SEARCH

SEARCH BY CITATION

Keywords:

  • Arabidopsis;
  • chlorophyll fluorescence imaging;
  • leaf senescence;
  • nonphotochemical quenching;
  • photoinhibition;
  • photoprotection;
  • sugar sensing

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    To prevent premature cell death and to allow efficient nutrient mobilization from senescing leaves, the photosynthetic apparatus has to be dismantled systematically. This requires temporal, spatial and metabolic regulation of photosynthetic function and photoprotection.
  • • 
    Conventional pulse-modulated fluorometry and chlorophyll fluorescence imaging were used to study age- and nutrient-dependent senescence patterns in Arabidopsis thaliana.
  • • 
    Nonphotochemical quenching (NPQ) rose during leaf maturation, indicating increased energy dissipation. During later stages of senescence, overall plant NPQ declined, but NPQ remained high in the base of rosette leaves. Other fluorescence parameters also showed spatial patterns, for example minimum fluorescence (F0) was temporarily increased in the tips of inner rosette leaves from where high F0 spread to the base, in a zone preceding cell death. Senescence-dependent changes in chlorophyll fluorescence characteristics were accelerated by growth on glucose-containing medium in combination with low, but not with high, nitrogen supply.
  • • 
    Our experiments revealed distinct spatial patterns of photosynthetic and photoprotective processes in senescing leaves and induction of these processes by high sugar-to-nitrogen ratios.

Abbreviations
F0

Minimum fluorescence

Fm

maximum fluorescence

Fv/Fm

maximum quantum efficiency of photosystem II photochemistry

inline image

quantum efficiency of excitation energy capture by open photosystem II centres

ΦPSII

quantum efficiency of photosystem II electron transport

NPQ

nonphotochemical quenching

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material

Seed of Arabidopsis thaliana (L.) Heinh. (Col-0) was suspended in 0.5% (w/v) low-melting agarose and pipetted onto compost (Murphy's Multi Purpose Compost; Murphy Garden Products, Ipswich, UK). After cold treatment for 3–4 d at 4°C, the pots were transferred into controlled environment growth chambers and the plants were grown at a photon flux density of approximately 100 mol m−2 s−1 for 12 h d−1 at a temperature of 22°C during the day and 18°C at night.

For growth on agar medium, seeds were sterilized in commercial bleach, washed, resupended in 0.7% low-melting agarose, and pipetted on agar (1% w/v) medium. For high-nitrogen treatments the medium consisted of a half-concentrated Murashige–Skoog (MS) medium containing 30 mm nitrogen (10.3 mm NH4+ and 19.7 mm NO3). For low-nitrogen treatments the nitrogen concentration was reduced to 4.7 mm (only NO3). After cold treatment the plates were transferred to growth chambers and grown in vertical orientation under the same conditions as described for the compost-grown plants, but with a daylength of 16 h to accelerate plant development and prevent effects caused by drying of the agar medium.

Determination of chlorophyll content

Relative chlorophyll content was determined using a Minolta SPAD chlorophyll meter (N-tester, Hydro Agri, Immingham, UK). Measurements were taken in the middle of each leaf and values for five outer rosette leaves per plant were averaged.

Chlorophyll fluorescence analysis

Chlorophyll a fluorescence was analysed using a pulse-modulated fluorometer (FMS-2, Hansatech, King's Lynn, UK) with the fibre optics pointing to the middle of a large outer rosette leaf (usually leaf 10). Minimum fluorescence (F0) was measured by exposing leaves of dark-adapted plants to modulated red light, before a saturating flash of white light was applied to record maximum fluorescence (Fm). Leaves were then illuminated with actinic light (225 mol m−2 s−1) and saturating flashes of 0.7 s duration were applied every 1.5 min. After 15 min illumination, maximum fluorescence of light-adapted leaves (inline image), steady-state fluorescence (Fs) and ground fluorescence (inline image) were recorded. The following equations were used for calculating photosynthetic parameters.

Maximum quantum efficiency of photosystem II photochemistry, Fv/Fm = (Fm − F0)/Fm; quantum efficiency of excitation energy capture by open photosystem II centres, inline image; quantum efficiency of photosystem II electron transport, inline image; photochemical quenching, inline image; nonphotochemical quenching, inline image.

Chlorophyll fluorescence images were captured with a pulse-modulated imaging fluorometer (FluorCam 700MF, Photon Systems Instruments, Brno, Czech Rebublic) as described by Nedbal et al. (2000). After measuring Fv/Fm in dark-adapted plants, the plants were illuminated with actinic light (100 mol m−2 s−1) and saturating flashes of 0.8 s duration were applied every 2 min to determine quenching parameters. For plants grown in compost, data were analysed for individual leaves (usually leaf 10) and averaged for the whole rosette. For plants grown on agar medium, data were averaged for the whole rosette.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Development of senescence in Arabidopsis rosettes

Senescence proceeded from the old (outer) to young (inner) rosette leaves (Fig. 1). In the Col-0 accession of Arabidopsis studied here, but not in Ws-2 plants (not shown), loss of chlorophyll was accompanied by an accumulation of anthocyanins. Cell death (visible collapse of cells) proceeded from tip to base of outer rosette leaves (Fig. 1). The inner rosette leaves stayed alive until late development.

image

Figure 1. Development of senescence in rosettes of Arabidopsis plants.

Download figure to PowerPoint

Overall changes in chlorophyll fluorescence parameters during leaf senescence

Chlorophyll content (Table 1) increased while the outer rosette leaves were still expanding (Fig. 1). At the same time, the quantum efficiency of photosystem II electron transport, ΦPSII, already started to decline, indicating that electron transport rates were reduced before full leaf expansion. At day 54, ΦPSII was reduced by 41% compared with leaves of young plants (day 34), while chlorophyll content was 5% higher. This initial decline in ΦPSII was mainly caused by a decrease in inline imageand thus was likely to be caused by a reduced efficiency of excitation capture, not by a change in the concentration of open photosystem II reaction centres (Genty et al., 1989). Nonphotochemical quenching (NPQ) increased until day 60, but declined during the final stages (Table 1). The maximum quantum efficiency of photosystem II photochemistry in dark-adapted leaves, Fv/Fm, was clearly reduced from day 49 onwards. The large standard deviation of fluorescence parameters during late senescence (Table 1) was probably caused by variations in the extent of senescence between and within individual leaves of a plant (Fig. 1).

Table 1.  Changes in chlorophyll content and fluorescence characteristics during leaf senescence in Arabidopsis
DayChlorophyll (relative units)Fv/FmΦPSIIinline imageNPQ
  1. Chlorophyll was measured in five outer rosette leaves per plant. Maximum quantum efficiency of photosystem II photochemistry (Fv/Fm), quantum efficiency of photosystem II electron transport (ΦPSII), quantum efficiency of excitation energy capture by open photosystem II centres (inline image) and nonphotochemical quenching (NPQ) were determined by conventional pulse-modulated fluorometry in one outer rosette leaf (usually leaf 10) per plant. Data are means ± SD of five plants.

34314 ± 180.824 ± 0.0120.653 ± 0.0140.767 ± 0.0050.40 ± 0.05
41392 ± 510.832 ± 0.0120.623 ± 0.0300.753 ± 0.0140.52 ± 0.08
46389 ± 180.804 ± 0.0150.528 ± 0.1300.699 ± 0.0820.67 ± 0.33
49330 ± 490.782 ± 0.0230.450 ± 0.0580.649 ± 0.0720.72 ± 0.24
54329 ± 730.728 ± 0.0620.388 ± 0.1660.534 ± 0.1500.77 ± 0.28
60225 ± 830.646 ± 0.1060.180 ± 0.0820.363 ± 0.1091.23 ± 0.52
63198 ± 310.454 ± 0.1550.183 ± 0.0200.336 ± 0.0570.95 ± 0.73
67 93 ± 410.337 ± 0.2150.173 ± 0.1200.303 ± 0.1880.37 ± 0.20

Spatial patterns in photosynthetic parameters

To study how the changes in photosynthetic parameters were distributed over individual leaves and the whole leaf rosette, photosynthesis was analysed in a separate experiment by imaging of chlorophyll fluorescence. Although the exact time course of fluorescence changes in this experiment was not identical with the experiment presented in Table 1, the overall changes were the same: ΦPSII declined early, accompanied by a temporary increase in NPQ, while Fv/Fm declined later (Fig. 2).

image

Figure 2. Changes in chlorophyll fluorescence characteristics during leaf senescence as determined by fluorescence imaging. (a) Maximum quantum efficiency of photosystem II photochemistry (Fv/Fm); (b) quantum efficiency of photosystem II electron transport (ΦPSII); and (c) nonphotochemical quenching (NPQ) were analysed for whole rosettes and individual outer rosette leaves (usually leaf 10). Data are means ± SD of four plants.

Download figure to PowerPoint

When data were averaged for the whole leaf rosette, Fv/Fm decreased to a much smaller extent than for individual outer rosette leaves (Fig. 2a). ΦPSII was highest in young rosettes and declined before the onset of senescence was detectable as a decline in Fv/Fm or as visible leaf yellowing. Until day 61, ΦPSII values were the same for whole rosettes and for outer rosette leaves (Fig. 2b). After this time point, ΦPSII declined drastically in outer rosette leaves but remained constant averaged over the whole rosette, probably because of the formation of additional young rosette leaves. NPQ initially rose during leaf development, both in the whole rosette and in outer rosette leaves (Fig. 2c), but declined when senescence became visible. Again the decline in outer rosette leaves was more pronounced than for the whole rosette.

Images revealed local changes in F0 (Fig. 3a) from day 61 onwards. While F0 declined in the outer rosette, it rose from tip to base of inner rosette leaves. F0 was highest in a band of cells separating green from dead areas of the leaves, shortly before cell death occurred. By contrast, Fm mainly paralleled the distribution of chlorophyll and reached highest values in the inner rosette leaves during late senescence (Fig. 3b). As expected, Fv/Fm declined in senescing leaves, showing the lowest values on the tips of the outer rosette leaves (Fig. 3c). Images of NPQ showed that values remained low in the cotyledons but increased almost uniformly in the rosette leaves until day 47 (Fig. 3d). During later stages of senescence, NPQ mainly declined in the leaf tips, but high NPQ was maintained the base of individual rosette leaves until day 74.

image

Figure 3. Images of chlorophyll fluorescence characteristics in senescing Arabidopsis plants. (a) False colour images of minimum fluorescence of dark-adapted plants (F0); (b) maximum fluorescence (Fm); (c) maximum quantum efficiency of photosystem II photochemistry (Fv/Fm); (d) nonphotochemical quenching (NPQ).

Download figure to PowerPoint

Effect of nitrogen and sugar supply on photosynthetic parameters during leaf senescence

To analyse the metabolic regulation of photosynthetic function during leaf senescence, plants were grown on agar medium with varied nitrogen and glucose supply. In this system the plants remained small, but flowered and produced seeds (Fig. 4). Compared with high nitrogen supply, growth at low nitrogen supply led to anthocyanin accumulation in the petioles and stems, but did not accelerate visible senescence. This may be because the chosen lower concentration of 4.7 mm nitrate was still quite high. However, when glucose was added to the low-nitrogen medium, senescence was clearly accelerated and anthocyanins accumulated in the leaf blades. It is possible that addition of glucose accelerated nitrogen utilization by the plants and thus led to faster nitrogen depletion from the medium. By contrast, plants grown on high-nitrogen medium plus glucose did not show accelerated senescence, but were darker green than plants grown in the absence of sugar. Addition of sorbitol and mannitol in combination with low nitrogen supply did not induce senescence, demonstrating that the effect of glucose was not merely osmotic (not shown). In plants grown on the 2% glucose/low-nitrogen medium, Fv/Fm first declined in the cotyledons followed by the rosette leaves. In the siliques, Fv/Fm remained high when the leaf rosette had already senesced (Fig. 4e), indicating that nutrients were exported out of the leaves and used for reproduction.

image

Figure 4. Effect of sugar and nitrogen supply on senescence of Arabidopsis plants. Plants were grown for 3 d on agar medium with high nitrogen (a,c) or low nitrogen (b,d) supply, without sugar (a,b) or with addition of 2% glucose (c,d). (e) False colour image of Fv/Fm in a plant grown at low nitrogen supply plus 2% glucose.

Download figure to PowerPoint

The effect of glucose and nitrogen supply on senescence of leaf rosettes was quantified by imaging of fluorescence parameters. Fv/Fm values confirmed the optical impression that only a combination of glucose with low nitrogen supply accelerated senescence (Fig. 5a,b). A low concentration of 2% glucose was equally as effective as 4% glucose, without causing the delay in early photosynthetic development that was detectable with 4% glucose. At high nitrogen supply the addition of sugar, especially 4% glucose, prevented the decline in ΦPSII that occurs in the absence of an external carbon source (Fig. 5c). At low nitrogen supply the fall in ΦPSII was initially also delayed by glucose, but only until day 20, after which glucose accelerated the decline in ΦPSII (Fig. 5d). NPQ rose until day 20 at high nitrogen supply (Fig. 5e). This increase was delayed in the presence of 4% glucose. At low nitrogen supply the increase in NPQ was not as pronounced (Fig. 5f), but NPQ declined earlier in the 2% glucose treatment.

image

Figure 5. Effect of sugar and nitrogen supply on chlorophyll fluorescence characteristics in senescing Arabidopsis plants as determined by chlorophyll fluorescence imaging. (a,b) Maximum quantum efficiency of photosystem II photochemistry (Fv/Fm); (c,d) quantum efficiency of photosystem II electron transport (ΦPSII); and (e,f) nonphotochemical quenching (NPQ) were determined in Arabidopsis plants grown on agar medium containing 30 mm nitrogen (HN, filled symbols) or 4.7 mm nitrogen (LN, open symbols) with or without addition of 2 or 4% glucose. Data are means ± SD of at least 10 plants.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To allow efficient reallocation of nutrients in senescing plants, photosynthetic processes have to be regulated at the following levels: (i) temporal regulation is required to activate photoprotective processes while photosynthetic proteins are being degraded; (ii) spatial regulation is required to protect those parts of the leaves that are essential for enzymatic and transport processes; (iii) metabolic regulation is required to integrate senescence-dependent nutrient recycling with environmental factors.

Temporal regulation of photosynthetic parameters

Based on our results, it is possible to divide senescence into an early phase that is characterized by a decrease in ΦPSII, increase in NPQ and high Fv/Fm values; and a late phase during which NPQ declines in parallel with Fv/Fm (Table 1; Figs 2, 3, 5).

The early decline in photosynthetic electron transport, as indicated by a decrease in ΦPSII, is in agreement with the early reduction in CO2 assimilation reported by Stessman et al. (2002) for Arabidopsis. Reduced energy consumption by CO2 assimilation, in combination with the high chlorophyll content during this phase, is potentially dangerous. An imbalance between energy capture and utilization can result in an over-reduction of the electron transport chain, photoinhibition and oxidative stress caused by photoreduction of oxygen to superoxide in the Mehler reaction (Badger, 1985). In addition, reactive singlet oxygen could be formed through reaction of oxygen with triplet chlorophyll, especially when chlorophyll is released by the breakdown of the chlorophyll–protein complexes in photosynthetic membranes (Merzlyak & Hendry, 1994). Formation of reactive oxygen species can lead to lipid peroxidation in senescing tissues (Berger et al., 2001; Leverentz et al., 2002), resulting in decompartmentalization and cell death.

To prevent damage, excess energy has to be dissipated, for example by NPQ (Horton et al., 1994; Müller et al., 2001). The results presented here confirm that NPQ is involved in photoprotection during early senescence. This is in agreement with results obtained by Lu & Zhang (1998) and Lu et al. (2001) for senescing leaves of maize and wheat. We also found a temporary increase in NPQ in glasshouse-grown tobacco plants (data not shown). Given the low growth photon flux density in this study, it may be surprising that NPQ did increase, and the response may have been stronger under high light conditions. As NPQ relies on xanthophyll cycle activity and formation of a proton gradient across the thylakoid membrane (Horton et al., 1994), it is not surprising that it declines during later stages of senescence. Analysis of senescence in Arabidopsis mutants with reduced NPQ, for example mutants lacking PsbS (Li et al., 2000), would show the extent to which NPQ is involved in preventing premature cell death.

In addition to NPQ, anthocyanin formation, as seen in plants grown in compost and on agar medium with a combination of glucose and low nitrogen supply (Figs 1, 4), could be involved in the protection of senescing leaves. Based on experiments with ‘red-senescing’ compared with ‘yellow-senescing’ leaves of Cornus stolonifera, Feild et al. (2001) suggested that optical masking of chlorophyll by anthocyanins may reduce the risk of photo-oxidative damage during leaf senescence. By contrast to the Col-0 accession of Arabidopsis studied here, anthocyanins did not accumulate during senescence in Ws-2 plants, which showed an earlier decline in Fv/Fm and accelerated cell death (data not shown).

Spatial regulation of photosynthetic parameters

Senescence usually proceeds from leaf tip to base, while the vascular bundles stay intact until the final stages (Feller & Fischer, 1994). During the early stages photosynthetic parameters were evenly distributed over the leaf rosette. However, spatial patterns became apparent later during the senescence process (Fig. 3). Whereas overall plant NPQ declined during late senescence, high NPQ was maintained in the base of individual rosette leaves. Photoprotection in the leaf base is probably important to allow nutrient export.

The decline in Fv/Fm in outer rosette leaves and from tip to base of inner rosette leaves shows that these parts of the rosette were no longer sufficiently protected against photoinhibition. A decrease in Fv/Fm is usually caused by a decrease in Fm, in combination with an increase in F0 (Ögren, 1991). In the outer rosette leaves, which had already lost most of their chlorophyll, the drastic decline in Fv/Fm was caused by lower Fm values in combination with reduced F0 values. In a band between the tip and base of inner rosette leaves, Fv/Fm decreased because of a strong increase in F0, combined with a decrease in Fm, whereas the leaf bases exhibited increased F0 in combination with increased Fm. This shows that at least two different factors independently affecting F0 and Fm are responsible for the decline in Fv/Fm in senescing leaves. Increased F0 could be caused by the release of free chlorophyll from protein–pigment complexes; while Fm is affected by the capacity to reduce the electron acceptor QA. It would not have been possible to detect these changes using conventional pulse-modulated fluorometry.

Metabolic regulation of leaf senescence

Our results demonstrate that the metabolic regulation of senescence can be studied by growing plants on agar medium (Fig. 4) in combination with simple and rapid monitoring of senescence using chlorophyll fluorescence imaging (Fig. 5). Although the plants grown on agar medium were cultivated under longer days than the plants grown in compost (16 h compared with 12 h), they showed similar fluorescence characteristics: an early decline in ΦPSII combined with a rise in NPQ and a late decline in Fv/Fm. There was no indication that senescence could be triggered by glucose in the presence of high nitrogen supply. Electron transport rates, as indicated by high ΦPSII values, even remained higher than in the absence of sugars, indicating that sugars may extend the lifespan of leaves in nitrogen-sufficient plants. By contrast, in combination with low nitrogen supply, glucose triggered visible senescence which was most closely associated with an early decline in Fv/Fm.

Senescence-like symptoms can also be triggered by feeding glucose to leaf discs (Wingler et al., 1998). Although external supply of sugars may seem artificial, there is evidence that it reflects processes occurring during natural leaf senescence. Sugar contents increase during senescence in Arabidopsis, tobacco and a range of other plant species (Noodén et al., 1997; Wingler et al., 1998; Quirino et al., 2001; Stessman et al., 2002), and a glucose-insensitive hexokinase-1 mutant has recently been shown to exhibit delayed senescence (Moor et al., 2003). Regulation of metabolic and developmental processes by sugars often depends on nitrogen supply, suggesting that the sugar and nitrogen signalling pathways interact (Paul & Driscoll, 1997; Nielsen et al., 1998; Martin et al., 2002). Our results clearly show that the regulation of senescence by sugars is nitrogen-dependent, supporting the view 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). This could also explain accelerated senescence in plants grown in elevated CO2 (Nie et al., 1995; Miller et al., 1997). In this scenario a high sugar-to-nitrogen ratio would signal a reduced requirement for investment in Rubisco and other photosynthetic proteins in the old leaves, releasing nitrogen that would become available for the growth of young leaves and for fruit and seed formation.

Using Arabidopsis as a model species, significant progress has already been made in understanding the control of leaf senescence (Buchanan-Wollaston et al., 2003). Work with mutants in sugar or nitrogen signalling could help unravel the interactions between sugar and nitrogen signalling pathways during senescence. The development of a simple Petri dish system in combination with monitoring of senescence using chlorophyll fluorescence imaging makes it possible to isolate mutants that are affected in sugar-regulated senescence.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by research grants from the BBSRC (31/P16341) and the Royal Society.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Badger MR. 1985. Photosynthetic oxygen exchange. Annual Review of Plant Physiology 36: 2753.
  • Berger S, Weichert H, Porzel A, Wasternack C, Kühn H, Feussner I. 2001. Enzymatic and non-enzymatic lipid peroxidation in leaf development. Biochimica et Biophysica Acta 1533: 266276.
  • Buchanan-Wollaston V, Earl S, Harrison E, Mathas E, Navabpour S, Page T, Pink D. 2003. The molecular analysis of leaf senescence – a genomics approach. Plant Biotechnology Journal 1: 322.
  • Dai N, Schaffer A, Petreikov M, Shahak Y, Giller Y, Ratner K, Levine A, Granot D. 1999. Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence. Plant Cell 11: 12531266.
  • Feild TS, Lee DW, Holbrook NM. 2001. Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red-osier dogwood. Plant Physiology 127: 566574.
  • Feller U, Fischer A. 1994. Nitrogen metabolism in senescing leaves. Critical Reviews in Plant Sciences 13: 241273.
  • Fryer MJ, Oxborough K, Mullineaux PM, Baker NR. 2002. Imaging of photo-oxidative stress responses in leaves. Journal of Experimental Botany 53: 12491254.
  • Genty B, Briantais JM, Baker NR. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990: 8792.
  • Ghosh S, Mahoney SR, Penterman JN, Peirson D, Dumbroff EB. 2001. Ultrastructural and biochemical changes in chloroplasts during Brassica napus senescence. Plant Physiology and Biochemistry 39: 777784.
  • Guiamét JJ, Tyystjärvi E, Tyystjärvi T, John I, Kairavuo M, Pichersky E, Noodén LD. 2002. Photoinhibition and loss of photosystem II reaction centre proteins during senescence of soybean leaves. Enhancement of photoinhibition by the ‘stay-green’ mutation cyt6. Physiologia Plantarum 115: 468478.
  • Hensel LL, Grbiaç V, Baumgarten DA, Bleecker AB. 1993. Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell 5: 553564.
  • Himelblau E, Amasino RM. 2001. Nutrients mobilized from leaves of Arabidopsis thaliana during leaf senescence. Journal of Plant Physiology 158: 13171323.
  • Horton P, Ruban AV, Walters RG. 1994. Regulation of light harvesting in green plants. Indication by nonphotochemical quenching of chlorophyll fluorescence. Plant Physiology 106: 415420.
  • Humbeck K, Krupinska K. 2003. The abundance of minor chlorophyll a/b-binding proteins CP29 and LHCI of barley (Hordeum vulgare L.) during leaf senescence is controlled by light. Journal of Experimental Botany 54: 375383.
  • Jang JC, Sheen J. 1994. Sugar sensing in higher plants. Plant Cell 6: 16651679.
  • Lee RH, Chen SCG. 2002. Programmed cell death during rice leaf senescence is nonapoptotic. New Phytologist 155: 2332.
  • Leipner J, Oxborough K, Baker NR. 2001. Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging. Journal of Experimental Botany 52: 16891696.
  • Leverentz MK, Wagstaff C, Rogers HJ, Stead AD, Chanasut U, Silkowski H, Thomas B, Weichert H, Feussner I, Griffiths G. 2002. Characterization of a novel lipoxygenase-independent senescence mechanism in Alstroemeria peruviana floral tissue. Plant Physiology 130: 273283.
  • Li XP, Björkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, Niyogi KK. 2000. A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403: 391395.
  • Lichtenthaler HK, Babani F, Langsdorf G, Buschmann C. 2000. Measurement of differences in red chlorophyll fluorescence and photosynthetic activity between sun and shade leaves by fluorescence imaging. Photosynthetica 38: 521529.
  • Lu C, Zhang J. 1998. Modifications in photosystem II photochemistry in senescent leaves of maize plants. Journal of Experimental Botany 49: 16711679.
  • Lu C, Lu Q, Zhang J, Kuang T. 2001. Characterization of photosynthetic pigment composition, photosystem II photochemistry and thermal energy dissipation during leaf senescence of wheat plants grown in the field. Journal of Experimental Botany 52: 18051810.
  • Martin T, Oswald O, Graham IA. 2002. Arabidopsis seedling growth, storage lipid mobilization, and photosynthetic gene expression are regulated by carbon : nitrogen availability. Plant Physiology 128: 472481.
  • Masclaux C, Valadier MH, Brugière N, Morot-Gaudry JF, Hirel B. 2000. Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211: 510518.
  • Masclaux-Daubresse C, Valadier MH, Carrayol E, Reisdorf-Cren M, Hirel B. 2002. Diurnal changes in the expression of glutamate dehydrogenase and nitrate reductase are involved in the C/N balance of tobacco source leaves. Plant, Cell & Environment 25: 14511462.
  • Meng Q, Siebke K, Lippert P, Baur B, Mukherjee U, Weis E. 2001. Sink–source transition in tobacco leaves visualized using chlorophyll fluorescence imaging. New Phytologist 151: 585595.
  • Merzlyak MN, Hendry GAF. 1994. Free radical metabolism, pigment degradation and lipid peroxidation in leaves during senescence. Proceedings of the Royal Society of Edinburgh 102B: 459471.
  • Miller A, Tsai CH, Hemphill D, Endres M, Rodermel S, Spalding M. 1997. Elevated CO2 effects during leaf ontogeny. A new perspective on acclimation. Plant Physiology 115: 11951200.
  • Moore B, Zhou L, Rolland F, Hall Q, Cheng WH, Liu YX, Hwang I, Jones T, Sheen J. 2003. Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science 300: 332336.
  • Müller P, Li XP, Niyogi KK. 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiology 125: 15581566.
  • Munné-Bosch S, Alegre L. 2002. Plant aging increases oxidative stress in chloroplasts. Planta 214: 608615.
  • Nedbal L, Soukupová J, Kaftan D, Whitmarsh J, Trtílek M. 2000. Kinetic imaging of chlorophyll fluorescence using modulated light. Photosynthesis Research 66: 312.
  • Nie GY, Long SP, Garcia RL, Kimball BA, Lamorte RL, Pinter PJ, Wall GW, Webber AN. 1995. Effects of free-air CO2 enrichment on the development of the photosynthetic apparatus in wheat, as indicated by changes in leaf proteins. Plant, Cell & Environment 18: 855864.
  • Nielsen TH, Krapp A, Röper-Schwarz U, Stitt M. 1998. The sugar-mediated regulation of genes encoding the small subunit of Rubisco and the regulatory subunit of ADP glucose pyrophosphorylase is modified by phosphate and nitrogen. Plant, Cell & Environment 21: 443454.
  • Noodén LD, Guiamét JJ, John I. 1997. Senescence mechanisms. Physiologia Plantarum 101: 746753.
  • Ögren E. 1991. Prediction of photoinhibition of photosynthesis from measurements of fluorescence quenching components. Planta 184: 538544.
  • Ono K, Terashima I, Watanabe A. 1996. Interaction between nitrogen deficit of a plant and nitrogen content in the old leaves. Plant and Cell Physiology 37: 10831089.
  • Paul MJ, Driscoll SP. 1997. Sugar repression of photosynthesis: the role of carbohydrates in signalling nitrogen deficiency through source : sink imbalance. Plant, Cell & Environment 20: 110116.
  • Quirino BF, Reiter WD, Amasino RD. 2001. One of two tandem Arabidopsis genes homologous to monosaccharide transporters is senescence-associated. Plant Molecular Biology 46: 447457.
  • Scholes JD, Rolfe SA. 1996. Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata): quantitative imaging of chlorophyll fluorescence. Planta 199: 573582.
  • Smeekens S. 2000. Sugar-induced signal transduction in plants. Annual Review of Plant Physiology and Plant Molecular Biology 51: 4981.
  • Stessman D, Miller A, Spalding M, Rodermel S. 2002. Regulation of photosynthesis during Arabidopsis leaf development in continuous light. Photosynthesis Research 72: 2137.
  • Stitt M, Krapp A. 1999. The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background. Plant, Cell & Environment 22: 583621.
  • Thomas H, Ougham HJ, Wagstaff C, Stead AD. 2003. Defining senescence and death. Journal of Experimental Botany 54: 11271132.
  • Wingler A, Von Schaewen A, Leegood RC, Lea PJ, Quick WP. 1998. Regulation of leaf senescence by cytokinin, sugars, and light. Effects on NADH-dependent hydroxypyruvate reductase. Planta 116: 329335.