PhotosynthesisResearch 45: 225-237, 1995. © 1995KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

Imaging of chlorophyll-a-fluorescence in leaves: Topography of photosynthetic oscillations in leaves of Glechoma hederacea Katharina Siebke & Engelbert Weis Institut fiir Botanik, Lehrstuhl fiir Botanik/Pflanzenphysiologie,

Schlossgarten 3, D-48149 Manster, Germany

Received 19 April 1995;acceptedin revisedform 8 August 1995

Key words: chlorophyll-a-fluorescence, imaging, oscillations, photosynthesis, minor veins

Abstract

Images of chlorophyll-a-fluorescenceoscillations were recorded using a camera-based fluorescence imaging system. Oscillations with frequencies around 1 per min were initiated by a transient decrease in light intensity during assimilation at an elevated CO2-concentration. The oscillation was inhomogenously distributed over the leaf. In cells adjacent to minor veins, frequency and damping rate was high, if there was any oscillation. In contrast, the amplitude was highest in Cells most distant from phloem elements (maximal distance about 300/2m). The appearance of minor veins in oscillation images is explained by a gradient in the metabolic control in the mesophyll between minor veins and by transport of sugar from distant cells to phloem elements. The potential of fluorescence imaging to visualize 'microscopic' source-sink interactions and metabolic domains in the mesophyll is discussed.

Abbreviations: Pi - inorganic phosphate; Fru2,6BP- fructose-2,6-bisphosphate; FBPase- fructose- 1,6-bisphosphatase; SPS - sucrose-phosphate synthetase; H P - hexosephosphate Introduction

Oscillations in the rate of photosynthesis are well known and have been the subject of extensive studies (see e.g. Van der Veen 1949; Walker 1992; RydePettersson 1992; Giersch 1995). Typically, they are sinusoidal with a period in the range of about one minute. In contrast to 'free-running' metabolic oscillations, such as glycolytic oscillations (Hess and Boiteux 1971), photosynthetic oscillations seem to be more or less damped and tend to die out after a number of periods. Oscillations may be initiated in leaves photosynthesizing at a high rate by various kinds of sudden change in external parameters such as light, atmospheric CO2 or O2-concentration. Photosynthetic oscillation seems to be a 'manifestation of over-reactions of regulatory mechanisms' (Walker 1992). The occurrence of oscillations and their parameters, such as amplitude, frequency and damping rate, depend on the metabolic state of the leaf mesophyll. Sequestering of Pi in the mesophyll stimulates

oscillations whilst feeding phosphate favours damping (Harris et al. 1983; Walker and Sivak 1985; Sivak and Walker 1987; Stitt and Schreiber 1988). Generally, oscillation can be initiated when the assimilation rate is controlled by triose-phosphate export and inadequate recycling of free Pi ('O2-insensitive assimilation'; Sharkey et al. 1986; Leegood and Furbank 1986). Therefore, it may be regarded as a symptom of the contribution of 'internal' metabolic factors to control of assimilation. Like glycolytic oscillations (Hess and Boiteux 1971; Schellenberger et al. 1985; Yuan et al. 1991; Das and Busse 1991), photosynthetic oscillations are accompanied by inverse oscillations in the ATP/ADP and NADPH/NADP-ratio, and in other metabolite pools related to these ratios (Furbank and Foyer 1986; Stitt 1986; Stitt et al. 1988a; Laisk et al. 1991; Giersch 1995). The electron transport chain and the glyceraldehyde-phosphate-dehydrogenasereaction are the two coupling points for the phosphorylation- and redox-state of the metabolites. Competition for ATP

226 by 3-phosphoglyceraldehyde-kinase and ribulose-5phosphate-kinase reactions in the Calvin cycle (Giersch 1986; Walker et al. 1983) or an imbalance between ATP- and NADPH-formation by electron transport (Laisk et al 1991; Giersch and Rover 1995) have been considered as of possible significance. The capacity of cytoplasmic sucrose synthesis is adjusted to sucrose demand and export (Foyer 1990). Imbalances between stromal reactions and cytosolic sucrose synthesis seem to play a major role in the initiation of oscillation (Sharkey et al. 1986; Leegood and Furbank 1986; Stitt et al. 1988a; Rover and Giersch 1995). The signal metabolite Fru2,6BP, which regulates sucrose synthesis at the cytosolic fructose-l,6bisphosphatase (Stitt et al. 1987), also regulates the glycolytic phosphofructokinase and the Fru2,6BP regulation system is considered to be involved in glycolytic (Schellenberg et al. 1985) as well as photosynthetic (Stitt et al. 1988a) oscillations. In this report, we record and characterize the topography of photosynthetic oscillations in leaves of a higher plant, Glechoma hederacea L. by means of chlorophyll-a-fluorescence imaging. Daley and coworkers (1989) were the first to derive quantitatively the spatial distribution of photosynthetic parameters from images of chlorophyll-a-fluorescence. Fluorescence imaging has been used to visualize the topography and dynamics of photosynthesis during stomata movement and stomata-related oscillations (Patzke 1990; Cordon et al. 1994; Genty and Meyer 1994; Siebke and Weis 1995) and after virus infection in leaves (Balachandran et al. 1994). Chlorophyll-a-fluorescence has proved to be a noninvasive optical tool with high diagnostic value in photosynthesis research (Krause and Weis 1991). There exists a complex but surprisingly robust empirical relationship between fluorescence quenching and the rate of electron transport, relative to light absorption in leaves (Weis and Berry 1987; Genty et al. 1989; Krause and Weis 1991). During photosynthetic oscillations, steady state fluorescence oscillates antiparallel to the rate of oxygen evolution, with a small phase shift, the

changes in fluorescence proceeding those in the oxygen evolution (Stitt and Schreiber 1988; Walker et al. 1983; Walker 1992). Variations in fluorescence are caused by 'photochemical' and 'non-photochemical' quenching. In this study, fluorescence images of a leaf were taken at 10 s time intervals by a digitized video camerasystem and analyzed pixel-by-pixel by computer processing. From these fluorescence images we derived images of oscillation parameters such as amplitude, frequency and damping rate and conclusions could be made about the spatial distribution of metabolic control of assimilation. The most intriguing result of this study is the appearance of the minor vein distribution in oscillation images. A network of minor veins is embedded between the palisade parenchyma and the spongymesophyll. It contains xylem and phloem elements. Sugar, mainly sucrose, produced in distant mesophyll cells diffuses to the vicinity of minor veins apoplasmic or symplasmic cell-to-cell via plasmodesmata ('short distance sugar transport'), and is then taken up actively by phloem elements, via companion cells. G. hederacea belongs to the family of Lamiaceae, where sugar transport is likely to occur symplasmic (Gamalei 1986; Gamalei et al. 1989; see Bush 1992; Turgeon and Bebe 1991). As the phloem loading zone represents a primary 'sink' for most photoassimilates produced in the mesophyll, the minor vein boundaries are of particular interest with respect to metabolic control of photosynthesis.

Material and m e t h o d s

Plants of Glechoma hederacea were harvested from a shady place in the botanical garden in September and tendrils were cut. Attached leaves were placed in a gas exchange chamber. Steady-state gas exchange was measured with a two channel gas flow system, essentially as described before (Siebke and Weis 1995). The gas flow

Fig. 1. (a) Oscillations in chlorophyll-a-fluorescence in a leaf of Glechoma hederacea L. The light intensity was 450 # m o l quanta m - 2 s - t. Oscillations were initiated by a decrease in light intensity from 450 to 150/zmol quanta m - 2 s - 1 for 5 min followed by a sudden return to 450 /zmol quanta m - 2 s - l . CO2-concentration (time: 0 s) 1750 # m o l m - 2 s - 1 , oxygen concentration 2%. In the steady state the assimilation rate was: 9.7 CO2 # m o l m - 2 s - 1 . The fluorescence yield is normalized to the fluorescence yield of the dark adapted leaf. The curve represents

average value of the entire leaf. (b). Fluorescenceimages. For a better optical comparison, the gray scale for fluorescenceintensity was

calibratedto meanvalue(m.v.)and standarddeviation(s.d., meanvalue + standarddeviation;-s.d., 2.s.d., -2-s.d., likewise).Left side: dark adapted leaf (meanvalue: 129+/-22). Right side: image taken about 10 s aftera fluorescencemaximum(50 s) as indicatedby the arrow in Fig. la. (meanvalue: 104+/-18).

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228 rate through the cuvette was 550 ml min -1, the leaf temperature was maintained at 23 °C, relative humidity at 72-73%. Concentration of atmospheric CO2 and 02 was 1750 #11 - l and 2%, respectively. The computer-controlled video-camera-system used for image processing is similar to that described recently (Siebke and Weis 1995). Images were taken from fluorescence elicitated by 'saturating light pulses' and from 'steady state fluorescence' elicitated by continuous illumination. Each image was calibrated against the emission of a solid fluorescent standard (Heinz Walz, Effeltrich, Germany) mounted into the leaf chamber. The video camera system (CCD b/w camera KP MIE/K Hitachi Denishi Ltd. Japan with zoom optic 18-108 mm; frame grabber: ITI OFG-KITC2-AT Stemmer PC-Systeme, Puchheim, Germany) has a resolution of 256 grey steps. The light source was a tungsten halogen projector lamp, which provided a homogeneous light. It was defined by the following filters: Schott KG1, OCLI heat mirror, Corning 9782 and Schott neutral density filters. The camera is protected by a Schott RG 665 filter. Continuous illumination with actinic light for photosynthesis was obtained by providing the light source with different neutral density filters. A 1 s saturating light pulse (SLP, 1000 #mol quanta m -2 s - l ) was given by rapidly removing a neutral density filter. For further details see Siebke and Weis (1995). For microscopic observations detached leaves of G. hederacea were fed with Naphthalene Black B (Serva, Heidelberg, Germany) via the petiol to stain the veins.

Results

The experiment shown in Fig. la was performed with a leaf of Glechoma hederacea L. placed in a gas exchange chamber. The temperature was kept at 23 ° C. CO2 and H20 exchange were recorded and fluorescence images were taken at 10 s intervals. At a light intensity of 450 #mol quanta m -2 s-~ the steady-state rate of assimilation was 9.7 #mol CO2 m -2 s-l (transpiration rate: 530 mmol H20 m -2 s-l). A decline in light intensity to 150 #mol m -2 s -1 for 1 to 5 min, followed by a rapid return to 450 #mol quanta m -2 s - l , induced a transient increase in fluorescence and subsequent oscillations. The fluorescence parameter Fs represents the fluorescence signal elicitated by continuous actinic light (450 #mol quanta m -2 s - l ) normalized to the maximal fluorescence signal dur-

ing a 'saturating light pulse' given to the dark adapted leaf, FM. The oscillations were sinusoidal and damped. After 5 or 6 periods, the oscillations had nearly died out and fluorescence returned to the original steadystate level. The trough level of periods remained close to the level of steady-state fluorescence and did not change very much. In other words, fluorescence oscillations described here could be regarded as periodic overshooting in the fluorescence level. The maximal amplitude of oscillation was about 30% of the steadystate signal (Fs = 0.22), which was close to the initial dark level of fluorescence, Fo (as observed with a PAM-fluorimeter; not shown). In a number of studies it was demonstrated quite clearly that a stable inverse relationship exists between oscillations in fluorescence and oscillations in the rate of oxygen evolution, the fluorescence signal proceeding the oxygen evolution by a small phase shift (Walker 1982). Thus, we can, as a first approximation, take oscillations in Fs as the reciprocal of oscillations in the rate of photosynthetic electron transport. Figure lb displays fluorescence images of the source leaf. The image on the left side was taken from the dark adapted leaf during a 'saturating light pulse'. In this state, fluorescence yield is assumed to be maximal and no fluorescence quenching related to electron transport processes is expected. The image exhibits a rather homogeneous distribution. Major veins appear as low-fluorescent (dark) lines, minor veins as thin weak lines with a fluorescence level not more than 15% lower than that in the surrounding area. The horizontal lines are artifacts caused by nylon strings in the cuvette. The right-hand image was taken during an oscillation, about 10 s after the first peak, as indicated by the arrow in Fig. la. Here, low fluorescence areas appear along minor veins, while cells further from minor veins, in the center of 'areols' (segments between minor veins), appeared as high fluorescent spots. To display details of images the camera lens was zoomed in (Fig. 2). Oscillations were again initiated by decreasing the light intensity for one minute to 150/zmol quanta m -2 s -I , followed by rapid increase to 450 #mol quanta m -2 s -1. In Fig. 2a, six images are displayed which were taken in 10 s time intervals between the second and the third fluorescence maximum, as indicated in Fig. 2c. Figure 2b represents cross section profiles of fluorescence, taken from all six images, along a line displayed in the image of the 110 s, normalized to the lowest fluorescence value recorded after 360 s. Each cross section in Fig. 2b is plotted from 30 image points and includes two minor

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b) n,

minor vein ..3 time, s 1 I -100 . . . . ..** -r-110 ..2 ==.. .‘ .:-._. _:. -.-._ 120 .i 1C=r,l+* -‘ . \ . ----130 T‘ h@ \ . ... ,