diffusion resistance. The induction of ETR at CO 2

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1 Sink source transition in tobacco leaves visualized using Blackwell Science Ltd chlorophyll fluorescence imaging Qingwei Meng 1, Katharina Siebke, Peter Lippert, Bernhard Baur, Ute Mukherjee and Engelbert Weis 1 Institut für Botanik, Universität Münster Schlossgarten 3, D Münster, Germany; Present address: College of Life Science, Shandong Agricultural University, Taian, Shandong Province, , PR China Summary Author for correspondence: Katharina Siebke Fax: Tel: siebke@rsbs.anu.edu.au Received: 2 January 2001 Accepted: 20 April 2001 The sink source transition of developing Nicotiana tabacum (tobacco) leaves was studied here using chlorophyll fluorescence imaging. In accordance with leaf development, the quantum efficiency of PSII, showed a steep gradient across the leaf with increasing values towards the tip. The linear electron transport rate (ETR) saturated at higher concentrations in the younger, than in the mature, part of the leaf, probably due to a lower Rubisco activity or a higher diffusion resistance. The induction of ETR at concentrations near the compensation point after long-term dark adaptation of the young leaf, showed distinct responses; ETR rose rapidly in the basal but more slowly in the apical regions. There was a correlation between fast induction and carbohydrate import, as measured by 14 C-translocation. In the basal regions, larger pools of metabolic intermediates are expected due to imported carbohydrates. These might be used in the Calvin cycle directly after dark light transition providing the electron acceptors for the faster induction of ETR. Additionally, a higher mitochondrial respiration can provide for the Calvin cycle in these regions. Key words: 14 C-translocation, chlorophyll a fluorescence, electron transport rate (ETR), imaging, leaf development, Nicotiana tabacum (tobacco), quantum efficiency of photosystem II, sink source transition. New Phytologist (2001) 151: Introduction In young expanding leaves, photosynthesis is low and carbohydrates are imported from fully developed leaves. Respiration and biosynthetic pathways are highly activated (Nelson, 1994). In dicotyledonous plants, the transition of the mesophyll from sink to a fully developed source tissue occurs in a basipetal direction over a period of several days. While cell proliferation mainly occurs at the leaf base, cell expansion and maturation proceeds in the middle part and towards the leaf tip (Turgeon & Webb, 1973; Fellows & Geiger, 1974). During expansion and maturation, the rate of photosynthesis gradually increases and the rate of dark respiration slowly decreases. The dependence on import of carbohydrates rapidly diminishes and the tissue begins to export metabolites from its own photosynthetic assimilation (Turgeon, 1989). The vein system is transformed during this transition from an importing to an exporting system. Roberts et al. (1997) established that the function of veins is related to their branching order. The largest veins, defined as first and second order veins, are involved in long distance transport only. The third order veins in young leaves are able to release imported carbohydrates into the mesophyll. Veins of higher orders, which develop later, are used for export. During the maturation of the mesophyll, the import from the third order veins decreases. In this study, we examined the transition from sink- to source-mesophyll in developing leaves of Nicotiana tabacum by means of chlorophyll fluorescence imaging. The chlorophyll fluorescence analysis is a nondestructive, quantitative measure of both photochemical and nonphotochemical energy dissipation processes in photosystem II (PSII). It can also be used to analyse photosynthetic flux and control of fluxes by metabolic processes in leaves (Weis & Berry, 1987; Genty et al., 1989; Weis & Lechtenberg, 1989; Krause & Weis, 1991). Kinetics of chlorophyll fluorescence have previously been imaged in developing cucumber leaves (Croxdale & Omasa, 1990). Recently, camera based systems for chlorophyll a fluorescence imaging have been developed which are capable of visualizing the New Phytologist (2001) 151:

2 586 distribution of the quantum efficiency of PSII (Φ PSII ) throughout a leaf. (Daley et al., 1989; Genty & Meyer, 1995; Rolfe & Scholes, 1995; Siebke & Weis, 1995). We examined photosynthetic electron transport rate (ETR), calculated from Φ PSII, and the induction of ETR in leaves that had been dark-adapted for a long period of time and subsequently illuminated in low concentrations. Materials and Methods Plant material Wild type tobacco (Nicotiana tabacum L., Samsun NN) plants were used in this study. Seeds were sown and germinated on wet paper. The seedlings were transferred to soil in plastic pots and grown in a temperature controlled glasshouse (23/18 C day/night between April and August) with additional light sources providing µmol m 2 s 1 photosynthetic photon flux density (PPFD) at the uppermost leaves. Young expanding leaves from 8 to 12-wk-old plants were used for the experiments. Gas exchange measurements Attached leaves were placed in a gas exchange chamber. Gas exchange was measured with a two-channel gas flow system, essentially as described before (Siebke & Weis, 1995). The gas flow rate through the cuvette was 1000 ml min 1, the leaf temperature was maintained at C, rh at 72 73%. The tip and the base of a leaf were measured separately by only enclosing the leaf tip or the base in the cuvette while the other part was protruding out of the chamber. Mapping of PSII photochemistry using chlorophyll a fluorescence imaging The computer controlled video-camera-system used for image processing was similar to that described in Siebke & Weis (1995) with changes made in the optical arrangement according to Genty & Meyer (1995) as described in Jensen & Siebke (1997). An image under continuous illumination (F ) and an image during a saturating light pulse with a PPFD of 3500 µmol m 2 s 1 (F M ) were used to calculate the quantum efficiency of PSII ((F M F )/F M ). The intensity of the saturating light pulse is not sufficiently saturating with high actinic PPFD. We therefore used actinic PPFDs up to 1000 µmol m 2 s 1 only for our study. From the quantum efficiency of PSII the linear electron transport rate (ETR) can be calculated, if the amount of absorbed photons are known (Genty et al., 1989). We assumed that half of the absorptance is attributed to PSII. Since we are not able to measure the distribution of light absorptance in the leaf nearly as accurately as the fluorescence distribution, we used a uniform value of 80% to calculate an approximate ETR, if not stated otherwise. We sometimes, however, measured absorptance values at the base as low as 65% meaning that this assumption introduces an error of up to 25% overestimation of ETR in the basal regions. Despite this error, we prefer to present the data here as estimated ETR rather than raw quantum efficiency of PSII, because otherwise the obtained results cannot be compared when we use different PPFD. The development of a single leaf was followed over several days. Each day we measured the photosynthetic activity at a PPFD of 350 µmol m 2 s 1 in 670 µl l 1 and 2% in a developing leaf. The plant was taken from the glasshouse and the leaf adapted for 60 min to the conditions within the cuvette. After the measurement the plant was returned to the glasshouse. Photosynthetic induction was obtained upon illumination of a leaf in 60 µl l 1 and 21% which had been covered overnight (from late afternoon until late in the next morning) for long-term dark adaptation (14 16 h). From the first images during the first s integral images were calculated with the following equation: integrated image (16bit) = Eqn 1 n k = 1 image k ( 8bit) + image k+ 1 ( 8bit) ( time k+ 1 time k ) time tot (time k, the time of image k ; and time tot, the total integration time.) The result represents an image of the sum of electrons transported during the induction time, if the scale values are multiplied with time tot and the absorbed photons. Light absorptance was measured using a tungsten halogen lamp using the same blue filter set as in the measuring light for the fluorescence measurement, a self-made Taylor integrating sphere, and a quantum sensor (LI-190 SA, Li-Cor, Lincoln, NE, USA). The leaf area measured was 0.5 cm labelling and autoradiography After images of chlorophyll fluorescence were taken from a young leaf, 14 pulse-chase experiments were carried out. A 10-min pulse of bq 14 was given to a different (source) leaf sealed in a 2-l leaf chamber. PPFD was 350 µmol m 2 s 1. After the pulse of 14 was given, the rest of 14 was sucked through soda lime to be absorbed and fresh air was allowed to stream in through a tube on the other side of the leaf chamber. The translocation period was 3 h. The young sink leaf, which had been imaged, was then cut from the plant and immediately placed between Whatman paper. The leaf was freeze dried, transferred to Hyperfilm (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) and exposed for 2 d at 70 C before developing (Turgeon & Webb, 1973). 3-phosphoglycerate (PGA) determination Young leaves from 8 to 10-wk-old plants were dark-adapted from late afternoon until the next morning for 16 h. With a New Phytologist (2001) 151:

3 587 razor blade, areas of c. 0.2 cm 2 in size, which did not include veins, were cut out from the tip and the base regions. Samples were weighed and ground in liquid nitrogen. 1 M perchloric acid (1 ml) was added. The mixture was allowed to thaw during grinding. Debris was removed from the sample, which was allowed to stand on ice for 30 min before centrifugation. The extract was neutralized with 5 M K 2 CO 3. The extract was then treated with charcoal (c. 5 mg ml 1 ) to remove inhibitors. In the obtained extract PGA was measured according to Michal (1984). Chlorophyll was determined according to Porra et al. (1989). Results Distribution of ETR in a developing leaf of Nicotiana tabacum We used chlorophyll a fluorescence to derive photosynthetic electron transport rates (Genty et al., 1989). To confirm the validity of this approach for young leaves, we compared the assimilation rate from gas exchange with ETR from fluorescence imaging. exchange and fluorescence were measured simultaneously in either the leaf tip or base at a concentration of 670 µl l 1 and in 2%. Under such conditions, photorespiration is minimal and electrons are largely consumed by assimilation. Different fluxes were established by varying PPFD. At the leaf tip, about 80 µmol e m 2 s 1 correspond to 18 µmol m 2 s 1 (Fig. 1). The values for the base of the leaf fall almost on the same line. The correlation is linear, but does not extrapolate to the origin. Figure 2 shows images of estimated photosynthetic ETR in a young expanding leaf (4.3 cm long) of Nicotiana tabacum under different and concentration or irradiance. For the calculation of ETR we used a value of 80% for light absorptance. This is a very good approximation for fully developed source tissue such as in expanded leaves and the tip of young leaves. We are aware that in the base of young leaves the absorptance is lower. We found minimal values of 65%. Therefore this approximation entails an error of up to 25% overestimation of ETR for the base. In low (close to the compensation point, Fig. 2a), ETR distribution was largely uniform with values c. 30 µmol e m 2 s 1. In ambient (340 µl l 1, Fig. 2b) the images revealed a large activity gradient with increased ETR towards the tip of the leaf ( µmol e m 2 s 1, Fig. 2b). When was increased to 670 µl l 1 (Fig. 2c) the maximal ETR of the leaf tip was only slightly stimulated ( µmol e m 2 s 1 ), while no significant stimulation was seen with further increasing the concentration (Fig. 2d). In 2%, ETR measured at the leaf tip would be comparable to a assimilation rate of 25 µmol m 2 s 1. In the middle of the leaf, ETR was around 30 µmol e m 2 s 1 at ambient, and was stimulated by high concentration (Fig. 2c &d). At the leaf base, the ETR remained at its minimal value (green zone) even at 2400 µl L 1. Electron transp. rate (µmol m 2 s 1 ) assimilation rate (µmol m m 2 s 1 ) Fig. 1 The relationship between the rate of fixation measured by gas exchange and estimated electron transport rate (ETR) calculated from chlorophyll fluorescence images (average of (F M values). Triangles, tip; circles, base of a sink leaf. Gas composition: 670 µl l 1, 2% with various photosynthetic photon flux densities (PPFDs) ranging from 100 to 570 µmol m 2 s 1. The means of light absorptance were used, 78% and 72% in leaf tip and base, respectively. Figure 2(e) shows an image taken at 2% (compare with Fig. 2c). In 2%, electrons are largely used for fixation, while in 21%, a variable proportion of electrons is used for photorespiration. Reducing from 21% to 2% had little effect at the leaf tip and base, but caused a small but significant depression in the middle zone (Fig. 2c,e). Figure 2(b,f h) shows images at ambient concentration and different PPFD. At the leaf base, ETR was saturated at 200 µmol m 2 s 1 PPFD (data not shown, but compare Fig. 3). At the leaf tip, 700 µmol m 2 s 1 PPFD was required for saturation of electron transport (Fig. 2b). Figure 3 displays ETR profiles from the leaf base to the tip. Values were averaged from 16 small areas alongside the mid-vein. These figures clearly indicate that photosynthesis at the leaf base was very low. The ETR was saturated by 200 µmol m 2 s 1 PPFD. A steep gradient in the ETR from about 30 at the base to 140 µmol m 2 s 1 at the tip was seen at a PPFD of 1000 µmol m 2 s 1 and a concentration of 1000 µl l 1 (Fig. 3a). The curves further demonstrate that in low PPFD the middle of the leaf was -sensitive, while the tip was light limited (Fig. 3c). At higher PPFD the tip also became -sensitive. Measurements at high PPFD (700 and 1000 µmol m 2 s 1 ) were made h before those made at low PPFD ( µmol m 2 s 1 ). The leaf developed a little during this time period and therefore the ETR near the base seemed to be lower in a PPFD of 1000 than at 400 µmol m 2 s 1 New Phytologist (2001) 151:

4 588 Fig. 2 Approximate electron transport rate (ETR) in a sink leaf (4.3 cm long) exposed to different external concentrations (a e), concentration (e) or photosynthetic photon flux density (PPFD) (f h) as indicated. PPFD in (a e): 700 µmol m 2 s 1, concentration in (f h): 340 µl l 1, oxygen concentration in (a d) and (f h): 21%, in (e): 2%. (Fig. 3a,c). When either the concentration or the concentration was increased from 340 µl l 1 and 2% to 1000 or 21%, ETR increased in a similar leaf zone. In low irradiance this zone was near the middle in high irradiance near the tip of the leaf (Fig. 3). Φ PSII images during leaf development Images of Φ PSII (670 µl l 1, 2%, PPFD: 350 µmol m 2 s 1 ) were followed during expansion of one leaf from a length of 3.9 cm to 8 cm over a period of 6 d (Fig. 4). The plant was taken from the glasshouse once a day for measurements. Initially (3.9 cm, Fig. 4a,b) relatively high Φ PSII ((F M = 0.42 equivalent to a assimilation rate of 12 µmol m 2 s 1 ) were restricted to small areas at the leaf tip, while the assimilation rate was low throughout the middle part (equivalent to 2 5 µmol m 2 s 1 ). As the leaf expanded, high assimilation rates were observed throughout most of the leaf, while low assimilation areas (green) were restricted to small distinct zones at the base and along the major veins (Fig. 4c f ). New Phytologist (2001) 151:

5 589 Photosynthetic induction in young leaves The induction of ETR during dark-light transition was analysed in a young leaf (5 6.5 cm long) in 60 µl l 1 and 21% (PPFD: 150 µmol m 2 s 1 ). Under these conditions ETR largely reflects photorespiratory cycling. The induction was performed after 14 h of over-night dark adaptation. In mature leaves the induction of ETR after such a long dark period was slow and uniform throughout the leaf (data not shown). In the young leaf, differential induction patterns were observed. ETR induction was faster at the leaf base than tip area, and faster near the first and second order veins than within the interveinal areas (Fig. 5a d, see also induction kinetics in Fig. 6). After 760 s the ETR was similar in both sink and source areas. Figure 5(e) shows the integral of the first 10 images taken within the first 400 s of illumination. This represents the sum of electrons, which had passed through PSII during the recorded induction time (multiply scale by µmol m 2 s 1 ) and can be taken as a relative measure of the induction velocity. The time courses of two individual locations marked in Fig. 5(e) (location 1 and 2) are shown in Fig. 6. Induction curves taken from areas at the leaf base exhibited a steeper initial slope than those from the tip. Steady state ETR took 3 4 min at the base and about 12 min at the tip. The even distribution of ETR throughout the leaf at 760 s (Fig. 5d) changed after switching gas composition (60 µl 1 /21% 670 µl 1 /2% ) and increasing irradiance ( µmol m 2 s 1 PPFD). This distribution is the reverse of that observed during the induction period, with low assimilation zones largely coinciding with fast induction zones at the leaf base (compare Fig. 5e,f ). The comparison with Fig. 6 shows that in the fast induction zones the ETR did not change with increased irradiance or, but due to the increased irradiance the same ETR was maintained with a lower quantum efficiency of PSII (green zones in Fig. 5f ). By contrast, the slow induction zones were associated with more mature tissue, which had a higher light saturation point and therefore had increased rates of ETR at high concentrations. When the oxygen concentration was increased from 2% (Fig. 5f ) to 21% (Fig. 5g), the ETR increased substantially in the middle-zones of the leaf, and slightly at the base, but stayed constant at the tip. This oxygen effect is similar to that shown in Fig. 2(c) in comparison with Fig. 2(e). Fig. 3 Estimated electron transport rate (ETR) alongside the mid-vein at various photosynthetic photon flux density (PPFD) and or concentrations. Numbers beside the curves indicate PPFD (µmol m 2 s 1 ). closed circles, open circles, external concentration; 340 µl l 1 ; closed squares, open squares, external concentration; 1000 µl l 1. Filled symbols: concentration 21%; open symbols, concentration 2%. ETR was corrected for differences in light absorbency by assuming a linear change between the measured values of the leaf base (66%) and tip (80%). New Phytologist (2001) 151:

6 590 Fig. 4 Images of (F M at steady state with 670 µl l 1, 2% and photosynthetic photon flux density (PPFD): 350 µmol m 2 s 1. Images of a single leaf were taken each day over several days. The leaf was (a), 3.9; (b), 4.5; (c), 5.1; (d), 6.0; (e), 7.1; and (f), 8.0 cm long, respectively. Comparison of fluorescence images and 14 C-autoradiographic images Figure 7(a) is an integrated image, which represents the first 10 images at the early stage of photosynthetic ETR induction (until 460 s). The PPFD chosen (350 µmol m 2 s 1 ) was higher than in Fig. 5, but the distribution of ETR induction was similar to the previous measurement in low PPFD (Fig. 5e). Induction of photosynthetic ETR was rapid (Fig. 7a) in the base and along the major veins. In the same region, the amount of imported 14 C was high, as can be seen from the blackening of the film (Fig. 7b) while the assimilation rate, measured at high and low (Fig. 7c), was low. Carbon import, PGA content and photosynthetic induction For the experiment in Fig. 8(a) only the young leaves were covered from late afternoon until the next morning for darkadaptation (16 h), while the rest of the plant remained uncovered, being illuminated in the evening and morning hours in the naturally lit glasshouse. In Fig. 8(b) the total plant was kept in the dark for 16 h overnight. Fig. 8(a,b) shows integrated induction images of the ETR representing the first 11 images (from 0 to 450 s after onset of illumination). The induction was performed with a PPFD of 200 µmol m 2 s 1 in 60 µl l 1 and 21%. In Fig. 8(a), the pattern is similar to that shown in Figs 5 and 7. In Fig. 8(b) the regions of fast induction were restricted to areas along the veins. Iodine staining was performed as described by Molisch (1914) and showed that only the big veins took up the stain, while the mesophyll remained unstained. In the leaf of Fig. 8(a), starch was present in the mid vein and secondary veins, in the leaf of Fig. 8(b), very little starch was present in the mid vein only (results not shown). For both treatments, the stain appeared denser near the base and in the central parts of the leaf and faded towards the tip or periphery. PGA was measured in several leaves, which were dark-adapted in the same way as shown in Fig. 8(a) (Table 1). The amount of PGA per unit chlorophyll was higher in the leaf base in comparison to the tip. The rate of dark respiration was also higher in the leaf base than in the leaf tip (Table 1). New Phytologist (2001) 151:

7 591 Fig. 5 Images of (F M recorded during photosynthetic induction and at steady state. Images (a) (b) (c) and (d) of a dark-adapted leaf (14 h) were taken during photosynthetic induction at photosynthetic photon flux density (PPFD) of 150 µmol m 2 s 1 in 60 µl l 1 and 21%. Image (e) represents the integral of nine images taken during photosynthetic induction from 0 to 400 s after start of illumination. The scale represents (F M or in (e) an estimate of total electrons transported during the first 400 s when multiplied by µmol m 2. Images (f) and (g) show (F M in the steady state in 670 µl l 1 and subsaturating PPFD (350 µmol m 2 s 1 ). Image (f) was taken in 2% and image (g) in 21%. New Phytologist (2001) 151:

8 % Electron transport rate (µmol m 2 s 1 ) µl l 1 2% PPFD: 350 µmol m 2 s Time (s) Fig. 6 Time course of mean estimated electron transport rate (ETR) of small areas of the leaf (1 and 2 shown in Figure 5e). Circles, area 1; squares, area 2. Induction of ETR at photosynthetic photon flux density (PPFD): 150 µmol m 2 s 1 in 60 µl l 1 and 21%. The concentration, concentration, and PPFD were changed as indicated. The closed and the open symbols mark the different PPFD. The last two points of the curve represent the images (f) and (g) in Figure 5. Fig. 7 Chlorophyll fluorescence images in comparison to whole-leaf autoradiography of 14 C-labelled import. (a) Integrated picture which represents 10 images taken during photosynthetic induction (from 0 to 460 s after onset of illumination, scale represents the average value of (F M F)F M during that time or an estimate of total amount of electrons transported, when values are multiplied by µmol m 2 ). Induction was performed on a dark-adapted leaf (14 h) illuminated with 350 µmol m 2 s 1 in 60 µl L 1 and 21%. (b) Autoradiography 14 C-labelled import. (c) Steady state measured at 670 µl l 1 and 2%, scale represents (F M. Discussion Photosynthesis images of young leaves, demonstrated in this study, coincided in a first approximation with the autoradiographic image of a classical 14 C-labelling experiment (Fig. 7). The autoradiographic image is fairly similar to the images published by Turgeon & Webb (1973). The tip remained unlabelled, indicating that it was not sink tissue, but rather source tissue. We observed a labelling gradient within the lower part of the leaf, indicating the presence of a gradient in sinkstrength within the base (Turgeon, 1987). The veins and the mesophyll along major veins were more strongly labelled than New Phytologist (2001) 151:

9 593 Fig. 8 The effect on induction of darkadapting for 16 h (a) only the sink leaves or (b) the total plant. The integrated images represent 11 images taken during photosynthetic induction (from 0 to 450 s after onset of illumination, scale represents average (F M F )/F M or an estimate of total amount of electrons transported, when values are multiplied by µmol m 2 ). Induction was performed with 200 µmol m 2 s 1 in 60 µl l 1 and 21%. Leaves were 5.8 (a) and 5.6 cm (b) long. Table 1 3-phosphoglycerate (PGA) content of young leaves measured after 16 h of dark adaptation (values ± SE, numbers in brackets are sample size), and mitochondrial respiration measured on a different set of plants with min dark adaptation PGA nmol mg FW PGA nmol µg Chl uptake (µmol m 2 s 1 ) 6.3 cm leaf tip 0.29 ± 0.01 (12) ± 0.4 (12) base 0.27 ± 0.01 (13) ± 0.2 (12) 4.3 cm leaf tip 0.40 ± 0.04 (6) ± 0.4 (12) base 0.30 ± 0.01 (6) ± 0.4 (12) the surrounding tissue. A similar pattern was seen in the assimilation and induction images (Fig. 7a,b). In a first approximation, assimilation and induction images were complementary: fast induction coincided with low assimilation zones and vice versa. However, the 14 C labelling and assimilation images do not coincide in a quantitative manner. The labelling is rather diffuse compared with the distinct distribution pattern of low and high assimilation zones. The distinct assimilation pattern of a young leaf may reflect different zones of the leaf anatomy and mesophyll development. At the leaf tip, even in very young leaves, ETR in ambient air is almost as high as usually observed in mature leaves (own observations). By contrast, ETR remains very low in the base of the leaf and is not stimulated by high. In this zone chloroplasts are not fully developed yet and electron flux may not be totally related to the assimilation of external. A different situation was observed in the middle part of young leaves. Here ETR depended on external, but the carboxylation efficiency, as can be seen from the low increase of ETR with increased in 2%, is lower than at the leaf tip. This may be the result of low Rubisco activity and/or low conductance. At the cellular level, conductance may be low, as the chloroplasts are embedded in a relatively thick cytoplasmic layer, so that the diffusion of in the liquid phase is not yet minimized. At the tissue level, due to the incomplete cell expansion, the intercellular air space is still underdeveloped and stomata are still developing (microscopic observations, not shown). The images suggest that the proximity of veins has an influence on the developmental state of the tissue. Photosynthesis was high in areas distant from major veins while it was low along the major veins. Croxdale & Omasa (1990) studied the pattern of chlorophyll fluorescence kinetics in developing cucumber leaves. They noted that the proximity of the vascular tissue did not influence the development of the photochemical function of PSII in chloroplasts. This suggests that the difference in ETR observed here was not due to the function of PSII, but to differences in carbon metabolism, which obviously depends on the proximity to the veins. There was a good correlation between steady state and induction images. In zones of low assimilation at the leaf base and along the major veins of the young leaf, the induction of photosynthetic ETR is rapid compared with the mature high assimilation zones. While the induction of fixation is well described (Edwards & Walker, 1983 Lan et al., 1992; Sassenrath-Cole & Pearcy, 1992, 1994; Woodrow & Mott, 1992), induction of ETR has been only studied in relation to fixation (Bro et al., 1996), but not in relation to Calvin cycle metabolism. Three main factors influence the induction of fixation: the pool sizes of metabolic intermediates; the activation of the Calvin cycle enzymes; and stomatal opening. New Phytologist (2001) 151:

10 594 The induction of ETR differs from that of fixation in that it can also increase without any net fixation. We made the measurements at the compensation point because then the induction of ETR occurs independent of stomatal opening. The early phase of induction depends on the supply of metabolic intermediates from reactions within the chloroplasts that occurred in the dark, such as the oxidative pentose phosphate cycle or starch degradation for export. Following the long dark period, carbohydrates have been either consumed or exported out of the leaf. This leads to low levels of chloroplast intermediates. Therefore, the regeneration of Calvin cycle metabolites takes longer, especially at low external concentrations, when it requires recycling of carbon from the photorespiratory cycle and mitochondrial respiration. In source regions, ETR induction is slow after long dark adaptation. By contrast in sink regions the induction of ETR is rapid even at concentrations near the compensation point. Obviously both, the reductive pentose phosphate cycle and the photorespiratory cycle with their related electron consumption can proceed immediately in the light in sink tissue. However, this fast induction depends on the supply of external carbohydrates as shown in Fig. 8. The reductive pentose phosphate cycle shares metabolites with the oxidative pentose phosphate cycle, which completely occurs in the chloroplast but incompletely in the cytosol (in spinach, Schnarrenberger et al., 1995). It supplies intermediates for biosynthetic pathways, and is highly activated during the early exponential growth of plant cell cultures for the synthesis of amino acids (Ganson & Jensen, 1987). Therefore, we propose that the fast induction of ETR indicates that the intermediates of the oxidative pentose phosphate cycle are high from imported carbohydrates in the sink tissue of young leaves, but not in the source tissue after long dark adaptation. During the dark-light transition the ETR can therefore rise much faster in sink than in source tissue. We observed some difference in the PGA concentration in the base and the tip supporting this hypothesis, but not as strong as we hoped. Interestingly, each individual leaf spot exhibits either a slow or a fast induction, that is we found little evidence of intermediate induction kinetics. This phenomenon may result from the autocatalytic nature of the Calvin cycle. The transition itself seems to be gradual, as shown by the gradient in the autoradiagraphic images or by the fact that, after inadequate carbon supply, sink cells close to the large veins and in the lower base imported carbon while more distant cells did not. Fast induction is not necessarily associated with low photosynthetic rates as in expanding leaves. In fully developed source leaves, fast induction spots could be initiated by local treatment with cytokinin (not shown) or by local infection with pathogens (Esfeld et al., 1995). Both the plant hormone treatment and elicitation are well known to stimulate cellular activities, such as cell cycle activation or defence. These processes also involve an activation of the chloroplast oxidative pentose phosphate cycle. The above interpretations are not the only possible explanation for high ETR rates. The Mehler reaction (Mehler, 1957) in which acts as an electron acceptor might be responsible for some of the observed ETR. However, Ruuska et al. (2000) have shown, using transgenic tobacco having a reduced amount of Rubisco, that the transfer of electrons to via the Mehler reaction is so small in intact leaves that it could not be distinguished from mitochondrial respiration. The Mehler reaction can only occur in leaves if the consumption of ATP is not accompanied by NADPH consumption. In chloroplasts, different metabolic pathways require different amounts of ATP and NADPH. The balance of ATP to NADPH consumption is unknown for developing chloroplasts. In addition, cyclic electron transport around PSII (Heber et al., 1979) could mimic PSII activity (Schreiber & Neubauer, 1990). Data supporting the existence of a cyclic electron transport pathway around PSII are scarce in intact leaves and have only been observed under extreme conditions in which the oxygen evolving complex from PSII was functionally disrupted (Canaani & Havaux, 1990; Havaux, 1998). Ohashi et al. (1989) measured chlorophyll fluorescence induction in isolated etiochloroplasts during the early phase of greening. Their measurements showed that PSII was functional. However variable fluorescence was not observed before linear electron transport was working. Therefore it is unlikely that we can assume that cyclic electron transport around PSII played a role in the observed PSII activity in our measurements. Acknowledgements We wish to thank Drs Oula Ghannoum and John Evans for critically reading the manuscript and giving helpful suggestions. References Bro E, Meyer S, Genty B Heterogeneity of leaf assimilation during photosynthetic induction. Plant, Cell & Environment 19: Canaani O, Havaux M Evidence for a biological role in photosynthesis for cytochrome b-559 a component of photosystem II reaction center. Proceedings of the National Academy of Sciences, USA 87: Croxdale JG, Omasa K Patterns of chlorophyll fluorescence kinetics in relation to growth and expansion in cucumber leaves. Plant Physiology 93: Daley PF, Raschke K, Ball JT, Berry JA Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence. 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