Diel changes in nitrogen and carbon resource status ... - Oxford Journals

Annals of Botany 103: 1025– 1037, 2009 doi:10.1093/aob/mcp043, available online at www.aob.oxfordjournals.org

Diel changes in nitrogen and carbon resource status and use for growth in young plants of tomato (Solanum lycopersicum) Ruth Huanosto Maganã, Ste´phane Adamowicz* and Loı¨c Page`s INRA, UR 1115 Plantes et Syste`mes de Culture Horticoles, F-84914 Avignon, France Received: 8 August 2008 Returned for revision: 10 November 2008 Accepted: 14 January 2009 Published electronically: 2 March 2009

† Background and Aims Modellers often define growth as the development of plant structures from endogenous resources, thus making a distinction between structural (WS) and total (W ) dry biomass, the latter being the sum of WS and the weight of storage compounds. In this study, short-term C and N reserves were characterized experimentally (forms, organ distribution, time changes) in relation to light and nutrition signals, and organ structural growth in response to reserve levels was evaluated. † Methods Tomato plants (Solanum lycopersicum) were grown hydroponically in a growth room with a 12-h 23 ). Three experiments were carried out 18 d after photoperiod and an adequate supply of NO2 3 (3 mol m 23 2 sowing: [NO3 ] was either maintained at 3 mol m , changed to 0.02 mol m23 or to 0 mol m23. Plants were sampled periodically throughout the light/dark cycles over 24–48 h. Organ WS was calculated from W together with the amount of different compounds that act as C and N resources, i.e. non-structural carbohydrates and carboxylates, nitrate and free amino acids. † Key Results With adequate nutrition, carbohydrates accumulated in leaves during light periods, when photosynthesis exceeded growth needs, but decreased at night when these sugars are the main source of C for growth. At the end of the night, carbohydrates were still high enough to fuel full-rate growth, as WS increased at a near constant rate throughout the light/dark cycle. When nitrate levels were restricted, C reserves increased, but [NO2 3] decreased progressively in stems, which contain most of the plant N reserves, and rapidly in leaves and roots. This resulted in a rapid restriction of structural growth. † Conclusions Periodic darkness did not restrict growth because sufficient carbohydrate reserves accumulated during the light period. Structural growth, however, was very responsive to NO2 3 nutrition, because N reserves were mostly located in stems, which have limited nitrate reduction capacity. Key words: Solanum lycopersicum, tomato, nitrogen, carbon, structural growth, reserves, nitrate, amino acids, carbohydrate, carboxylate.

IN T RO DU C T IO N Plant growth is dependant on external resources, with carbon and nitrogen being amongst the most important. However, there is a discrepancy between the continuous demand for resources due to growth and their fluctuating acquisition by plants. For example, patterns of CO2 exchange rates are determined by light-dependent photosynthesis. Thus, dry biomass accumulation follows cycles of marked increases during the day and null gains at night (or even slightly negative values due to respiratory losses), from which it is inferred that nightly growth draws C from previously accumulated reserves. Large changes in nitrogen uptake rates are observed when it becomes depleted in the rooting medium, and also during day/night cycles even when N is constantly available (Clement et al., 1978; Ca´rdenas-Navarro et al., 1998; Matt et al., 2001a), leading to the question of how growth responds to such variations. Growth can be defined as the build-up of plant structures (metabolic or not) from endogenous resources in defined ratios. Storage of C and N is crucial because it enables the continuous growth of new structures and buffers against significant changes in uptake rates. Thus, storage means that * For correspondence. E-mail [email protected]

resource use for growth is uncoupled from resource acquisition. This uncoupling is sometimes neglected in long-term growth models, but it should be taken into consideration when several possibly limiting resources are involved (Thornley, 1977; Dewar, 1993) and also in the day/night cycle (Gary, 1988b; Ca´rdenas-Navarro et al., 1998). Theoretical growth models, however, remain abstract and difficult to relate to reality, or even to parameterize, without knowledge of the nature of these resources and their respective amounts. Indeed, experimental evidence is necessary to answer three questions: (1) which molecules are the most involved in storage? (2) how are these allocated to different parts of the plant? and (3) how much do their concentrations vary, particularly during the day/night cycle, and what are the consequences for growth? In the literature, non-structural carbohydrates, i.e. soluble sugars and starch (Gary, 1988b; Lim et al., 1990) have been identified as major C resources because their accumulation in leaves is directly related to photosynthesis. However, carbohydrates alone do not account for the entire C store. Indeed, the plant carboxylate content, mostly malate, is clearly linked to NO2 3 nutrition (Ben Zioni et al., 1971; Kirkby and Knight, 1977; Touraine et al., 1988; Stitt et al., 2002; Urbanczyk-Wochniak and Fernie, 2005) and shows marked diurnal variations (Scheible et al., 1997). Thus, it has been

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Huanosto Maganã et al. — Growth and diel N and C status in tomato seedlings

suggested that both non-structural carbohydrates and carboxylates should be considered in growth models (Bijlsma and Lambers, 2000). Nitrate is an important N growth resource because it balances the role played by carbohydrates and, presumably, carboxylates in growth. Internal nitrate concentrations have been reported to be related to that of the nutrient solution (Steingro¨ver et al., 1986; Chen et al., 2004; Richard-Molard et al., 2008), and also to follow diurnal variations (Delhon et al., 1995; Ca´rdenas-Navarro et al., 1998; Matt et al., 1998, 2001a, b; Sto¨hr and Ma¨ck, 2001) associated with changing nitrate reduction activities (Lillo et al., 2004). Free amino acids can also be considered as N stores. In leaves, amino acids pools contain as much N as NO2 3 , and show marked daily variations opposed to that of NO2 3 , with net accumulations during the day followed by a decrease at night (Matt et al., 2001a; Masclaux-Daubresse et al., 2002; Lea et al., 2006). However, most published studies have focused on plant leaves, and a comprehensive view of all vegetative organs is needed in order to compare the importance of nitrate and amino acids as N stores. Depending on the plant species, other molecules may also be involved in the storage of C (e.g. fructosans) and N (e.g. ureides, vegetative storage proteins); however, to our knowledge, these compounds have not been identified in tomato. The degradation of alkaloids, which are involved in plant defence, releases N. A few alkaloids have been detected in tomato (Moco et al., 2006; Lijima et al., 2008), but only the concentration of tomatine has been reported in vegetative tissues (Friedman, 2002) and it appears to be too low for a significant N source for growth. In this work, the short-term growth of young tomato plant parts (leaves, stems and petioles, roots) is inferred from the changes in structural dry weight and compared to total dry weight. Growth was monitored over the day/night cycle and following changes in the solution [NO2 3 ] in order to assess growth sensitivity to light and nutritional signals. The importance of the main C and N stores (carbohydrates, carboxylates, nitrate and free amino acids) is compared with the needs of growth.

the experiment. The average PPFD (sensor LI-190; LICOR, Lincoln, NE) at plant level was 320 mmol m22 s21 (+ 22 s.d.), humidity and air temperature were set to 85 % and 25 8C from days 1 to 8, then to 75 % and 20 8C, respectively. Nutrient solutions were regulated at air temperature. The nutrient solution (3 mol m23 NO2 3 ) used to grow the plants from sowing until the experimental period (18 – 19 DAS) was made up with deionized water and the following pure salts, in mol m23: KH2PO4, 1.0; K2SO4, 1.0; Ca(NO3)2, 1.5; CaSO4, 2.0; MgSO4, 1.5; EDTA-Fe, 0.043. Other trace elements, following the Kanieltra formula 6-Fe (Hydro Azote, France), were added at 1 1024 m3 m23. During the experimental periods, solutions with lower NO2 3 concentrations (0 or 0.02 mol m23 NO2 3 ) were obtained by replacing Ca(NO3)2 with equivalent amounts of CaSO4, thus leaving phosphate and cation concentrations unchanged. Sulphate was preferred to chloride as a replacement ion, because the latter interferes with NO2 3 determination in the nutrient solution. Furthermore, there have been reports that Cl2 (unlike SO22 4 ) inhibits short-term nitrate influx (Deane-Drummond and Glass, 1982). Solution changes were performed without disturbing the plants. For this, the NFT system was disconnected from the solution tank and rinsed with 0.02 m3 of a nitrate-free solution (regulated at 20 8C), which was discarded. The NFT system was then connected to a second tank containing the new nutrient solution regulated at 20 8C. The whole process lasted for about 10 min and the flow of solution to roots was never interrupted for more than 2 min. The [NO2 3 ] in the solution was monitored using a UV method (Vercambre and Adamowicz, 1996), at 30– 180 min intervals during the growth period, and 5 – 30 min intervals during experiments. Automatic injections of NO2 3 salts (containing, in moles: 58 % K, 29 % Ca, 13 % Mg) by precision syringes maintained solutions within 5 % of the set concentrations. Solution volume (0.06 m3) and pH (5.0) were also automatically maintained at the same levels by deionized water and sulfuric acid injections, respectively. Electroconductivity was 1.20 mS.

Experiments

M AT E R IA L S A ND M E T HO DS Plant material and growth conditions

Tomato seeds (Solanum lycopersicum L., ‘Rondello’; De Ruiter Seeds, Bleiswijk, Holland) were sown directly in a nutrient-film technique (NFT) set-up inside a growth room, as described in Ca´rdenas-Navarro et al. (1998). Ten days after sowing (DAS), seedlings were thinned out to leave only one seedling out of three. Plantlets were selected according to visible homogeneity criteria based on the length of the first true leaf. In order to avoid any mechanical stress, plants were never manipulated until harvested. The system was set up in a growth room maintained under continuous darkness for 3 d, after which a 12-h photoperiod was applied. The photosynthetic photon flux density (PPFD) was increased progressively by switching on an increasing proportion of fluorescent lamps: 25 % from day 4, 50 % from day 5, 75 % from day 7, and full light from day 8 until the end of

Three experiments were carried out. The first one was designed to establish long-term growth parameters, whilst the other two monitored short-term growth throughout the light/dark cycle under varied N regimes. Experiment I. Two independent groups of seedlings were grown successively to monitor their long-term growth with 23 adequate N nutrition (3 mol NO2 ). Harvests of eight 3 m (first group: at 12, 15, 16, 17, 18 and 19 DAS) or 12 (second group: at 17, 19, 20 and 21 DAS) randomly sampled plants were performed during the last 30 min of the dark period. Because axillary buds and floral pieces appeared at 21 DAS, the experimental stage for the following experiments was set at 18– 19 DAS. Experiment II. Two independent groups of seedlings were grown successively to monitor their short-term (over one light-and-dark cycle, at 18 DAS) growth and composition changes with adequate N nutrition (3 mol NO3- m23). On day 18, from the beginning of the light period (time zero for

Huanosto Maganã et al. — Growth and diel N and C status in tomato seedlings the experiment) onwards, 6 – 8 randomly sampled plants were harvested at seven time intervals: 0, 4, 8, 12, 16, 20 and 24 h. Experiment III. Two independent groups of seedlings were grown successively to monitor their short-term (over two light/dark cycles, at 18– 19 DAS) growth and composition 23 changes with restricted N nutrition, i.e. ,0.3 mol NO2 , 3 m which restricts long-term growth (Adamowicz and Le Bot, 2008). For both groups, at 18 DAS the solution containing the sufficient N concentration that had been used during growth was changed during the last 10 min before the light period (time zero of the experiment). The new solution 23 contained 0.02 mol NO2 (first group) and no NO2 3 m 3 (second group). Six plants were sampled randomly at the following times: 0, 3, 6, 9, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 h; thus measurements extended over two full light/ dark cycles.

Plant analyses

After harvesting, each plant was separated into roots, stems with petioles, and leaf laminae. Roots were rinsed with deionized water and spin-dried (2 min at 2800 g). Plant material was weighed on an analytical balance, frozen with liquid N2, and stored at 280 8C until being freeze-dried (GENESIS 25ES; Virtis Company, Gardiner, NY). Dry material was weighed. Because stem and root biomasses were not enough to perform all the required chemical analyses, samples of the same plant parts were pooled together, ground to a fine powder (model MM301; Retsch, Haan, Germany) under liquid N2 and stored at 220 8C until analysis. Total C and N were measured in dry powders according to the Dumas method (elemental analyser, ANA 1500; Carlo Erba, Milano, Italy). Nitrate was determined on water-extracts of the dried material, in an auto-analyser (AQUATEC 5500; Tecator, Ho¨gana¨s, Sweeden) using a colorimetric assay to measure nitrite (Griess reaction) after nitrate reduction by cadmium. Free NH4 was extracted in a 2 % solution of 5-sulfosalicylic acid and determined by the phenol hyperchlorite colorimetric method (Berthelot reaction). Free amino acids were analysed by reverse-phase high performance liquid chromatography (HPLC) after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and detected by fluorimetry (Waters Corp, Milford, MA). Peak areas (Millenium software, Waters Corp.) were compared to standards obtained from Sigma (St. Louis, MO). Sugars and organic acids were determined after extraction with a water/methanol/chloroform mixture as described by Gomez et al. (2002). Soluble sugars (glucose, fructose and sucrose) and starch were measured by micro-enzymatic assay (microplate reader ELx800UV; Bio-Tek Instruments, Winooski, VT) following the method of Gomez et al. (2007). Organic acids, malate and citrate were also determined from micro-enzymatic assays, as described in Gautier et al. (2009). Free SO22 was determined at a companion laboratory 4 (INRA – EMMAH, domaine St-Paul, site agroparc, 84914 Avignon cedex 9, France) on water-extracts by capillary electrophoresis, using an electrolyte made of chromate (4.6 mM), OFM-OH (0.5 mM) and boric acid to obtain a pH value of 8.

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Gas exchange measurements

Net photosynthesis and transpiration were measured on intact leaves using a portable system (CIRAS-1 version 2.7, differential CO2/H2O infra-red gas analyser; PP Systems, Hitchin, UK) equipped with a 2.5-cm2 leaf cuvette and quartz halogen light unit. Conditions were set to the ambient conditions of the growth room: PPFD and temperature in the cell were 377 mmol m2 s21 and 20 8C, respectively, [CO2] and water pressure at the outlet were 370 ppm and 14 mbar, respectively. Data processing

In this study, total dry weight (W, in g plant21) is assumed to have two biomass components, structural (WS) and nonstructural (WNS), such that: W ¼ WS þ WNS

ð1Þ

The non-structural biomass (WNS) was computed as the sum of the weights of organic and mineral non-structural molecules. The organic molecules that were analysed were non-structural carbohydrates (starch, glucose, fructose and sucrose), nonstructural carboxylates (malate, citrate) and 20 free amino 2 22 acids. Minerals analysed were free NHþ 4 , NO3 and SO4 . Cations are problematic, because they compensate for the negative charges of both structural (e.g. pectate) and nonstructural (e.g. nitrate) biomass. We considered non-structural Kþ as the equivalent amount necessary to compensate for the negative charges of non-structural mineral anions. Emission spectrophotometry was carried out on a few samples from all plant parts in order to verify that the computed non-structural Kþ was less than the actual Kþ. In a similar way, total C and N (C, N; mmol g21 d. wt) may be described as two components, structural (CS, NS) and nonstructural (CNS, NNS), such that: C ¼ CS þ CNS ; and N ¼ NS þ NNS

ð2Þ

To calculate CNS, we added together the C content of nonstructural organic molecules (carbohydrates, carboxylates and free amino acids); to calculate NNS, we summed the N conþ tained in NO2 3 , NH4 and each free amino acid. In order to interpret changes in dry biomass throughout light/dark cycles, the data were fitted using least-square piecewise linear regression with breakpoints when the light regime changed. For example, the typical formula to describe the diel changes of W over time, t, starting from W0 at time zero, and light/darkness transition at time tt is: If t tt ; W ¼ W0 þ a0 t else W ¼ W0 þ tt ða0 a1 Þ þ a1 t ð3Þ with a0 and a1 being the slopes of W over time before and after tt, respectively. It is easy to verify that for t ¼ tt, both expressions in eqn (3) give W ¼ W0 þ a0tt. Computations were performed using the procedure nls in R software (R Project for Statistical Computing, http://www.R-project. org). The statistical significance of slopes was determined,


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F I G . 1. Dry biomass (W, g plant21) accumulation over time in tomato plants. Growth of two independent groups of seedlings (open and closed symbols) was monitored in a growth room with the same environmental conditions: 12/12 h photoperiod, 20 8C air and solution temperature, 320 mmol m22 s21 PPFD, 3 mol m23 NO2 3 . Symbols and vertical bars indicate the mean and s.e. for eight (open symbols) and 12 (closed symbols) randomly sampled plants. The curve is the common exponential best fit: W ¼ 0.001e 0299DAS.

and when they were not significantly different (P . 0.05, Student’s t-test) a unique linear regression was computed over the whole period.

RES ULT S Dry biomass accumulation Adequate N nutrition. We first characterized the long-term vegetative growth with adequate N nutrition (3 mol 23 NO2 3 m ) of two independent groups of tomato seedlings. Figure 1 shows the accumulation of biomass for whole plants (W ). The replicates showed a high level of reproducibility, indicating that the culture system is suitable to study and compare plants from successive experiments. Under our experimental conditions, biomass accumulation was described by a smooth exponential curve with a relative growth rate (RGR) almost equal to 0.3 d21. The short-term experiment, however, showed that, at 18 DAS, W could not be represented by a smooth exponential curve through the course of the light/dark cycle (Fig. 2A-1). In these replicated experiments, also carried out with adequate N nutrition, W increased by a mean of 0.12 g plant21, which represents a RGR value of 0.33 d21, close to that obtained from the long-term experiment. A linear fit of W over time yielded a significant positive regression (r ¼ 0.874, P , 0.001) with unevenly distributed residuals. Thus, considering that biomass increments result mostly from net photosynthesis during the light period, we also fitted a piecewise linear regression of W over time with a break point at the time when the light regime changed (Fig. 2A-1). This improved the distribution of residuals and increased the goodness-of-fit (R 2 ¼ 0.968), with the slope of W over time being significant in the light (9 1023 g plant21 h21, P , 0.001) but not in

the dark (5 1024 g plant21 h21, P . 0.5). At the organ level, the same pattern of W accumulation was also observed for leaf laminae (Fig. 2A-2). Stems and petioles (Fig. 2A-3), displayed different light/dark characteristics, with W increasing significantly during the dark period, albeit more slowly than during the light period. The slopes of W over time for roots (Fig. 2A-4), however, were not statistically different during light and dark periods, suggesting that roots accumulated biomass at a constant rate throughout the whole cycle. In this study, WNS was measured as the biomass of molecules considered as endogenous stores of C and/or N used for structural growth. Figure 2A-1 shows that in whole plants the pattern of WNS with time was also phasic over the light/ dark cycle, with a marked increase during the light period and a statistically significant decrease in the dark. Leaf laminae (Fig. 2A-2) showed the same pattern; however, stems and petioles (Fig. 2A-3) and roots (Fig. 2A-4) differed because WNS remained constant in the dark. Structural dry biomass WS was calculated as the difference between W and WNS. Figure 2A-1 shows that in whole plants with adequate N nutrition, WS increased constantly during both the light and dark periods (3.3 1023 g plant21 h21; r ¼ 0.959, P , 0.001), with no significant difference between light and drak period slopes. Similar patterns of WS were also observed for each separate organ (Fig. 2A-2– 4). Thus, W and WS showed different, organ-dependent, patterns. As a consequence, the root:shoot ratio (RSR, data not shown) computed from W showed significant diel patterns (0.171 and 0.146 at the onset of light and dark periods, respectively), whereas when it was computed from WS data it remained constant (0.235) throughout the entire experiment. This difference occurred mostly because leaf laminae contain most of the plant reserves. Indeed, as shown in Fig. 2A-2, overall, WNS and WS were similar in leaves, whereas in roots large differences were observed, with WNS values around five times lower than for WS. Restricted N nutrition. Short-term tomato growth with restricted nutrition was characterized over a 48-h period from day 18, 23 either by applying 0.02 mol NO2 (Fig. 2B) or 0 mol 3 m 23 23 2 NO3 m (Fig. 2C). At 0.02. mol m , RGR was unchanged during the first 24-h period (0.31 d21), but it decreased the following day (0.15 d21). RGR was strongly reduced in N-free conditions from the first day (0.17 d21). It should be stressed that we did not find significant differences in net photosynthesis measured on leaves before and during the experiments for plants treated with different N regimes (data not shown), except during the second day without NO2 3 where it fell from 11 to 7 mmol CO2 m22 s21. In whole plants (Fig. 2B-1, C-1), W showed roughly the same diel phasic patterns as those observed with adequate nutrition. Experimental errors were visibly higher in the 0.02 mol m23 experiment, but W increased significantly during the two light periods (P , 0.001 for each) while the observed decreases in the dark were not significant (P . 0.2). Root W was again observed to follow a less phasic pattern than leaf W (Fig. 2B-2, B-4). However, the most striking observation concerns root biomass at 0 mol m23 (Fig. 2C-4). Compared to adequate nutrition conditions (Fig. 2A-4), initial root growth rates were severely restricted

Leaf biomass (g plant–1d. wt)

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Huanosto Maganã et al. — Growth and diel N and C status in tomato seedlings 0·5

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23 F I G . 2. N nutrition effects on diel dry biomass accumulation in organs and whole tomato plants. Plants were grown with 3 mol NO2 (12 h photoperiod). At 3 m 18 DAS (0 h on time axis), plants were sampled at 3– 4 h intervals over a 24–48 h period to determine total (W ), non-structural (WNS) and structural (WS ¼ W – 23 23 (A-1– 4; two independent experiments), or changed to 0.02 mol NO2 WNS) dry weights. At time 0, nutrition was either maintained at 3 mol NO2 3 m 3 m 23 (B-1–4) or 0 mol NO2 (C-1–4). Dark periods are indicated by shading. Each symbol is the mean of 6– 8 plants and lines are piecewise linear regressions, 3 m as described in the Materials and Methods.

from day one; however, W increased at a higher rate from one half-photoperiod to the next, which led to an inverse growth pattern whereby the rate during the dark was higher than during the preceding light period. This meant that roots

gradually recovered and even grew at rates that significantly exceeded those of roots exposed to 3 mol m23 at 18 DAS. The 0.02 mol m23 N concentration did not change the diel patterns of WNS at the plant (Fig. 2B-1) or organ level

Huanosto Maganã et al. — Growth and diel N and C status in tomato seedlings Leaves

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Amino acids Carboxylates Carbohydrates NO3– Minerals WS

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F I G . 3. Whole-plant dry biomass (W ) and organ components expressed on a percentage basis. Non-structural biomass (WNS) is divided into free amino acids, carboxylates (malate þ citrate), carbohydrates (starch þ soluble þ and SO22 sugars), NO2 3 and other minerals (non-structural K 4 ). Structural biomass (WS) was calculated as the difference between total and non-structural dry weights (WS ¼ W 2 WNS). Tomato plants in the vegetative growth stage 23 (18–19 DAS) with adequate nutrition (3 mol NO2 3 m ) are compared to plants grown in a N-free solution for 48 h. Data are the mean of 6– 8 plants, sampled at the end of the dark period.

(Fig. 2B-2 – 4). When N was no longer present in the growth solution, however, the overall WNS accumulation rates as well as the amplitude of their phasic patterns were strongly reduced (Fig. 2C-1 – 4). Whole-plant WNS, as a percentage of W, was strongly reduced at 0 mol m23 and this was also true for leaf laminae and stems (Fig. 2C). In terms of structural biomass, the N concentration was also observed to affect growth rates (Fig. 2A-1– C-1). At 3 mol m23, WS increased at a mean rate of 2.9 1023 g plant21 h21, whereas it fell to 2.1 1023 and 1.8 1023 at 23 0.02 and 0 mol NO2 3 m , respectively, and the differences were statistically significant. The same trend was observed for leaves (Fig. 2A-2– C-2); however, for stems, although N restriction led to a significantly lower structural growth rate, the mean rates at 0.02 and 0 mol m23 were not significantly different (Fig. 2A-3 – C-3). On the other hand, root structural growth rates did not differ significantly at 3 and 0.02 mol m23 (0.5 1023 and 5.7 1024 g plant21 h21, respectively; Fig. 2A-4 – C-4), but were significantly reduced at 0 mol m23 (4.4 1024 g plant21 h21). It is important to note, however, that while structural growth for roots occurred at a constant rate throughout the experiment at 0.02 1022 mol m23, when no N was present in the medium root growth increased gradually, as described above for W. Resources. The main effects of restricted N are highlighted in Fig. 3, which shows biomass components (in %) following the dark period for plants fed with either 3 or 0 mol 23 NO2 for 48 h. There was an overall increase in the 3 m NO2 3

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F I G . 4. Diel changes in the content (mmol N g d. wt) of (A) whole tomato plants, (B) leaves, (C) stems and petioles, and (D) roots. Experimental details are the same as in Fig. 2, with nutritional treatments as indicated. Dark periods are indicated by shading. Each symbol is the mean of 6– 8 plants.

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percentage of non-structural biomass at the whole-plant level, which could be ascribed to non-structural carbohydrates. This mostly reflects the content of leaf laminae, which form the main carbohydrate reservoir and which responded markedly to N nutrition. The carbohydrate content was considerably lower in other organs, but it also responded strongly to N restriction. The carboxylate content was unchanged at the whole-plant level, but this resulted from inverse trends in different organs: a decrease in leaf laminae was countered by an increase in stems and roots. In fact, it appears that in stems and petioles carboxylates are the main C reservoir, instead of carbohydrates as in leaf laminae. N restriction led to a decrease in the percentage of free amino acids (including ammonium) and nitrate content. In plants with adequate nutrition, in any organ NO2 3 accounted for more of the dry weight than amino acids, and in stems it contributed even more weight than carbohydrates and carboxylates. In fact, stems were the only organ that still retained a measurable NO2 3 content after 48 h of N restriction. Replacement of NO2 3 by equivalent in the nutrient solution in order to induce amounts of SO22 4 N restriction also greatly increased plant SO22 4 . Thus, it appeared that other minerals in the medium could interfere in the N-nutrition experiments, and we considered SO22 4 and 22 Kþ as counter ions of NO2 3 and SO4 , respectively, to be nonstructural minerals. As can be seen in Fig. 3, non-structural minerals cannot be neglected, particularly in stems and roots. As a consequence, the percentage of C, which seemed low in terms of total dry biomass (39.2, 32.4 and 36.5 % in leaves, stems and roots, respectively), was normal when estimated on a structural-biomass basis (41.3, 39.7 and 42.1 % in leaves, stems and roots, respectively). In the following sections, non-structural biomass is considered in terms of endogenous N and C resources.

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Two pools of nitrogen were considered as endogenous N resources used for plant growth: nitrate and free amino acid (free ammonium being grouped with amino acids). In order to allow comparisons, both were computed as N equivalents (mmol N g21 d. wt). As can be seen in Fig. 4, large diurnal changes in endogenous nitrate were observed under adequate nutrition, at the plant (Fig. 4A) and organ levels (Fig. 4B –D), with a general decrease in the light, followed by a roughly equivalent increase in the dark. The relative amplitude of this pattern was largest in the leaf laminae (Fig. 4B) and weakest in the roots, but the largest absolute difference was clearly in stems, at 370 mmol N g21 (leaf laminae ¼ 170 mmol N g21; roots ¼ 110 mmol N g21). In fact, the mean diel [NO2 3 ] reached its highest level in stems and petioles at 1100 mmol N g21, compared with 100 mmol N g21 in leaves and 800 mmol N g21 in

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F I G . 5. Diel changes in the free amino acid N content (mmol N g21 d. wt) of (A) whole tomato plants, (B) leaves, (C) stems and petioles, and (D) roots. Experimental details are the same as in Fig. 2. N content was calculated from the free amino acid composition determined by HPLC; nutritional treatments as indicated. Dark periods are indicated by shading. Each symbol is the mean of 6–8 plants. Free ammonium was grouped with the amino acid pool.


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roots. Taking the respective biomasses into account, leaves, stems and roots contained 17, 54 and 29 % of total plant nitrate, respectively, thus highlighting the role of stems as a nitrate reservoir. When plants were grown with 0.02 mol 23 NO2 in the nutrient solution, the diel pattern remained 3 m phasic for the whole plant (Fig. 4A) and for the individual organs (Fig. 4B– D); however, the dark period [NO2 3 ] increase occurred at a slower rate under nitrate limitation. Thus, the resulting long-term trend was a decrease in plant [NO2 3 ]. When plants were grown without N, the phasic pattern changed because [NO2 3 ] no longer increased in the dark. Instead, it remained roughly constant during the dark period in leaf laminae (Fig. 4B), but decreased in other organs (Fig. 4C, D), albeit at a slower rate than in the preceding light periods. In the light, [NO2 3 ] decreased in all organs at a higher rate than in plants with ample nutrition. At the end of the experiment using the N-free solution, stems and petioles were the only organs that still contained significant nitrate. The free amino acid N content followed diurnal changes under adequate nutrition in a similar fashion to nitrate at both the plant (Fig. 5A) and organ level (Fig. 5B– D). It differed from nitrate, however, in three ways: (1) it followed an inverse pattern, with an increase in the light and a decrease in the dark; (2) the absolute amplitude of the diel variation was higher in leaf laminae (68 mmol amino-N g21 d. wt) than in stems and petioles (51 mmol amino-N g21) and roots (7 mmol amino-N g21); and (3) there was far less difference in the mean diel N content of all organs (110, 140 and 120 mmol amino-N g21 in leaves, stems and roots, respectively). Taking the biomasses into account, leaves, stems and roots contained on average 63, 23 and 14 % of plant amino-N, respectively, showing that leaves are the main reservoir for this N pool. Although the average plant amino-N concentration (119 mmol g21 d. wt) was much less than the 21 average [NO2 ), in leaves the concentrations 3 ] (390 mmol N g of the two pools were very similar (110 and 97 mmol g21 for amino- and nitrate-N, respectively). When plants were grown 23 with 0.02 mol NO2 in the nutrient solution, the diel 3 m pattern was still phasic for amino-N in the whole plant (Fig. 5A) and leaves (Fig. 5B), but it differed from full nutrition because peak concentrations were observed before the onset of darkness, and minimum concentrations were observed before the light was turned on. In other organs (Fig. 5C, D) the patterns were unclear. For unknown reasons, the initial free amino-N concentration (time zero in Fig. 5) was higher with 23 0.02 mol NO2 than with full nutrition, and this high 3 m level appeared to be the long-term trend. When plants were grown without N in the solution, the phasic pattern was still observed for whole plants (Fig. 5A) and leaves (Fig. 5B) during the first day, but it vanished on the second day. The long-term trend was a decrease in whole plants (Fig. 5A), leaf laminae (Fig. 5B), and stem and petioles (Fig. 5C), but levels remained stable in roots (Fig. 5D). As a result, after

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F I G . 6. Diel changes in starch C content (mmol C g21 d. wt) of (A) whole tomato plants, (B) leaves, (C) stems and petioles, and (D) roots. Experimental details are the same as in Fig. 2, with nutritional treatments as indicated. Dark periods are indicated by shading. Each symbol is the mean of 6 –8 plants.


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48 h in the N-free solution, roots had the highest amino-N concentration, which represented 93 % of their non-structural N. Significant amounts of amino-N remained in all organs, in contrast to what was observed for nitrate.


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Four pools were considered as endogenous C resources for plant growth: starch, non-structural soluble sugars, nonstructural carboxylates, and free amino acids. In order to allow comparisons, these were computed as C equivalents (mmol C g21 d. wt). The quantitative importance of these molecules differed greatly: in whole plants grown with 3 mol 23 NO2 3 m , starch accounted for 70 % of non-structural carbon, soluble sugars for 7 %, carboxylates for 18 % and amino-acids for 4 %. Leaves were the main source for all these compounds and contained 97, 75, 69 and 62 % of plant starch, soluble sugars, carboxylates and amino acids, respectively. In some organs, however, starch was not the dominant C resource: in stems and petioles, carboxylates accounted for 55 % of non-structural C (compared with 20 % for starch); in roots, non-structural C was more evenly distributed between soluble sugars (33 %), carboxylates (34 %) and amino acids (27 %). Figure 6 shows that there were large diurnal variations for starch-C in plants with adequate or limiting N nutrition. A pattern of an increase during the light period followed by a decrease in the dark was found at both the plant (Fig. 6A) and organ (Fig. 6B– D) levels. The starch content was about one order of magnitude higher in leaves than in stems, and one order of magnitude higher in stems than in roots. Unfortunately, the initial starch concentration in leaves differed between different experiments, but when N was limited 23 (0.02 and 0 mol NO2 3 m ), the long-term trend appeared to be an increase. The C content in starch from stems and petioles was not noticeably affected by nutritional treatments, in contrast to the starch content in roots in N-free solution (Fig. 6D), which increased greatly during the second day. Soluble sugars showed clear responses to both light and N-nutrition signals. As can be seen in Fig. 7, marked diurnal patterns were observed at both the plant (Fig. 7A) and organ level (Fig. 7B – D) regardless of solution N content. N limitation led to increased soluble sugar concentrations in all organs. As previously noted, soluble sugars generally contain far less C than starch. In roots, however, the absolute amplitude of changes in response to light and nutrition were far greater for soluble sugars than for starch. A distinctive characteristic of the soluble sugar diel pattern was observed for leaves (Fig. 7B): the peaks and troughs preceded transitions in the light regime. Figure 8 illustrates that there were no clear diurnal patterns for carboxylate content in plants with adequate N nutrition, except in roots (Fig. 8D) where carboxylates accumulated in

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F I G . 7. Diel changes in soluble-sugar C content (mmol C g21 d. wt) of (A) whole tomato plants, (B) leaves, (C) stems and petioles, and (D) roots. Experimental details are the same as in Fig. 2, with nutritional treatments as indicated. C content was measured by analysing glucose, fructose and sucrose. Dark periods are indicated by shading. Symbols are the means of 6– 8 plants.


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the light and decreased in the dark with an absolute amplitude of 290 mmol C g21 d. wt (compare with soluble sugars at 390 mmol C g21). In other organs (Fig. 8B, C) and whole plants (Fig. 8A) N nutrition effects were unclear due to large initial differences in carboxylate concentrations, and due to unexplained divergence between the effects of reduced N and zero N nutrition. In leaves, for example (Fig. 8B), the carboxylate concentration remained stable during the first light 23 period when either no N or 3 mol NO2 was present in 3 m the medium, but it increased quickly to nearly double the initial concentration at the intermediate N regime (0.02 mol 23 NO2 ). Even the long-term trend appears puzzling, 3 m because the whole-plant carboxylate content (Fig. 8A) was stable without N, whereas an inverse relationship between nitrate nutrition and organic acid content would be expected. The results for C in free amino acids (Fig. 9) showed patterns very similar to those for N (Fig. 5). This is easily explained, because the C:N ratio of the amino acid pool (mean plant C:N ¼ 3.7 mol mol21 under adequate N nutrition) did not change much with light (diel amplitude: 0.4) or nutri23 tion (C:N increase ¼ 0.5 after 48 h at 0 mol NO2 ). 3 m


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In this study we aimed to describe short-term plant growth and C/N status in response to light and N-nutrition signals. Growth, as described by dry biomass accumulation with time (Fig. 2), showed phasic patterns over the light/dark cycle, stopping immediately in the dark as expected. Carbon, the acquisition of which depends exclusively on photosynthesis, amounted to approx. 40 % of the dry matter. Interestingly, the same pattern was observed in leaves, which fulfil the photosynthetic needs for the whole plant. In contrast, non-photosynthetic organs (stems, roots) are continuously fuelled by C from the phloem and did not show such contrasting diurnal patterns of growth. Dry biomass was not as responsive to N nutrition as it was to light. Previous long-term experiments under similar conditions have shown that the critical [NO2 3 ] that limits growth is around 0.3 mol m23 for young tomato plants (Adamowicz and Le Bot, 2008). In our short-term experiments, however, very lownitrate solutions (0.02 mol m23) did not have an effect on the first day of application, and only a slight effect on the second day. Furthermore, photosynthesis measured in leaves was not affected, implying that N limitation did not affect the efficiency of C acquisition. When plants were grown in a nitrate-free solution, accumulation of biomass was restricted, but did not stop completely. For a proper interpretation of these findings, resource acquisition (e.g. photosynthesis), which provides an endogenous resource store of carbon, and resource use for structural growth (Thornley, 1977; Gent and Enoch, 1983) must be considered separately. Both growth as structural biomass

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F I G . 8. Diel changes in the non-structural carboxylate C content (mmol C g21 d. wt) of (A) whole tomato plants, (B) leaves, (C) stems and petioles, and (D) roots. Experimental details are the same as in Fig. 2, with nutritional treatments as indicated. The C content was determined by analysing malate, citrate and oxalate. Dark periods are indicated by shading. Each symbol is the mean of 6– 8 plants.


Amino-C (mmol g–1 d. wt)

accumulation, and the status of endogenous C and N resources as the contents of corresponding non-structural metabolites were therefore determined. We did not take into account secondary metabolites (e.g. phenylpropanoids), which do not contribute to either structures or resources, and assumed that this did not create a significant bias in the evaluation of structural biomass. Growth, as estimated by structural biomass accumulation, did not show any significant response to light in plants with adequate N nutrition (Fig. 2). This finding was the same in all the plant organs considered because endogenous stores, rather than immediate photosynthesis, are used for growth. This does not imply that the growth rate should necessarily be the same for day and night. Gary (1988a) modelled the rate of growth of young tomato plants, and its associated respiration rate, as a saturable function of shoot non-structural carbohydrates (starch þ soluble sugars) with a Michaelis constant equal to 6 % shoot d. wt. This is far below the levels measured in our experiments with adequate nutrition, which varied from 20 – 30 % of shoot d. wt (dawn to dusk), implying that the C status was always saturating the needs of shoot growth. Such a high level was due to the irradiance/temperature combination in our experiments (12-h photoperiod, 320 mmol m22 s21 PPFD, 20 8C), as Gent (1986) showed that the tomato carbohydrate content increased with the PPFD:temperature ratio. In his trials, which matched our experimental conditions, carbohydrates amounted to 15– 25 % of whole-plant d. wt, close to the 18– 28 % found in our experiments. At the whole-plant level, from carbon exchange rates and average leaf area ratio measurements (1.92 1024 m2 g21), we estimated that the daily amount of photosynthesis with adequate nutrition was 9100 mmol CO2 g21 d. wt. Thus, the C stored in carbohydrates and carboxylates at dusk (Figs. 6 – 8) amounts to more than a full day of photosynthesis. We also computed, on a C basis, the daily structural growth and C reserves at dusk (18 DAS, adequate nutrition), which were 2400 and 12 000 mmol C plant21, respectively. Thus, assuming a 75 % yield of growth (Ruget, 1981), this C store was enough to feed 3.8 d of growth in the dark. Unfortunately, we could not find any relevant respiration/growth data in the literature in relation to root C status. Indeed, the root carbohydrate content at adequate nutrition (1.5 – 3.0 % d. wt) was low compared to the shoot contents and to the cited Michaelis constant, which raises the question of whether root growth can be limited by the carbohydrate status. The structural growth rate was more responsive to N than to light. Low [NO2 3 ] in the solution raised the plant carbohydrate content to relatively high levels (Fig. 3), in agreement with the literature (Ball et al., 1998; de Groot et al., 2002). It is thus unlikely that the C status mediated this response of growth, at least in leaf laminae. Instead, the endogenous N store decreased dramatically in all organs (Fig. 3). However, we


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F I G . 9. Diel changes in the free amino acid C content (mmol C g21 dw) of (A) whole tomato plants, (B) leaves, (C) stems and petioles, and (D) roots. Experimental details are the same as in Fig. 2, with nutritional treatments as indicated. C content was calculated from the free amino acid composition determined by HPLC. Dark periods are indicated by shading. Each symbol is the mean of 6– 8 plants.

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measured two components of this store, crude nitrate and free amino acids, and these behaved differently. Nitrate was the most important and most variable component, reaching near-zero values in leaves and roots after 48 h in N-free solution (Fig. 4B, D). However, the stem, which was by far the most important reservoir, still contained significant NO2 3 (Fig. 4C), explaining why whole-plant structural growth continued until the end of the experiment (Fig. 2A-1). Daily structural growth and the NO3-N reserves at dawn were also calculated (on an N basis; 18 DAS, adequate nutrition) and were 299 and 147 mmol N plant21, respectively. Thus, the nitrate store alone was only sufficient to sustain half a day of full-rate growth. In any case, the rate of structural growth is more likely to be directly related to the free amino acid content, not to [NO2 3 ]. Indeed, Fig. 2A-2 shows that the diel leaf growth at adequate nutrition occurred at a constant rate, despite the extremely large diel changes in the leaf nitrate content (Fig. 4B). These changes are clearly related to the photosynthetic nature of NO2 3 assimilation in the leaves (Pearson et al., 1981; Ourry et al., 1996). When plants were fed with a N-free solution, the leaf [NO2 3 ] only fell significantly in the light while in roots, where nitrate assimilation is respiration dependent, it also decreased in the dark (Fig. 4D). Nitrate assimilation produces the necessary amino acids for structural growth. In contrast to nitrate, the free amino-N pool was present at significant levels in all organs throughout the 48 h with N-free solution (Fig. 5B– D); this observation probably explains why none of the organs stopped growing. Root growth decreased immediately with N-free nutrition, but then accelerated to such a point that roots were growing faster than other organs at the end of the experiment. As a result, the structural root:shoot ratio changed gradually from 0.166 to 0.228. As the amino acid content was not greatly changed in the roots, we suggest that they responded to their carbohydrate rather than to their amino acid status. In all organs, the amino-N concentration proved sensitive both to light and N nutrition. However, the free amino acid content is the balance between NO2 3 assimilation, protein decay and synthesis, and import from and export to other organs, resulting in intricate patterns that require modelling. CO NCL USI ON S This study shows that structural growth can be evaluated by monitoring organ dry weights and the amounts of a few metabolites. It highlights the difference between dry weight accumulation and growth: the former reflects photosynthesis with marked diel phasic patterns in leaves, while the latter shows no or only gradual changes in any given organ because it depends only on endogenous reserves, not on instant resource acquisition from the environment. Thus, the comparison of short-term total and structural biomass offers a new way to study the sensitivity of growth to fluctuating environmental variables. Under our experimental conditions, C reserves provided much greater growth autonomy to plantlets than N reserves (albeit having previously received adequate nutrition), suggesting that growth was more sensitive to N- than to C-acquisition changes. This difference may be explained

partly by the higher level of C reserves compared to N reserves. However, another factor may also be important: most tomato N reserves are nitrate ions stored in stems and petioles, and nitrate assimilation is necessary prior to N utilization for structural growth. It is thought that all plant organs participate in nitrate reduction and assimilation but, because this ion is transported only via xylem sap, roots are rapidly depleted of nitrate in cases of deficiency and are not involved in nitrate reduction. Furthermore, stems seem to have low nitrate reductase activities (Andrews et al., 1984; Deroche and Babalar, 1987) and it is likely that nitrate transport limits the assimilation rate by leaves (Gojon et al., 1991). Other growth conditions that modify the levels of C and N reserves would help to further explain growth sensitivity to the environment. For example, solution [NO3] and [NH4] have an effect on N stores through root uptake, while PPFD, photoperiod and air [CO2] directly affect net daily C exchange rates and the build-up of C stores. In contrast, temperature mostly influences structural growth rate and the depletion of both C and N stores. Thus, it is likely that a broad range of growth autonomies may be obtained according to culture conditions. ACK N OW L E DG E M E N T S We thank J. Fabre and J. Hostalery for conducting the experiments; V. Serra and J. Le Bot for help with harvests; and D. Bancel, E. Rubio, V. Diakou and G. Se´venier for chemical analyses. During this research project, R. Huanosto Maganã received a Mexican CONACYT fellowship. L I T E R AT U R E C I T E D Adamowicz S, Le Bot J. 2008. Altering young tomato plant growth by NO3 and CO2 preserves the proportionate relation linking long-term organic-N accumulation to intercepted radiation. New Phytologist 180: 663 –672. Andrews M, Sutherland JM, Thomas RJ, Sprent JI. 1984. Distribution of nitrate reductase activity in six legumes: the importance of the stem. New Phytologist 98: 301–310. Ball RA, Sabbe WE, DeLong RE. 1998. Starch and nitrogen status in soybean during shading and nutrient deficiency. Journal of Plant Nutrition 21: 665–685. Ben Zioni A, Vaadia Y, Lips SH. 1971. Nitrate uptake by roots as regulated by nitrate reduction products of the shoot. Physiologia Plantarum 24: 288– 290. Bijlsma RJ, Lambers H. 2000. A dynamic whole-plant model of integrated metabolism of nitrogen and carbon. 2. Balanced growth driven by C fluxes and regulated by signals from C and N substrate. Plant and Soil 220: 71–87. Ca´rdenas-Navarro R, Adamowicz S, Robin P. 1998. Diurnal nitrate uptake in young tomato (Lycopersicon esculentum Mill.) plants: test of a feedback-based model. Journal of Experimental Botany 49: 721– 730. Chen B-M, Wang Z-H, Li S-X, Wang G-X, Song H-X, Wang X-N. 2004. Effects of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables. Plant Science 167: 635– 643. Clement CR, Hopper MJ, Jones LHP, Leafe EL. 1978. The uptake of nitrate by Lolium perenne from flowing nutrient solution. II. Effect of light, defoliation, and relationship to CO2 flux. Journal of Experimental Botany 29: 1173–1183. Deane-Drummond CE, Glass ADM. 1982. Studies of nitrate influx into barley roots by the use of 36ClO3 as a tracer for nitrate. 1. Interactions with chloride and other ions. Canadian Journal of Botany 60: 2147–2153. Delhon P, Gojon A, Tillard P, Passama L. 1995. Diurnal regulation of NO2 3 uptake in soybean plants. I. Changes in NO3- influx, efflux, and N

Huanosto Maganã et al. — Growth and diel N and C status in tomato seedlings utilization in the plant during the day/night cycle. Journal of Experimental Botany 46: 1585– 1594. Deroche M-E, Babalar M. 1987. In vivo nitrate reductase activities in different organs of lucerne (Medicago sativa) plants: effects of combined nitrogen during first vegetative growth and after shoot harvest. Physiologia Plantarum 70: 90–98. Dewar RC. 1993. A root– shoot partitioning model based on carbon–nitrogen–water interactions and Mu¨nch phloem flow. Functional Ecology 7: 356– 368. Friedman M. 2002. Tomato glycoalkaloids: role in the plant and in the diet. Journal of Agricultural and Food Chemistry 50: 5751–5780. Gary C. 1988a. Relation entre tempe´rature, teneur en glucides et respiration de la plante entie`re chez la tomate en phase ve´ge´tative. Agronomie 8: 419– 424. Gary C. 1988b. Un mode`le simple de simulation des relations microclimatbilan carbone´ chez la tomate en phase ve´ge´tative. Agronomie 8: 685– 692. Gautier H, Massot C, Stevens R, Se´rino S, Geńard M. 2009. Regulation of tomato fruit ascorbate content is more highly dependent on fruit irradiance than leaf irradiance. Annals of Botany 103: 495– 504. Gent MPN. 1986. Carbohydrate level and growth of tomato plants. II. The effect of irradiance and temperature. Plant Physiology 81: 1075– 1079. Gent MPN, Enoch HZ. 1983. Temperature dependence of vegetative growth and dark respiration: a mathematical model. Plant Physiology 71: 562– 567. Gojon A, Wakrim R, Passama L, Robin P. 1991. Regulation of NO2 3 assimilation by anion availability in excised soybean leaves. Plant Physiology 96: 398 –405. ´ , Auge´ M. 2002. A new procedure for extraction and Gomez L, Rubio E measurement of soluble sugars in ligneous plants. Journal of the Science of Food and Agriculture 82: 360– 369. Gomez L, Bancel D, Rubio E, Vercambre G. 2007. The microplate reader: an efficient tool for the separate analysis of sugars in plant tissues – validation of a micro-method. Journal of the Science of Food and Agriculture 87: 1893–1905. de Groot CC, Marcelis LFM, van den Boogaard R, Lambers H. 2002. Interactive effects of nitrogen and irradiance on growth and partitioning of dry mass and nitrogen in young tomato plants. Functional Plant Biology 29: 1319–1328. Kirkby EA, Knight AH. 1977. Influence of the level of nitrate nutrition on ion uptake and assimilation, organic acid accumulation, and cation– anion balance in whole tomato plants. Plant Physiology 60: 349– 353. Lea US, Leydecker M-T, Quillere´ I, Meyer C, Lillo C. 2006. Posttranslational regulation of nitrate reductase strongly affects the levels of free amino acids and nitrate, whereas transcriptional regulation has only minor influence. Plant Physiology 140: 1085– 1094. Lijima Y, Nakamura Y, Ogata Y, et al. 2008. Metabolite annotations based on the integration of mass spectral information. The Plant Journal 54: 949– 962. Lillo C, Meyer C, Lea US, Provan F, Oltedal S. 2004. Mechanism and importance of post-translational regulation of nitrate reductase. Journal of Experimental Botany 55: 1275– 1282. Lim JT, Wilkerson GG, Raper CDJr, Gold HJ. 1990. A dynamic growth model of vegetative soya bean plants: model structure and behaviour under varying root temperature and nitrogen concentration. Journal of Experimental Botany 41: 229– 241. Masclaux-Daubresse C, Valadier M-H, Carrayol E, Reisdorf-Cren M, Hirel B. 2002. Diurnal changes in the expression of glutamate

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dehydrogenase and nitrate reductase are involved in the C/N balance of tobacco source leaves. Plant, Cell and Environment 25: 1451– 1462. Matt P, Schurr U, Klein D, Krapp A, Stitt M. 1998. Growth of tobacco in short-day conditions leads to high starch, low sugars, altered diurnal changes in the nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis. Planta 207: 27–41. Matt P, Geiger M, Walch-Liu P, Engels C, Krapp A, Stitt M. 2001a. Elevated carbon dioxide increases nitrate uptake and nitrate reductase activity when tobacco is growing on nitrate, but increases ammonium uptake and inhibits nitrate reductase activity when tobacco is growing on ammonium nitrate. Plant, Cell and Environment 24: 1119–1137. Matt P, Geiger M, Walch-Liu P, Engels C, Krapp A, Stitt M. 2001b. The immediate cause of the diurnal changes of nitrogen metabolism in leaves of nitrate-replete tobacco: a major imbalance between the rate of nitrate reduction and the rates of nitrate uptake and ammonium metabolism during the first part of the light period. Plant, Cell and Environment 24: 177– 190. Moco S, Bino RJ, Vorst O, et al. 2006. A liquid chromatography–mass spectrometry-based metabolome database for tomato. Plant Physiology 141: 1205–1218. Ourry A, Macduff JH, Prudhomme M-P, Boucaud J. 1996. Diurnal variation in the simultaneous uptake and ‘sink’ allocation of NHþ 4 and NO2 3 by Lolium perenne in flowing solution culture. Journal of Experimental Botany 47: 1853– 1863. Pearson CJ, Volk RJ, Jackson WA. 1981. Daily changes in nitrate influx, efflux and metabolism in maize and pearl millet. Planta 152: 319–324. Richard-Molard C, Krapp A, Brun F, Ney B, Daniel-Vedele F, Chaillou S. 2008. Plant response to nitrate starvation is determined by N storage capacity matched by nitrate uptake capacity in two Arabidopsis genotypes. Journal of Experimental Botany 59: 779 –791. Ruget F. 1981. Respiration de croissance et respiration d’entretien: me´thodes de mesure, comparaison des re´sultats. Agronomie 1: 601–610. Scheible W-R, Gonza´lez-Fontes A, Morcuende R, et al. 1997. Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase. Planta 203: 304– 319. Steingro¨ver E, Ratering P, Siesling J. 1986. Daily changes in uptake, reduction and storage of nitrate in spinach grown at low light intensity. Physiologia Plantarum 66: 550–556. Stitt M, Mu¨ller C, Matt P, et al. 2002. Steps towards an integrated view of nitrogen metabolism. Journal of Experimental Botany 53: 959–970. Sto¨hr C, Ma¨ck G. 2001. Diurnal changes in nitrogen assimilation of tobacco roots. Journal of Experimental Botany 52: 1283–1289. Thornley JHM. 1977. Growth, maintenance and respiration: a re-interpretation. Annals of Botany 41: 1191–1203. Touraine B, Grignon N, Grignon C. 1988. Charge balance in NO2 3 fed soybean. Estimation of Kþ and carboxylate recirculation. Plant Physiology 88: 605– 612. Urbanczyk-Wochniak E, Fernie AR. 2005. Metabolic profiling reveals altered nitrogen nutrient regimes have diverse effects on the metabolism of hydroponically-grown tomato (Solanum lycopersicum) plants. Journal of Experimental Botany 56: 309–321. Vercambre G, Adamowicz S. 1996. Dosage de l’ion nitrate en solution nutritive et en pre´sence de polye´thyle`ne glycol par spectrome´trie uv. Agronomie 16: 73–87.