C : N Ratio Increases in the Phloem Sap During Floral Transition of the Long-Day Plants Sinapis alba and Arabidopsis thaliana

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1 Plant Cell Physiol. 43(6): (2002) JSPP 2002 C : N Ratio Increases in the Phloem Sap During Floral Transition of the Long-Day Plants Sinapis alba and Arabidopsis thaliana Laurent Corbesier 1, Georges Bernier and Claire Périlleux Laboratoire de Physiologie Végétale, Département des Sciences de la Vie, Université de Liège, Sart Tilman B22, B4000 Liège, Belgium In plants of Sinapis alba and Arabidopsis thaliana, leaf exudate (phloem sap) was analysed during and after a single long day inducing flowering and in control short days. The amounts of carbohydrates and amino acids were measured to estimate the organic C : N ratio. In both species, the C : N ratio of the phloem sap increased markedly and early during the inductive treatment, suggesting that an inequality in organic C and N supply to the apical meristem may be important at floral transition. Keywords: Arabidopsis thaliana C : N ratio Floral stimulus Flowering Phloem sap Sinapis alba. Abbreviations: FMOC, 9-fluorenylmethoxycarbonyl chloride; HPLC, high performance liquid chromatography; LD, long day; SD, short day. For almost a century the plant nutritional status was believed to be an essential factor in the control of flowering time. For Klebs (1913), a high endogenous ratio of carbohydrates to nitrogen, the so-called C : N ratio, promotes flowering whereas a high nitrogen availability resulting in a low C : N ratio promotes vegetative growth. This idea was inferred from the facts that (a) conditions favouring photosynthetic CO 2 fixation generally accelerate flowering and (b) a high nitrogen supply (fertilizer) might delay or reduce reproductive development in some plants (reviewed in Bernier et al. 1981). Later, Sachs and Hackett (1977) postulated that, irrespective of the environmental factors involved (day length, vernalization, etc.), floral induction consists of the modification of source : sink relationships in such a way that the shoot apical meristem (SAM) receives a better supply of assimilates (mainly carbohydrates). On the other hand, Raper et al. (1988) and Rideout et al. (1992) hypothesized that floral transition is stimulated by an imbalance in the relative availability of carbohydrates and N in the SAM. In day-neutral tobacco, these authors found that flowering may be accelerated by different stress (low temperature, nitrogen withdrawal, restriction of nitrogen uptake) and these treatments result in an inequality in the relative availability of carbohydrates and N in the SAM, the increase in carbohydrates being higher than the increase in N. ; The importance of an appropriate C : N ratio for flowering was also observed in vitro, for example in Torenia fournieri (Tanimoto and Harada 1981), Pharbitis nil (Ishioka et al. 1991) and tomato (Dielen et al. 2001). For Baeria, Chenopodium, Glycine, Lemna, Nicotiana and Perilla, culture media prepared for flower production must contain more sucrose than media used to grow vegetative plants, and a high N concentration inhibits flowering (reviewed in Dickens and van Staden 1988). Photoperiod-promoted flowering is thought to involve movement within the plants of signals from the induced organs the leaves to the target: the SAM (Lang 1965, Bernier et al. 1981). The nature of this floral stimulus moving in the phloem has been debated for a very long time but observations have accumulated indicating that this stimulus may include assimilates, together with other factors such as hormones (Bernier et al. 1993, Bernier et al. 1998). In one of the bestinvestigated species, the long-day (LD) plant Sinapis alba, it was shown that the supply of both C-assimilates (carbohydrates) and N-assimilates (amino acids) toward the SAM increases at floral transition (Lejeune et al. 1991, Corbesier et al. 2001). The same situation was observed in Arabidopsis thaliana (Corbesier et al. 1998, Corbesier et al. 2001). In addition, sucrose was also found to rescue most late-flowering mutants of A. thaliana grown in vitro, provided the concentration of this carbohydrate was not too high (Araki and Komeda 1993, Roldan et al. 1999, Ohto et al. 2001). Although these experiments support the idea that flowering time is influenced by the C and N status of the plants, whether this reflects the relative availability of C- and N- assimilates remains to be shown. Since organic C and N are essentially supplied to the SAM by the phloem sap, our aim here was to provide data concerning changes of the phloem organic C : N ratio during the transition to flowering in S. alba and A. thaliana both induced to flower by one LD. Analyses of leaf exudates were performed for the first time on the same samples collected simultaneously during the inductive LD and in control short days (SDs) for both species (Fig. 1). Our purpose was to estimate the quantitative balance between the main C- and N-assimilates that are translocated in the phloem and have been previously found to be putative components of the floral stimulus in S. alba and A. thaliana, 1 Corresponding author: , laurent.corbesier@ulg.ac.be; Fax,

2 C : N ratio and flowering in S. alba and A. thaliana 685 Fig. 1 Experimental design. Leaf exudates were collected during 4-h or 8-h periods in S. alba and A. thaliana, respectively. Exudation started at various times after the beginning of a standard 8-h SD or the inductive 22-h LD. Photoperiodic conditions are shown as white (light) and black (night) bars. namely carbohydrates and amino acids. Although many other substances move in the phloem of which some are also florigenic such as cytokinins and polyamines they contribute very little to the C :N status of that sap because of their low abundance. It is also the case for organic acids that mostly move through the xylem (Martinoia and Rentsch 1994, Peoples and Gifford 1997). Consequently, they were not investigated in this study. The nature and amounts of carbohydrates and amino acids detected were similar to those previously found in separate sampling experiments (Lejeune et al. 1991, Corbesier et al. 1998, Corbesier et al. 2001). Briefly, in both species, sucrose and glutamine were the major assimilates transported. In SD, sucrose export was low during the light period, exhibited a Table 1 Variation in the organic C : N ratio of the leaf exudates of SD-grown and LD-induced (a) S. alba and (b) A. thaliana plants (a) Sinapis alba Exudation period in SD (h) Exp #1 Exp #2 Exp #3 SD LD SD LD SD LD (b) Arabidopsis thaliana Exudation period in SD (h) Exp #1 Exp #2 Exp #3 SD LD SD LD SD LD For the light conditions of the exudation periods, see Fig. 1. maximum at the beginning of the night and decreased thereafter; the export of amino acids remained almost constant during the 24-h cycle but tended to increase during the light period of the SD. In LD-induced plants, a large increase in the sucrose and glutamine exports was observed during the LD and the following SD, before they returned to control levels. In SD, the C : N ratio was higher during the night (8 24 h) than during the light (24 32 h) period, in both S. alba and A. thaliana (Table 1). The increased C : N ratio during the night in both species comes from the fact that sucrose export in SDs is higher during the night than during the light period (see above). This trend was clearly seen in all three experiments despite the variation detected between the experiments. An important variation was also observed for samples collected on plants induced to flower by one LD (Table 1), especially in S. alba. This situation is frequently described and can be easily explained by the fact that this plant material is not genetically uniform. Moreover, even if the LD synchronizes the population and induces all the individuals to flower, our 4-h path for harvesting is quite narrow and may suffer timing differences (the variations are weaker for A. thaliana with an 8-h path). It is clear, however, that the fluctuations from one experiment to the other are of the same order of magnitude in LD and in SDs, thus we decided to calculate relative values (Fig. 2) and this indeed highlights reproducible and statistically significant trends. In S. alba, the C : N ratio of the phloem sap rose in LDinduced plants as compared to SD-controls: there was a first transient increase during the extension period of the LD: the C : N ratio was about doubled between 12 and 16 h after the start of the experiment (Fig. 2). Then the C : N ratio remained high or decreased but then peaked again after the LD, at 24 and 28 h. At that time, the difference between LD and SD was statistically significant. In A. thaliana, the situation was similar: the C : N ratio was higher in induced plants during and after the LD, but the kinetics were not sufficiently precise to assess whether the increase during the LD was transient or not. However, the increases observed were statistically significant at h and h. In both species, the amplitude of the

3 686 C : N ratio and flowering in S. alba and A. thaliana Fig. 2 Changes in the organic C : N ratio of the leaf exudates of LD-induced S. alba (a) and A. thaliana (b) plants. Relative values are given and were calculated as (C : N ratio in LD). (C : N ratio in SD) 1. Labels on X-axis correspond to the start of the exudation periods; for their timing and the light conditions, refer to Fig. 1. *, statistically different from SD controls (P = 0.05). changes was similar: the C : N ratio was 2 4 times higher in LD than in SD. Translocation of the floral stimulus in fact movement of its slowest component if the floral stimulus is multifactorial can be followed by defoliation experiments. This approach enabled us to estimate that in both S. alba and A. thaliana, export of the floral stimulus out of the leaves occurs during the extension period of the LD and is completed during the following 8-h light period of the SD (Bernier 1989, Corbesier et al. 1996). Since an increased C : N ratio in the leaf exudate was observed at that time, we may stress the correlation between an increased C : N ratio in the phloem sap and the induction of flowering. Previous experiments showed that both carbohydrate and amino acid contents of the phloem sap increase in S. alba and A. thaliana during floral transition. Thus the increase in the C : N ratio reported here suggests an imbalance in favour of C- compounds directed toward the SAM. It was indeed shown that changes observed in the leaf exudates are similar to those observed in apical exudates collected just below the SAM in S.

4 C : N ratio and flowering in S. alba and A. thaliana 687 alba (Lejeune et al. 1991, Corbesier et al. 2001). Interestingly, we observed a transient increase of the C : N ratio in S. alba during the LD which suggests an early signalling role while the second increase observed after the LD could be caused by the increased sink activity of the SAM. Our observation is rather similar to that of Rideout et al. (1992) who showed that treatments accelerating flowering of tobacco result in an inequality in the relative availability of carbohydrates and N in the SAM with a higher increase of carbohydrates than of N. Beside C- and N- assimilates, involvement of other factors as putative components of the floral stimulus has been well documented (reviewed in Bernier et al. 1993, Levy and Dean 1998), as well as cross-talks between sugar-, nitrogen-, and hormone-signalling (reviewed in Coruzzi and Zhou 2001, Gibson 2000). Thus, progress towards understanding the mechanisms of floral induction will require an integrated approach of all these multiple factors. Growth conditions for S. alba and A. thaliana ecotype Columbia were as described elsewhere (Corbesier et al. 1996, Lejeune et al. 1988). Briefly, plants of S. alba were sown and grown on a mixture of perlite and vermiculite (1 : 1) in 8-cm pots and were watered alternatively with demineralized water and a complete Hoagland solution. Seeds of A. thaliana were first vernalized in the dark at 2 C for 6 weeks, then sown and grown on a mixture of leaf mould, clay and sand. Six plants were grown per tray containing 1 liter of substrate and were watered daily with tap water. All plants were grown in controlled cabinets. Light was provided by fluorescent tubes (Very High Output Sylvania, Zaventem, Belgium) at an irradiance of 150 and 48 mol m 2 s 1 (PAR) for S. alba and A. thaliana, respectively. Day and night temperature was 20 C and relative humidity was about 80%. After 65 or 56 d of culture in 8-h SDs, respectively, plants of S. alba and A. thaliana were induced to flower by a single 22-h LD, then returned to the SD regimen. The photoperiodic extension was given at the same irradiance as during standard SDs. Dissection of shoot apices 2 weeks after the experiment showed that 100% of the S. alba and A. thaliana plants had initiated flowers in response to the LD while control plants continuously kept in SDs remained vegetative. Leaf exudates were collected using the EDTA-method (King and Zeevaart 1974) as previously described by Lejeune et al. (1988) for S. alba and Corbesier et al. (1998) for A. thaliana. Briefly, the uppermost five leaves below the halfexpanded one of S. alba plants were placed together in a 250- ml beaker containing 20 ml of 20 mm EDTA (ph 7.5). For A. thaliana, the seven youngest mature leaves were collected per plant and placed in a 500- l microcentrifuge tube containing 400 l of 10 mm EDTA (ph 8.5). Ten plants of each species were used at each sampling time. Then, the vessels containing the leaves were enclosed in airtight clear chambers containing water to ensure maximum relative humidity and to prevent EDTA uptake by the leaves. The duration of exudation was 4 h for S. alba and 8 h for A. thaliana. During this period, the exuding leaves were subjected to light-dark cycles as experienced by intact plants (Fig. 1). After collection, exudates were stored at 20 C until analysis. Three independent experiments were conducted with each species. To separate and identify carbohydrates, exudates were passed through an AG2X8 column (Bio-Rad, Brussels, Belgium) before analysis by high performance liquid chromatography (HPLC) and refractometry on an Aminex HPX-87C column (Bio-Rad, Brussels, Belgium) as described in Corbesier et al. (1998). Amino acids were analysed as previously described in Corbesier et al. (2001). Briefly, samples were pre-column derivatized using M 9-fluorenylmethoxycarbonyl chloride (FMOC, Merck-Belgolabo, Overijse, Belgium) dissolved in acetone. The excess of FMOC was quenched by M 1- adamantamine (in 75% acetone). Samples were then diluted in ultrapure water prior to analysis by reverse phase HPLC on a Spherisorb C18 analytical column (100 2 mm i.d., Achrom, Zulte-Machelen, Belgium) and fluorescence detection (excitation, 265 nm; emission, 313 nm). Amounts of carbohydrates, essentially sucrose, were converted to equivalent C atoms (12 atoms of C per molecule of sucrose). The same method was used to transform all identified amino acids into their equivalent N atoms. The organic C : N ratio was then calculated from these data. Analysis of the significance of the changes observed was examined using the Chi-Square distribution of the LD as relative to the SD control values of each independent experiment with P = Acknowledgments L.C. is grateful to the F.N.R.S. for the award of a Postdoctoral Research Fellowship. This research was supported by grants from the European Community Contract BI04 CT ; the Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister s Office Federal Office for Scientific, Technical and Cultural Affairs; P4/15) and the University of Liège (Fonds Spéciaux). References Araki, T. and Komeda, Y. (1993) Flowering in darkness in Arabidopsis thaliana. Plant J. 4: Bernier, G. (1989) Events of the floral transition of meristems. In Plant Reproduction: From Floral Induction to Pollination. Edited by Lord, E. and Bernier, G. pp The American Society of Plant Physiologists Symposium Series Vol. 1., Rockville. Bernier, G., Corbesier, L., Périlleux, C., Havelange, A. and Lejeune, P. 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