- Top of page
- Materials and Methods
The main function of leaf senescence is the recycling of nutrients. For example, > 80% of the nitrogen contained in Arabidopsis leaves is exported during senescence (Himelblau & Amasino, 2001). To allow mobilization and transport of nutrients, cell death has to be prevented until senescence has been completed. Indeed, membrane integrity and cellular compartmentalization are maintained until late senescence (Lee & Chen, 2002), supporting the view that senescence is a nonapoptotic transdifferentiation process (Thomas et al., 2003).
Mobilization of nitrogen from photosynthetic proteins, such as Rubisco, results in a decline in photosynthetic CO2 assimilation. In Arabidopsis photosynthesis declines early, before the leaves are fully expanded, whereas the chlorophyll content remains high until later stages of development (Stessman et al., 2002). A combination of high chlorophyll content and low CO2 assimilation could potentially result in photo-oxidative damage caused by an imbalance between energy capture and dissipation. For example, it has recently been demonstrated that a ‘staygreen’ mutant of soybean exhibits increased susceptibility to photoinhibition (Guiamét et al., 2002). Furthermore, oxidative stress in chloroplasts typically increases with increasing leaf age (Munné-Bosch & Alegre, 2002). To prevent photo-oxidative processes that could lead to lipid peroxidation and cell death, the photosynthetic apparatus has to be dismantled in an ordered manner. For example, photosystem II activity declines before photosystem I activity in Brassica napus cotyledons (Ghosh et al., 2001) and adjustments of the amount of minor light-harvesting complexes may prevent photo-oxidative damage in senescing barley leaves (Humbeck & Krupinska, 2003). Protective processes, such as nonphotochemical quenching (NPQ) are also likely to play a role in preventing damage during senescence. In maize and wheat, NPQ increases in combination with an accumulation of xanthophyll cycle carotenoids, indicating increased dissipation of excess excitation energy as heat (Lu & Zhang, 1998; Lu et al., 2001). By contrast, a decline in NPQ was found in senescing soybean leaves (Guiamét et al., 2002). This discrepancy may be caused by different growth conditions or differences in the stage of senescence analysed. In addition, whether recorded values of photosynthetic parameters, such as NPQ, are increased or decreased may depend on where within a senescing leaf fluorescence is measured. Leaf senescence usually proceeds from the tip to the base of a leaf, while the veins stay alive until the final stages (Feller & Fischer, 1994). It is therefore likely that photosynthetic parameters show heterogeneous spatial patterns.
Spatial patterns of photosystem II processes can be analysed using chlorophyll a fluorescence imaging. This technique is now commonly applied for measuring photosystem II processes in heterogeneous systems. Imaging has been used to study photosynthetic responses to pathogen infection (Scholes & Rolfe, 1996); ozone-induced perturbations of photosynthesis (Leipner et al., 2001); photo-oxidative stress (Fryer et al., 2002); and light adaptation (Lichtenthaler et al., 2000). The sink–source transition in young leaves has also been characterized using chlorophyll fluorescence imaging (Meng et al., 2001), but we do not know of any study where this technique has been applied to analyse photosynthetic parameters during leaf senescence.
In addition to allowing spatial analysis of photosynthetic processes, imaging can be used as a fast and convenient method for studying photosynthetic changes in a large number of small plants, e.g. Arabidopsis grown on media with varied nitrogen and carbon supply. There is increasing evidence that senescence is regulated by the carbon–nitrogen balance in leaves (Ono et al., 1996; Stitt & Krapp, 1999; Masclaux et al., 2000; Masclaux-Daubresse et al., 2002). Whereas it had previously been suggested that leaf senescence is triggered by an age-dependent decline in photosynthesis (Hensel et al., 1993), it now seems more likely that the senescence-related decline in photosynthesis is a consequence of sugar accumulation, especially during early stages of senescence (Noodén et al., 1997; Wingler et al., 1998; Masclaux et al., 2000). Sugar sensing has been demonstrated to regulate a large number of metabolic and developmental processes, some of which involve hexokinase as a sugar sensor (Jang & Sheen, 1994; Smeekens, 2000). Hexokinase may also be responsible for the sugar-dependent regulation of leaf senescence. Tomato plants overexpressing hexokinase-1 from Arabidopsis show accelerated senescence (Dai et al., 1999), while senescence is delayed in hexokinase-1 mutants of Arabidopsis (Moore et al., 2003). Further work is required to unravel the interactions of sugar and nitrogen signalling during the regulation of senescence.
The aim of this study was to analyse spatial and temporal patterns in photosynthetic function during leaf senescence, especially with respect to the regulation by sugar and nitrogen supply.