Planta (1992, 188:39~7
P l a n t a 9 Springer-Verlag1992
Light-regulated expression of the nitrate-reductase and nitrite-reductase genes in tomato and in the phytochrome-deficient a u r e a mutant of tomato Thomas W. Becker 1, Christine Foyer 2, and Michel Caboche 1. Laboratoires de 1 BiologieCellulaire, and du 2 M6tabolismeet de la Nutrition des Plantes, INRA, Centre de Versailles, Route de Saint-Cyr, F-78026 Versailles Cedex, France Received 2 December 1991; accepted 18 March 1992 Abstract. The phytochrome-deficient a u r e a mutant of tomato ( L y c o p e r s i c o n e s c u l e n t u m (L.) Mill) was used to investigate if phytochrome plays a role in the regulation of nitrate-reductase (NR, EC 1.6.6.1) and nitrite-reductase (NiR, EC 1.7.7.1) gene expression. We show that the expression of the tomato NR and NiR genes is stimulated by light and that this light response is mediated by the photoreceptor phytochrome. The red-light response of the NR and NiR genes was reduced in etiolated a u r e a seedlings when compared to isogenic wild-type cotyledons. The relative levels of NR mRNA and NiR transcripts and their diurnal fluctuations were identical in mature white-light-grown leaves of the wild-type and of the a u r e a mutant. The transcript levels for cab and R b c S (genes for the chlorophyll-a/b-binding protein of PSII and the small subunit of the enzyme ribulose-l,5-bisphosphate carboxylase/oxygenase, respectively) in a u r e a leaves grown in white light were indistinguishable from the respective transcript levels in the leaves of the wildtype grown under the same conditions. Despite a severe reduction in the chlorophyll content, the rate of net CO2 uptake by leaves of the a u r e a mutant was only slightly reduced when compared to the rate of net photosynthesis of wild-type leaves. This difference in the photosynthetic performances of wild-type and a u r e a mutant plants disappeared during aging of the plants. The increase in zeaxanthin and the concomitant decrease in violaxanthin in leaves of the aurea mutant compared with the same pigment levels in leaves of the wild-type indicate that the activity of the xanthophyll cycle is increased in a u r e a leaves as a consequence of the reduced CO2-fixation capacity of the mutant leaves.
* To whomcorrespondenceshould be addressed; FAX (0) 1/30833111 Abbreviations: Chl-chlorophyll;NR=nitrate reductase; NiR=nitrite reductase; cab = gene for the chlorophyll-a/b-bindingproteins of PS II; RbcS = gene for the small subunit of the enzymeribulose1,5-bisphosphate carboxylase/oxygenaseRuBPCase= ribulose-1,5bisphosphate carboxylase/oxygenase
Key words: Gene expression - L y c o p e r s i c o n - Mutant (tomato, aurea) - Nitrate reductase - Nitrite reductase Phytochrome
Introduction Light-regulated gene expression in higher plants is mediated by several photoreceptors including phytochrome (Mohr 1966), a dimeric chromoprotein (Lagarias and Mercurio 1985) that can exist in two spectrally distinct forms. In etiolated tissue, phytochrome is synthesized in the physiologically inactive, red-light-absorbing form (Pr). Red light induces the reversible coversion of Pr into the active, far-red-light-absorbing form (Pfr; Lagarias 1985). This photoconversion leads to the induction of the transcription of light-responsive genes such as those for the chlorophyll-a/b-binding protein of PSII ( c a b ) and the small subunit of the enzyme ribulose-l,5bisphosphate carboxylase/oxygenase ( R b c S ) (Tobin and Silverthorne 1985). Light induces the transcription of the nitrate-reductase (NR, EC 1.6.6.1) genes of tobacco (Deng et al. 1989) and stimulates mustard nitrite-reductase (NiR, EC 1.7.7.1) gene expression (Rajasekhar and Mohr 1986). The enzymes NR and NiR catalyze the reduction of nitrate to ammonia within the pathway of primary nitrogen assimilation. In this study, we have analyzed the NR mRNA pool in dark-grown tomato and upon irradiation of etiolated tomato cotyledons with different light qualities to determine if phytochrome is involved in mediating the lightresponse of the tomato NR gene. A role of phytochrome in NR gene regulation has been suggested by the red/farred modulation of both NR activity and NR protein in etiolated squash cotyledons (Rajasekhar et al. 1988). A phytochrome-deficient mutant of tomato, the aurea mutant (Koornneef et al. 1981) has been isolated. Etiolated tissue of this tomato mutant contains at least 95 % less phytochrome than the isogenic wild-type tomato
40 strain ( K o o r n n e e f et al. 1985; P a r k s et al. 1987). W e have a d d r e s s e d the q u e s t i o n o f w h e t h e r the t o m a t o aurea m u t a n t c a n be u s e d as a t o o l to test the i n v o l v e m e n t o f the p h o t o r e c e p t o r p h y t o c h r o m e in the l i g h t - i n d u c t i o n o f gene e x p r e s s i o n using the N i R gene(s) as an example. E v i d e n c e f o r p h y t o c h r o m e - c o n t r o U e d N i R gene induct i o n was p r o v i d e d b y the increase in N i R t r a n s c r i p t s u p o n i r r a d i a t i o n o f d a r k - g r o w n m u s t a r d seedlings with red light (Schuster a n d M o h r 1990b). D e s p i t e the effects c a u s e d b y the p h y t o c h r o m e deficiency (see Peters et al. 1991 for a review), the aurea m u t a n t is viable a n d fertile w h e n g r o w n in white light. This o b s e r v a t i o n l e a d s to several q u e s t i o n s : Is there a difference with respect to the e x p r e s s i o n o f light (acting via p h y t o c h r o m e ) - r e g u l a t e d genes b e t w e e n the w i l d - t y p e a n d the aurea m u t a n t w h e n b o t h t o m a t o strains are g r o w n in white light? H o w does the s t r o n g l y r e d u c e d c h l o r o p h y l l c o n t e n t o f w h i t e - l i g h t g r o w n aurea m u t a n t leaves affect the p h o t o s y n t h e t i c p e r f o r m a n c e o f this t o m a t o m u t a n t ? In the case o f a r e d u c e d C O z - f i x a t i o n c a p a c i t y o f the m u t a n t leaves, is the a c t i v i t y o f the x a n t h o p h y l l cycle i n c r e a s e d in o r d e r to p r e v e n t o v e r - e x c i t a t i o n o f the P S I I r e a c t i o n center? Is the b a l a n c e b e t w e e n lighta n d d a r k - r e a c t i o n s o f p h o t o s y n t h e s i s m a i n t a i n e d in aurea leaves w h e n the p l a n t s are r a i s e d u n d e r g r e e n h o u s e c o n d i t i o n s ? D o e s the m a g n i t u d e o f the i m p a c t o f the aurea m u t a t i o n o n g r o w t h a n d d e v e l o p m e n t o f the m u t a n t p l a n t s c h a n g e in r e l a t i o n to i n c r e a s i n g age o f the p l a n t s ? In this p a p e r , we p r e s e n t the results o f experim e n t s d e s i g n e d to a n s w e r these questions.
Materials and methods Plants and growth conditions. Wild-type tomato ( Lycopersicon esculentum L. cv. Moneymaker; INRA, Versailles, France) and the tomato aurea mutant strain W616 (auwauw; Koornneefet al. 1981)
were grown as previously described by Galangau et al. (1988) in a controlled chamber with a 16-h photoperiod under light from white fluorescent lamps providing a photosynthetically active irradiance (PAR) of 250 lamol photons-m - 2 . s -1. The temperature was 25~ C during the day and 18~ C during the night. Etiolated seedlings were generated by germinating and growing seeds of the wild-type or the aurea mutant in continuous darkness on three layers of Whatman (Maidstone, Kent, UK) filter paper soaked with a nutrient solution (Hoagland and Arnon 1938). This nutrient solution contained 11 mM nitrate. Test experiments showed that best results with respect to the germination of our seeds were achieved when the temperature was 30~ C. In general, about 50% of the seeds of the mutant strain germinated. Ten-day-old seedlings of each tomato strain were subjected to irradiation with different light qualities as described in the legend of Fig. 1. Isolation of R N A and Northern blot analysis. Ribonucleic acid was
extracted from plant tissue as described by Verwoerd et al. (1989). The isolated RNA was precipitated twice with 4 M LiC1 for 60 min at 0 ~ C each to remove traces of DNA and small RNA species. Total RNA (5-15 lag) sample~, each dissolved in 5 lal of a solution containing 25 mM EDTA and 0.1% SDS (sodium dodecyl sulfate), were denatured by adding 25 lal of denaturing buffer consisting of 750lal formamide, 150lal 2 M Mops (3-(N-(morpholino)propanesulfonic acid) buffer (pH 7) containing 0.5 M sodium acetate and 0.1 M EDTA, 240 lal formaldehyde, 100 lal water, 100 lal glycerol and 0.02% bromophenol blue followed by incubation for 15 min at 65 ~ C.
T.W. Becker et al. : Expression of nitrate and nitrite reductase genes For staining, 1 lal of a solution containing 1 mg" ml-1 of ethidium bromide was added to each of the denatured RNA samples prior to electrophoretic separation on gels made of 1.2% agarose and 0.37 M formaldehyde in 0.2 M Mops buffer (pH 7), 50 mM sodium acetate and 10 mM EDTA. After electrophoresis, the RNA was transferred onto "Zeta Probe" (Bio-Rad-Laboratoties, Richmond, Calif. USA) blotting membranes in the presence of 1.5 M NaC1 and 1.65 M sodium citrate (pH 7). Hybridizations were performed at 65 ~ C under constant shaking for 16 h in the following buffer consisting of 0.5 M NazHPO4 (pH 7.2), 7 % SDS (w/v), and 1 mM EDTA. The membranes were then washed twice at 65 ~ C for 20 min with a solution containing 0.125 M NazHPO4 (pH 7.2) and 2% SDS (w/v). This was followed by two further washings for 30 min each with a buffer consisting of 25 mM NazHPO4 (pH 7.2) and 1% SDS (w/v). The wet membranes were finally exposed to a Kodak XAR film at - 8 0 ~ C using an intensifying screen (Dupont, Wilmington, Dal., USA). Plasmid DNA preparations. The "Bluescript" (Stratagene, La Jolla,
Calif., USA) plasmid was used as the cloning vector. Plasmid DNA for cloning and for the isolation of the eDNA inserts used as probes was prepared as described by Maniatis et al. (1982). Restriction enzymes were purchased from Boehringer (Mannheim, FRG) and used according to the manufacturer's instructions. Enzyme assays. Nitrate-reductase enzyme activity was calculated
from the increase of nitrite during the assay. The nitrite was colorimetrically determined at 540 nm after azo-coupling with sulfanilamide and naphthylethylenediamine dihydrochloride (Hageman and Flesher 1960). The benzyl-viologen-dependent activity of nitrite reductase was calculated from the decrease of the substrate nitrite during the assay (Joy and Hageman 1965). The carboxylase activity of ribulose-l,5-bisphosphate carboxylase/oxygenase was calculated from the incorporation of 14C into the substrate ribulose1,5-bisphosphate during the assay (Parry et al. 1988). Pigment analysis. Chlorophyll (Chl) was determined according to Arnon (1949). Carotenoid pigments were extracted following a 30-min irradiation with white light (1000 lamol photons - m - 2 , S - 1 ) in air and separated by non-aqueous reversed-phase high-pressure liquid chromatography using a gradient of 3-40 % dichloromethane in a mixture of acetonitrile and methanol (70/30 (v/v); Foyer et al. 1989). Gas-exchange measurements. The COz-assimilation measurements
were made with an ADC 225-MK3 infrared gas-analyzing system (Analytical Developement Co. Hoddesdon, Hefts., UK)
Results L i g h t - i n d u c t i o n o f N R gene transcription in etiolated wildtype a n d aurea m u t a n t cotyledons. I n the presence o f
nitrate, t r a n s c r i p t s c o r r e s p o n d i n g to the gene e n c o d i n g N R were d e t e c t e d a t low levels in e t i o l a t e d tissue o f w i l d - t y p e t o m a t o (Fig. 1, lane 1; Fig. 2, lane 1 in p a n e l A). H i g h e r m R N A levels o f N R were d e t e c t e d after i l l u m i n a t i o n o f the e t i o l a t e d t o m a t o c o t y l e d o n s w i t h eit h e r white light (Fig. 1, lane 2) o r r e d light (Fig. 1, lane 4; Fig. 2, lane 2 in p a n e l A). F a r - r e d light t r e a t m e n t i m m e d i a t e l y after the red light i l l u m i n a t i o n c a u s e d r e d u c e d N R m R N A levels (Fig. 1, lane 3). E t i o l a t e d c o t y l e d o n s o f the aurea m u t a n t c o n t a i n e d N R m R N A at a similar level to t h a t o f d a r k - g r o w n w i l d - t y p e c o t y l e d o n s (Fig. 2, lane 1 in p a n e l B a n d p a n e l C). I n c o n t r a s t to the s i t u a t i o n in the w i l d - t y p e (Fig. 1, lane 4), a red light pulse d i d n o t i n d u c e N R gene t r a n -
T.W. Becker et al.: Expression of nitrate and nitrite reductase genes
Fig. 1. Accumulation of NR transcripts in etiolated wild-type tomato cotyledons upon illumination with white light (lane 2) or red light (lane 4). Total RNA (15 ~tg) samples were isolated from dark-grown (lane 1) tomato and also after the transfer of the etiolated plants into permanent white light for 12 h (lane 2) or at 12 h after illumination of the etiolated tomato seedlings for 5 min with red light immediately followed by transfer into continuous darkness (lane 4). The RNA sample analyzed in lane 3 was isolated 12 h after the etiolated tomato seedlings had been consecutively irradiated for 5 min with red light and for 15 min with far-red light and then placed in continuous darkness. The denatured RNA samples were fractionated on a formaldehyde-agarose gel (1.2% agarose) and transferred onto a "Zeta Probe" membrane. The RNA blot was probed with the 250 bp HinclI-SalI fragment cloned by us from the third intron of the tomato NR gene (Vedele et al. 1989)
scription in etiolated aurea cotyledons (Fig. 2, lane 2 in panel C). H o w e v e r , 3 d after etiolated aurea cotyledons h a d been transferred into p e r m a n e n t red light, an increase in the N R transcript level was detectable (Fig. 2, lane 2 in panel B). T h e N R transcript p o o l in these etiolated aurea cotyledons, which h a d been exposed to red light for 3 d, was 70-80% lower than the identical transcript pool determined in etiolated wild-type cotyledons after illumination with red light for 72 h (Fig. 2, lane 2 in panel A). Light-induction o f N i R 9ene transcription in etiolated wild-type and aurea mutant cotyledons. Nitrite-reductase
Fig. 2. Northern blot analysis of NR mRNA levels in etiolated seedlings of both the wild-type (lane 1 in panel A) and the aurea mutant strains (lanes 1 in panels B and C) of tomato, and at 72 h after the etiolated plants had been transferred into continuous red light (wild-type: lane 2 in panel A, aurea mutant: lane 2 in panel B). Lane 2 of panel C shows the NR mRNA levels in a total RNA sample extracted from etiolated aurea mutant cotyledons which had been exposed to red light for 5 min and then transferred into darkness for 12 h. Panels D and E show the NR mRNA level in etiolated
41
Fig. 3. Northern blot analysis of NiR mRNA levels in etiolated seedlings of both the wild-type (lane I in panel A) and the aurea mutant strain (lane 1 in panel B) of tomato and at 3 d after the transfer of the etiolated plants into continuous red light (lane 2 in each panel). Northern blots were performed as described in the legend of Fig. 1 and probed with the partial 1.6 kbp copy DNA (cDNA) clone encoding tobacco NiR (data not shown)
m R N A was detected in etiolated c o t y l e d o n s o f b o t h the wild-type a n d the aurea m u t a n t strain o f t o m a t o (Fig. 3, lane 1 in each panel). The N i R transcript levels increased u p o n illumination with either red light (Fig. 3, lane 2 in panel A) or white light (data n o t shown). H o w e v e r , the N i R m R N A level in etiolated aurea cotyledons irradiated with red light for 3 d was 70% lower than the N i R transcript level f o u n d in d a r k - g r o w n wild-type cotyledons exposed to red light for 72 h (Fig. 3, lane 2 in panel B). Nitrate-dependent expression o f the N R 9ene in etiolated wild-type and aurea mutant cotyledons. I n the absence o f nitrate, no N R m R N A was detectable in etiolated cotyledons o f either the wild-type or the aurea m u t a n t strain o f t o m a t o (Fig. 2, lanes 1 in panels D a n d E). Nitratereductase transcripts did n o t a p p e a r u p o n illumination
cotyledons of the wild-type and the aurea mutant strain of tomato that were germinated in the absence of nitrate (lane I in each panel). Lanes 2 in panels D and E show the NR mRNA level in etiolated wild-type and aurea mutant seedlings, respectively, after illumination with red light for 72 h in the absence of nitrate. Northern blots were performed as described in the legend of Fig. 2 and probed with the 250 bp HinclI-SalI fragment cloned by us from the third intron of the tomato NR gene (Vedele et al. 1989)
Fig. 4. Northern blot analysis of N R (panel A), NiR (panel B), RbeS (panel C), cab (panel D), and 13-ATPase m R N A levels (panel E) in leaves of the wild-type (I) and the aurea mutant strain (II) of tomato during a diurnal cycle. The beginning of the 16-h photoperiod was at 8 a.m. Denatured total R N A samples (10 ~tg) isolated from wild-type or aurea mutant leaves at 8 a.m. (lanes 1), 10 a.m. (lanes 2), 2 p.m. (lanes 3), 8 p.m. (lanes 4), 2 a.m. (lanes 5), 6 a.m. (lanes 6), or 8 a.m. (lanes 7) were subjected to Northern blot analysis as described in the legend of Fig. 1 and probed with
either the 250-bp HinclI-SalI fragment cloned by us from the third intron of the N R gene of tomato (Vedele et al. 1989; panel A), the partial 1.6-kbp e D N A clone encoding tobacco NiR the 1.6-kbp e D N A clone encoding RbcS of Nieotiana sylvestris (Pinck et al. 1984; panel C, the 470 bp SaclI-ApaI fragment of the coding region of the maize gene cab-m7; panel D), or the 1.6 kbp e D N A clone encoding the 13-subunit of rnitochondrial ATPase of tobacco (Boutry and Chua 1986 ; panel B). The black and white bars under (I) and (II) indicate the light conditions
T.W. Becket et al. : Expression of nitrate and nitrite reductase genes Table 1. Activity expressed as lamol 9g Fw - 1. h- 1 of the enzymes NR and NiR during the greening of etiolated cotyledons of both the wild-type and the aurea mutant strain of tomato in white light (A), and in mature leaves of both tomato strains grown in white
Wild-type NR NiR
43 light during a diurnal cycle (B). The beginning of the 16-h photoperiod was at 8 a.m. Each time point represents the average of four experiments. Standard errors are given for each time point. The phenotypes of the white-light-grown plants are shown in Fig. 5
A etiolated
12 h light
8 a.m.
12 p.m.
8 p.m.
2 a.rn.
n.d. ~ 4.5+0.23
1.7 9 0.02 10.1:62.48
3.5+0.05 8.8+0.38
4.2+0.06 7.5+0.53
2.7+0.15 9.3+0.46
3.1~0.11 7.8~0.31
n.d. a 3.9 • 0.44
0.2-4-0.09 6.3 -4-0.26
2.2+0.02 _b
2.6+0.03 4.7+0.12
1.7+0.02 4.9•
1.9+0.04 _b
B
aurea mutant
NR NiR "Not detectable b Not determined
of dark-grown wild-type seedlings, or etiolated aurea mutant cotyledons with continuous red light when nitrate was not present (Fig. 2, lanes 2 in panels D and E). Diurnal m R N A fluctuations in wild-type and aurea leaves 9rown in white light. The N R gene-transcript level showed
a diurnal fluctuation with the maximum concentration reached at the beginning of the 16-h light period (Fig. 4, panel A). No N R m R N A was detected at 8 p.m. or at 2 a.m. (Fig. 4, panel A). Our data do not, however, allow a more exact localization o f the minimum N R transcript level during the diurnal cycle. A pattern of diurnal fluctuation was observed for the N i R m R N A level (Fig. 4, panel B) with the maximum and the minimum concentrations reached at the beginning and at 4 h prior to the end of the light period, respectively (Fig. 4, panel B). However, the amplitude of this diurnal NiR-transcript fluctuation was lower than the amplitude of N R - m R N A oscillation. N o differences with respect to the relative transcript levels and the diurnal fluctuation patterns of the N R and NiR transcripts were found when wild-type and aurea leaves were compared (Fig. 4, panels A and B). The transcript pool derived from the photoresponsive Rbes genes showed no clearly detectable diurnal fluctuation in leaves of either the wild-type or the aurea mutant strain of tomato grown in white light (Fig. 4, panel C). Furthermore, the relative Rbes m R N A concentration was the same in the wild-type and aurea leaves grown in white light (Fig. 4, panel C). We do not know why the hybridization signal at the first time point for the wildtype is so low. This anomaly o f a single time point does not, however, compromise the conclusion that the R b e S m R N A levels in leaves of the wild-type and the aurea mutant are similar. In leaves of the wild-type and aurea tomato plants, the cab transcript pool exhibited a diurnal fluctuation. The minimum pool size was detected at the beginning of the 16-h light period, while the maximum cab m R N A concentration was observed at 6 h after the beginning of the photoperiod (Fig. 4, panel D). Furthermore, no difference with respect to the diurnal oscillation pattern of cab transcripts in wild-type and aurea leaves existed (Fig. 4, panel D).
The transcript pool corresponding to the fl-subunit o f mitochondrial ATPase, which we used as a control, remained unchanged during a day/night cycle (Fig. 4, panel E). The non-photoresponsive gene(s) encoding [3-ATPase are known to be constitutively expressed in leaves (Boutry and Chua 1986). E n z y m e activities. In contrast to N i R activity, no N R
activity could be measured in etiolated wild-type tomato cotyledons germinated in the presence of nitrate (Table 1). U p o n exposure of dark-grown tomato seedlings for 12 h to white light, N R activity appeared and N i R activity increased 2.2-fold. Both N R and N i R activity increased at a lesser rate upon transfer of etiolated aurea mutant cotyledons into continuous light (Table 1). In the case o f the NiR activity, however, this result may not be significant since the calculated standard errors o f the measured activity values are relatively high. Activities o f both N R and N i R expressed as gmol 9 g fresh weight -~- h -~ were 1.6-fold higher in mature leaves of the wild-type than in the leaves of the aurea mutant strain of tomato (Table 1). When these activity values are expressed as ~tmol 9mg Chl-a 9h-~, however, both the in-vitro N R and N i R activities are identical in the leaves of the wild-type and the aurea mutant strain o f tomato. In the leaves of both the tomato wild-type and aurea strain the N R activity changed during the light period o f the diurnal cycle: N R activity was 1.6-fold higher at 4 h after the beginning o f the photoperiod than at 12 h after the beginning o f the light period. No such change in NiR activity was detected during the photoperiod (Table 1). No difference in the RuBPCase activity between the wild-type and the aurea mutant o f tomato was detected when the enzymatic activity of R b c S was measured relative to the leaf Chl content. Values obtained were 11.1 g m o l . m i n -~ -mg Ch1-1 in the wild-type and 12.7 gmol 9min -1 mg 9 Chl -a in the aurea mutant. Characterization o f the tomato aurea m u t a n t Pigment content. The yellow-green leaves (Fig. 5) of fourweek-old tomato aurea mutant plants contained about
44
T.W. Becker et al.: Expression of nitrate and nitrite reductasegenes
Fig. 5. Comparison of the phenotypesof four-week-old tomato aurea mutant plants (au), and wild-typetomato plants (wt) of the same age. The plants were raised in a growth chamber as described in Materials and methods Table 2. Pigment (chlorophyll,Chl; xanthophyll, X; violaxanthin,V; zeaxanthin, Z; neoxanthin,A) compositionof mature leaves of the wild-typeand of the aurea mutant strain of tomato grown in white light. The phenotypes of the plants are shown in Fig. 5
Cht (rag- g FW- I)
Cht a Chl b
Chl Xanthophyll V x 100 carotenoid 13-carotene X
(A+Z) x t00 X
Wild-type 1.43
4.5
5.1
4.5
15.4
12.4
0.87
5.1
5.0
4.3
10.8
19.8
aurea
mutant
40% less Chl than the green leaves of the wild type at the same stage of development. Furthermore, the mutant leaves contained 12% less Chl b in relation to Chl a than the wild-type leaves (Table 2). These results are similar to those originally reported by Koornneef et al. (1981). In this study, we show that the ratio of leaf Chl content to the total carotenoid content was the same in wild-type and a u r e a mutant leaves (Table 2). The zeaxanthin content, however, was 1.6-fold higher in a u r e a leaves than in wild-type leaves (Table 2). A concomitant decrease in the level of violaxanthin in a u r e a leaves was observed (Table 2). P h o t o s y n t h e t i c p e r f o r m a n c e . The a u r e a leaves had only a limited
reduced Chl level in the effect on the measured light-saturation curve of photosynthesis. The maximum photosynthetic capacity of four-week-old a u r e a mutant leaves in air was reduced by t5-20% (Fig. 6A). The calculated intercellular CO2 concentration was higher in mutant leaves than in wild-type leaves at irradiations above 100 gmol photons, m -2. s-1 (Fig. 6B). No differences in stomatal resistance were observed between a u r e a and wild-type leaves (data not shown). The photosynthetic performances of both wild-type and a u r e a mutant leaves were identical when the plants had reached the flowering state (Fig. 6C, D). Discussion
The tomato gene encoding the enzyme NR is lightregulated and this light regulation is mediated by the
photoreceptor phytochrome. This conclusion is supported by the strong increase in the in-vitro activity of NR during the greening of etiolated tomato cotyledons in white light, and by the red/far-red photoreversible low-energy response of the NR transcript level in etiolated tomato cotyledons. Besides the red/far-red photoreversible low-energy response, the far-redmediated high-irradiance response also represents a criterion for the involvement of phytochrome in the regulation of light-inducible genes. Rajasekhar et al. (1988) previously showed that, in squash, nitrate-reductase expression is also inducible by far-red high irradiance. The reported result that the tomato NR gene promoter confers light-inducibility upon a reporter gene in transgenic N i c o t i a n a p l u m b a g i n i f o l i a (Dorbe et al. 1991) indicates that the phytochrome-mediated changes in NR mRNA levels observed by us in tomato are very likely the result of transcriptional modulation. The reduced red-light response of the NR mRNA level in etiolated cotyledons of the a u r e a mutant can be correlated with the phytochrome-deficiency of the a u r e a mutant. It is attractive to consider the reduced red-light response of a light-inducible gene in etiolated a u r e a mutant cotyledons as evidence for the involvement of the photoreceptor phytochrome in the light-regulation of this gene. The reduced red-light response of the NiR transcript level in dark-grown seedlings of the a u r e a mutant may, therefore, indicate that the NiR mRNA level is under the control of the photoreceptor phytochrome. The detection of both NiR transcripts and NiR activity in etiolated tomato cotyledons indicates a basic level
T.W. Becker et al. : Expression of nitrate and nitrite reductase genes 400
18
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16
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ii
B
9
:l.
14
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+= ._m
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)
P l a n t a 9 Springer-Verlag1992
Light-regulated expression of the nitrate-reductase and nitrite-reductase genes in tomato and in the phytochrome-deficient a u r e a mutant of tomato Thomas W. Becker 1, Christine Foyer 2, and Michel Caboche 1. Laboratoires de 1 BiologieCellulaire, and du 2 M6tabolismeet de la Nutrition des Plantes, INRA, Centre de Versailles, Route de Saint-Cyr, F-78026 Versailles Cedex, France Received 2 December 1991; accepted 18 March 1992 Abstract. The phytochrome-deficient a u r e a mutant of tomato ( L y c o p e r s i c o n e s c u l e n t u m (L.) Mill) was used to investigate if phytochrome plays a role in the regulation of nitrate-reductase (NR, EC 1.6.6.1) and nitrite-reductase (NiR, EC 1.7.7.1) gene expression. We show that the expression of the tomato NR and NiR genes is stimulated by light and that this light response is mediated by the photoreceptor phytochrome. The red-light response of the NR and NiR genes was reduced in etiolated a u r e a seedlings when compared to isogenic wild-type cotyledons. The relative levels of NR mRNA and NiR transcripts and their diurnal fluctuations were identical in mature white-light-grown leaves of the wild-type and of the a u r e a mutant. The transcript levels for cab and R b c S (genes for the chlorophyll-a/b-binding protein of PSII and the small subunit of the enzyme ribulose-l,5-bisphosphate carboxylase/oxygenase, respectively) in a u r e a leaves grown in white light were indistinguishable from the respective transcript levels in the leaves of the wildtype grown under the same conditions. Despite a severe reduction in the chlorophyll content, the rate of net CO2 uptake by leaves of the a u r e a mutant was only slightly reduced when compared to the rate of net photosynthesis of wild-type leaves. This difference in the photosynthetic performances of wild-type and a u r e a mutant plants disappeared during aging of the plants. The increase in zeaxanthin and the concomitant decrease in violaxanthin in leaves of the aurea mutant compared with the same pigment levels in leaves of the wild-type indicate that the activity of the xanthophyll cycle is increased in a u r e a leaves as a consequence of the reduced CO2-fixation capacity of the mutant leaves.
* To whomcorrespondenceshould be addressed; FAX (0) 1/30833111 Abbreviations: Chl-chlorophyll;NR=nitrate reductase; NiR=nitrite reductase; cab = gene for the chlorophyll-a/b-bindingproteins of PS II; RbcS = gene for the small subunit of the enzymeribulose1,5-bisphosphate carboxylase/oxygenaseRuBPCase= ribulose-1,5bisphosphate carboxylase/oxygenase
Key words: Gene expression - L y c o p e r s i c o n - Mutant (tomato, aurea) - Nitrate reductase - Nitrite reductase Phytochrome
Introduction Light-regulated gene expression in higher plants is mediated by several photoreceptors including phytochrome (Mohr 1966), a dimeric chromoprotein (Lagarias and Mercurio 1985) that can exist in two spectrally distinct forms. In etiolated tissue, phytochrome is synthesized in the physiologically inactive, red-light-absorbing form (Pr). Red light induces the reversible coversion of Pr into the active, far-red-light-absorbing form (Pfr; Lagarias 1985). This photoconversion leads to the induction of the transcription of light-responsive genes such as those for the chlorophyll-a/b-binding protein of PSII ( c a b ) and the small subunit of the enzyme ribulose-l,5bisphosphate carboxylase/oxygenase ( R b c S ) (Tobin and Silverthorne 1985). Light induces the transcription of the nitrate-reductase (NR, EC 1.6.6.1) genes of tobacco (Deng et al. 1989) and stimulates mustard nitrite-reductase (NiR, EC 1.7.7.1) gene expression (Rajasekhar and Mohr 1986). The enzymes NR and NiR catalyze the reduction of nitrate to ammonia within the pathway of primary nitrogen assimilation. In this study, we have analyzed the NR mRNA pool in dark-grown tomato and upon irradiation of etiolated tomato cotyledons with different light qualities to determine if phytochrome is involved in mediating the lightresponse of the tomato NR gene. A role of phytochrome in NR gene regulation has been suggested by the red/farred modulation of both NR activity and NR protein in etiolated squash cotyledons (Rajasekhar et al. 1988). A phytochrome-deficient mutant of tomato, the aurea mutant (Koornneef et al. 1981) has been isolated. Etiolated tissue of this tomato mutant contains at least 95 % less phytochrome than the isogenic wild-type tomato
40 strain ( K o o r n n e e f et al. 1985; P a r k s et al. 1987). W e have a d d r e s s e d the q u e s t i o n o f w h e t h e r the t o m a t o aurea m u t a n t c a n be u s e d as a t o o l to test the i n v o l v e m e n t o f the p h o t o r e c e p t o r p h y t o c h r o m e in the l i g h t - i n d u c t i o n o f gene e x p r e s s i o n using the N i R gene(s) as an example. E v i d e n c e f o r p h y t o c h r o m e - c o n t r o U e d N i R gene induct i o n was p r o v i d e d b y the increase in N i R t r a n s c r i p t s u p o n i r r a d i a t i o n o f d a r k - g r o w n m u s t a r d seedlings with red light (Schuster a n d M o h r 1990b). D e s p i t e the effects c a u s e d b y the p h y t o c h r o m e deficiency (see Peters et al. 1991 for a review), the aurea m u t a n t is viable a n d fertile w h e n g r o w n in white light. This o b s e r v a t i o n l e a d s to several q u e s t i o n s : Is there a difference with respect to the e x p r e s s i o n o f light (acting via p h y t o c h r o m e ) - r e g u l a t e d genes b e t w e e n the w i l d - t y p e a n d the aurea m u t a n t w h e n b o t h t o m a t o strains are g r o w n in white light? H o w does the s t r o n g l y r e d u c e d c h l o r o p h y l l c o n t e n t o f w h i t e - l i g h t g r o w n aurea m u t a n t leaves affect the p h o t o s y n t h e t i c p e r f o r m a n c e o f this t o m a t o m u t a n t ? In the case o f a r e d u c e d C O z - f i x a t i o n c a p a c i t y o f the m u t a n t leaves, is the a c t i v i t y o f the x a n t h o p h y l l cycle i n c r e a s e d in o r d e r to p r e v e n t o v e r - e x c i t a t i o n o f the P S I I r e a c t i o n center? Is the b a l a n c e b e t w e e n lighta n d d a r k - r e a c t i o n s o f p h o t o s y n t h e s i s m a i n t a i n e d in aurea leaves w h e n the p l a n t s are r a i s e d u n d e r g r e e n h o u s e c o n d i t i o n s ? D o e s the m a g n i t u d e o f the i m p a c t o f the aurea m u t a t i o n o n g r o w t h a n d d e v e l o p m e n t o f the m u t a n t p l a n t s c h a n g e in r e l a t i o n to i n c r e a s i n g age o f the p l a n t s ? In this p a p e r , we p r e s e n t the results o f experim e n t s d e s i g n e d to a n s w e r these questions.
Materials and methods Plants and growth conditions. Wild-type tomato ( Lycopersicon esculentum L. cv. Moneymaker; INRA, Versailles, France) and the tomato aurea mutant strain W616 (auwauw; Koornneefet al. 1981)
were grown as previously described by Galangau et al. (1988) in a controlled chamber with a 16-h photoperiod under light from white fluorescent lamps providing a photosynthetically active irradiance (PAR) of 250 lamol photons-m - 2 . s -1. The temperature was 25~ C during the day and 18~ C during the night. Etiolated seedlings were generated by germinating and growing seeds of the wild-type or the aurea mutant in continuous darkness on three layers of Whatman (Maidstone, Kent, UK) filter paper soaked with a nutrient solution (Hoagland and Arnon 1938). This nutrient solution contained 11 mM nitrate. Test experiments showed that best results with respect to the germination of our seeds were achieved when the temperature was 30~ C. In general, about 50% of the seeds of the mutant strain germinated. Ten-day-old seedlings of each tomato strain were subjected to irradiation with different light qualities as described in the legend of Fig. 1. Isolation of R N A and Northern blot analysis. Ribonucleic acid was
extracted from plant tissue as described by Verwoerd et al. (1989). The isolated RNA was precipitated twice with 4 M LiC1 for 60 min at 0 ~ C each to remove traces of DNA and small RNA species. Total RNA (5-15 lag) sample~, each dissolved in 5 lal of a solution containing 25 mM EDTA and 0.1% SDS (sodium dodecyl sulfate), were denatured by adding 25 lal of denaturing buffer consisting of 750lal formamide, 150lal 2 M Mops (3-(N-(morpholino)propanesulfonic acid) buffer (pH 7) containing 0.5 M sodium acetate and 0.1 M EDTA, 240 lal formaldehyde, 100 lal water, 100 lal glycerol and 0.02% bromophenol blue followed by incubation for 15 min at 65 ~ C.
T.W. Becker et al. : Expression of nitrate and nitrite reductase genes For staining, 1 lal of a solution containing 1 mg" ml-1 of ethidium bromide was added to each of the denatured RNA samples prior to electrophoretic separation on gels made of 1.2% agarose and 0.37 M formaldehyde in 0.2 M Mops buffer (pH 7), 50 mM sodium acetate and 10 mM EDTA. After electrophoresis, the RNA was transferred onto "Zeta Probe" (Bio-Rad-Laboratoties, Richmond, Calif. USA) blotting membranes in the presence of 1.5 M NaC1 and 1.65 M sodium citrate (pH 7). Hybridizations were performed at 65 ~ C under constant shaking for 16 h in the following buffer consisting of 0.5 M NazHPO4 (pH 7.2), 7 % SDS (w/v), and 1 mM EDTA. The membranes were then washed twice at 65 ~ C for 20 min with a solution containing 0.125 M NazHPO4 (pH 7.2) and 2% SDS (w/v). This was followed by two further washings for 30 min each with a buffer consisting of 25 mM NazHPO4 (pH 7.2) and 1% SDS (w/v). The wet membranes were finally exposed to a Kodak XAR film at - 8 0 ~ C using an intensifying screen (Dupont, Wilmington, Dal., USA). Plasmid DNA preparations. The "Bluescript" (Stratagene, La Jolla,
Calif., USA) plasmid was used as the cloning vector. Plasmid DNA for cloning and for the isolation of the eDNA inserts used as probes was prepared as described by Maniatis et al. (1982). Restriction enzymes were purchased from Boehringer (Mannheim, FRG) and used according to the manufacturer's instructions. Enzyme assays. Nitrate-reductase enzyme activity was calculated
from the increase of nitrite during the assay. The nitrite was colorimetrically determined at 540 nm after azo-coupling with sulfanilamide and naphthylethylenediamine dihydrochloride (Hageman and Flesher 1960). The benzyl-viologen-dependent activity of nitrite reductase was calculated from the decrease of the substrate nitrite during the assay (Joy and Hageman 1965). The carboxylase activity of ribulose-l,5-bisphosphate carboxylase/oxygenase was calculated from the incorporation of 14C into the substrate ribulose1,5-bisphosphate during the assay (Parry et al. 1988). Pigment analysis. Chlorophyll (Chl) was determined according to Arnon (1949). Carotenoid pigments were extracted following a 30-min irradiation with white light (1000 lamol photons - m - 2 , S - 1 ) in air and separated by non-aqueous reversed-phase high-pressure liquid chromatography using a gradient of 3-40 % dichloromethane in a mixture of acetonitrile and methanol (70/30 (v/v); Foyer et al. 1989). Gas-exchange measurements. The COz-assimilation measurements
were made with an ADC 225-MK3 infrared gas-analyzing system (Analytical Developement Co. Hoddesdon, Hefts., UK)
Results L i g h t - i n d u c t i o n o f N R gene transcription in etiolated wildtype a n d aurea m u t a n t cotyledons. I n the presence o f
nitrate, t r a n s c r i p t s c o r r e s p o n d i n g to the gene e n c o d i n g N R were d e t e c t e d a t low levels in e t i o l a t e d tissue o f w i l d - t y p e t o m a t o (Fig. 1, lane 1; Fig. 2, lane 1 in p a n e l A). H i g h e r m R N A levels o f N R were d e t e c t e d after i l l u m i n a t i o n o f the e t i o l a t e d t o m a t o c o t y l e d o n s w i t h eit h e r white light (Fig. 1, lane 2) o r r e d light (Fig. 1, lane 4; Fig. 2, lane 2 in p a n e l A). F a r - r e d light t r e a t m e n t i m m e d i a t e l y after the red light i l l u m i n a t i o n c a u s e d r e d u c e d N R m R N A levels (Fig. 1, lane 3). E t i o l a t e d c o t y l e d o n s o f the aurea m u t a n t c o n t a i n e d N R m R N A at a similar level to t h a t o f d a r k - g r o w n w i l d - t y p e c o t y l e d o n s (Fig. 2, lane 1 in p a n e l B a n d p a n e l C). I n c o n t r a s t to the s i t u a t i o n in the w i l d - t y p e (Fig. 1, lane 4), a red light pulse d i d n o t i n d u c e N R gene t r a n -
T.W. Becker et al.: Expression of nitrate and nitrite reductase genes
Fig. 1. Accumulation of NR transcripts in etiolated wild-type tomato cotyledons upon illumination with white light (lane 2) or red light (lane 4). Total RNA (15 ~tg) samples were isolated from dark-grown (lane 1) tomato and also after the transfer of the etiolated plants into permanent white light for 12 h (lane 2) or at 12 h after illumination of the etiolated tomato seedlings for 5 min with red light immediately followed by transfer into continuous darkness (lane 4). The RNA sample analyzed in lane 3 was isolated 12 h after the etiolated tomato seedlings had been consecutively irradiated for 5 min with red light and for 15 min with far-red light and then placed in continuous darkness. The denatured RNA samples were fractionated on a formaldehyde-agarose gel (1.2% agarose) and transferred onto a "Zeta Probe" membrane. The RNA blot was probed with the 250 bp HinclI-SalI fragment cloned by us from the third intron of the tomato NR gene (Vedele et al. 1989)
scription in etiolated aurea cotyledons (Fig. 2, lane 2 in panel C). H o w e v e r , 3 d after etiolated aurea cotyledons h a d been transferred into p e r m a n e n t red light, an increase in the N R transcript level was detectable (Fig. 2, lane 2 in panel B). T h e N R transcript p o o l in these etiolated aurea cotyledons, which h a d been exposed to red light for 3 d, was 70-80% lower than the identical transcript pool determined in etiolated wild-type cotyledons after illumination with red light for 72 h (Fig. 2, lane 2 in panel A). Light-induction o f N i R 9ene transcription in etiolated wild-type and aurea mutant cotyledons. Nitrite-reductase
Fig. 2. Northern blot analysis of NR mRNA levels in etiolated seedlings of both the wild-type (lane 1 in panel A) and the aurea mutant strains (lanes 1 in panels B and C) of tomato, and at 72 h after the etiolated plants had been transferred into continuous red light (wild-type: lane 2 in panel A, aurea mutant: lane 2 in panel B). Lane 2 of panel C shows the NR mRNA levels in a total RNA sample extracted from etiolated aurea mutant cotyledons which had been exposed to red light for 5 min and then transferred into darkness for 12 h. Panels D and E show the NR mRNA level in etiolated
41
Fig. 3. Northern blot analysis of NiR mRNA levels in etiolated seedlings of both the wild-type (lane I in panel A) and the aurea mutant strain (lane 1 in panel B) of tomato and at 3 d after the transfer of the etiolated plants into continuous red light (lane 2 in each panel). Northern blots were performed as described in the legend of Fig. 1 and probed with the partial 1.6 kbp copy DNA (cDNA) clone encoding tobacco NiR (data not shown)
m R N A was detected in etiolated c o t y l e d o n s o f b o t h the wild-type a n d the aurea m u t a n t strain o f t o m a t o (Fig. 3, lane 1 in each panel). The N i R transcript levels increased u p o n illumination with either red light (Fig. 3, lane 2 in panel A) or white light (data n o t shown). H o w e v e r , the N i R m R N A level in etiolated aurea cotyledons irradiated with red light for 3 d was 70% lower than the N i R transcript level f o u n d in d a r k - g r o w n wild-type cotyledons exposed to red light for 72 h (Fig. 3, lane 2 in panel B). Nitrate-dependent expression o f the N R 9ene in etiolated wild-type and aurea mutant cotyledons. I n the absence o f nitrate, no N R m R N A was detectable in etiolated cotyledons o f either the wild-type or the aurea m u t a n t strain o f t o m a t o (Fig. 2, lanes 1 in panels D a n d E). Nitratereductase transcripts did n o t a p p e a r u p o n illumination
cotyledons of the wild-type and the aurea mutant strain of tomato that were germinated in the absence of nitrate (lane I in each panel). Lanes 2 in panels D and E show the NR mRNA level in etiolated wild-type and aurea mutant seedlings, respectively, after illumination with red light for 72 h in the absence of nitrate. Northern blots were performed as described in the legend of Fig. 2 and probed with the 250 bp HinclI-SalI fragment cloned by us from the third intron of the tomato NR gene (Vedele et al. 1989)
Fig. 4. Northern blot analysis of N R (panel A), NiR (panel B), RbeS (panel C), cab (panel D), and 13-ATPase m R N A levels (panel E) in leaves of the wild-type (I) and the aurea mutant strain (II) of tomato during a diurnal cycle. The beginning of the 16-h photoperiod was at 8 a.m. Denatured total R N A samples (10 ~tg) isolated from wild-type or aurea mutant leaves at 8 a.m. (lanes 1), 10 a.m. (lanes 2), 2 p.m. (lanes 3), 8 p.m. (lanes 4), 2 a.m. (lanes 5), 6 a.m. (lanes 6), or 8 a.m. (lanes 7) were subjected to Northern blot analysis as described in the legend of Fig. 1 and probed with
either the 250-bp HinclI-SalI fragment cloned by us from the third intron of the N R gene of tomato (Vedele et al. 1989; panel A), the partial 1.6-kbp e D N A clone encoding tobacco NiR the 1.6-kbp e D N A clone encoding RbcS of Nieotiana sylvestris (Pinck et al. 1984; panel C, the 470 bp SaclI-ApaI fragment of the coding region of the maize gene cab-m7; panel D), or the 1.6 kbp e D N A clone encoding the 13-subunit of rnitochondrial ATPase of tobacco (Boutry and Chua 1986 ; panel B). The black and white bars under (I) and (II) indicate the light conditions
T.W. Becket et al. : Expression of nitrate and nitrite reductase genes Table 1. Activity expressed as lamol 9g Fw - 1. h- 1 of the enzymes NR and NiR during the greening of etiolated cotyledons of both the wild-type and the aurea mutant strain of tomato in white light (A), and in mature leaves of both tomato strains grown in white
Wild-type NR NiR
43 light during a diurnal cycle (B). The beginning of the 16-h photoperiod was at 8 a.m. Each time point represents the average of four experiments. Standard errors are given for each time point. The phenotypes of the white-light-grown plants are shown in Fig. 5
A etiolated
12 h light
8 a.m.
12 p.m.
8 p.m.
2 a.rn.
n.d. ~ 4.5+0.23
1.7 9 0.02 10.1:62.48
3.5+0.05 8.8+0.38
4.2+0.06 7.5+0.53
2.7+0.15 9.3+0.46
3.1~0.11 7.8~0.31
n.d. a 3.9 • 0.44
0.2-4-0.09 6.3 -4-0.26
2.2+0.02 _b
2.6+0.03 4.7+0.12
1.7+0.02 4.9•
1.9+0.04 _b
B
aurea mutant
NR NiR "Not detectable b Not determined
of dark-grown wild-type seedlings, or etiolated aurea mutant cotyledons with continuous red light when nitrate was not present (Fig. 2, lanes 2 in panels D and E). Diurnal m R N A fluctuations in wild-type and aurea leaves 9rown in white light. The N R gene-transcript level showed
a diurnal fluctuation with the maximum concentration reached at the beginning of the 16-h light period (Fig. 4, panel A). No N R m R N A was detected at 8 p.m. or at 2 a.m. (Fig. 4, panel A). Our data do not, however, allow a more exact localization o f the minimum N R transcript level during the diurnal cycle. A pattern of diurnal fluctuation was observed for the N i R m R N A level (Fig. 4, panel B) with the maximum and the minimum concentrations reached at the beginning and at 4 h prior to the end of the light period, respectively (Fig. 4, panel B). However, the amplitude of this diurnal NiR-transcript fluctuation was lower than the amplitude of N R - m R N A oscillation. N o differences with respect to the relative transcript levels and the diurnal fluctuation patterns of the N R and NiR transcripts were found when wild-type and aurea leaves were compared (Fig. 4, panels A and B). The transcript pool derived from the photoresponsive Rbes genes showed no clearly detectable diurnal fluctuation in leaves of either the wild-type or the aurea mutant strain of tomato grown in white light (Fig. 4, panel C). Furthermore, the relative Rbes m R N A concentration was the same in the wild-type and aurea leaves grown in white light (Fig. 4, panel C). We do not know why the hybridization signal at the first time point for the wildtype is so low. This anomaly o f a single time point does not, however, compromise the conclusion that the R b e S m R N A levels in leaves of the wild-type and the aurea mutant are similar. In leaves of the wild-type and aurea tomato plants, the cab transcript pool exhibited a diurnal fluctuation. The minimum pool size was detected at the beginning of the 16-h light period, while the maximum cab m R N A concentration was observed at 6 h after the beginning of the photoperiod (Fig. 4, panel D). Furthermore, no difference with respect to the diurnal oscillation pattern of cab transcripts in wild-type and aurea leaves existed (Fig. 4, panel D).
The transcript pool corresponding to the fl-subunit o f mitochondrial ATPase, which we used as a control, remained unchanged during a day/night cycle (Fig. 4, panel E). The non-photoresponsive gene(s) encoding [3-ATPase are known to be constitutively expressed in leaves (Boutry and Chua 1986). E n z y m e activities. In contrast to N i R activity, no N R
activity could be measured in etiolated wild-type tomato cotyledons germinated in the presence of nitrate (Table 1). U p o n exposure of dark-grown tomato seedlings for 12 h to white light, N R activity appeared and N i R activity increased 2.2-fold. Both N R and N i R activity increased at a lesser rate upon transfer of etiolated aurea mutant cotyledons into continuous light (Table 1). In the case o f the NiR activity, however, this result may not be significant since the calculated standard errors o f the measured activity values are relatively high. Activities o f both N R and N i R expressed as gmol 9 g fresh weight -~- h -~ were 1.6-fold higher in mature leaves of the wild-type than in the leaves of the aurea mutant strain of tomato (Table 1). When these activity values are expressed as ~tmol 9mg Chl-a 9h-~, however, both the in-vitro N R and N i R activities are identical in the leaves of the wild-type and the aurea mutant strain o f tomato. In the leaves of both the tomato wild-type and aurea strain the N R activity changed during the light period o f the diurnal cycle: N R activity was 1.6-fold higher at 4 h after the beginning o f the photoperiod than at 12 h after the beginning o f the light period. No such change in NiR activity was detected during the photoperiod (Table 1). No difference in the RuBPCase activity between the wild-type and the aurea mutant o f tomato was detected when the enzymatic activity of R b c S was measured relative to the leaf Chl content. Values obtained were 11.1 g m o l . m i n -~ -mg Ch1-1 in the wild-type and 12.7 gmol 9min -1 mg 9 Chl -a in the aurea mutant. Characterization o f the tomato aurea m u t a n t Pigment content. The yellow-green leaves (Fig. 5) of fourweek-old tomato aurea mutant plants contained about
44
T.W. Becker et al.: Expression of nitrate and nitrite reductasegenes
Fig. 5. Comparison of the phenotypesof four-week-old tomato aurea mutant plants (au), and wild-typetomato plants (wt) of the same age. The plants were raised in a growth chamber as described in Materials and methods Table 2. Pigment (chlorophyll,Chl; xanthophyll, X; violaxanthin,V; zeaxanthin, Z; neoxanthin,A) compositionof mature leaves of the wild-typeand of the aurea mutant strain of tomato grown in white light. The phenotypes of the plants are shown in Fig. 5
Cht (rag- g FW- I)
Cht a Chl b
Chl Xanthophyll V x 100 carotenoid 13-carotene X
(A+Z) x t00 X
Wild-type 1.43
4.5
5.1
4.5
15.4
12.4
0.87
5.1
5.0
4.3
10.8
19.8
aurea
mutant
40% less Chl than the green leaves of the wild type at the same stage of development. Furthermore, the mutant leaves contained 12% less Chl b in relation to Chl a than the wild-type leaves (Table 2). These results are similar to those originally reported by Koornneef et al. (1981). In this study, we show that the ratio of leaf Chl content to the total carotenoid content was the same in wild-type and a u r e a mutant leaves (Table 2). The zeaxanthin content, however, was 1.6-fold higher in a u r e a leaves than in wild-type leaves (Table 2). A concomitant decrease in the level of violaxanthin in a u r e a leaves was observed (Table 2). P h o t o s y n t h e t i c p e r f o r m a n c e . The a u r e a leaves had only a limited
reduced Chl level in the effect on the measured light-saturation curve of photosynthesis. The maximum photosynthetic capacity of four-week-old a u r e a mutant leaves in air was reduced by t5-20% (Fig. 6A). The calculated intercellular CO2 concentration was higher in mutant leaves than in wild-type leaves at irradiations above 100 gmol photons, m -2. s-1 (Fig. 6B). No differences in stomatal resistance were observed between a u r e a and wild-type leaves (data not shown). The photosynthetic performances of both wild-type and a u r e a mutant leaves were identical when the plants had reached the flowering state (Fig. 6C, D). Discussion
The tomato gene encoding the enzyme NR is lightregulated and this light regulation is mediated by the
photoreceptor phytochrome. This conclusion is supported by the strong increase in the in-vitro activity of NR during the greening of etiolated tomato cotyledons in white light, and by the red/far-red photoreversible low-energy response of the NR transcript level in etiolated tomato cotyledons. Besides the red/far-red photoreversible low-energy response, the far-redmediated high-irradiance response also represents a criterion for the involvement of phytochrome in the regulation of light-inducible genes. Rajasekhar et al. (1988) previously showed that, in squash, nitrate-reductase expression is also inducible by far-red high irradiance. The reported result that the tomato NR gene promoter confers light-inducibility upon a reporter gene in transgenic N i c o t i a n a p l u m b a g i n i f o l i a (Dorbe et al. 1991) indicates that the phytochrome-mediated changes in NR mRNA levels observed by us in tomato are very likely the result of transcriptional modulation. The reduced red-light response of the NR mRNA level in etiolated cotyledons of the a u r e a mutant can be correlated with the phytochrome-deficiency of the a u r e a mutant. It is attractive to consider the reduced red-light response of a light-inducible gene in etiolated a u r e a mutant cotyledons as evidence for the involvement of the photoreceptor phytochrome in the light-regulation of this gene. The reduced red-light response of the NiR transcript level in dark-grown seedlings of the a u r e a mutant may, therefore, indicate that the NiR mRNA level is under the control of the photoreceptor phytochrome. The detection of both NiR transcripts and NiR activity in etiolated tomato cotyledons indicates a basic level
T.W. Becker et al. : Expression of nitrate and nitrite reductase genes 400
18
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16
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B
9
:l.
14
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+= ._m
,.=+ ,+
8
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o 0
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(gmol 9m-2.s-1)
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