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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 distribution of the quantum efficiency of PSII (ΦPSII) 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 ΦPSII, and the induction of ETR in leaves that had been dark-adapted for a long period of time and subsequently illuminated in low CO2 concentrations.
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Photosynthesis images of young leaves, demonstrated in this study, coincided in a first approximation with the autoradiographic image of a classical 14C-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 sink-strength within the base (Turgeon, 1987). The veins and the mesophyll along major veins were more strongly labelled than 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 14C 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 CO2. In this zone chloroplasts are not fully developed yet and electron flux may not be totally related to the assimilation of external CO2.
A different situation was observed in the middle part of young leaves. Here ETR depended on external CO2, but the carboxylation efficiency, as can be seen from the low increase of ETR with increased CO2 in 2% O2, is lower than at the leaf tip. This may be the result of low Rubisco activity and/or low CO2 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 CO2 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 CO2 fixation is well described (Edwards & Walker, 1983Lan et al., 1992; Sassenrath-Cole & Pearcy, 1992, 1994; Woodrow & Mott, 1992), induction of ETR has been only studied in relation to CO2 fixation (Bro et al., 1996), but not in relation to Calvin cycle metabolism. Three main factors influence the induction of CO2 fixation: the pool sizes of metabolic intermediates; the activation of the Calvin cycle enzymes; and stomatal opening. The induction of ETR differs from that of CO2 fixation in that it can also increase without any net CO2 fixation. We made the measurements at the CO2 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 CO2 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 CO2 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 O2 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 O2 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.