions do not coincide with areasof increasing relative growth rate. Two of these regions lie at theleaf edge and their divergent kinetics may be related to structuraland physiological features present at this position. A third areawith different kinetics, in the basal region of the leaf, is spatiallyconsistent with primordial regions that are clonal during development. The correspondence between areas of clonal growthand specific fluorescence kinetics indicates that cells of common
ancestry show functional uniformity. No evidence was found thatthe proximity of the vascular tissue influenced development ofphotochemical function.

Plant Materials and Leaf Area Measurements

Cucumber (Cucumis sativus L. cv Hokushin) plants weregrown as previously described (10) for up to 5 weeks. For
fluorescence and gas exchange measurements, plants werebrought from the controlled-environment greenhouse to thelaboratory and allowed to acclimate for about 2 h. Plants were
dark-incubated for at least 30 min before fluorescence induction. No appreciable differences in induction kinetics wereobserved with longer dark periods. Leaves monitored for
fluorescence and assimilation were intact and remained attached until all measurements on the plant were completed.
Leaf area was measured with a LI 3100 area meter (LI-COR,
Lincoln, NE).
Chi a Fluorescence Measurements
A portable Kautsky apparatus (Richard Branker ResearchLtd., Plant Productivity Meter, SF-20) was used to monitor
fluorescence on both leaf surfaces at a position midway between the margin and midvein. Light of 670 nm at 40 ,umol
photons m-2 s-' was used for excitation and fluorescence wasmeasured at >710 nm. Data were collected at 30 ms intervals
over a period of 400 s, stored, and analyzed by computer.

The attainment of photosynthetic competency in angiosperm leaves is regulated by light and by developmental

factors. Unlike monocots such as wheat (2), cucumber leaves
are not photochemically competent when first exposed tolight, but acquire competency over an extended time period,
6 d or more in the light (5). Within these leaves photochemical
activity is not homogeneous, but develops basipetally as
shown by Chl a fluorescence transients. These data indicate

Imaging System for Measuring Chl FluorescenceInduction
As previously described in detail (10), this system consistsof a video camera with a charge coupled device imager andappropriate filters interfaced to a time code generator andvideo tape recorder. Images of the entire leaf were collected
at intervals of 17 ms, digitized by an analog to digital convertor, stored on an optical disc, and then analyzed by computer.
Two xenon lamps (380-620 nm) were used to illuminate
evenly (± 10%) the entire upper leaf surface at a flux densityof 150 ,umol photons m-2 s-'; fluorescence was measured at
683 nm. Five data points from each measured area wereanalyzed and the data averaged. The kinetics of each data
point were examined prior to averaging in order that spatial
deviations in kinetics were not overlooked.

that the primary regulation of the development of photochemical activity lies with developmental factors, not light (5).
Although the attainment of characteristic fluorescence transients progresses in a basipetal direction, mirroring the general
direction of expansion and growth of the leaf (5), the influenceof developmental factors from cells and tissues within theleaves has not been studied. The objective of the present
research was to determine if the spatial pattern of photochemical development coincided with leaf expansion and relative
growth rates, intercalary cell division, or position relative tothe vascular tissue. With the availability of dynamic imaging
of Chl a fluorescence (10), a precise, nondestructive means ofstudying these influences exists.

Fluorescence Data

The kinetic data are displayed as single curves on a log timescale. While this time basis changes the overall appearance of'Supported by a National Science Foundation grant (JGC).

1083

1 084

Plant Physiol. Vol. 93,1990

CROXDALE AND OMASA

fluorescence curves, the presentation gives good resolution of

Leaf 5
K7.5

150

CM2

the fast phases of fluorescence and compresses the slow phasesof the signal, but without loss of important characteristics offluorescence induction. The terminology of Lavorel and Ettienne (10) is used to identify the consecutive phases of thefluorescence induction signals (I, D, P, S, M, T2) and theirinterpretation is based on established criteria (4, 7, 8, 11, 19,

100
..

50

27).

Assimilation Measurements
0.

Measurements were made by an apparatus previously describedf Leaf-CO2\83cmr?
100 -in(1 7). Briefly, air was mixed from N2, 02, and 3% v/v
airusing massflow

controllers. Some of this synthetic

air passed through an aluminum leaf chamber. The air flow

nf

..0

. . :

-

_ A

through the chamber was controlled by a mass flow controller.

Some of the synthetic air and air from the leaf chamber wascompared for water content and C02 content with an IR gas
analyzer. Cross-sensitivity of the CO2 measuring section towater was eliminated by condensing the water out of the airas the air passed from the water measuring tube to the CO2
measuring tube. Leaf temperature was measured with a cop- - - - -per-constantan thermocouple probe. Calculations of assimi0 -..
lllationmade according to
Caemmerer and Farquhar
150(26). Light was provided by a quartz halogen lamp and flux

50

were

210cm2e(1)

density was the same as that used for fluorescence measurements. Water content was measured with a Dew 10 (General
Eastern) dew point hygrometer.

a)

100

RESULTSa) 50
C.)The development of photochemical activity in the Hoku-

C

U)O
0

-------------------------------

Leaf2

150

e
196cm200
Ushin cultivar of cucumber (Cucumis sativus L.) was estabby measuring fluorescence transients on the upper
lower leaf surfaces of 3-week-old plants (Fig. upper traces
are from the upper leaf surfaces). Three-week-old plants have
5 leaves expanded away from the main bud. Only the firstleaf produced, leaf 1, has completed its expansion; the re-0-lished__________

a)

a)

von

^-\_

maining

10.

and

1,

leavesstill

growing. Only on the oldest

leaf (Fig.

characteristic fluores
cence transients. The slow and fast phases of fluorescence1,leafl) did both leaf surfaces show

kinetics appeared more quickly on the lower surface of thisleaf than on the upper surface; however, the initial level of50

fluorescence and its peak value were greater on the upper sideof the leaf.
0-- - - -In developing leaves (Fig. 1, leaves 5 to 2), characteristic150

2Abbreviations: I, D, P, S, M, T, inflection points of Chl
fluorescencekinetics where I is intermediary level, D is dip, P is peak,
S is quasi-stationary level, M is second maximum, and T is terminal

Leaf 1

a

>
34
cm2

9

100

.

.

100

f
*

~level.

*~~~~~~~~~~~~-~~~

50

Figure 1. Fluorescence induction transients on the upper and lower

Cucumis sativus L.
cv. Hokushin

-

0
0.001

0.1

1.0

Time [SI10dark-incubated for at leastmphotons

30 minbefore

40

Jmol
-2
S-1;
upper traces are from the upper leaf surfaces. An
measurement

outline of each measured leaf appears with its leaf number and area

----

0.01

-d-old cucumber plant. Plants were

surfaces in the five leaves of a 21100

to the left of its fluorescence induction traces; the outlined images
are reductions, but not to scale.

'~". A-~ . ~ .

PATTERNS OF FLUORESCENCE AND GROWTH IN DEVELOPING LEAVES

200>~ 1 0
Un

C),
0

C

C)

o 2C
0 2

B~~~~~~~~~~~~~~~~~~~~~~

AA

D

10 -0
-....-.-..-......-.-..-.. -I

.

..

!:.

A

0
0.01

0.1

1.0

Figure 2. Diagram of a developing leaf (theeighth leaf from the bottom of a 31 -d-old cucumber plant) in which Chi a fluorescence was dynamically imaged; the sample points within areas
are marked with solid circles on the diagram.
The regions with areas of common kinetics (A,B, C, and D) are differentially shaded on the
diagram. A curve representative of each region
appears below the leaf diagram; the area undereach curve has been shaded for ease in distinguishing the kinetics among regions. The cucumber plant was dark-incubated for 30 min prior
to measurement at 150 Mmol photons m-2 S-1.

.:.::..:... . :. . : . . .

..-.-....-

a)
co

iC

1 085

'

10.0

100

1000

0.01

O.1

.

.

1.0

........

.

10.0

100

1and

Time [S]fluorescence transients developed slowly on each surface withthe upper side achieving typical fluorescence kinetics sooner
than the lower surface. On the upper surface the kinetics firstconsisted of I, D, and a broad P on the smallest leaf and werethen followed by the presence of an extended S region and an
expanded M peak on leaf 4. In the next older leaf, P was
quenched more rapidly, but M remained broad. The twooldest foliage leaves showed an increase in the time to P anda slight decrease in the rate of rise; the S to M to T transientswere typical of mature leaves. On the lower surface, P was
broad in the beginning and showed slow quenching throughout development. Typical transients including M were present
only on the oldest leaf. Variable fluorescence was greater on
the upper surface throughout leaf development (Fig. 1) withleaf 3 showing the largest difference between the two leaf
surfaces.
The regional differences in fluorescence response of developing leaves known in the Natsusairaku cultivar of cucumber(5) were studied in this variety by dynamic imaging. A single
leaf (similar to leaf 4 in Fig. 1) was used to determine theexact locations of variation in fluorescence kinetics. Analysis
of approximately 150 points within representative areas of
the upper side of the leaf showed four areas with different
kinetics (Fig. 2). The different transients occurred at the tips
(Fig. 2A), in a submarginal position (Fig. 2B), in the distal
two-thirds of the blade (Fig. 2C), and in the basal one-thirdof the blade (Fig. 2D).
In the tip areas, at the terminus and along the leaf margin(Fig. 2A), the fluorescence response consisted of a single,

broad P peak. These extreme tip regions may never become
photochemically competent because equivalent areas of older,mature leaves showed the same kinetics (data not shown). In
the submarginal position (Fig. 2B), the I, D, P, and M transients were present, but the initial level of fluorescence was
reduced relative to the tip areas, although peak fluorescence
was approximately the same. The interveinal areas in the
distal portion ofthe leaf (Fig. 2C) showed typical fluorescencekinetics, with all transients present. However, the proximal
region of the leaf (Fig. 2D) was characterized by kinetics
without a well-defined M peak; I and D were also absent.
To examine the development of the spatial and temporal
patterns of fluorescence in greater detail, a single area of ayoung leaf from a plant 33 d old was imaged on 3 consecutived (Fig. 3). The leaf was the tenth from the base and appearedat the edge of the main bud when the plant was between 28
and 31 d old. The leaf area measured was 20% of full
expansion on the first day it was imaged and 35% expandedon the third day of imaging. Thirteen sampling regions wereselected for analysis and the sample points were relocatedeach day on the digitized images (Fig. 3). Within the area
imaged, data are presented from three of the thirteen regions
based on their position with respect to the leaf tip, the midvein, and the margin. Data from the other ten regions showed
the same pattern of changing fluorescence kinetics. There
were no discernible changes in kinetics that were position
dependent.
The three regions showed similar kinetics on each day andsimilar changes from day to day. On the 1st d, the transients1 086

CROXDALE AND OMASA

Plant Physiol. Vol. 93,1990

consisted of a single, broad peak, P (Fig. 3, d 1). Additionaltransients began to appear on the following day, but were notclearly distinguishable (Fig. 3, d 2). By the 3rd d, all transientswere evident (Fig. 3, d 3). Figure 4 is a series of frames
showing representative fluorescence transients (I, P, M, T)from the videotape images of the fluorescing leaf diagrammedin Figure 3. Assimilation measured in an equivalent leaf
increased linearly from 2 to 10 ,umol m-2 s-' as it expanded
over a 5 d period (Fig. 5).
DISCUSSION

The fluorescence kinetics were different on the two surfacesof developing cucumber leaves of plants ranging in age from
5 to 30 days old (Fig. 1, 30 plants measured). Each surface
showed changes in fluorescence kinetics similar to the threestep sequence reported in another cultivar of cucumber (5).
Similar changes in variable fluorescence, increases in I and Dlevels, and an increase in the DP rise caused by activation of
the water splitting enzyme system are apparent during leaf
development (5).
The upper leaf surface showed characteristic kinetics earlier
and developed greater peak fluorescence than did the lower
surface. Previous measurements have shown that the lower
side has greater Fo and FM (3, 16) and that fluorescence
response on the lower surface is like that of sun leaves while
the upper surface is similar to shade leaves. The differencesamong these species may be taxa specific, or result from the

Figure 4. Electron images of the 1, P, M, and T transients of the leaf
in Figure 3 on their 3rd day of imaging. The Gray scale on the righthand side of the figure represents relative fluorescence.

developmental age of the samples and their growth environment or from changes in the measuring environment.
The greater fluorescence response on the upper surface of
cucumber leaves may be the result of increased light absorption by chlorophyll as a consequence of light scattering. Actual

7s'z sativulity-loku sr.

Figure 3. Diagram of a young leaf tip (the 10th
leaf from the bottom of a plant 33 d old) that
underwent dynamic imaging of Chl a fluorescence transients on 3 consecutive d; the sample
points within regions are marked with solid circles. Three regions have been shaded and theirfluorescence kinetics appear below; the regions
were selected based on their position relative tothe leaf tip, the midvein and the margin. The
plant was dark-incubated for 30 min prior tomeasurement at 150 /smol photons m-2 Si1.

I/

111.

I/--

PATTERNS OF FLUORESCENCE AND GROWTH IN DEVELOPING LEAVES

10-Expanding Leaf

180 cm2

(I)

E
NE

86 -

C:
0
co

4 10 cm2cn

2 -Cucumis sativus L.
cv. Hokushin

1

2

3

4

5

Day
Figure 5. Carbon assimilation measured at the same intracellular
C02 partial pressure over a 5 d period in a single developing leaf
which changed in area from 10 to 180 cm2.

measurements in the interior of cucumber cotyledons (24)and bifacial Medicago sativa leaves (25) support this view.
The initial level of fluorescence was always greater on theupper side of developing cucumber leaves. This could occur
if the number of antenna molecules were greater or if energy
distribution between the reaction centers and photosystems
favored an increased density of excited PSII pigments (17).
As cucumber leaves expanded, the time to P increased as
the rate of the rise to P declined, indicating changes in PSII
acceptors (7, 15). The maximum changes in the time and riseto P coincided with the narrowing of the M peak in leaves 1
and 2 (Fig. 1). In contrast, in developing pea leaves the timeto P declines linearly with age (20). Quenching mechanisms
in cucumber developed over an extended time period on bothsides of expanding leaves (Fig. 1), but were more protracted
on the lower leaf surface. The delay may be a response to thespectral environment seen by these chloroplasts (25) andwould be consistent with photosynthetic properties of chloroplasts isolated from successive, vertical regions of the mesophyll (2 1-23).
The fixation of CO2 was minimal or lacking in younger
cucumber leaves, even though they had been exposed to lightfor one or more days. Evidence of this was shown by theabsence of M (Figs. 1, 2, and 4) (27) and the direct measurement of carbon assimilation (Fig. 5). The M transient appeared first on the upper side of leaf 4 and later on the lower
surface in the older leaves, 2 and 3. Young pea leaves alsolack M (20). However, once present the time to M declines
with leaf age in both peas and cucumber (Fig. 1). While M
appears and/or oscillates under certain physiological conditions in mature leaves (19, 27), its presence is also a normal

1 087

feature of leaf development and a useful indicator of carbon
metabolism.
The pattern of fluorescence kinetics within a developing
cucumber leaf (Fig. 2) does not accurately coincide with thepattern of dicot leaf expansion and growth. Four regions withdifferent fluorescence responses occur in this leaf, but theseregions do not correspond spatially to areas of growth. Although a comparison of the two large interveinal areas (Fig.
2, C and D) showed that the distal region achieved characteristic kinetics prior to the proximal area signifying basipetal
development, two other areas of fluorescence (Fig. 2, A andB) were present at the leaf margin and each extended longitudinally from the tip to the base. Studies of dicot leaf
expansion (6, 18), including cucumber (1), show that sectors
differing in growth rate extend horizontally across the entire
blade and frequently curve basally at the leaf margin. Thus,
there are qualitative differences between the location of maxima in fluorescence kinetics and in relative growth in the leaf.
The marginal areas of the leaf which developed photochemical activity according to a program independent of the central
blade region have spatial correspondence with known patternsof division within leaves (12, 14). In a clonal analysis of
tobacco leaf development, selected clones are restricted to theleaf edge and contributed to elongation growth at the marginand not growth in blade width. These clonal regions occupy
the same location as the marginal areas with different fluorescence induction kinetics (Fig. 2B). The tips that are foundalong the entire length of the leaf margin never developed thefluorescence response characteristic of mature leaves (Fig.
2A). This is probably related to the structural differences oftissue at the leaf edge. The two-tiered mesophyll region at theinterior of the leaf gradually changes to a more or less homogenous group of cells without intercellular spaces at theleaf margin. The venation and stomatal distribution changes
at the leaf edge may also serve to isolate the tips from CO2
and nutrients carried by the conducting tissue. The lack of acharacteristic fluorescence response in the leaf tips may be areflection of the changes in structural and physiological aspects in this area.
The proximal area of the developing cucumber leaf (Fig. 4)
is reminiscent of primordial areas occurring between thelateral veins of young tobacco leaves (12). The homogeneityofthis region in cucumber may reflect a clone of cells specified
to function early in leaf development. The specification offunction may be carried through cell division to yield a cellgroup of photochemical uniformity. Although all groups eventually achieve characteristic fluorescence responses, during
development several regions of different photochemical activity appear on a single leaf. According to this scheme, occasionally adjacent cells should be functionally asynchronous
because each belongs to a different clone and each was originally specified at a different time. This could be explicitly
demonstrated using a cucumber mutant with variegated foliage leaves or by inducing clones on developing leaves (13).
Differences in the micro growth environment within these
expanding leaves may also be involved in the spatial development of photochemical activity. Mesophyll near vascularbundles or the leaf margin are likely locations for such differences. Light intensity, pigment composition and concentra-

1 088CROXDALE AND OMASA

tion, water potential and stomatal function undoubtedly differ
in cells from different regions of the leaf, thus contributing to
the spatial differences in photochemical activity. Water potential must be different throughout developing leaves in orderthat immature areas can continue to extend. Some of these
physiological parameters may be coupled to cell ancestry anddevelopment of the photochemical apparatus, while others
are regulated differently and not in synchrony with photosynthetic development.
There was no evidence that the proximity of the vascular
tissue influenced development of photochemical function as
in corn (9). Imaging of the tip of a young cucumber leaf onconsecutive days showed homogeneity in kinetics in the interveinal areas with similar changes occurring at all points on
a day to day basis (Fig. 4). The fluorescence responses did notvary with distance from leaf veins but were uniform throughout the intercostal areas. Thus, position influences or positional sensing appears not to modify the attainment offunction.
Dicotyledonous leaves have complex patterns of photochemical activity during their expansion and maturation.
These patterns do not coincide spatially with areas of relative
growth rate in leaves, although there is a basipetal progression
in the acquisition of the characteristic phases of fluorescenceinduction. The configuration of areas with comparable fluorescence responses shows the greatest resemblence to cellclones established early in leaf growth. The resulting mosaic
of photochemical activity indicates that cells of common
ancestry show functional uniformity.
ACKNOWLEDGMENTS
The authors acknowledge Mr. Takashi Machida and Mrs. Hatsu
Morioka of the National Institute for Environmental Studies and Mr.
Keilchiro Chiba of Nihon University for their assistance with theimaging aspects of this study, Tom Sharkey for his advice and use of
his gas exchange equipment, and Kandis Elliott for preparation ofillustrations.

LITERATURE CITED

1. Archer R, Lockhart J, Deshmukh R (1973) On the distributionof leaf area growth. Technical Report No. EM73-2. Engineering Research Institute, University of Massachusetts, Amherst,
MA
2. Baker NR, Webber AN, Bradbury M, Markwell JP, Baker M,
Thornber JP (1984) Development of photochemical competence during growth of the wheat leaf. In JP Thornber, LA
Staehelin, RB Hallick, ed, Biosythesis of the Photosynthetic
Apparatus: Molecular Biology, Development and Regulation.
R. Liss, New York, pp 237-255
3. Bjorkman 0, Demmig B (1987) Photon yield and oxygen evolution and chlorophyll fluorescence characteristics at 77K among
vascular plants of diverse origins. Planta 170: 489-504
4. Bradbury M, Baker NR (1981) Analysis of the slow phases of
the in vivo chlorophyll fluorescence induction curve. Changes
in redox state of photosystem electron acceptors and fluorescence emission from photosystems I and II. Biochim Biophys Acta 635: 542-551
5. Croxdale JG, Omasa K (1990) Chlorophyll a fluorescence andcarbon assimilation in developing leaves of light-grown cucumber. Plant Physiol 93: 1078-1082
II

Plant Physiol. Vol. 93,1990

6. Erickson RO (1966) Relative elemental rates and anisotropy ofgrowth in area: a computer programme. J Exp Bot 17: 390403
7. Krause GH, Briantais J-M, Vernotte C (1981) Two mechanismsof reversible fluorescence quenching in chloroplasts. In G
Akoyunoglou, ed, Photosynthesis. I. Photophysical ProcessesMembrane Energization. Balaban International Science Services, Philadelphia, pp 575-593
8. Krause GH, Weis E (1984) Chlorophyll fluorescence as a tool inplant physiology. II. Interpretation of fluorescence signals.
Photosynth Res 5: 139-157
9. Langdale J, Zelitch I, Miller E, Nelson T (1988) Cell positionand light influence C4 versus C3 patterns of photosynthetic
gene expression in maize. EMBO J 7: 3643-3651
10. Lavorel J, Ettienne AL (1977) In vivo chlorophyll fluorescence.
In J Barber, ed, Primary Processes of Photosynthesis. Elsevier,
Amsterdam, pp 203-268
11. Omasa K, Shimazaki K-I, Aiga I, Larcher W, Onoe M (1987)
Image analysis of chlorophyll fluorescence transients for diagnosing the photosynthetic system of attached leaves. Plant
Physiol 84: 748-752
12. Papageorgiou G (1975) Chlorophyll fluorescence: An intrinsic
probe of photosynthesis. In Govindjee, ed, Bioenergetics of
Photosynthesis. Academic Press, New York, pp 319-371
13. Poethig RS, Sussex IM (1985) The developmental morphology
and growth dynamics of the tobacco leaf. Planta 165: 158-169
14. Poethig RS, Sussex IM (1985) The cellular parameters of leaf
development in tobacco: a clonal analysis. Planta 165: 170184
15. Schreiber U, Fink R, Vidaver W (1977) Fluorescence inductionin whole leaves: differentiation between the two leaf sides andadaptation to different light regimes. Planta 133: 121-129
16. Sharkey TD, Seemann JR, Berry JA (1986) Regulation of ribulose-1,5-bisphosphate carboxylase in response to changing partial pressureof 02 and light in Phaseolus vulgaris. Plant Physiol
81: 788-791
17. Shimazaki K, Ito K, Kondo N, Sugahara K (1984) Reversible
inhibition of the photosynthetic water-splitting enzyme system
by S02-fumigation assayed by chlorophyll fluorescence andEPR signal in vivo. Plant Cell Physiol 25: 795-803
18. Silk WK (1983) Kinematic analysis of leaf expansion. In JE Dale,
FL Milthorpe, eds, The Growth and Functioning of Leaves.
Cambridge Press, Cambridge, pp 89-108
19. Sivak M, Walker DA (1985) Chlorophyll a fluorescence: can it
shed light on fundamental questions in photosynthetic carbon
dioxide fixation? Plant Cell Environ 8: 439-448
20. Somersalo S, Aro E-M (1987) Fluorescence induction in pea
leaves of different ages. Photosynthetica 21: 29-35
21. TerashimaI, InoueY (1984) Comparative photosynthetic properties of palisade tissue chloroplasts and spongy tissue chloroplasts of Camellia japonica L.: functional adjustment of thephotosynthetic apparatus to the light environment within aleaf. Plant Cell Physiol 25: 555-563
22. Terashima I, Inoue Y (1985) Palisade tissue chloroplasts andspongy tissue chloroplasts: biochemical and ultrastructural differences. Plant Cell Physiol 26: 63-75
23. TerashimaI, InoueY (1985) Vertical gradient in photosynthetic
properties of spinach chloroplasts dependent on intra-leaf lightenvironment. Plant Cell Physiol 26: 781-785
24. Vogelmann TC, Knapp AK, McClean TM, Smith WK (1988)
Measurement of light within thin plant tissues with fiber optic
microprobes. Physiol Plant 72: 623-630
25. Vogelmann TC, Borman JF, Josserand S (1989) Photosynthetic

light gradients and spectral regimen within leaves of Medicago
sativa. Philos Trans R Soc Lond B 323: 411-421
26. von CaemmererS, Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchangeof leaves. Planta 153: 376-387
27. Walker DA (1981) Secondary fluorescence kinetics of spinach

leaves in relation to the onset of photosynthetic carbon assimilation. Planta 153: 273-278