Abstract. Diurnal changes in carbohydrates and nitratereductase (NR) activity were compared in tobacco(Nicotiana tabacum. L.cv. Gatersleben) plants growingin a long (18 h light/6 h dark) and a short (6 h light/18 hdark) day growth regime, or after short-term changes inthe light regime. In long-day-grown plants, source leavescontained high levels of sugars throughout the light anddark periods. In short-day-grown plants, levels ofsucrose and reducing sugars were very low at the endof the night and, although they rose during the lightperiod, remained much lower than in long days anddeclined to very low levels again by the middle of thenight. Starch accumulated more rapidly in short-day-than long-day-grown plants. Starch was completely re-mobilised during the night in short days, but not in longdays. A single short day/long night cycle su�ced tostimulate starch accumulation during the following lightperiod. In long-day-grown plants, the Nia transcriptlevel was high at the end of the night, decreased duringthe day, and recovered gradually during the night. Inshort-day-grown plants, the Nia transcript level wasrelatively low at the end of the night, decreased to verylow levels at the end of the light period, increased to amarked maximum in the middle of the night, anddecreased during the last 5 h of the dark period. In long-day-grown plants, NR activity in source leaves rose by2- to 3-fold in the ®rst part of the light period anddecreased in the second part of the light period. In short-day-grown plants, NR activity was low at the end of thenight, and only increased slightly after illumination.Dark inactivation of source-leaf NR was partiallyreversed in long-day-grown plants, but not in shortday-grown plants. In both growth regimes, mutants withone instead of four functional copies of the Nia gene hada 60% reduction in maximum NR activity in the source

leaves, compared to wild-type plants. The diurnalchanges in NR activity were almost completely sup-pressed in the mutants in long days, whereas the mutantsshowed similar or slightly larger diurnal changes thanwild-type plants in short days. When short-day-grownplants were transferred to long-day conditions for 3 d,NR activity and the diurnal changes in NR activityresembled those in long-day-grown plants. Phloemexport from source leaves of short-day-grown plantswas partially inhibited by applying a cold-girdle for onelight and dark cycle. The resulting increase in leaf sugarwas accompanied by an marked increase in the Niatranscript level and a 2-fold increase in NR activity atthe end of the dark period. When wild-type plants weresubjected to a single short day/long night cycle ofincreasing severity, NR activity in source leaves at theend of the night decreased when the endogenous sugarsdeclined below about 3 lmol hexose (g FW))1. In sinkleaves in short-day conditions, sugars were higher andthe light-induced rise in NR activity was much largerthan in source leaves on the same plants. The sourceleaves of wild-type plants in short-day conditionscontained very high levels of nitrate, very low levels ofglutamine, low levels of total amino acids, and lowerprotein and chlorophyll, compared to long-day-grownplants. Plants grown in short days had relatively highlevels of glutamate and aspartate, and extremely lowlevels of most of the minor amino acids in their sourceleaves at the end of the night. Illumination led to adecrease in glutamate and an increase in the minoramino acids. A single short day/long night cycle led toan increase in glutamate, and a large decrease in theminor acids at the end of the dark period, and re-illumination led to a decrease in glutamate and anincrease in the minor amino acids. It is proposed thatsugar-mediated control of Nia expression and NRactivity overrides regulation by nitrogenous compoundswhen sugars are in short supply, resulting in a severeinhibition of nitrate assimilation. It is also proposed thatsugars exert a global control on amino acid metabolism.The importance of sugars for the regulation of nitrogenmetabolism is strikingly illustrated by the ®nding that

Abbreviations: Nia � the gene encoding nitrate reductase; NR �nitrate reductase

Correspondence to: M. Stitt;E-mail: mstitt@botanik1.bot.uni-heidelberg.de;Fax: 49(6221)545859

Planta (1998) 207: 27±41

Growth of tobacco in short-day conditions leads to high starch,low sugars, altered diurnal changes in the Nia transcript and low nitratereductase activity, and inhibition of amino acid synthesis

Petra Matt, Uli Schurr, Diana Klein, Anne Krapp, Mark Stitt

Botanisches Institut, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany

Received: 6 January 1998 /Accepted: 27 April 1998

tobacco is carbon and nitrogen limited when it is grownin short-day conditions.

Key words: Amino acids ± Nicotiana ± Nitrate reductase± Short days ± Starch ± Sugars

Introduction

Nitrate assimilation depends on and in some conditionscompetes with photosynthetic carbon metabolism. Ni-trate assimilation and amino acid synthesis utilisesreducing equivalents and ATP from the photosyntheticelectron chain (Huppe and Turpin 1994). Further,carbon is diverted away from carbohydrate synthesisin order to synthesise organic acids that act as counteranions for nitrate, and as carbon skeletons in theglutamate synthase (GOGAT) pathway and duringamino acid synthesis (Martinoia and Rentsch 1994;Heldt 1996; Scheible et al. 1997a). In many conditions,the carbon that is required during nitrate assimilationcan be obtained by reducing the rate of starch accumu-lation or by re-mobilising starch (Waring et al. 1985;Fichtner and Schulze 1992; Scheible et al. 1997a). Acontinuation of nitrate assimilation when carbohydratesare in short supply, however, could lead to a metabolicimbalance due to accumulation of ammonium, ashortfall of organic acids to counteract acidi®cation, ora lack of carbon for export or respiration. To preventthis, it will presumably be important to inhibit nitrateassimilation when the carbohydrate supply is low.

Nitrate assimilation is subject to transcriptional andpost-translational regulation at the step catalysed bynitrate reductase (NR). Signals from nitrogen metabo-lism play an important role in regulating the expressionof the nitrate reductase gene Nia, in particular, Nia isinduced by nitrate and repressed by glutamine (Ho�et al. 1994). The Nia transcript and NR activity alsoincrease in the light (Deng et al. 1990). Althoughphytochrome is implicated in the light-regulation ofNR in germinating seedlings (Mohr et al. 1992;Bergarache et al. 1994; Chandok and Sopory 1996),light still leads to an increase in Nia transcript and NRactivity in source leaves of the phytochrome-de®cientaurea mutant of tomato (Becker et al. 1992; Venkatesh-war and Sharma 1994). The e�ect of light in matureleaves can be mimicked by feeding sugars in the dark(Cheng et al. 1992; Vincentz et al. 1993). Furtherevidence for a role for sugars in regulating Nia expres-sion and NR activity is provided by the observationsthat the Nia transcript increases after cooling the petioleto increase endogenous sugar levels (Krapp and Stitt1995), and that the decline in NR activity in roots ofdarkened plants is reversed by feeding sugars (Botreland Kaiser 1997; Sivasankar et al. 1997).

Sugars are also implicated in the post-translationalregulation of NR. Darkening leads to phosphorylationof ser543 and magnesium-dependent binding of aninhibitory 14-3-3 protein (Bachmann et al. 1996; Moor-

head et al. 1996). Dark-inactivation is reversed ifsugars are supplied to leaves in the dark (Kaiser andBrendle-Behnisch 1991; Kaiser and Huber 1994), andlight-activation does not occur if carbon ®xation isprevented by water stress or low carbon dioxide (Kaiserand FoÈ rster 1989; Kaiser and Brendle-Behnisch 1991;Lejay et al. 1997).

There have not yet been any detailed comparisons ofthe changes in endogenous sugar levels with the changesin the Nia transcript level, NR activity and NR activa-tion leaves in di�erent conditions. Although there havebeen numerous studies of the diurnal regulation of NRin plants growing in intermediate or long-day conditions,it is di�cult to disentangle the contribution of sugarsfrom the contribution of nitrate and glutamine, as allthree are vary simultaneously (Scheible et al. 1997b). Theclearest evidence for an e�ect of sugars on Nia transcriptand NR activity is based on feeding experiments inwhich rather high levels of sugars were supplied(Vincentz et al. 1993; Botrel and Kaiser 1997; Sivasankaret al. 1997), and on petiole cooling experiments whichalso lead to a larger accumulation of sugars than occursduring a normal diurnal period (Krapp and Stitt 1995).Although there was a dramatic decline in NR activity inantisense rbcS tobacco lines, this only occurred in lineswith a large reduction in Rubisco activity, photosynthe-sis and sugars (Stitt and Schulze 1994).

Information is also needed with respect to thepossible e�ects of the sugar supply on amino acidsynthesis. The transcript for cytosolic glutamine synthe-tase also increases in the light, and this increase can bemimicked by adding sugars in the dark (Lam et al.1996). Prolonged darkness leads to induction of aspar-agine synthetase in leaves (Lam et al. 1996) and roots(Brouquisse et al. 1992) and this increase is prevented ifsugars are added (Chevalier et al. 1996; Lam et al. 1996),indicating that asparagine synthetase is activated incarbon starvation conditions when it may contribute toamino acid catabolism. The observation that sugarstarvation led to a general decrease in amino acids insugar cane cells (Veith and Komor 1993) indicates,however, that sugars may have more far-reaching e�ectson amino acid metabolism.

While growing tobacco plants under short-day con-ditions, we noticed that they had very low NR activityand contained high levels of nitrate and very low levelsof amino acids. We therefore investigated the starch andsugar levels in tobacco growing in short-day conditionsand long-day conditions, and compared them with thediurnal changes in the Nia transcript level, NR activity,and the pools of nitrate and various amino acids.Growth in di�erent day length regimes will a�ect manyother features of plant growth and physiology, inaddition to carbohydrate levels. We therefore alsoinvestigated the e�ect of transferring plants fromshort-day to long-day conditions, of exposing plants toa single extended night, and of inhibiting phloem exportin short-day conditions to provide evidence that the lowNR activity and low levels of amino acids in short-dayconditions are at least in part due to a shortfall ofcarbohydrate.

28 P. Matt et al.: Nitrogen metabolism in short days

Materials and methods

Plant growth. Wild-type tobacco (Nicotiana tabacum L.cv.Gatersleben; provided by M. Caboche, INRA, Versailles, France)and F23 and F22 mutants with one instead of four functionalcopies of the Nia gene (Scheible et al. 1997a, b) were germinated ina 12 h light/12 h dark (400 lmol m)2 s)1) regime. After 10 d, theseedlings were transferred into individual 1.4-L pots and grown incontrolled climate chambers as in Scheible et al. (1997b) except forthe use of either an 18 h light/6 h dark regime, or a 6 h light/18 hdark regime in which the 18-h dark period was interrupted after13 h darkness by 10 min of light. The irradiance is given in the®gure legends (300±600 lmol m)2 s)1). The plants were irrigateddaily with full nutrient medium (Scheible et al. 1997a, 1997b)containing 12 mM nitrate and 6 mM ammonium as nitrogensources for the ®rst 5 d, and 12 mM nitrate as the sole nitrogensource from day 5 onwards (Scheible et al. 1997a, b). The volumeof the nutrient medium was 0.1 L per day per plant until the plantswere 45 d old, 0.2 L until they were 50 d old, 0.3 L until they were55 d old, and 0.5 L when they were older. Plants were harvested foranalyses of Nia transcript, NR activity, and nitrogen and carbonmetabolites after 46 d in long-day conditions, and after 46±61 d(for details see text and ®gure legends) in short-day conditions.

In one experiment, plants were grown in 6 h light/18 h darkregime for 46 d, transferred to a 14 h light/10 h dark regime for3 d, some of the plants were harvested in the course of the fourthdiurnal cycle, and others subjected on the following day to a singleshort day/long night cycle before harvest on the ®fth day. To dothis, groups of plants were darkened after 4, 6, 8 10 and (control)14 h illumination left in the dark for 20, 18, 16, 14, and 10 h,respectively, re-illuminated at the time point corresponding to thestart of the light period in the control plants, and harvested 4 hlater. In this experiment, the dark period was not interrupted withlight.

To investigate the e�ect of decreased export on plants growingin short day conditions, wild-type plants were grown for 45 d in a6 h light/18 h dark regime, and a water-cooled metal block (Krappand Stitt 1995) was then attached around the petiole of theyoungest fully expanded source leaf of half the plants at the start ofthe light period. As controls, an identical metal block was attachedto the ®rst fully expanded leaf of the other plants but the coolingwater circuit was not turned on. The leaves were harvested 23 hlater at the end of the following dark period.

In all experiments, the youngest fully expanded leaf washarvested for investigation of source leaf metabolism. The sinkleaves were about 5 cm long, which is about one-third of the lengthof a fully expanded leaf in these conditions.

Extraction and analytic techniques. All leaf material was frozen inliquid nitrogen under the ambient light intensity. RNA wasextracted and separated and Nia transcript detected as in Scheibleet al. (1997a). The NR was extracted and activity determined in thepresence and absence of magnesium as in Scheible et al. (1997b).Leaf material was extracted in ethanol and analysed for starch,sucrose, reducing sugars, nitrate, total amino acids, individualamino acids, chlorophyll and protein as in Scheible et al. (1997b).

Results

Plant growth, protein and chlorophyll. Tobacco grewmore slowly (data not shown) in a 6 h light/18 h darkregime than in a 18 h light/6 h dark regime. Eventhough the short light period would be expected to leadto a carbohydrate shortage, tobacco plants growing inshort days also contained lower levels of chlorophyll andprotein in their leaves. In 46-d-old plants, chlorophylllevels in the youngest fully expanded leaf decreased from0.85 � 0.08 to 0.60 � 0.1 mg Chl (g FW))1, and

protein decreased from 18.27 � 2.45 to 13.26 �2.45 mg protein (g FW))1 in short days, compared tolong days (data not shown, mean � SE of 30 plants).The F23 mutants with one instead of four functional Niagene copies (Vaucheret et al. 1990; Scheible et al. 1997b)contained similar levels of protein and chlorophyll towild-type plants in both growth regimes (data notshown, see also Scheible et al. 1997a, 1997b).

Nitrate reductase in source leaves in long days. The NRactivity and sugar levels were investigated in theyoungest fully expanded leaf of 46-d-old plants growingin a 18 h light/6 h dark regime. There was a 2-foldincrease in NR activity during the ®rst 2 h of thephotoperiod, followed by a decline later in the photo-period (Fig. 1A). The F23 mutants contained 60% lessNR activity than wild-type plants in the middle of thephotoperiod, and NR activity did not decline during thesecond part of the photoperiod or the night. A similardiurnal rhythm of NR activity in wild-type plants and asimilar modi®cation of the rhythm in F23 were found ina 12 h light/12 h dark regime (Scheible et al. 1997b).

Post-translational regulation of NR can be moni-tored by assaying NR in the presence of magnesium(Kaiser and Huber 1994). As expected, the apparentactivation state of NR (the activity in the presence ofmagnesium as a percentage of the activity in the absenceof magnesium) was very high in the light (Fig. 1B).However, whereas NR is inactivated in wild-type plantsin the dark in intermediate photoregimes (Scheible et al.1997b), the dark inactivation of NR was partiallyreversed in long days (Fig. 1B). Further, whereas thedark inactivation of NR is only partially reversed in F23in a 12 h light/12 h dark irradiance regime (Scheible et al.1997b), it is almost completely reversed in long-dayconditions (Fig. 1B).

Sucrose accumulated gradually during the lightperiod and declined during the dark period (Fig. 1C).Glucose (Fig. 1D) accumulated during the ®rst part ofthe light period and declined to low levels during thesecond part of the light period. In this and allsubsequent experiments, fructose (data not shown)changed in parallel to glucose. Starch declined duringthe ®rst 1±2 h in the light, accumulated in the secondpart of the light period, and remained high during thenight (Fig. 1E). The decline in the reducing sugars (seeabove) coincides with the onset of starch accumulation.The diurnal changes in sugars and starch showed similartrends in wild-type plants and F23 (Figs. 1C±E). Similarchanges in sugars and starch are seen in a 12 h light/12 hdark irradiance regime, except that more starch isaccumulated (data not shown).

Nitrate reductase activity in a short-day regime. Thesource leaves of short-day-grown (6 h light/18 h dark)plants contained very low NR activity (Fig. 2a). The NRactivity at the end of the night [8 lmol (g FW))1 h)1;Fig. 2A] was only 30±50% of the activity at the end of thedark period in a 12 h light/12 h dark [15 lmol(g FW))1 h)1; Scheible et al. 1997b] or a 18 h light/6 hdark regime [24 lmol (g FW))1 h)1; Fig. 1A]. Illumination

P. Matt et al.: Nitrogen metabolism in short days 29

only produced a slight increase in NR activity in short-day conditions (Fig. 2A) whereas it produced a 2-to 3-fold increase in plants grown in a longer lightperiod (Fig. 1A and Scheible et al. 1997b). The maxi-

mum activity after 3±4 h illumination [about 12 lmol(g FW))1 h)1] is only a quarter of that found after 3±4 hillumination in a 12 h light/12 h dark or a 18 h light/6 hdark regime (Scheible et al. 1997b and Fig. 1A). Theweak rise in NR activity during the photoperiod inshort-day conditions is not due to the light period beingtoo short to give time for NR activity to rise, becauseNR activity increases by 20±30 lmol (g FW))1 h)1

during the ®rst 3±4 h of the photoperiod in leaves ofplants growing in a longer photoperiod (Fig. 1A;Scheible et al. 1997b).

Experiments with two further batches of plantscon®rmed that short days lead to low NR activity anddampen the diurnal changes in NR activity (Fig. 3 anddata not shown). In the experiment of Fig. 3, the diurnalchanges in NR activity in F23 and F22 were alsoinvestigated. Both mutants contain one instead of fourfunctional Nia gene copies. Unexpectedly, in short-dayconditions, the diurnal changes in NR activity in F22and F23 were as large as in wild-type plants (Fig. 3 anddata not shown). This contrasts with long-day condi-tions, where the diurnal changes are almost completelydampened in F22 and F23 (Fig. 1A and Scheible et al.1997b).

When wild-type plants were grown in short-dayconditions, NR was almost fully activated in the light(Fig. 2B), and inactivated during the dark period(Figs. 2B, 3B). In F22 and F23 mutants, NR waspartially activated in the night in short-day conditions(Fig. 3B). This contrasts with plants grown in a long-dayhigh-irradiance regime, where NR was partially activat-ed during night in wild-type plants and almost fullyactivated in F23 (Fig. 1B).

Diurnal changes in the Nia transcript level. Earlier studiesin a 12 h light/12 h dark regime have shown that levelsof the transcript for Nia are quite high at the end of thedark period, rise further immediately after illumination,decline to a low level during the rest of the photoperiod,and recover during the dark period (Galangau et al.1988; Scheible et al. 1997b). A similar response was seenin a long-day (14 h light/10 h dark) regime (Fig. 4B). Inshort days, this pattern was modi®ed (Fig. 4A). Tran-script was present at relatively low levels at the end of thenight and during the ®rst part of the short light period,declined to very low levels at the end of the photoperiod,increased to a maximum in the middle of the night, anddecreased during the last 5 h of the night. Transcript inthe short-day plants in the middle of the dark period wasalmost as high as in long-day-grown plants at the start ofthe light period (compare Figs. 4A and 4B).

Transfer from short days to long days. Short days lead towidespread changes in the growth, morphology andphysiology of plants which could complicate the inter-pretation of our experiments. We therefore investigatedwhether transfer of plants back to long-day conditionsresulted in the re-establishment of high NR activity anda normal diurnal rhythm of NR activity in pre-formedsource leaves. On the day after the samples were takenfor the experiment of Fig. 2, the remaining plants were

Fig. 1A±E. Changes in NR activity (NRA), NR activation andamount of carbohydrates in tobacco grown in long days under highirradiance. Wild-type (d) and F23 mutants (n) were grown at500 lmol m)2 s)1 irradiance in a 18 h light/6 h dark photoregime.Samples were harvested from the ®rst fully expanded leaf of 46-d-oldplants to analyse NR activity (A), NR activation estimated as NRactivity assayed in the presence of magnesium and NR activity in theabsence of magnesium (B), sucrose (C), reducing sugars (D) andstarch (E). The results are given as the mean � SE (n � 3)

30 P. Matt et al.: Nitrogen metabolism in short days

transferred into a 14 h light/10 h dark regime, and thediurnal changes in NR re-investigated 3 d later (Fig. 5).Absolute NR activities were increased by about 4-fold(compare Fig. 5A with Fig. 2A) and large diurnalchanges in NR activity were re-established (compareFig. 5A with Fig. 1A).

Levels of carbohydrates in short-day plants. Source leavesof short day-grown plants contained extremely lowlevels of sucrose (Fig. 2C) and reducing sugars (Fig. 2D)at the end of the night. The total sugar content was lessthan 0.5 lmol hexose (g FW))1. Sugars increasedslightly during the short light period, and declined to

low levels by the middle of the night (Fig. 2C,D).Compared to plants in long-day conditions (Fig. 1C,D),the sugars in short-day-grown plants were 10- to 20-foldlower at the end of the night, and 3- to 4-fold lowerduring the day. Transfer of the plants to a 14 h light/10 h dark regime for 3 d led to an increase in sucrose(Fig. 5C) and reducing sugars (Fig. 5D) to levels similarto those found in plants growing in long-day conditions.

In short-day conditions, large amounts of starch wereaccumulated during the light period (Fig. 2E) andremobilised during the dark period (Fig. 2E). Similarresults were obtained in two further batches of plants(data not shown). Starch accumulation started soon

Fig. 2A±J. Nitrate reductaseactivity, NR activation, andamounts of carbohydrates, ni-trate and amino acids in thesource leaves of tobacco grow-ing under high irradiance inshort-day conditions. Wild-typetobacco plants were grown at600 lmol m)2 s)1 in a 6 h light/18 h dark photoregime for 46 d,and samples taken from the ®rstfully expanded leaf to assay NRactivity (A), NR activation (B),sucrose (C), reducing sugars(D), starch (E), nitrate (F), totalamino acids (G), glutamine (H),glutamate (I), and the minoramino acids (sum of phenylala-nine, tryptophan, tyrosine, va-line, isoleucine, leucine, lysine,arginine, histidine, J). The re-sults are given as themean � SE (n � 3)

P. Matt et al.: Nitrogen metabolism in short days 31

after illumination in short-day-grown plants. In con-trast, there is a lag of 3±4 h before starch accumulationbegins in a 12 h light/12 h dark regime (Scheible et al.1997b), and starch remobilisation continues during the®rst 3±4 h light in a 18 h light/6 h dark regime (Fig. 1E).The rate of starch accumulation in a 6 h light/18 h darkregime (estimated by comparing the amount of starch atthe beginning and end of the photoperiod) was about5.1 lmol hexose (g FW)1) h)1, which is 5±10 times fasterthan during the second part of the light period in a long-day regime (Fig. 1E). When short-day-grown plantswere transferred to a 14 h light/10 h dark regime for 3 d,the leaves contained high starch at the end of the nightand accumulated starch more slowly during the day[about 3.6 lmol hexose (g FW))1 h)1, see Fig. 5E].

E�ect of petiole-cooling on source leaf metabolism inshort-day-grown plants. To check that the dramaticchanges in the Nia transcript level and NR activity inshort-day-grown plants were due to changes in carbo-hydrate levels, the export of photoassimilate from leaveswas decreased, and the e�ect of the resulting increase inleaf carbohydrates on nitrogen metabolism investigated(Fig. 6). To do this, cooled metal blocks were ®ttedround the petioles of source leaves of wild-type plantsgrowing in short-day conditions at the beginning of onelight period, and the leaves harvested at the end of thefollowing dark period.

Compared to control leaves that were ®tted with ametal block at ambient temperature, petiole cooling ledto a large increase in the starch content at the end of thenight, from 1.4 lmol (g FW))1 in control leaves to22.7 lmol (g FW))1 in cold-girdled leaves (data notshown). Sugar levels increased from about 1 lmol (g

FW))1 to 5 lmol (g FW))1 in the cold-girdled leaves(Fig. 6A). This was accompanied by a 2-fold increase inNR activity (Fig. 6B) and by a marked increase in theNia transcript level (Fig. 6C) at the end of the 18-h darkperiod.

Nitrate in short-day plants. When tobacco is grown in a12 h light/12 h dark regime, nitrate decreases from 30±40 lmol (g FW))1 in the morning to 5±10 lmol (gFW))1 at the end of the light period (Scheible et al.1997b). In short-day conditions, source leaves containedvery high levels of nitrate at the end of the dark period,and nitrate only decreased slightly during the lightperiod (Fig. 2F). High nitrate contents were seen in twofurther experiments in short-day conditions. In olderleaves of short-day-grown plants, the daily changes innitrate were totally abolished (data not shown). After3 d in a 14 h light/10 h dark regime, nitrate was lowerand there was a pronounced decrease during thephotoperiod and recovery during the night (Fig. 5F).

Amino acids in short days. Source leaves of short-day-grown plants contained low levels of amino acids at theend of the night [about 4 lmol (g FW))1], and aminoacids only rose to about 8 lmol (g FW))1 during thelight period (Fig. 2G). After transfer to a 14 h light/10 hdark regime for 3 d, the leaves contained 10 lmol (gFW))1 amino acids at the end of the night, and aminoacids rose to over 25 lmol (g FW))1 at the end of thelight period (Fig. 5G). Similar levels and a similaraccumulation of amino acids were seen in source leavesof tobacco growing in a 12 h light/12 h dark regime(Scheible et al. 1997b).

Fig. 3A,B. Diurnal changes in NR activity (A) and NR activation(B) in the source leaves of wild-type tobacco plants and low-NRmutants. Wild-type plants (d), and F23 (n) and F22 (h) mutantswere grown as described in the legend of Fig. 2. The results are themean � SE (n � 3 separate plants)

Fig. 4A,B. Diurnal changes in the Nia transcript in source leaves. ALong-day-grown plants. B Short-day plants. All plants were grown at600 lmol m)2 s)1 irradiance. For each daylength regime, RNA wasextracted and analysed separately from three individual leaves. Similaramounts (20 lg) of RNA were applied and the blots were carried outand developed in triplicate. The lanes show representative samplesfrom single leaves. For the short-day regime each of the blots wasquanti®ed by phosphoimager (Fujix BAS-1000, Fuji). The averagedresults are given under the representative northern blot (mean � SE,normalised on the maximum signal at 02.30)

32 P. Matt et al.: Nitrogen metabolism in short days

There were striking di�erences in the response to daylength of di�erent amino acids (see Fig. 2H,I andTable 1). At the end of the night, plants in short dayscontained 30% less glutamate (Fig. 2I), 50% lessaspartate and alanine, 70% less serine and glycine,75% less asparagine (Table 1), and 90% less glutamine(Fig. 2H) than plants after 3 long days in a 14 h light/10 h dark regime (Fig. 5H,I and Table 1). Illuminationled to a marked increase in glutamine and a smallincrease in asparagine in short- and long-day conditions.Unexpectedly, glutamate decreased by 50% after illumi-nation of short-day-grown plants (Fig. 2I). Glutamateincreased slightly after illumination in long-day condi-tions (Fig. 5I, see also Scheible et al. 1997b). The

decrease in glutamate after illumination of short-day-grown plants was con®rmed in a second experiment(data not shown). Alanine, threonine, serine and glycineincreased in the light in short- and long-day conditions,although the increases were smaller in short days(Table 1). Aspartate decreased after illumination inshort- and long-day conditions (Table 1).

The changes in the individual minor aromatic aminoacids (phenylalanine, tryptophan, tyrosine), aliphaticamino acids (valine, leucine, isoleucine), arginine, histi-dine, and lysine are given in Table 1, and the changes inthe summed minor amino acids in Fig. 2J and Fig. 5J.Most minor amino acids were below the detection level[about 0.01 lmol (g FW))1] at the end of the dark period

Fig. 5A±J. Nitrate reductaseactivity, NR activation, andamounts of carbohydrates, ni-trate and amino acids in thesource leaves of tobacco, 3 dafter transfer from a short-dayphotoregime to a 14 h light/10 h dark photoregime. Wild-type tobacco plants grown at600 lmol/m)2 s)1 in a 6 h light/18 h dark photoregime for 46 das in Fig. 2, were transferred toa 14 h light/10 h dark for 3 d,and samples taken from the ®rstfully expanded leaf to assay NRactivity (A), NR activation (B),sucrose (C), reducing sugars(D), starch (E), nitrate (F), totalamino acids (G), glutamine (H),glutamate (I), and the minoramino acids (sum of phenylala-nine, tryptophan, tyrosine, va-line, isoleucine, leucine, lysine,arginine, histidine, J). The re-sults are given as themean � SE (n � 3)

P. Matt et al.: Nitrogen metabolism in short days 33

in a short-day regime, whereas they could be readilydetected at the end of the night in long-day conditions.The minor amino acids increased in the light, but werestill much lower in short-day conditions.

Activity of NR and levels of carbohydrates in sink leavesof short-day-grown plants. Starch accumulated duringthe day and was remobilised during the night in sinkleaves of short-day-grown plants. (Fig. 7A). Sugarsshowed less marked diurnal variations in sink leavesthan in source leaves. The sucrose level in sink leaves atthe end of the night was 10-fold higher than in sourceleaves at this time (compare Fig. 7B and Fig. 2C).Sucrose in sink leaves rose to about 9 lmol sucrose (gFW))1 during the day, which is 2-fold higher than in

source leaves, and remained at this value for most of thedark period (Fig. 7B). Glucose and fructose were alsopresent at higher levels in sink leaves than in sourceleaves (compare Fig. 7C and Fig. 2D), and did notdecline so rapidly in the dark. Similar changes incarbohydrates were found in sink leaves of the mutantsF23 and F22 (Figs. 7A±D).

The activity of NR in sink leaves of short-day-grownplants was low at the end of the night, increased 3- to 4-fold after illumination (Fig. 7D), and decreased gradu-ally during the night. This contrasts to source leaves,where the diurnal changes in NR activity were very small(see Figs. 2A, 3A). In short day-grown plants, sinkleaves contained higher NR activity than source leaves(compare Fig. 7D with Figs. 2A and 3 A). This con-trasts with plants growing in a 12 h light/12 h dark(Geiger et al. 1998) or in a 18 h light/6 h dark cycle (datanot shown), where sink leaves contain 50% and 80±90%less NR activity than source leaves after 4 h illuminationand during the night, respectively. Activation of NR washigh in sink leaves of short-day plants in the light, anddecreased gradually during the dark period (Fig. 7E).Nitrate was lower than in source leaves, and decreasedduring the light period and increased during the night(data not shown).

In F23 and F22, maximum NR activity in sink leavesduring the photoperiod was lower than in wild-typeplants. During the night, NR activity in sink leaves ofthe mutants was similar to or slightly higher than inwild-type plants (Fig. 7D), and NR was more stronglyactivated than in wild-type plants (Fig. 7E).

E�ect of a single extended night on source leaf metabo-lism. To investigate the e�ect of a short-term depletionof sugars on NR activity and amino acid levels, plantswere exposed to a single short day/long night cycle ofvarying severity. Four sub-groups of plants were dark-ened after 4, 6, 8, and 10 h light, and re-illuminated after20, 18, 16, and 14 h darkness, respectively. A ®fthcontrol group was left in the 14 h light/10 h dark regime(Fig. 8). In this experiment, the long dark period wasnot interrupted by short exposure to low light. In orderto have a starting material in which starch was relativelyhigh at the start of all the dark incubations, theexperiment was carried out with plants which had beentransferred from short-day conditions to a 14 h light/10 h dark regime for 4 d (see Fig. 5E).

A single short day/long night treatment led to areduction of starch (Fig. 8A), sucrose (Fig. 8B) andreducing sugars (Fig. 8C) at the end of the dark period.The changes were largest after 4 h light/20 h dark and6 h light/18 h dark, where sucrose fell to about 2 lmol(g FW))1 and reducing sugars to under 0.5 lmol(g FW))1. The levels of sucrose and reducing sugarswere still higher than in plants that were growing inshort-day conditions (compare Fig. 8B,C andFig. 2C,D). After illuminating the plants for 4 h, sucroselevels rose (Fig. 8B). The accumulation of reducingsugars that is typically seen in tobacco during the ®rsthalf of the light period (Fig. 5D, was inhibited after asingle short day/long night treatment (Fig. 8C).

Fig. 6A±C. E�ect of cold-girdling on the levels of sugars, NR activityand the level of theNia transcript in the source leaves at the end of thedark period in short-day conditions. Tobacco plants were grown for70 d in a 6 h light/18 h dark regime at 300 lmol m)2 s)1 irradiance.One hour after the start of the light period, a water-cooled metal blockwas ®tted to the petiole of the youngest fully expanded leaves. Thetemperature was reduced to ca. 4 °C with circulating water in half ofthe blocks, and left at ambient temperature in the other half. Theleaves were harvested 22 h later, at the end of the following 18-h darkperiod, and analysed for sucrose (h), glucose ( ) and fructose ( ) (A),NR activity measured in the absence of magnesium (B) and the levelof Nia transcript after 1 h illumination (C) before cooling the petiole(control, 9:00), and at the end of the following dark period in cold-girdled leaves (cold, 7:00) and in controls where the metal block was atroom temperature (warm, 7:00). The results in A and B are themean �SE (n � 4 separate plants), and C shows one representativeresult from 4 replicate extracts from separate plants

34 P. Matt et al.: Nitrogen metabolism in short days

A single short day/long night treatment led to amarked change in the rate of starch accumulation in the®rst 4 h of the following light period (Fig. 8A). Netstarch accumulation during the ®rst 4 h in the light wasnegligible in control plants (see also Figs. 1E, 5E andScheible et al. 1997b), about 6 lmol hexose (gFW))1 h)1 after a 10 h light/14 h dark or 8 h light/16 h dark treatment, and about 10 lmol hexose (gFW))1 h)1 after a 6 h light/18 h dark or a 4 h light/20 hdark treatment.

Sugar levels at the end of a single prolonged night(compare Figs. 8B,C with Figs. 2C,D) and NR activitywere both still higher than in short-day-grown plants(compare Fig. 8D with Figs. 2A and 3A). Nevertheless,the 6 h light/18 h dark and 4 h light/ 20 h darktreatments led to a small decrease in NR activity(Fig. 8D) and NR activation (Fig. 8E) at the end ofthe dark period. In these two treatments, total leaf sugarat the end of the dark period had decreased to under3 lmol (g FW))1. There was a small but non-signi®cantincrease in NR activity at the end of the night after a10 h light/14 h dark or a 8 h light/16 h dark treatment.After these treatments, the sugars did not decrease asmuch and (see below) and glutamine was lower than inthe controls. In all cases, re-illumination led to increasedNR activity and NR activation. Similar NR activitiesand activation were found in the controls and all fourtreatments after 4 h illumination.

A single short day and extended night treatmentresulted in a markedly increased level of nitrate at theend of the dark period (Fig. 8F). The total amino acid

content did not change signi®cantly (Fig. 8G), but therewere dramatic changes in the levels of the individualamino acids. Glutamine (Fig. 8H), glycine and serine(Table 2) decreased by 50±80%, asparagine, aspartateand alanine remained unaltered (Table 2), and gluta-mate increased (Fig. 8I) after a single short day andextended night. There was also a dramatic decrease inthe level of most of the minor amino acids (Fig. 8J),including the aromatic amino acids, the aliphatic aminoacids, arginine, and lysine (see Table 2 for details). Anincrease in glutamate and a decrease in the minor aminoacids was already apparent in response to a smallincrease in the duration of the dark period. After re-illumination, glutamate (Fig. 8I) and aspartate (Table 2)decreased, and all the minor amino acids increased(Fig. 8J and Table 2).

A single short day/long night treatment also led to asigni®cant decrease in the amount of chlorophyll. Thisdecrease was reversed after 4 h illumination after the10 h light/14 h dark and 8 h light/16 h dark treatments,but not after the more extreme 6 h light/18 h dark and4 h light/20 h dark treatments (Fig. 8K).

Discussion

Short days lead to low NR activity and a severe restrictionof nitrate assimilation. When tobacco is grown in a short-day regime, NR activity is severely reduced in the sourceleaves. The NR activity is very low at the end of the darkperiod and the light-induced increase in NR activity is

Table 1. Amino acid levels in source leaves of tobacco growing in short days, and 4 d after transfer to long days. The plants were grown at600 lmol m)2 s)1 in a 6 h light/18 h dark photoregime for 46 d as in Fig. 2, and then transferred to a 14 h light/10 h dark for 3 d as in Fig.3. For plants in short days, amino acids were assayed in samples harvested 30 min and 3.5 h after the start of illumination, 0.5 h before theend of the light period, and 9.5 h into the dark period. For plants in long days, amino acids were assayed in samples harvested 30 min and12.5 h after the start of illumination, 0.5 h before the end of the light period, and 6.5 h into the dark period. Cit, Citrullin; Gaba, c-aminobutricacid. The results are given as lmol (g FW))1 and as the mean � SE (n = 3)

Photoperiod 6 h light/18 h dark Photoperiod 14 h light/10 h dark

Time 7:30 10:30 12:30 22:30 7:30 13:30 20:30 3:30

Asp 0.82 � 0.03 0.74 � 0.04 0.76 � 0.04 2.42 � 0.12 1.62 � 0.26 2.28 � 0.25 2.00 � 0.28 4.15 � 0.77Glu 1.57 � 0.20 0.72 � 0.11 0.76 � 0.09 1.79 � 0.15 2.50 � 0.53 3.18 � 0.63 2.88 � 0.40 3.39 � 0.27Asn 0.10 � 0.04 0.16 � 0.02 0.16 � 0.04 0.21 � 0.06 0.41 � 0.19 0.57 � 0.12 0.62 � 0.19 0.63 � 0.12Ser 0.20 � 0.04 0.30 � 0.02 0.45 � 0.06 0.24 � 0.09 0.57 � 0.50 2.25 � 0.94 2.77 � 0.35 1.42 � 0.46Gln 0.28 � 0.03 1.37 � 0.15 2.34 � 0.55 0.70 � 0.08 2.48 � 0.41 5.41 � 2.67 6.86 � 0.61 3.28 � 1.39Gly 0.13 � 0.02 0.78 � 0.17 1.47 � 0.24 0.12 � 0.03 0.44 � 0.10 2.42 � 1.43 2.82 � 0.35 0.77 � 0.39Thr 0.12 � 0.01 0.15 � 0.01 0.20 � 0.02 0.11 � 0.03 0.27 � 0.06 0.60 � 0.13 0.81 � 0.12 0.47 � 0.13His n.d. n.d. 0.05 � 0.01 n.d. 0.15 � 0.02 0.17 � 0.08 0.18 � 0.06 0.20 � 0.07Cit n.d. 0.01 � 0.02 0.03 � 0.00 n.d. 0.10 � 0.00 0.24 � 0.06 0.23 � 0.02 0.12 � 0.05Ala 0.29 � 0.06 0.64 � 0.04 0.63 � 0.08 0.19 � 0.05 0.59 � 0.17 2.97 � 1.29 2.33 � 0.26 0.71 � 0.28Arg n.d. 0.07 � 0.01 0.09 � 0.03 0.01 � 0.02 0.12 � 0.05 0.31 � 0.13 0.48 � 0.05 0.19 � 0.14Gaba n.d. n.d. 0.03 � 0.05 n.d. 0.21 � 0.08 0.42 � 0.14 0.39 � 0.29 0.25 � 0.11Tyr n.d. 0.03 � 0.02 0.05 � 0.00 0.01 � 0.02 0.04 � 0.03 0.11 � 0.03 0.15 � 0.02 0.08 � 0.04Val 0.01 � 0.01 0.03 � 0.00 0.05 � 0.00 0.01 � 0.01 0.05 � 0.02 0.12 � 0.04 0.15 � 0.01 0.04 � 0.02Met n.d. 0.02 � 0.00 0.02 � 0.00 n.d. n.d. 0.04 � 0.02 0.04 � 0.01 0.01 � 0.01Trp n.d. n.d. n.d. n.d. n.d. 0.05 � 0.05 0.11 � 0.01 0.06 � 0.05Phe 0.01 � 0.02 0.15 � 0.03 0.24 � 0.07 0.03 � 0.03 0.19 � 0.04 0.94 � 0.30 0.97 � 0.11 0.29 � 0.14Ile n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.01 � 0.02Leu 0.02 � 0.03 0.05 � 0.01 0.08 � 0.01 0.03 � 0.03 0.05 � 0.04 0.16 � 0.06 0.21 � 0.04 0.08 � 0.04Lys n.d. n.d. 0.01 � 0.03 0.02 � 0.03 0.04 � 0.04 0.08 � 0.07 0.11 � 0.03 0.06 � 0.06

n.d. = not detected

P. Matt et al.: Nitrogen metabolism in short days 35

almost totally suppressed. The very high levels of nitrateand low levels of amino acids in leaves of short-day-grown plants indicate that the low NR activity is leadingto a severe inhibition of nitrate assimilation. Short dayshave also been reported to decrease NR activity in potatoplants (Ezekiel and Bhargava 1993). This inhibition ofnitrate assimilation is likely to exacerbate the inhibitionof growth in short-day conditions because it leads to adecline in protein and chlorophyll, which will furtherrestrict carbon gain during the short light period.

The regulation of Nia expression and NR activity bynitrate and glutamine is overridden in short-day condi-tions. Changes in nitrate and glutamine play an impor-tant role in the diurnal regulation of Nia expression andNR activity in tobacco growing in intermediate or long-day conditions (Galangau et al. 1988; Scheible et al.1997b; Geiger et al. 1998). In these conditions, the Niatranscript level increases during the dark period anddecreases during the light period (Fig. 4B), and NRprotein and NR activity rise during the ®rst 4 h of thephotoperiod and then decline (Fig. 1A). The diurnalchanges in the Nia transcript level in wild-type plantscorrelate well with the changes in leaf nitrate and

glutamine (Scheible et al. 1997b; Geiger et al. 1998), anda high Nia transcript level is found throughout the entirediurnal cycle in NR-de®cient or low-NR transformantsthat contain high nitrate and low glutamine (Vaucheretet al. 1990; Ho� et al. 1994; Scheible et al. 1997b). Thedecreased level of NR protein and NR activity duringthe light period in wild-type plants correlates with anaccumulation of glutamine (Galangau et al. 1988; Geigeret al. 1998), and this decline in NR protein and NRactivity is decreased or abolished in NR mutants andtransformants with decreased expression of NR(Fig. 1A; see also Ho� et al. 1994; Scheible et al.1997b) where glutamine accumulated more slowlyduring the light period (Scheible et al. 1997b).

Two lines of evidence show that, in-short-day condi-tions, the diurnal regulation of nitrate assimilation isdominated by a further signal that overrides these well-characterised signals from nitrate and nitrogen metab-olism. Firstly, NR activity is very low in the sourceleaves of short-day-grown tobacco, even though theleaves contain high levels of nitrate and the low levels ofglutamine which would be expected to result in high NRactivity. Secondly, in short-day conditions mutants withdecreased expression of NR exhibit diurnal changes inNR activity similar to or even slightly larger than thoseof wild-type plants (see Fig. 3A). In intermediate orlong-day growth regimes, the diurnal changes in NRactivity in these low-NR mutants are strongly dampened(see Fig. 1A) because the lower NR activity delays thedepletion of nitrate and the accumulation of glutamine(Scheible et al. 1997b), and weakens or delays thefeedback signals (see above) that are responsible for thedecreased NR activity later in the light period. Theappearance of a marked diurnal rhythm of NR activityin these low-NR mutants in short days shows that asignal that is unrelated to nitrogen metabolism makes adominant contribution to the regulation of NR activityin short-day conditions.

The inhibition of Nia expression and NR activity in short-day-grown plants may be due to low levels of sugars. It isknown that sugar-feeding leads to an increase in the Niatranscript (see Introduction). Several lines of evidenceindicate that the low endogenous sugar levels areresponsible for the inhibition of Nia expression andNR activity in short-day-grown tobacco. Firstly, the lowNR activity in source leaves of short-day-grown plantswas correlated with low levels of sucrose and reducingsugars, especially during the last part of the dark period(Fig. 2). Secondly, transfer to long days led, within 3 d,to a recovery of the sugar contents and a to a completerecovery of NR activity, showing that the low NRactivity is not an indirect e�ect due to the changedmorphology or life history of short-day plants (Fig. 3).Thirdly, when the leaf petiole was cooled to decreaseexport from the leaf and slightly increase the pool ofsugars at the end of the night in short-day-grown plants,there was a marked increase in the level of the Niatranscript and a 2-fold increase in NR activity (Fig. 6).Fourthly, exposing plants to a single short day/longnight treatment led to a decreased NR activity at the end

Fig. 7A±E. Nitrate reductase activity, NR activation, and amounts ofcarbohydrates in the sink leaves of tobacco growing in a short-dayregime. The plants were grown at 400 lmol m)2 s)1 in a 6 h light/18 h dark photoregime for 61 d, and samples taken from a sink leaf(length ca. 5 cm, equivalent to one-third of a fully expanded leaf) toassay starch (A), sucrose (B), reducing sugars (C), NR activity (D),and NR activation (E). The results are given as the mean �SE(n � 3)

36 P. Matt et al.: Nitrogen metabolism in short days

of the dark period (Fig. 8). Fifthly, short days did notlead to such a dramatic decrease of sugars in sink leaves,and NR activity was relatively high in sink leaves andshowed a large increase after illumination (Fig. 7). Adependence of NR expression on adequate levels ofsugars at the end of the night would also explain anearlier observation that starch-de®cient Arabidopsismutants have low NR activity (Schulze et al. 1994). Inthese genotypes, sugars are high during the day but arerapidly depleted during the dark period (Caspar et al.1986; Schulze et al. 1991).

When plants were exposed to a single extended night,NR activity did not decrease until the leaf sugar level atthe end of the dark period declined to under 3 lmolhexose/g FW (Fig. 8). This is much lower than the sugar

levels in source leaves at the end of the dark period inplants growing in a 12 h light/12 h dark (Scheible et al.1997b) or long-day conditions (Fig. 1C,D). Further, NRactivity was restored after 4 h illumination (Fig. 8), eventhough sugar levels were still lower than in plants in anuninterrupted long-day light regime. These results sug-gest that quite low levels of sugars su�ce to maintainhigh NR activity. It should be noted, however, thatleaves contain low glutamine and high nitrate in short-day conditions, or after an extended dark period. Thismay sensitise the response to sugars. When sugar, nitrateand glutamine are supplied to detached leaves via thetranspiration stream, sugar acts additively with nitrateand antagonistically to glutamine to increase NRactivity, and much higher internal levels of sugars are

Fig. 8A±J. Impact of a singleshort day/long night cycle onNR activity, NR activation,and amounts of carbohydrates,nitrate and amino acids insource leaves. Wild-type tobac-co plants were grown at600 lmol/m)2 s)1 in a 6 h light/18 h dark photoregime for 46 d,and transferred to a 14 h light/10 h dark regime for 5 d andthen divided into 5 sub-groups.One subgroup was left in the14 h light/10 h dark regime.The other subgroups weredarkened prematurely after 4, 6,8 or 10 h illumination, and wereleft in the dark for 20, 18, 16 or14 h and then all re-illuminatedat the start of the normal lightperiod. Samples were takenfrom the ®rst fully expandedleaf 30 min before illumination,and after 4 h illumination toassay starch (A), sucrose (B),reducing sugars (C), NR activ-ity (D), NR activation (E),nitrate (F), total amino acids(G), glutamine (H), glutamate(I), and the minor amino acids(sum of phenylalanine, tryp-tophan, tyrosine, valine, isoleu-cine, leucine, lysine, arginine,histidine, J). The results aregiven as the mean � SE(n � 4)

P. Matt et al.: Nitrogen metabolism in short days 37

Table

2.Im

pact

ofasingleshortdayandlongnightonthelevelsofaminoacidsin

thesourceleaf.Wild-typeplantsweregrownat600

lmolm

)2s)

11in

a6hlight/18hdark

photoregim

efor46d,andtransferredto

a14hlight/10hdark

for5d,andthen

divided

into

5sub-groups.Onesubgroupwasleftin

the14hlight/10hdark

regim

e.Theother

subgroupsweredarkened

prematurely

after

4,6,8or10hillumination,andwereleftin

thedark

for20,18,16or14handthen

allre-illuminatedatthestartofthenorm

allightperiod.Samplesweretaken

from

the

®rstfullyexpanded

leaf30min

before

illumination,andafter

4hillumination.Theresultsare

given

as

lmol(gFW))

1andasthemean�

SE

(n=

3)

Endofdark

treatm

ent

4hlightafter

dark

treatm

ent

20hdark

18hdark

16hdark

14hdark

10hdark

20hdark

18hdark

16hdark

14hdark

10hdark

Asp

4.06�

1.14

3.69�

0.23

3.00�

0.72

2.70�

0.60

2.88�

0.69

2.19�

0.52

1.87�

0.58

2.31�

0.35

1.72�

0.20

1.81�

0.15

Glu

4.17�

0.78

4.38�

0.32

3.83�

0.13

3.71�

0.65

3.08�

0.26

1.70�

0.40

1.53�

0.15

2.12�

0.40

2.09�

0.26

3.15�

0.39

Asn

1.24�

0.37

0.62�

0.22

0.73�

0.26

0.63�

0.20

0.79�

0.40

0.60�

0.14

0.36�

0.11

0.55�

0.31

0.46�

0.07

0.40�

0.08

Ser

0.96�

0.07

0.99�

0.11

1.22�

0.31

1.07�

0.54

1.49�

0.56

1.27�

0.17

1.14�

0.71

1.70�

0.49

1.20�

0.57

1.30�

0.49

Gln

1.71�

1.05

0.65�

0.13

2.69�

1.67

2.34�

1.28

3.63�

2.31

3.75�

0.48

2.99�

0.98

5.21�

2.77

5.02�

1.83

2.59�

0.35

Gly

0.30�

0.07

0.44�

0.17

0.74�

0.39

0.64�

0.54

0.87�

0.27

1.79�

0.07

1.82�

1.32

2.65�

1.47

2.26�

1.80

0.65�

0.19

Thr

0.33�

0.01

0.35�

0.02

0.33�

0.08

0.31�

0.13

0.41�

0.03

0.47�

0.02

0.42�

0.16

0.58�

0.11

0.50�

0.15

0.47�

0.09

His

0.10�

0.02

0.07�

0.06

0.11�

0.02

0.10�

0.04

0.28�

0.11

0.14�

0.01

0.11�

0.03

0.15�

0.06

0.15�

0.06

0.19�

0.08

Cit

0.04�

0.04

0.06�

0.01

0.10�

0.02

0.08�

0.07

0.10�

0.02

0.15�

0.03

0.12�

0.04

0.15�

0.04

0.18�

0.04

0.13�

0.02

Ala

0.66�

0.04

0.57�

0.10

0.78�

0.21

0.72�

0.34

0.71�

0.27

1.73�

0.19

1.55�

0.73

2.06�

0.38

1.70�

0.69

1.18�

0.20

Arg

0.07�

0.07

0.03�

0.02

0.14�

0.06

0.19�

0.17

0.40�

0.22

0.21�

0.02

0.17�

0.14

0.24�

0.17

0.28�

0.14

0.29�

0.14

Gaba

0.46�

0.21

0.30�

0.18

0.37�

0.12

0.27�

0.09

0.49�

0.21

0.27�

0.05

0.27�

0.16

0.35�

0.03

0.24�

0.02

0.27�

0.08

Tyr

n.d.

n.d.

0.02�

0.03

0.02�

0.03

0.11�

0.05

0.05�

0.01

0.06�

0.07

0.08�

0.03

0.07�

0.02

0.06�

0.01

Val

0.02�

0.00

0.03�

0.00

0.03�

0.03

0.03�

0.03

0.07�

0.04

0.08�

0.01

0.08�

0.05

0.10�

0.04

0.10�

0.04

0.07�

0.01

Met

n.d.

n.d.

0.01�

0.01

0.00�

0.01

0.02�

0.01

n.d.

0.01�

0.01

0.02�

0.01

0.02�

0.02

0.00�

0.01

Trp

n.d.

n.d.

n.d.

n.d.

0.05�

0.04

n.d.

n.d.

0.02�

0.04

0.04�

0.03

0.06�

0.01

Phe

0.02�

0.04

0.05�

0.05

0.06�

0.00

0.07�

0.00

0.14�

0.04

0.38�

0.07

0.30�

0.13

0.47�

0.13

0.41�

0.16

0.24�

0.06

Ile

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

0.02�

0.04

Leu

0.06�

0.01

0.07�

0.01

0.09�

0.04

0.06�

0.06

0.21�

0.16

0.11�

0.02

0.13�

0.13

0.14�

0.07

0.13�

0.07

0.07�

0.02

Lys

n.d.

n.d

0.05�

0.04

0.07�

0.06

0.17�

0.06

n.d.

0.05�

0.08

0.04�

0.08

0.09�

0.04

0.10�

0.03

n.d.=

notdetected

38 P. Matt et al.: Nitrogen metabolism in short days

needed to maintain high NR activity when nitrate is low,or glutamine is high (Morcuende et al. 1998).

Our experiments were not designed to investigatewhich sugar is sensed, or to analyse the mechanism ofsugar sensing. However, they indicate that sugars maya�ect the expression and activity of NR at severaldi�erent levels. (i) The Nia transcript rises to very highlevels during the ®rst part of the dark period in short-day-grown tobacco, as would be expected from the highnitrate and low glutamine levels in the leaves (see above),and then decreases in the last part of the dark periodafter the overall sugar levels have fallen to below 3 lmol(g FW))1. These results indicate that high nitrate andlow glutamine lead to an increase in the level of the Niatranscript until sugars fall to a critical level, below whichNia transcription is inhibited and/or the Nia transcript israpidly degraded. (ii) Sugars may also a�ect the trans-lation or stability of NR. In short days, NR activity onlyrises slightly after illumination even though some Niatranscript is still present at the start of the light period(Figs. 2A, 3A and 4A). In long days, the increase in NRactivity does not commence until after illumination eventhough the Nia transcript reaches a high level during thelast part of the dark period (Figs. 1A and 4B, see alsoScheible et al. 1997b). Morcuende et al. (1998) foundthat sugar feeding alters NR activity in detached leaves,but has no marked e�ect on the Nia transcript level.However, it will be necessary to measure the rate of de-novo synthesis of NR protein to con®rm this interpre-tation. (iii) Sugars also a�ect the post-translationalregulation of NR. The overall endogenous sugar levelscorrelate well with NR activation, when short-day-grown plants are compared to long-day-grown plants(compare Fig. 1B with Figs. 2B and 3B), and whenplants are exposed to a single short day/long night cycleof di�ering severity (Fig. 8E).

Although Nia is induced by red light and repressed byfar-red light in seedlings, studies with mature plantsshow that phytochrome is not essential for the light-regulation of Nia in greened leaves on older plants (seeIntroduction). In our experiments, the short-day-grownplants were always exposed to a brief period of lightafter 13 h of the dark period. It is therefore unlikely thatphytochrome-mediated photoperiod sensing is responsi-ble for the low NR activity in short-day plants in ourexperiments. However, our experiments do not excludethe possibility that mid- or long-term changes inhormones or further physiological signals also contrib-ute to the changes in regulation of Nia expression inshort day-grown plants.

Low sugars may lead to a general inhibition of amino acidsynthesis. Following the incorporation of ammoniuminto glutamine by glutamine synthetase, the aminogroup is transferred to oxoglutarate to form glutamate.Aspartate and alanine are formed by direct transamina-tion of metabolites formed in primary metabolism, andglycine and serine are formed during photorespiration.The other amino acids are synthesised via speci®cbiosynthetic pathways in which glutamate, asparatateand, occasionally, glutamine act as amino donors (Heldt

1996). Tobacco plants growing in short days had verylow levels of the minor amino acids, especially at the endof the night. This may be partly due to the low NRactivity. However, the changes in the individual aminoacid pools indicate that the sugar supply may also a�ectamino acid metabolism.

It is striking in short-day plants (Fig. 2, Table 1) that(i) at the end of the night there were still substantialpools of glutamate, aspartate and alanine whereas thepools of most of the minor amino acids were belowdetection and (ii) illumination led to an increase in theminor amino acids whereas glutamate decreased andaspartate remained unaltered. It is also striking in plantsthat were subjected to a single short day and extendednight (Fig. 8, Table 2) that (i) at the end of the darkperiod the minor amino acids were very low whereasglutamate and aspartate were unaltered or higher thanin control plants and (ii) re-illumination led to anincrease in the minor amino acids and a simultaneousdecrease in glutamate and asparatate. A decrease in theminor amino acids and an increase in glutamate werefound after even a moderate lengthening of the night,which did not decrease NR activity or NR activation(Fig. 8I,J), indicating that amino acid metabolism mayrespond more sensitively to a shortfall of sucrose thannitrate assimilation itself. A similar reciprocal decreasein glutamate and aspartate and increase in the minoramino acids was found after feeding sucrose to detachedtobacco leaves (Morcuende et al. 1998).

These reciprocal changes in the amino donors and theminor amino acids indicate that transient changes in thesugar supply lead to a rapid and reversible modi®cationof amino acid metabolism. The simplest explanation forthese results would be that the amino acid biosynthesispathways are inhibited when sugars are low. Although itis also possible that amino acid catabolism is increased,this would be expected to lead to an increased level ofasparagine (see Introduction) and the opposite was foundin short-day-grown plants (Table 1) and most of theextended-night treatments (Table 2). Amino acid bio-synthesis is subject to feedback regulation by speci®camino acids (Lea and Forde 1994). In mammals andfungi, amino acid synthesis is also regulated by thegeneral nitrogen control system, in which GNC4 acts asa transcription factor to promote transcription of morethan 30 amino acid biosynthesis genes in response tostarvation for a single amino acid (Dever et al. 1992;Pain 1994). There is evidence that Opaque-2 is afunctional plant homolog of GNC4 (Mauri et al. 1993)although its function in processes other than seedstorage-protein synthesis has not been investigatedin detail (Maddaloni et al. 1996). Less is known aboutthe regulation of amino acid synthesis in response tochanges in the carbon supply. Further studies are neededto investigate the e�ect of sugars on the transcriptionand activity of a wide range of enzymes in the aminoacid biosynthesis pathways.

Short days lead to a strong stimulation of starchmetabolism. Short days lead to a strong stimulation ofstarch synthesis in tobacco source leaves (compare

P. Matt et al.: Nitrogen metabolism in short days 39

Figs. 1E and 2 E). A similar stimulation of assimilatorystarch synthesis in short days is found in the sourceleaves of other species, including pea (Stitt et al. 1978),soybean (Chatterton and Silvius 1979), corn, pangola,spinach and sugar beet (Chatterton and Silvius 1980).This stimulation of starch synthesis in short-day condi-tions may be one of the reasons why sugars remainrelatively low during the light period.

Remobilisation of assimilatory starch supports su-crose export and leaf metabolism during the darkperiod. Starchless mutants show an increasingly stronginhibition of growth in short days (Caspar et al. 1986;Schulze et al. 1991; Huber and Hanson 1992), showingthat starch remobilisation is essential for survival duringa long dark period. Several lines of evidence indicatethat the starch is needed to support growth in sinkorgans: the levels of sugars in the sink leaf of short-dayplants are not as strongly a�ected as the pools in thesource leaves (Fig. 7B,C), leaf expansion was inhibitedduring the night but not during the day in starch-de®cient tobacco (Huber and Hanson 1992), andstarchless Arabidopsis mutants showed a particularlystrong inhibition of seed formation (Schulze et al. 1994).

The high rates of starch accumulation in short-day-grown plants raise intriguing questions with respect tothe regulation of starch synthesis. Starch accumulationis normally increased when photosynthesis is rapid andsugars are high (Stitt 1996; Stitt et al. 1987). Strikingly,the high rates of starch accumulation during the lightperiod in short day-grown plants are associated withvery low levels of sugars (see above). Starch accumula-tion is normally decreased after adding nitrate, whichrepresses agpS2 and induces genes required to divertcarbon to organic acid synthesis (Scheible et al. 1997a).In short days, however, starch accumulates to highlevels, even though nitrate is very high. This implies thatshort days stimulate starch synthesis via a signal thatoverrides the known mechanisms for regulating starchsynthesis. Chatterton and Silvius (1981) showed that thehigh rates of starch accumulation during a short lightperiod were not prevented when the light period wasextended by a period of very low light, and we found amarked change in partitioning in short days even thoughthe dark period was interrupted by 10 min high light.These results indicate that the increased rates of starchsynthesis in short days may be triggered by a change inthe sink-source balance of the plant, rather than byphotoperiod sensing. The signal might be depletion ofsugars in the leaves, in transport routes, or in growingorgans. It is striking that a single cycle of a short dayand long night su�ces to stimulate starch synthesis andto decrease the levels of sugars, especially glucose,during the ®rst 4 h of the following light period(Fig. 8A±C).

In conclusion, growth in short days leads to low levelsof sugars in the source leaves of tobacco, especially inthe second part of the dark period. Whereas the level ofthe Nia transcript rises throughout the dark period andNR activity rises 2-fold after illumination in long days,in short days there is a dramatic decline in the level ofthe Nia transcript during the second part of the dark

period, and NR activity only increases slightly afterillumination. Growth in short days also leads to adepletion of minor amino acids, even though theglutamate and aspartate remain high, which indicatesthat amino acid biosynthesis is regulated by sugars. Thedependence of nitrogen metabolism on the sugar supplyis strikingly illustrated by the ®nding that tobacco plantsgrown in very short days are simultaneously carbon andnitrogen de®cient. The implications for the survival andcompetitiveness of plants in short light regimes appearto be far-reaching, and require further investigation.

This research was supported by the Deutsche Forschungsgemeins-chaft (SFB 199).

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