Plant Physiol. (1994) 106: 1145-1149 Induction and Turnover of Nitrate Reductase in Zea mays' 6 Influence of Light Xiu-Zhen Li and Ann Oaks* Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Both light and NO3- are necessary for the appearanceof nitrate reductase (NR) activity (NRA) in photosynthetic tissues. To define the light effect more precisely, we examined the response to light/ dark transitions on NRA, NR protein (NRP), and NR mRNA in 6-d- old maize (Zea mays cv W64A x W182E) seedlings that had been grown in a lightldark regime for 5 d and then induced with 5 mM KNOB for 24 h. The decay of NRA and NR mRNA in the shoot was immediate, but there were only minor changes in NRP during the initial 4 h in the dark. In root tissues, in contrast, there was a 4-h delay in the loss of NRA, NRP, and NR mRNA after transfer to the dark. When the seedlings were returned to light after a 2-h interval in the dark, shoot NRA reached 92% of the initial levels within 30 min of illumination. These results indicate that in the shoots (a) NR message production requires light and (b) the NRP that appears with light treatment and that is active is inactivated in the dark. The NRP can be reactivated when the light is turned on after short periods of darkness (2 h). Root tissues, on the other hand, probably respond to the supply of photosynthetically produced metabo- lites rather than to immediate products of the light reactions of photosynthesis. Early work by Hageman and Flesher (1960) demonstrated that both light and NOs- were important environmental signals for the appearance of NRA. Since then it has been observed in many studies that Nos- reduction is accelerated in green tissues in the presence of light (reviewed by Srivas- tava, 1980). Light has been shown to enhance the synthesis of NR via the development of an active protein-synthesizing apparatus (Travis et al., 1970; Travis and Key, 1971), and when etiolated plants are used it has been shown to act through the phytochrome system (Jones and Sheard, 1972; Rajasekhar et al., 1988; Melzer et al., 1989).At a physiological level light plays a role in the provision of reductant for NR (Beevers and Hageman, 1972; Naik et al., 1982). It increases membrane permeability of the cells and thus enhances the availability of NOa- (Beevers et al., 1965; Rao and Rains, 1976), and it promotes the release of NO3- stored in the vacuoles to the metabolic pool (Aslam et al., 1976; Stulen and Bosgraaf, 1985). A number of heterotrophic organisms and nonphotosyn- thetic tissues in higher plants also assimilate NO3- (Aslam and Oaks, 1975; Marzluf, 1981), but the requirement for light 'This work was supported by grant A2818 to A.O. from the * Corresponding author; fax 1-519-767-1991. Natural Science and Engineering Research Council of Canada. has not been clearly defined. Root tissues have the capacity to use reducing power generated by the oxidation of photo- synthate translocated from green tissues. Thus, whereas light may supply reductant or other factors directly in leaf tissues, in heterotrophic tissues it may influence the assimilation of NOs- in an indirect manner. In some cases light also promotes a reversible dephosphorylation of the NRP, a reaction that results in an activation of that protein (Huber et al., 1992; Kaiser et al., 1992; MacKintosh, 1992). At the molecular level, light has been shown to induce both NRP and NR mRNA (Somers et al., 1983; Remmler and Campbell, 1986; Melzer et al., 1989). Melzer et al. (1989) found, for example, that light enhanced NR mRNA accu- mulation in both etiolated and green leaves. In their experi- ments the induction in etiolated leaves was under phyto- chrome control, whereas in green leaves it was not. In this study, hydroponically grown seedlings, which had been exposed to a 16-h light/S-h dark regime, were used to investigate the effects of a light/dark transition on the levels of NRA, NRP, and NR mRNA. When they were transferred to the dark after a 24-h induction with 5 m NOs-, there was a rapid loss of NRA and NR mRNA in leaves but an interval of 4 h before there was a significant loss of NRP. A 30-min reillumination of the seedlings after a 2-h dark inter- val resulted in an immediate and complete reactivation of shoot NRA. Roots, on the other hand, showed only a minor response to light/dark transitions. MATERIALS AND METHODS Growth Conditions of Plants Maize kemels (Zea mays L. cv W64A X Wl82E), purchased from the Wisconsin Seed Foundation (Madison, WI) were germinated and grown as described previously (Li and Oaks, 1993). On d 5 after planting, 5 m KN03 was added to the medium to induce NR. After a 24-h induction, the seedlings were transferred to the dark for the time required. Control plants were maintained under the normal light/dark regime for the whole time. Unless otherwise stated, all treatments were initiated 2 h after dawn. Shoots above the coleoptile or the whole root system were harvested, washed with deion- ized water, and blotted dry and then were rapidly frozen in Abbreviations: NR, nitrate reductase; NRA, nitrate reductase ac- tivity; NRP, nitrate reductase protein. 1145
Transcript
Plant Physiol. (1994) 106: 1145-1149
Induction and Turnover of Nitrate Reductase in Zea mays' 6
Influence of Light
Xiu-Zhen Li and Ann Oaks* Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Both light and NO3- are necessary for the appearance of nitrate reductase (NR) activity (NRA) in photosynthetic tissues. To define the light effect more precisely, we examined the response to light/ dark transitions on NRA, NR protein (NRP), and NR mRNA in 6-d- old maize (Zea mays cv W64A x W182E) seedlings that had been grown in a lightldark regime for 5 d and then induced with 5 mM KNOB for 24 h. The decay of NRA and NR mRNA in the shoot was immediate, but there were only minor changes in NRP during the initial 4 h in the dark. In root tissues, in contrast, there was a 4-h delay in the loss of NRA, NRP, and NR mRNA after transfer to the dark. When the seedlings were returned to light after a 2-h interval in the dark, shoot NRA reached 92% of the initial levels within 30 min of illumination. These results indicate that in the shoots (a) NR message production requires light and (b) the NRP that appears with light treatment and that i s active i s inactivated in the dark. The NRP can be reactivated when the light i s turned on after short periods of darkness (2 h). Root tissues, on the other hand, probably respond to the supply of photosynthetically produced metabo- lites rather than to immediate products of the light reactions of photosynthesis.
Early work by Hageman and Flesher (1960) demonstrated that both light and NOs- were important environmental signals for the appearance of NRA. Since then it has been observed in many studies that Nos- reduction is accelerated in green tissues in the presence of light (reviewed by Srivas- tava, 1980). Light has been shown to enhance the synthesis of NR via the development of an active protein-synthesizing apparatus (Travis et al., 1970; Travis and Key, 1971), and when etiolated plants are used it has been shown to act through the phytochrome system (Jones and Sheard, 1972; Rajasekhar et al., 1988; Melzer et al., 1989). At a physiological level light plays a role in the provision of reductant for NR (Beevers and Hageman, 1972; Naik et al., 1982). It increases membrane permeability of the cells and thus enhances the availability of NOa- (Beevers et al., 1965; Rao and Rains, 1976), and it promotes the release of NO3- stored in the vacuoles to the metabolic pool (Aslam et al., 1976; Stulen and Bosgraaf, 1985).
A number of heterotrophic organisms and nonphotosyn- thetic tissues in higher plants also assimilate NO3- (Aslam and Oaks, 1975; Marzluf, 1981), but the requirement for light
'This work was supported by grant A2818 to A.O. from the
* Corresponding author; fax 1-519-767-1991. Natural Science and Engineering Research Council of Canada.
has not been clearly defined. Root tissues have the capacity to use reducing power generated by the oxidation of photo- synthate translocated from green tissues. Thus, whereas light may supply reductant or other factors directly in leaf tissues, in heterotrophic tissues it may influence the assimilation of NOs- in an indirect manner. In some cases light also promotes a reversible dephosphorylation of the NRP, a reaction that results in an activation of that protein (Huber et al., 1992; Kaiser et al., 1992; MacKintosh, 1992).
At the molecular level, light has been shown to induce both NRP and NR mRNA (Somers et al., 1983; Remmler and Campbell, 1986; Melzer et al., 1989). Melzer et al. (1989) found, for example, that light enhanced NR mRNA accu- mulation in both etiolated and green leaves. In their experi- ments the induction in etiolated leaves was under phyto- chrome control, whereas in green leaves it was not.
In this study, hydroponically grown seedlings, which had been exposed to a 16-h light/S-h dark regime, were used to investigate the effects of a light/dark transition on the levels of NRA, NRP, and NR mRNA. When they were transferred to the dark after a 24-h induction with 5 m NOs-, there was a rapid loss of NRA and NR mRNA in leaves but an interval of 4 h before there was a significant loss of NRP. A 30-min reillumination of the seedlings after a 2-h dark inter- val resulted in an immediate and complete reactivation of shoot NRA. Roots, on the other hand, showed only a minor response to light/dark transitions.
MATERIALS AND METHODS
Growth Conditions of Plants
Maize kemels (Zea mays L. cv W64A X Wl82E), purchased from the Wisconsin Seed Foundation (Madison, WI) were germinated and grown as described previously (Li and Oaks, 1993). On d 5 after planting, 5 m KN03 was added to the medium to induce NR. After a 24-h induction, the seedlings were transferred to the dark for the time required. Control plants were maintained under the normal light/dark regime for the whole time. Unless otherwise stated, all treatments were initiated 2 h after dawn. Shoots above the coleoptile or the whole root system were harvested, washed with deion- ized water, and blotted dry and then were rapidly frozen in
liquid N2, ground to a fine powder, and stored in a sealed container at -7OOC until required.
NRA Assay and Protein Analysis
Frozen samples (1 g of powder) were extracted with 4 mL of Tris-HC1 buffer (pH 8.5) and centrifuged, and the super- natant solution was assayed for NRA as described previously (Li and Oaks, 1993). The results were expressed as pmol NO2- produced h-' g-' fresh weight. NRP was determined by the westem blot technique as described by Li and Oaks (1993). Equal volumes of extract (shoot, 10 pL; root, 15 pL) were loaded in each lane and subjected to electrophoresis on a 7.5% SDS-polyacrylamide gel. The protein was transferred to nitrocellulose paper, and the NRP was detected with anti- NR serum raised against purified maize shoot NR (see Long and Oaks, 1990, for details).
RNA Isolation and Hybridization
Total RNA was extracted from 2 g of frozen shoot or root powder as described by Jones et al. (1985). RNA samples (20 pg) were denatured by incubating at 6OoC for 5 min in 50% formamide, 7% formaldehyde, and 25 m~ Mops, pH 7.0, and size fractionated by electrophoresis in a 1% agarose gel containing 50 m~ Mops, pH 7.0, and 2.2 M formaldehyde. RNA was then transferred onto Zeta-probe membranes (Bio- Rad) by capillary action (Maniatis et al., 1982). The probe used for detecting shoot NR mRNA was the insert of a partial maize leaf NR cDNA clone, pCIB831. Root NR mRNA se- quences were assayed using the insert of a partial maize root NR cDNA clone, p1501. These NR cDNA clones were as described by Long et al. (1992) and were provided by Dr. S.J. Rothstein (Department of Molecular Biology and Genetics, University of Guelph). The fragments were labeled by ran- dom priming using a randomly primed DNA-labeling kit (Amersham Intemational) and [ ( U - ~ ~ P I ~ C T P (Amersham, Oakville, Ontario, Canada) to a specific activity of about 1 X 10' cpm/pg DNA. Membranes were prehybridized for 6 to 24 h at 42OC in a buffer containing 4X SSC; 50 m~ Na- phosphate, pH 7.4; 0.2% SDS; 50% formamide; 0.1 mg/mL poly(A); 0.25 mg/mL tRNA; 0.02% PVP/Ficoll; and 0.2 mg/ mL BSA. Hybridization was carried out at 42OC for 36 to 40 h in a fresh buffer as described above but in the presence of about 2 X lo7 cpm of the denatured probe. Membranes were washed twice with 2X SSC containing 0.1% SDS for 15 min each at room temperature and then with O. 1 X SSC containing 0.1% SDS at 65OC for 10 min to remove nonspecific 32P bindings. The membranes were air dried and sealed in a plastic bag and then subjected to autoradiography on Kodak OMAT-AR film for the required time at -7OOC.
RESULTS
Time Course of the Effect of Darkness on NR
When 5-d-old maize seedlings grown in a normal light/ dark (16 h of light/8 h of dark) regime were induced with 5 m~ NO3- for 24 h, NRA reached 8 and 6 pmol h-' g-' fresh weight in the shoot and root, respectively (Li and Oaks, 1993). When these seedlings were transferred to darkness,
there was an immediate loss of shoot NRA, which continued for about 8 h after the transfer. As shown in Figure 1, 8% of the original activity remained 24 h after the transfer. A decline in root NRA was seen only after a 4-h dark interval, and after 24 h about 25% of the initial root NRA reqained.
NRP levels, as measured by the westem blot technique, showed a similar pattem in both shoot and root issues (Fig. 2A). There was no apparent change in NRP during the first 4 h of darkness, and a minor amount of protem was still present after 24 h. These results indicate that the shoot NRA was much more sensitive to light deprivation than NRP. A similar phenomenon has been seen with NO3- depletion (Li and Oaks, 1993).
In contrast to the NRP levels, which were relatively stable, shoot NR mRNA decreased as soon as the seedings were transferred to the dark (Fig. 2B). In the root tissues, on the other hand, NR mRNA did not change during thli initial 4 h in the dark, and even after 24 h in the dark significant levels were still present. Levels of NR mRNA increased in both shoot and root tissues during the second 24-h period in the light.
Influence of Short-Term Darkness on the Reinduidion of NR by light
When seedlings were reexposed to the light after 2 h of darkness, shoot NRA increased to almost the initial level (92% of the value seen after a 24-h induction) within 30 min (Fig. 3A). After 'a 4-h dark interval there was a lag before NRA increased with reillumination, and in this case only 86% of the original value was recovered even after 1.5 h in the light. Root NRAs, in contrast, showed no significant
100 -
EO -
60 - Q CK Z
40 -
--t ROOT NADH-NR
o ' b k b ;2 16 ;O i4-
Time in Dark (h)
Figure 1. Decay of NRA in t h e dark after a 24-h induction. Seedlings were induced with 5 mM KNO, for 24 h (O time) before transfer to the dark. Shoot and root tissues were harvested at appropriate intervals after the transfer to the dark. NR activities were determined as described in "Materials and Methods." Experiments were re- peated three times. The O time values were 8.32 f 0.89 for shoot NRA, 6.25 f 0.86 for root NADH:NRA, and 3.86 f 0.51 pmol h-' g-' fresh weight for root NADPH:NRA. These values are the 100% control values.
Effect of Light on Maize Nitrate Reductase 1147
SHOOT
Figure 2. A, Western blots showing the loss of NRP with time inthe dark. There was a 24-h induction with 5 mM NO3~ before theexperiment was initiated. Shoot (top) and root (bottom) were har-vested 24 (0) or 48 h (24 hL) after the addition of 5 mM KNO3.Numbers above the lanes refer to hours after transfer to dark (D).Arrowhead shows the 116-kD NR subunit band. B, Northern blotsshowing the effect of darkness on the levels of NR mRNA after a24-h induction with 5 mM KNO3. Total RNA was extracted from theshoots (top) or roots (bottom) sampled at the beginning (0) and atdifferent intervals after transfer to the dark. The lane on the rightside is a control that was kept in NO3~ for an additional 24 h afterthe 24-h induction. The positions of NR mRNA band for shoot androot are indicated by the arrowhead.
change either during the dark interval or when reexposed tolight.
The response of leaf NRA to light is in contrast to obser-vations made when NO3~ was added back to the system aftera short interval of deprivation (Fig. 3C; Li and Oaks, 1993),when there was a lag before the reinduction of NRA in root.In the shoot there was no change in NRA during the initial4 h after the addition of NO3~. This indicates that after 2 hof darkness the NRP can be activated by light but that after4 h there is a mixed response, activation, and de novosynthesis.
When the seedlings were reexposed to the light after long-term darkness (48 h), the reinduction of NRA was similar (inshoot) or slower (in root) than the initial induction withNO3~, when NRA reached about 30% of the maximum valuesin 4 h (Li and Oaks, 1993). Reinduction of root NRA afterthe 48 h of darkness was minor (4% of the original control)during the initial 4 h of light (Fig. 3B). These observationsindicate a de novo synthesis of the NRP in response to NOi~and to light after a 48-h dark interval.
There were no apparent changes in NRP levels as shownon western blot in either shoot or root tissues after 2 h ofdarkness or after 0.5 or 1.5 h of reillumination (Fig. 4A).Thus, the reinduction of NRA seen after a short-term transferto the dark could be related to an enzyme inactivation/reactivation mechanism.
Levels of NR mRNA in the root also showed no changeduring the 2-h dark period and the following 0.5 or 1.5-hreillumination (Fig. 4B). In the shoot, however, NR mRNAdecreased in the dark and increased again upon reillumina-tion. The resynthesis in the light was very fast, and near
control levels of NR mRNA were seen after the initial 30 minof reillumination following a 2-h dark period.
DISCUSSION
Extractable levels of NR decline when plants are trans-ferred to the dark (Hageman and Flesher, 1960; Kaiser et al.,1992; Riens and Heldt, 1992; De Cires et al., 1993; Fig. 1).The time-course experiments, shown in Figures 1 and 2,
Figure 3. A, Effect of short-term darkness on the reinduction ofNRA by light. Seedlings were induced with 5 mM KNO3 for 24 h (0)before transfer to the dark for different intervals (•). Some seedlingswere reexposed to light for 0.5 or 1.5 h after 2 or 4 h of darkness(O). B, Effect of long-term darkness (48 h) on the reinduction ofNRA by light. Seedlings were induced with 5 mM KNO3 for 24 h(•) before transfer to the dark for 48 h (0). Seedlings were thenreexposed to light for different intervals (O). These results are themeans of two different experiments. The SE is shown by the bars.C, Effect of NOr withdrawal on the reinduction of NRA by read-dition of NO3~. Seedlings were induced with 1 mM KNO3 for 24 h(0) before transfer to NO3"-free medium for different intervals (•).Four hours after the removal of NO3", some seedlings were broughtback to NO3~ medium for 1, 2, or 4 h to determine the reappearanceof NRA (O) (adapted from Li and Oaks [1993]). The results are themeans of three different experiments, and SE values are shown bythe bars, gfw, Crams fresh weight.
1148 Li and Oaks Plant Physiol. Vol. 106, 1994
SHOOT
ROOT
SHOOT
ROOT
Figure 4. A, Effect of short-term darkness and reillumination on thelevels of NRP. After a 24-h induction with 5 mM KNO3 (0), plantswere transferred to the dark for 2 h (2hD). Some seedlings werereilluminated for 30 min or 1.5 h after 2 h (2hD+30'L, 2hD+1.5hL)darkness. Arrowheads show the 116-kD NR subunit band. Top,Shoot; bottom, root. B, Effect of short-term darkness and reillumi-nation on the levels of NR mRNA. Arrowheads show the NR mRNAband. Experiments were repeated twice, and representative dataare shown.
indicate that there is an immediate decay of NRA and NRmRNA in maize shoot tissues but that the NRP is relativelystable. In the root, however, there is a 4-h delay before a lossin NRA, NRP, or NR mRNA levels could be detected. Thus,NOs~ assimilation in the root appears to be relatively insen-sitive to light and as indicated previously (Li et al., 1993;X.-Z. Li, unpublished results) appears to be responding to adifferent set of environmental cues.
NRA levels in shoots decreased in the dark and increasedagain immediately after reillumination of the plants (Fig. 3A).Although the level of NR mRNA had declined significantlywithin 2 h of darkness (Figs. 2B and 4B), the NRP levels werenot visibly altered (Figs. 2A and 4A). Therefore, it seems thatthe loss of NRA in the dark could be caused by an inactivationof NRP and not by its degradation. After a short period ofdarkness (2 h), the inactivated protein was almost completelyreactivated upon reexposure to light.
The results of northern blot analyses (Fig. 4B) showed thatafter 30 min or 1.5 h of reillumination after a 2-h period inthe dark, NR mRNA in the shoot was resynthesized. Thus,the increase in NRA could be caused by the activation of apreexisting protein or by the renewed synthesis of the NRP.
Since NRA reached 92% of the control value, from 68%,within 30 min, it is unlikely that this restoration was causedby a newly synthesized protein. For example, after 48 h ofdarkness, NRA (Fig. 3B), NRP, or NR mRNA (data notshown) had totally disappeared, and, in fact, a de novosynthesis of both NR message and protein was detectedbefore the reappearance of NRA. In the case of NOs" depri-vation, NO3~ reintroduction did not cause an immediatereappearance of NRA (Fig. 3C). We interpret this lag to a denovo synthesis of the enzyme, a conclusion reinforced by the
fact that cycloheximide, an inhibitor of protein synthesis,inhibited the development of NRA (Li and Oaks, 1993). Theimmediate increase in NRA seen when the seedlings werereexposed to light after a short period of darkness (2 h)suggests the reactivation of a preexisting protein. Transfer tothe dark and the addition of cycloheximide before the reex-posure to light was too complicated to yield valid results, andin our hands experiments with excised shoots resulted in verysmall losses of NRA when the shoots were transferred to thedark. Such fluctuations are apparent with excised spinachleaves (Kaiser and Spill, 1991), and in spinach a reversiblephosphorylation/dephosphorylation mechanism appears tobe involved in the activation/inactivation of the NRP (Kaiserand Spill, 1991; Huber et al., 1992; Kaiser et al., 1992;MacKintosh, 1992). A phosphorylation/dephosphorylationhas been postulated for maize (Redinbaugh et al., 1993), butthe properties of this system have not been as clearly estab-lished as in the spinach system. The activation/inactivationin maize NRA could be explained by a phosphorylation/dephosphorylation mechanism as seen in spinach or by anoxidation/reduction as seen in Chlorella (Pistorius et al., 1976)and in wheat (Aryan et al., 1983). Experiments designed toresolve these possibilities are currently in progress.
ACKNOWLEDGMENT
We would like to thank Dr. S.J. Rothstein, Department of Molec-ular Biology and Genetics, University of Guelph, for the cDNAclones used in our experiments.
Received May 2, 1994; accepted July 27, 1994.Copyright Clearance Center: 0032-0889/94/106/1145/05.
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