Introduction

One of the most important functions of leaf senescence is the remobilisation of nitrogen from the old leaves (Himelblau and Amasino 2001; Hörtensteiner and Feller 2002). Nitrogen deficiency can accelerate leaf senescence, especially when photosynthetic carbon supply is high (Ono et al. 1996; Stitt and Krapp 1999). High carbon/nitrogen ratios in leaves could signal a decreased requirement for investment of nitrogen in the photosynthetic apparatus, thereby increasing the amount of nitrogen available for growth and reproduction. Several lines of evidence suggest that sugars act as a signal for high carbon availability during the regulation of leaf senescence. Leaf sugar contents typically increase during senescence (Wingler et al. 1998; Masclaux et al. 2000; Quirino et al. 2001; Stessman et al. 2002) and senescence-like symptoms, such as leaf yellowing, can be triggered by sugars (Wingler et al. 1998). This correlative evidence is supported by work with mutants and transgenic plants. Tomato plants overexpressing the sugar sensor hexokinase-1 from Arabidopsis exhibit accelerated senescence (Dai et al. 1999), whereas senescence is delayed in the Arabidopsis glucose-insensitive2 mutant lacking hexokinase-1 (Moore et al. 2003). Furthermore, prolonged leaf senescence has been linked to decreased source strength in Rubisco antisense plants of tobacco (Miller et al. 2000), which contain a reduced amount of sugars (Quick et al. 1991).

We have recently shown that growth on medium containing 111 mM glucose in combination with low, but not high nitrogen supply results in accelerated senescence without affecting early photosynthetic development (Wingler et al. 2004). Visible senescence correlates with a decline in maximum photosynthetic efficiency (F v/F m), which can be monitored rapidly and non-destructively using chlorophyll fluorescence imaging. However, it remained unclear whether this response is caused by cross-talk of the nitrogen and sugar signalling pathways or by metabolic interactions.

In addition to sugars, treatment with abscisic acid (ABA) can induce leaf senescence (Yang et al. 2003). Moreover, the majority of sugar-insensitive Arabidopsis mutants are either ABA insensitive (abi mutants) or ABA deficient (aba mutants), demonstrating that ABA plays an important role in sugar responses. If ABA is required for the sugar-dependent induction of senescence, aba mutants would be expected to display delayed senescence in the presence of glucose. However, most sugar response mutants have been isolated in screens based on decreased sugar sensitivity during seedling development and little is known about the interactions of sugar and ABA signalling in mature or senescing plants.

Here, we analyse the effects of sugar and nitrogen supply on senescence in abi and aba mutants of Arabidopsis. abi4-1 is allelic to the sugar response mutants gin6 (Arenas-Huertero et al. 2000), sun6 (Huijser et al. 2000), sis5 (Laby et al. 2000), isi3 (Rook et al. 2001) and the salt-tolerant mutant sañ5 (Quesada et al. 2000). In addition to being glucose-insensitive, abi4-1 is moderately resistant to mannose (Laby et al. 2000). The ABI4 gene encodes an APETALA2 domain transcription factor (Finkelstein et al. 1998) and is sugar inducible in young seedlings, but not in older seedlings or mature leaves (Finkelstein et al. 1998; Söderman et al. 2000; Arroyo et al. 2003). Nevertheless, altered sugar and CO2 sensitivity has also been reported for mature abi4 (sun6) mutants (Van Oosten et al. 1997; Oswald et al. 2001). In contrast to ABI4, ABI5, which encodes a basic leucine-zipper transcription factor (Finkelstein and Lynch 2000), is also glucose inducible in vegetative tissue during later stages of development (Brocard et al. 2002; Arroyo et al. 2003). The abi5-1 mutant is moderately glucose and mannose insensitive during seedling development (Arenas-Huertero et al. 2000; Laby et al. 2000), but it has not been isolated in sugar-insensitivity screens. Two additional abi mutants that are not glucose insensitive, abi2-1 and abi1-1 (Arenas-Huertero et al. 2000), were used as controls in our experiments.

In addition, we used the ABA-deficient and glucose-insensitive aba1-1, aba2-1 and aba3-1 mutants (Arenas-Huertero et al. 2000) to analyse the role of ABA synthesis in the regulation of senescence. The ethylene-insensitive and sugar-hypersensitive ein2-1 mutant (Cheng et al. 2002), which is allelic to mutants with delayed senescence (Oh et al. 1997), was included to analyse the interactions between ethylene and sugar signalling during senescence.

Our results show that sugar signalling during senescence is not ABA dependent and does not require ABI4. However, the ABI5-dependent sugar response pathway appears to be involved in the regulation of senescence by sugars.

Materials and methods

Plant material

Seed of Arabidopsis thaliana (L.) Heynh. was obtained from the Nottingham Arabidopsis Stock Centre (Nottingham, UK). The following mutants were used: abi1-1, abi2-1 (Koornneef et al. 1984), abi4-1, abi5-1 (Finkelstein 1994), aba1-1 (Koornneef et al. 1982), aba2-1, aba3-1 (Léon-Kloosterziel et al. 1996) and ein2-1 (Bleecker et al. 1988). After sterilisation in commercial bleach, the seeds were washed in water, resuspended in 0.7% low-melting-point agarose and pipetted onto agar (1% w/v) medium (approx. 10 plants per plate). For high nitrogen (HN) treatments, half-strength Murashige and Skoog (MS) basal salt mixture (M 5524; Sigma–Aldrich, Gillingham, Dorset, UK) containing 30 mM nitrogen (10.3 mM NH4 + and 19.7 mM NO3 −) was used. For low nitrogen (LN) treatments, the nitrogen concentration was lowered to 4.7 mM nitrogen (NO3 − only) by using quarter-strength Murashige and Skoog basal salt mixture without NH4NO3 (M 2909: Sigma–Aldrich). The effect of this treatment was the same as for growth on non-commercial MS medium containing 4.7 mM NO3 − and all other nutrients at half strength. After cold treatment for 2–4 days at 4°C, the plates were transferred to growth chambers and grown in vertical orientation at a photon flux density of 100 μmol m−2 s−1 for 16 h per day and a temperature of 22°C during the day and 18°C at night.

Chlorophyll fluorescence analysis

Chlorophyll a fluorescence was analysed using a pulse-modulated imaging fluorometer (FluorCam 700MF; Photon Systems Instruments, Brno, Czech Republic). After dark-adaptation for at least 20 min, minimum fluorescence (F 0) was measured by exposing the plants to modulated red light, before a saturating flash of white light (0.8 s duration) was applied to record maximum fluorescence (F m) and to determine the maximum photosynthetic efficiency (F v/F m) in whole leaf rosettes as described by Wingler et al. (2004).

Sugar analysis

Shoots of plants were harvested between 7 and 8 h into the photoperiod, washed thoroughly in water and extracted in 80% ethanol at 80°C. Sugars were determined enzymatically according to Stitt et al. (1989) using an ELX 808UI microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Results

Effect of glucose and nitrogen supply on sugar contents during senescence

We have shown previously that visible leaf senescence is correlated with a decline in maximum photosynthetic efficiency (F v/F m), a parameter that can be monitored rapidly and non-destructively using chlorophyll fluorescence imaging (Wingler et al. 2004). Senescence-dependent leaf yellowing and the decline in F v/F m are accelerated by glucose in combination with low, but not with high nitrogen supply (Wingler et al. 2004). In contrast to glucose, sorbitol and mannitol did not induce leaf yellowing (not shown) or an early decline if F v/F m in the leaf rosettes of nitrogen-deficient plants (Fig. 1), demonstrating that induction of senescence is not caused by the osmotic potential of the growth medium.

Fig. 1
figure 1

Effect of glucose, sorbitol and mannitol supply on senescence. Senescence was monitored as a decline in maximum photosynthetic efficiency (F v/F m) in whole leaf rosettes of Arabidopsis thaliana (Col-0) plants grown on low nitrogen (4.7 mM nitrate) medium without sugar (LN; closed circles) or with addition of 111 mM glucose (LN+Glc; open triangles), mannitol (LN+Mtl; open squares) or sorbitol (LN+Stl; open diamonds). Data are means of at least 20 plants + SD

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To determine the effect of nitrogen and glucose supply on internal sugar accumulation, we measured glucose, fructose and sucrose contents of Col-0 plants at four different developmental stages (Fig. 2a–c). On day 23, no signs of senescence were visible; on day 30, plants grown on glucose with a low nitrogen supply showed the first signs of senescence-dependent leaf yellowing; on day 37, senescence was clearly visible on this medium; on day 44, most plants of this treatment were dead. Glucose, fructose and sucrose accumulated strongly in plants growing on medium with glucose in combination with low nitrogen supply. Sugar contents rose during senescence (until day 37) and remained high during the final stages (day 45). In contrast, growth on 111 mM glucose in combination with high nitrogen supply only led to a moderate accumulation of sugars in the leaves. For example, fructose content was equally as high in plants grown on low nitrogen medium without glucose as in those grown on high nitrogen medium plus glucose. These results show that, especially during senescence, sugar accumulation not only depends on external glucose supply but also on the availability of nitrogen.

Fig. 2a–c
figure 2

Effect of glucose and nitrogen supply on glucose (a), fructose (b) and sucrose (c) contents in leaf rosettes of senescing Arabidopsis (Col-0) plants. The plants were grown on agar medium containing 30 mM nitrogen (HN; closed symbols) or 4.7 mM nitrogen (LN; open symbols) without sugar (HN, LN; circles) or with addition of 111 mM glucose (HN+Glc, LN+Glc; triangles). Data are means of four samples ± SD

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Effect of glucose and nitrogen supply on senescence in the sugar-insensitive abi4-1 and abi5-1 mutants

No difference was found in the regulation of the senescence-dependent decline in F v/F m between the abi4-1 mutant and its wild type Col-0 (Fig. 3a,b). In both genotypes, senescence was accelerated to the same extent by growth on medium containing glucose combined with low nitrogen supply. No decline in F v/F m was detected on media with high nitrogen supply or without glucose over the course of the experiment. Addition of glucose in the presence of high nitrogen supply generally resulted in higher F v/F m values.

Fig. 3a, b
figure 3

Effect of glucose and nitrogen supply on senescence in wild-type Arabidopsis (Col-0; closed symbols) and the sugar-insensitive abi4-1 mutant (open symbols). Senescence was monitored as a decline in maximum photosynthetic efficiency (F v/F m) in whole leaf rosettes of plants grown on agar medium containing 30 mM nitrogen (HN; a) or 4.7 mM nitrogen (LN; b) without sugar (HN, LN; circles) or with addition of 111 mM glucose (HN+Glc, LN+Glc; triangles). Data are means of at least 10 plants + SD

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On medium with glucose and low nitrogen supply, senescence was delayed in abi5-1 compared to wild-type (Ws-2) plants (Fig. 4a,b). This effect was statistically significant (e.g. P=0.0015; t-test for day 56). No clear differences between abi5-1 and Ws-2 were detected during growth on the other media over the course of the experiment.

Fig. 4a, b
figure 4

Effect of glucose and nitrogen supply on senescence in wild-type Arabidopsis (Ws-2; closed symbols) and in the sugar-insensitive abi5-1 mutant (open symbols). Senescence was monitored as a decline in maximum photosynthetic efficiency (F v/F m) in whole leaf rosettes of plants grown on agar medium containing 30 mM nitrogen (HN; a) or 4.7 mM nitrogen (LN; b) without sugar (HN, LN; circles) or with addition of 111 mM glucose (HN+Glc, LN+Glc; triangles). Data are means of at least 10 plants + SD

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Effect of glucose and nitrogen supply on senescence in other ABA-insensitive, ABA-deficient or ethylene-insensitive mutants

In addition to being ethylene-insensitive, ein2 has been shown to display delayed senescence, as well as sugar hypersensitivity. In our experiments, F v/F m was generally reduced in ein2-1, especially until day 30 (Fig. 5a–d). However, there was no indication that the regulation of senescence by sugars was affected in ein2-1, suggesting either that EIN2 is not involved in the sugar-dependent regulation of senescence or that delayed senescence can override the effect of sugar hyper-sensitivity. In addition to being ABA deficient, the aba2 and aba3 mutants are sugar insensitive during seedling development. However, neither of these mutants showed delayed senescence in the presence of glucose. Instead, senescence was significantly accelerated in aba2-1 (P=0.0006; t-test for day 56), but not in aba3-1 grown on medium with low nitrogen supply plus glucose. On other media with either high nitrogen supply or without glucose, F v/F m in aba2-1 was not different from wild-type values.

Fig. 5a–d
figure 5

Effect of glucose and nitrogen supply on senescence in wild-type Arabidopsis (Col-0; closed circles) and in the ein2-1, aba3-1 and aba2-1 mutants (open symbols). Senescence was monitored as a decline in maximum photosynthetic efficiency (F v/F m) in whole leaf rosettes of plants grown on agar medium containing 30 mM nitrogen without sugar (HN; a) or with addition of 111 mM glucose (HN+Glc; b) and on 4.7 mM nitrogen without sugar (LN; c) or with addition of 111 mM glucose (LN+Glc; d). Data are means of at least 10 plants + SD

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Another ABA-deficient mutant, aba1-1, also showed a significantly accelerated decline in F v/F m on the medium with low nitrogen supply plus glucose (P=0.0024; t-test for day 56) but not on the other media tested (Fig. 6 a–d). Two additional mutants, abi1-1 and abi2-1, which are both ABA insensitive but not glucose insensitive, did not have any detectable senescence phenotype.

Fig. 6a–d
figure 6

Effect of glucose and nitrogen supply on senescence in wild-type Arabidopsis (Ler-0; closed circles) and in the abi1-1, abi2-1 and aba1-1 mutants (open symbols). Senescence was monitored as a decline in maximum photosynthetic efficiency (F v/F m) in whole leaf rosettes of plants grown on agar medium containing 30 mM nitrogen without sugar (HN; a) or with addition of 111 mM glucose (HN+Glc; b) and on 4.7 mM nitrogen without sugar (LN; c) or with addition of 111 mM glucose (LN+Glc; d). Data are means of at least 10 plants + SD

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Effect of glucose and nitrogen supply on visible senescence in the abi5-1, aba2-1 and aba1-1 mutants

The results of the F v/F m measurements were confirmed by the optical impression of the plants (Fig. 7). On medium with low nitrogen supply plus glucose, visible leaf yellowing was delayed in abi5-1 and accelerated in aba2-1 and aba1-1 compared to their wild types. abi5-1 and aba2-1 did not show a visible senescence phenotype on any of the other media. Leaf yellowing was also visible in the old leaves of aba1-1 plants grown on low nitrogen medium without glucose and on high nitrogen medium with glucose.

Fig. 7
figure 7

Effect of glucose and nitrogen supply on visible senescence in the abi5-1, aba2-1 and aba1-1 mutants of Arabidopsis and their respective wild-types (Ws-2, Col-0 and Ler-0) grown for 42 days on agar medium containing 30 mM nitrogen without sugar (HN) or with addition of 111 mM glucose (HN+Glc) and on 4.7 mM nitrogen without sugar (LN) or with addition of 111 mM glucose (LN+Glc)

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Effect of glucose and nitrogen supply on flowering in the abi5-1, aba2-1 and aba1-1 mutants

Flowering time was determined to test if altered senescence in abi5-1, aba2-1 or aba1-1 was caused by overall changes in development, as indicated by early or late flowering. In the presence of glucose, Ws-2 and abi5-1 plants flowered earlier on low compared to high nitrogen medium (Fig. 8a). There was no difference in flowering time between abi5-1 and its wild type Ws-2 on medium with high nitrogen plus glucose. However, flowering was slightly delayed in abi5-1 plants on medium with low nitrogen plus glucose. Flowering was clearly accelerated in aba2-1 plants grown on medium with low nitrogen plus glucose compared to its wild-type Col-0 (Fig. 8b). It is thus likely that early senescence on this medium was linked to accelerated development. In aba1-1, flowering was slightly delayed rather than accelerated on medium with low nitrogen plus glucose (Fig. 8c). Early senescence was thus not caused by accelerated plant development.

Fig. 8a–c
figure 8

Flowering of the abi5-1 (a), aba2-1 (b) and aba1-1 (c) mutants (open symbols) and their respective wild types (Ws-2, Col-0 and Ler-0; closed symbols) grown on agar medium containing 30 mM nitrogen with addition of 2% glucose (HN+Glc; triangles) and 4.7 mM nitrogen with addition of 2% glucose (LN+Glc; circles). Data are means of plants grown on 4–5 separate Petri dishes

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Discussion

Senescence is induced by sugar accumulation in nitrogen-deficient plants

Leaf senescence can be induced by glucose in combination with low nitrogen supply, resulting in an early decline in F v/F m (Wingler et al. 2004). Interactions of sugars and nitrogen in the regulation of leaf senescence could either be caused by cross-talk between the sugar and nitrogen signalling pathways or by metabolic interactions, e.g. by an effect of nitrogen supply on sugar accumulation. Our results show that the extent of sugar accumulation during growth on glucose-containing medium is nitrogen dependent (Fig. 2). Sugar accumulation was much stronger on medium with low compared to high nitrogen supply in all genotypes analysed (Ws-2, abi5-1, Col-0, aba2-1, Ler-0 and aba1-1; data not shown). Increased sugar accumulation in nitrogen-deficient plants could be due to decreased sugar utilisation for the synthesis of amino acids and protein. Increased sugar contents have also been reported for Arabidopsis seedling germinated in the presence of glucose in combination with low nitrogen supply (Martin et al. 2002), Arabidopsis plants grown hydroponically with low nitrogen supply (Sun et al 2002), tobacco plants after nitrogen withdrawal (Paul and Driscoll 1997) and tobacco seedlings grown on low-nitrogen agar medium (Nielsen et al. 1998). However, glucose, fructose and sucrose contents were reduced in leaves of tobacco after growth at low nitrogen supply (Scheible et al. 1997, 2000) and growth in elevated CO2 only led to sugar accumulation in nitrogen-sufficient, but not in nitrogen-deficient plants (Geiger et al. 1999). These discrepancies could result from differences between species or in the developmental stage analysed. For example, nitrogen and sugar contents were negatively correlated during early senescence, but not in non-senescing leaves of sugar maple (Schaberg et al. 2003). In our experiments, the effect of nitrogen limitation on sugar accumulation was also strongest in senescing plants.

It is likely that early senescence on medium containing glucose in combination with low nitrogen supply was caused by increased sugar contents, rather than by enhanced sugar sensitivity of nitrogen-deficient plants. Although senescence was not a response to the osmolarity of the growth medium (Fig. 1), an osmotic effect caused by the high sugar accumulation within the plants (Fig. 2) cannot be ruled out as the cause of accelerated senescence. Moreover, the accumulation of sugars could have triggered pathogen-defence responses that lead to the initiation of senescence (Yoshida et al. 2002).

Interactions of carbon and nitrogen metabolism that suggest an involvement of sugars in the regulation of leaf senescence have also been reported for other species. In sunflower, low nitrogen in combination with high light results in an accumulation of sugars and triggers an early decline in leaf nitrogen content (Ono et al. 1996). Sugar signalling may also be responsible for the shift in leaf ontogeny and accelerated senescence observed in plants growing in elevated CO2 concentrations (Nie et al. 1995; Miller et al. 1997). However, Ludewig and Sonnewald (2000) found no correlation between sugar content and the senescence-dependent down-regulation of photosynthetic gene expression in elevated CO2, suggesting that CO2 effects are independent of sugar accumulation.

The ABI5-dependent, but not the ABI4-dependent, sugar signalling pathway is involved in the regulation of senescence by sugars

Having established that sugar accumulation can trigger symptoms of leaf senescence, we were interested to find out if this response is attenuated in sugar response mutants. However, most sugar response mutants of Arabidopsis have been isolated based on insensitivity to very high sugar concentrations during seedling germination. In mature plants, sugar-insensitive phenotypes have only been established in a few cases, e.g. delayed senescence and decreased leaf expansion in the gin2-1 mutant (Moore et al. 2003) and reduced feedback inhibition of photosynthesis and altered expression of the plastocyanin gene in the sun6 (=abi4) mutant (Van Oosten et al. 1997; Oswald et al. 2001). However, the ABI4 gene is mainly expressed in seeds and in young seedlings, whereas expression in vegetative tissue is very low (Finkelstein 1994; Arroyo et al. 2003). It is therefore not surprising that we could not find any effect of the abi4-1 mutation on sugar-induced leaf senescence (Fig. 3). In addition, senescence was not affected in compost-grown abi4-1 plants (data not shown). ABI4 therefore does not appear to be involved in the induction of leaf senescence by sugars.

In contrast to the abi4-1 mutant, senescence was delayed in the abi5-1 mutant (Fig. 4), demonstrating that ABI5, a basic leucine-zipper transcription factor, is involved in sugar responses during leaf senescence. Expression of the ABI5 gene is very low in vegetative tissue compared to seeds (Finkelstein and Lynch 2000) and is only ABA inducible in young seedlings (Lopez-Molina et al. 2001). However, more recent evidence shows that ABI5, in contrast to ABI4, can also be induced by glucose during later stages of development (Brocard et al. 2002; Arroyo et al. 2003). It would be interesting to analyse if ABI5 is also induced during leaf senescence. There are two principal ways in which ABA and sugar signalling pathways could interact. Either sugars and ABA share components of the same signalling pathway or sugars induce ABA synthesis, e.g. due to their osmotic effect (Rook et al. 2001; Price et al. 2003). Growth of the abi5-1 mutant at elevated CO2 or high light would show to what extent internal sugar formation affects leaf senescence in this mutant.

Induction of senescence by sugars does not require ABA synthesis, but ABA deficiency can result in accelerated senescence

To investigate whether or not the effect of sugars during senescence is dependent on ABA synthesis, we studied leaf senescence in the ABA-deficient and sugar-insensitive aba1-1, aba2-1 and aba3-1 mutants. As senescence can be induced by ABA (Yang et al. 2003), delayed senescence could theoretically also be caused directly by ABA deficiency. However, senescence was not delayed in any of the aba mutants (Figs. 5, 6), showing that induction of senescence by sugars does not require ABA synthesis. While aba3-1 showed no senescence phenotype, senescence was even accelerated in the aba2-1 and aba1-1 mutants. Senescence was also accelerated in the aba1-1 mutant when it was grown in compost (data not shown). Acceleration of leaf senescence by ABA deficiency could be caused by enhanced osmotic stress due to impaired stomatal regulation, resulting in a wilty phenotype (León-Kloosterziel et al. 1996; Merlot et al. 2002). The difference between aba1-1/aba2-1 and aba3-1 can be explained by differences in the extent of ABA deficiency. aba3-1 contains more ABA and loses less water through transpiration than the other aba mutants (León-Kloosterziel et al. 1996). As aba1 mutants are impaired in epoxy-carotenoid biosynthesis and thus in xanthophyll cycle activity (Rock and Zeevaart 1991), osmotic stress may have been exacerbated by decreased photoprotection. Likewise, the Arabidopsis aba1-3 mutant shows increased sensitivity to salinity in combination with high light (Cramer 2002). Interestingly, aba mutants exhibit decreased NaCl sensitivity during seedling germination (León-Kloosterziel et al. 1996; Gonzáles-Guzmán et al. 2002). Again, this shows that effects on seedling development are quite different from effects during later developmental stages. In mature plants, sugar accumulation probably increases stress in the aba1-1 and aba2-1 mutants, thereby inducing early senescence.