Plant Physiol. (1990) 93, 1078-1082
0032-0889/90/93/1 078/05/$01 .00/0

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

Chlorophyll a Fluorescence and Carbon Assimilation in
Developing Leaves of Light-Grown Cucumber1
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.)
ABSTRACTin development, and that the remainder of the leaf becomescompetent and may increase in efficiency over time (6).
Within developing leaves this is indicated by their patterns ofcarbon import and export (1 1, 28) and lack of synchrony in
Chl content and Hill activity (8) or Chl and rubisco activity(2, 27). However, a direct demonstration of changes in photochemical activity within regions of a leaf has been lacking.
The objective of this work was to determine if developing
cucumber (Cucumis sativus L. cv Natsusairaku) leaves andthe regions within them acquire the capacity for photochemical function simultaneously or sequentially in a pattern related to their expansion and maturation. Imaging of Chl afluorescence of entire leaves (16) now makes it possible tomonitor photochemical activity nondestructively and compare induction kinetics in selected leaf areas. The goal was tolocalize in the developing leaf the first occurrence of characteristic phases of fluorescence kinetics and to complement
this with gas exchange measurements on equivalent leaves togive information on carbon assimilation.

The development of photochemical activity and carbon assimilation in light-grown cucumber (Cucumis sativus L. cv Natsusairaku) leaves was studied to determine the pattern of acquisition
and its relationship to leaf growth and expansion. Measurementsof chlorophyll a fluorescence showed that leaves acquire photochemical function over a period of 6 or more days, and gas
exchange studies showed increases in carbon assimilation overa parallel time period. As leaves expand and mature, they
undergo a sequential, three-step series of changes in fluorescence response. The initial kinetics show the absence of whollyfunctional quenching mechanisms. Dynamic imaging of fluorescence kinetics showed that a temporal series of changes occurred within defined areas of individual developing leaves. The
spatial acquisition of photochemical activity in leaves was basipetal as is their directional expansion, development of air spaces
and stomata, and the cessation of imported carbon.

Biochemical and physiological studies of developing photosynthesis in monocot leaves show that young cells just above
the basal meristem are photosynthetically competent as soon
as they are in the light (1). The reaction centers of bothphotosystems are functional, energy passes from PSII to PSI
through the chain of electron carriers, and water is split
photochemically. Although the basal cells of 4-d-old leaves,
but not 8-d-old leaves, are unable to perform state I to state
II transitions, photosynthetic activity is otherwise homogeneous throughout the leaf (1). The functional homogeneity
may be related to the single locus of origin of most leaf cells,
the basal meristem.
Typical dicot leaves grow in a more complex fashion than
monocots (15). Cells within the leaf blade have numerous
origins and the blade exhibits mosaic growth, i.e. meristematic
cells scattered throughout the leaf divide and expand to produce clusters of cells until expansion is complete (19). In spiteof this mosaic growth, dicot leaves expand anisotropically inbanded-sectors. The magnitude of the relative growth rate ineach sector increases basipetally (23).
This pattern of leaf growth results in heterogeneity inchloroplast and photosynthetic development within each leaf,
consequently making their study more difficult than monocots (14). Older studies have established that the capacity forphotosynthesis is attained by certain regions of the leaf early'Supported by a National Science Foundation grant (JGC).

MATERIALS AND METHODS
Plant Materials and Leaf Area Measurements

Cucumber (Cucumis sativus L. cv Natsusairaku) plants
were grown as previously described (16) for up to 8 weeks.
For fluorescence and gas exchange measurements, plants werebrought from the controlled-environment greenhouse to thelaboratory and allowed to acclimate for about 2 h at 25 C.
Plants were dark-incubated for at least 30 min before fluorescence induction. 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 the upper leaf surfaces in interveinal areas
midway between the midvein and leaf margin. Light of 670
nm at 40 jLmol photons m-2 s-' was used for excitation andfluorescence was measured at >710 nm. Data were collected
at 30 ms intervals over a period of 400 s, stored, and analyzed
by computer.
1078

Chl FLUORESCENCE IN DEVELOPING CUCUMBER LEAVES

Imaging System for Measuring Chi FluorescenceInduction
As previously described in detail (16), 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 evenly illuminate (± 10%) the entire upper leaf surface at fluxes of 80 and
150 /tmol m-2 s-', depending on leaf size; fluorescence wasmeasured at 683 nm. Five data points from each measured
area (less than 2 mm-2) were analyzed and the data averaged.

Fluorescence Data

The kinetic data are displayed as single curves on a log timescale. While this time basis changes the overall appearance offluorescence curves, the presentation gives good resolution of
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 (13) is used to identify the consecutive phases of thefluorescence induction signals (I, D, P, S, M, T2), and theirinterpretation is based on established criteria ( 12, 17, 24, 30).
Assimilation Measurements

Measurements were made by an apparatus previously described (21). Briefly, air was mixed from N2, 02, and 3% v/v
CO2 in air using mass flow controllers. Some of this synthetic
air passed through an aluminum leaf chamber. The air flow
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 CO2 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 copper-constantan thermocouple probe. Calculations of evaporation, conductance to gas exchange, photosynthesis, andintercellular CO2 partial pressure were made according to vonCaemmerer and Farguhar (29). Light was provided by a quartz
halogen lamp and flux was measured with a LiCor quantum
sensor. Water content was measured with a Dew 10 (General
Eastern) dew point hygrometer.

displayed an asymmetric curve consisting of a single, broad P
peak, quenched over a 400 sec interval. No other transientswere present. The next two leaves showed increased variablefluorescence, a steeper rise to P, but continued slow quenchingof P as in the youngest leaf; other transients were absent. The
fourth leaf showed the typical pattern of fluorescence kinetics
with increased variable fluorescence and the presence of I, D,
S, and M transients. The area of the leaves measured in these
reproductive plants ranged from 1.5 to more than 300 cm2.
Dynamic Imaging of Fluorescence Transients within
Developing Leaves
Four-week-old cucumber plants also showed progressivechanges in kinetics; the changes occurred among leaves andwithin the base, middle, and tip regions of leaves (Fig. 3).
Beginning with leaf 9, just expanded away from the bud,different kinetics were found in the three leaf regions. The
leaf tip showed the largest variable fluorescence and thepresence of P and an indication of M. The middle and basal
regions of this leaf had less fluorescence and only a single,
broad P peak that was quenched slowly. The next two older
leaves, 8 and 7, showed similar patterns of fluorescence transients, with increases in variable fluorescence, especially inthe tip area, but also in the middle region of leaf 7. The P, S,
and M transients were more evident and the D to P rise wassteeper in leaf 7 than in the younger leaves. In leaf 6, thevariable fluorescence decreased in the tip, but the characteristic kinetics remained. The base and middle area of this leaf
continued to show a curve with a single peak. In leaf 3, alarge, mature leaf of more than 400 cm2, the characteristic
fast and slow phases of Chl a fluorescence were present andsimilar in the middle and basal regions of the leaf. Variablefluorescence declined in the leaf tip. Imaged leaves ranged inarea from 10 to 400 cm2.

Assimilation in Developing Leaves
The upper surface of cucumber leaves had modest levels of
assimilation regardless of their developmental age and posi-

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Chi a Fluorescence of Developing Leaves

Mature leaves of 8-week-old reproductive plants showed
typical Chl a fluorescence transients, with I, D, P, S, M, andT clearly evident (Fig. 1). However, these points could not be
distinguished in young leaves (Fig. 2). The youngest leaf
Abbreviations: I, D, P, S, M, T, inflection points of Chl afluorescence kinetics where I is intermediary level, D is dip, P is peak,
S is quasi-stationary level, M is second maximum, and T is terminal
level.

050

Cucumis staivus L.
cv. Natsusairaku
0

0.01

0.1

I
1.0

10

100

Time [S]2Figure 1. Chl a fluorescence transients measured in midregion of amature leaf, greater than 300 cm2 in area, on an 8-week-old cucumber
plant.

T,5

1 080

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Plant Physiol. Vol. 93, 1990

CROXDALE AND OMASA

200

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Cucumis sativus L.
cv. Natsusairaku

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Time [S]Figure 2. Fluorescence induction transients in the four youngest
leaves of an 8-week-old, reproductive cucumber plant. The areas ofleaves A to D were 1.5, 3.5, 8, and 70 cm2, respectively; fluorescencemeasurements were made in the middle of each leaf.

-----.

100
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DISCUSSION
Cucumber leaves were not photochemically competent assoon as exposed to light. Characteristic Chl a fluorescencekinetics developed (Fig. 2) during a period of six or moredays. Since the leaves are fully illuminated during this time,developmental factors rather than light play the major role inregulating the development of photochemical activity. The
lengthy development of photochemical activity in cucumber
is difficult to reconcile with the expression of photosynthetic
genes which is temporally regulated initially and begins whenleaves are within the bud and shielded from light (3, 5, 9, 10,20). Pea leaves also require several days of light before fluorescence transients are typical of mature leaves (26). In cucumber, this pattern of development was found in vegetative,
4-week-old plants (Fig. 3) and reproductive, 8-week-old plants(Fig. 2), indicating that the development ofthe photochemical
apparatus follows the same course regardless of plant age or
reproductive status.
Changes in Chl a fluorescence in developing leaves occurredin a three-step sequence. First, only a single, broad peak, P,
was present. The shallow rise to P was accompanied by alengthy quenching period. Due to the functional connectionsof PSII with other photosynthetic components, this mightsignify limited development and function of PSII components( 17), a deficiency of one or more PSII acceptors or plastoquinone (12), and the absence or inefficiency of quenchingmechanisms (12).
Since the photoreduction of Q through reductant fromwater is reflected in the DP rise (12, 17) and a diminished risecan be induced by inhibiting the water-splitting enzyme sys-~~~~~~eaf 7C:

0.1200tion on these vegetative plants (Fig. 4). In contrast, assimilation on lower leaf surfaces was minimal in the youngest leaf,
then rose to a peak in the middle leaves, and declined inolder, fully expanded leaves.

~

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200

100

Tip

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415
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MiddleLeaf

100-------

Cucumis sativus

0
0.01

0.1

1.0

L.

10

cv.

Base

Natsusairaku
100Time [SIFigure 3. Dynamic imaging of Chl a fluorescence in the tip, middle,
and base regions of leaves along the stem of a 4-week-old, vegetativeplant of cucumber. Leaves are numbered according to age with leaf

9 being the youngest leaf, just expanded away from the bud. Plants
were dark-incubated for a minimum of 30 min prior to imaging offluorescence transients. Leaves 9 to 6 were exposed to 150 timol

1081

Chi FLUORESCENCE IN DEVELOPING CUCUMBER LEAVES

tem with SO2 fumigation (22), the slow rise in developing
cucumber leaves indicated the absence of the enzyme systemor its incomplete function. The PS decline is due to photochemical and nonphotochemical quenching (1, 12). The slow
decline of this transient in cucumber indicated an inhibitionof electron flow from Q to PSI and the lack of a transthylakoid proton gradient because of the missing or incompletely developed water-splitting enzyme system. The absenceof M in these fluorescence responses may indicate that carbon
fixation is not occurring (24, 30).
The second step in the sequence was an increase in variablefluorescence attended by a steeper rise to P and the appearanceof the remaining characteristic transients (I, D, S, M). In the
final step of the sequence, the level of variable fluorescence
declined, but the kinetics remained characteristic. Declines influorescence may be caused by fluorescence reabsorption by
the increased chlorophyll content in mature leaves (4).
The development of fluorescence kinetics varies among
dicot species (25, 26). In pea the time to P and M decreases
as leaves complete their expansion (26), while in cucumber
the time to P increased (Fig. 3). This occurred as the DP riseand variable fluorescence increases. An increased time to M
has been reported in cucumber (25), but did not occur inleaves of this study (Fig. 3). The quenching of P in young pea
leaves initially increases and then declines as they age (26),whereas in developing sunflower (25) and cucumber leaves(Figs. 2 and 3), the rate of quenching after P continuously
increases. The variation among species indicates that there
are differences in the synthesis of individual components,
their assembly into functional photochemical units, and/orinterrelations between photochemical and assimilatory elements.
The ordered changes in the kinetics of fluorescence transients occurred among and within leaves, with one or morestages in the transition being present in a single leaf. Thus,
photochemical activity was not acquired uniformly withinthese dicot leaves as it is in monocots (1). Instead kinetics
within developing leaves were heterogeneous. Since theseleaves are still developing, water potential, internal CO2 partialpressure, and capacity for stomatal function may not be
uniform throughout. However, porometer measurements
made on equivalent leaves of single plants (data not shown)indicated that stomatal resistance declined as the temperature
decreased and light availability increased. Therefore, at least
a population of stomata on the leaf was functional.
Characteristic kinetics appear first at the leaf tip, then in
the middle region, and finally at the base. This progression offluorescence response follows the basipetal course of leaf
expansion and the development of air spaces (7, 15) andstomata ( 18), and parallels the cessation of carbon import tothe leaf (l 1, 28).
Gas exchange measurements cannot be made with thespatial resolution equivalent to dynamic imaging, but the
progressive increases in assimilation follow the same develphotons m-2 s-1 of exciting light and leaf 3 was exposed to 80
iAmol photons m-2 s-' of light. The decrease in flux density for leaf
3 was necessitated by its large size and the requirement for evenillumination.

'i9
8

E

0

Co

E
2

Ccunmis sativus L.
cv. Natsusairaku

9

8

7

6

5

4

3

2

Leaf number

Figure 4. Carbon assimilation measurements in a cucumber plantwith 9 leaves, equivalent to the plant imaged in Figure 3. Data forboth surfaces of each leaf are presented with the exception of thesmallest leaf for which a single value represents total assimilationfrom both surfaces. Photon flux density and air temperature were
the same as for fluorescence measurements; leaf numbering as in
Figure 3.

opmental pattern as the attainment of characteristic fluorescence transients. However, until the spatial pattern of competent stomata is known, we cannot know if the availabilityof CO2 or the photochemical machinery is limiting carbon
fixation. Based on the presence of the S to M to T transients,
the majority of assimilation was due to the leaf tip alone whenleaves are 2% of full expansion (leaf 9 of Figs. 3 and 4) anddue to the middle and tip region until the leaf was more than
30% expanded (leaf 7 of Figs. 3 and 4).
These results demonstrate that the appearance of photochemical function in dicots is fundamentally different frommonocot leaves. In dicot leaves, cells are not photochemicallycompetent when first exposed to light nor is photochemical
activity homogeneous within a leaf until late in its expansion.
Dynamic imaging of fluorescence kinetics showed that photochemical activity develops sequentially over time and progressively within defined leaf areas. The attainment of characteristic Chl a fluorescence transients and the increased
assimilation of carbon dioxide in cucumber parallels the
directional expansion of the leaf and the growth of spongy
parenchyma cells and the presence of air spaces. However,the directional extension of the leaf is superimposed over acomplex, mosaic pattern of cell division and growth within
the blade. Thus, detailed analyses of fluorescence images may
show that photochemical function is spatially distributed toreflect this intercalary growth or position with respect to thevascular tissue.
ACKNOWLEDGMENTS
The authors thank Mr. Takashi Machida and Mrs. Hatsu Moriokaof the National Institute for Environmental Studies and Mr. KeiIchiro Chiba of Nihon University for their assistance in the imaging

CROXDALE AND OMASA

1 082

aspects this study, Tom Sharkey for his advice and use of gas exchangeequipment, and Kandis Elliott for preparation of manuscript figures.

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