The Plant Cell, Vol. 4, 383-388,April 1992 O 1992 American Society of Plant Physiologists
RESEARCH ARTICLE
The Pea Ferredoxin I Gene Exhibits Different Light Responses in Pea and Tobacco Maria Gallo-Meagher,ai’ Dolores A. Sowinski,b and William F. Thompsonaibi2 a
Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695 Department of Botany, North Carolina State University, Raleigh, North Carolina 27695
We monitored Fed-7 (encoding ferredoxin I) mRNA levels in etiolated transgenic tobacco seedlings containing the intact pea Fed-7 gene to determine if the characteristiclight responses of this gene in pea seedlings are also observed in transgenic tobacco. Fed-7 transcript levels in transgenic tobacco seedlings closely paralleled those of the native gene in pea buds when etiolated seedlings were transferred to white light. However, the response to red light was much smaller in tobacco than in pea and was not efficiently reversed by far-red light. The red light response of endogenoustobacco ferredoxin transcripts is closely comparable to that of the Fed-7 transgene, with a similar lack of photoreversibility. Thus, the pea Fed-7 transgene responds normally to tobacco gene-regulatory factors, but these factors are less influenced by phytochrome in tobacco cotyledons than in pea buds.
INTRODUCTION The light responses of the pea Fed-7 gene (encoding ferredoxin I) exhibit a number of unusual properties (Kaufman et al., 1985a, 1985b; Kaufman et al., 1986; Dobres et al., 1987). Fed-7 transcript levels in the buds of etiolated pea seedlings increase dramatically in response to white light and show an unusually rapid response to red light treatment, reaching nearly maximum levels within 1 hr. This increase in Fed-7 mRNAcould be fully reversed by far-red light given as long as 7 hr after the inductive red light pulse, indicating an unusually prolonged requirement for Pfr. In sharp contrast, severa1 other transcripts, including ribulose bisphosphate carboxylase small subunit (RbcS) and chlorophyll a/b binding (Cab)mRNAs, increased in abundance much more gradually and lost far-red light sensitivity more rapidly. The relative rapidity of the Fed-7 response in pea buds and its more prolonged requirement for Pfr indicate that one or more biochemical steps in phytochrome regulation of Fed-7 mRNA abundance differ from those regulating the RbcS and Cab transcript levels in this species. To gain a further understanding of the mechanisms governing Fed-7 light responses, the pea Fed-7 gene was isolated and the expression of various chimeric derivatives was characterized in transgenic tobacco (Elliott et al., 1989a, 1989b). Dark adaptation experiments showed that light is required for high-leve1expression in green leaf tissue. However, it is not known whether peaFed-7 expression is regulated by phytochrome in transgenic tobacco. Current address: Texas Agricultura1 Experiment Station, Weslaco,
TX 78596. To whom correspondence should be addressed.
In this study, we examined the light responsivenessof Fed-7 in etiolated transgenic tobacco seedlings. We monitored changes in transcript levels in response to continuous illumination with white light and to treatment with short pulses of red and far-red light designed to stimulate the phytochrome system. We showed that the white light response of Fed-7 mRNA in tobacco seedlings closely parallels that in pea seedlings. However, red light alone is less effective in tobacco than it is in pea. Thus, photoreceptors other than phytochrome appear to play a larger role in the tobacco response.
RESULTS White Light lnduces Rapid and Extensive Fed-7 mRNA Accumulation in Both Pea and Transgenic Tobacco Seedlings We first compared a time course for white light induction of Fed-7 transcripts in tobacco seedlings containing the intact Fed-7 gene construct (Elliott et al., 1989a) to a similar time course for the native Fed-7 gene in pea. Six-day-oldetiolated seedlings were transferred to light and harvested after 2, 4, or 24 hr. Controls were harvested after 6 and 7 days in complete darkness. Total RNA from pea buds and whole tobacco seedlings was analyzed on RNA gel blots. Figure 1 shows the result of this time course for seedlings of pea and two independent tobacco transformants containing the intact Fed-7 gene. The etiolated seedlings of both pea
384
The Plant Cell
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24C24
120
104
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24Cj4
and Cab mRNAs in response to white light. Transgenic tobacco seedlings carrying the intact pea Fed-1 gene were treated as in Figure 1, and total RNA was isolated from whole seedlings. Figure 2 shows that generally similar patterns of accumulation were seen for the Fed-1 and RbcS mRNAs. Both transcripts continued to increase up to 24 hr, although the Fed-1 mRNA showed a more rapid response at the early time points. Tobacco Cab transcripts showed the fastest rate of accumulation, reaching maximum levels after only 4 hr of continuous white light. Overall, each set of transcripts had a unique pattern of accumulation, with the pea Fed-1 response more closely resembling that of tobacco flbcS transcripts.
Phytochrome Does Not Induce High Levels of Fed-1 mRNA in Etiolated Tobacco Seedlings
2
4
24
C24
Hours of Light
Figure 1. Time Course of Pea Fed-1 mRNA Accumulation in Etiolated Pea and Transgenic Tobacco Seedlings. Total RNA was isolated from the buds of etiolated pea seedlings and from whole tobacco seedlings derived from two different transformants (plants 104 and 120) containing the intact pea Fed-1 gene. In each case, seedlings were grown in continuous darkness for 6 days and then exposed to continuous white light for 0, 2, 4, or 24 hr. C0, 6-day-old etiolated seedlings; C24, 7-day-old etiolated seedlings. (A) Fed-1 mRNA detected on gel blots of total cellular RNA (5 ng per lane) hybridized with a 32P-labeled RNA probe as described in Methods. (B) Histogram depicting the net counts of ^P-label hybridized to Fed-1 RNA in each lane of the gel blot shown in (A) measured from a radioanalytical scan as described in Methods.
and the transgenic tobacco contained low amounts of Fed-1 mRNA on day 6 (Figure 1, CQ). However, Fed-1 mRNA increased dramatically after only 2 hr of continuous white light, and by the 4-hr time point, the level was already more than half of that accumulated by 24 hr. The extent of Fed-1 mRNA accumulation over this time course was quite similar in both pea and tobacco, with both species showing an approximately 25-fold increase in Fed-1 mRNA abundance relative to the starting point (C0) and a sixfold increase over the 7-day dark control (C24). Thus, the accumulation of Fed-1 mRNA in etiolated tobacco seedlings containing the intact Fed-1 gene was a rapid, strongly light-dependent phenomenon similar to that found in pea seedlings. To compare the response of the Fed-1 transgene to those of other light-regulated genes in the tobacco system, we also measured the accumulation of the endogenous tobacco FlbcS
In etiolated pea seedlings, phytochrome has a major influence on Fed-1 mRNA accumulation. A single red light pulse induces a large increase in the amount of transcript, and the importance of phytochrome as the predominant photoreceptor in this response is clearly established by its far-red reversibility (Kaufman et al., 1985a, 1986). However, Figure 3 shows that a red light pulse that is able to induce a strong response in pea buds gives only marginal induction in tobacco seedlings. In pea, Fed-1 transcripts reached levels nearly half as high as those of the white light controls, whereas transgene mRNA
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RESEARCH ARTICLE
The Pea Ferredoxin I Gene Exhibits Different Light Responses in Pea and Tobacco Maria Gallo-Meagher,ai’ Dolores A. Sowinski,b and William F. Thompsonaibi2 a
Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695 Department of Botany, North Carolina State University, Raleigh, North Carolina 27695
We monitored Fed-7 (encoding ferredoxin I) mRNA levels in etiolated transgenic tobacco seedlings containing the intact pea Fed-7 gene to determine if the characteristiclight responses of this gene in pea seedlings are also observed in transgenic tobacco. Fed-7 transcript levels in transgenic tobacco seedlings closely paralleled those of the native gene in pea buds when etiolated seedlings were transferred to white light. However, the response to red light was much smaller in tobacco than in pea and was not efficiently reversed by far-red light. The red light response of endogenoustobacco ferredoxin transcripts is closely comparable to that of the Fed-7 transgene, with a similar lack of photoreversibility. Thus, the pea Fed-7 transgene responds normally to tobacco gene-regulatory factors, but these factors are less influenced by phytochrome in tobacco cotyledons than in pea buds.
INTRODUCTION The light responses of the pea Fed-7 gene (encoding ferredoxin I) exhibit a number of unusual properties (Kaufman et al., 1985a, 1985b; Kaufman et al., 1986; Dobres et al., 1987). Fed-7 transcript levels in the buds of etiolated pea seedlings increase dramatically in response to white light and show an unusually rapid response to red light treatment, reaching nearly maximum levels within 1 hr. This increase in Fed-7 mRNAcould be fully reversed by far-red light given as long as 7 hr after the inductive red light pulse, indicating an unusually prolonged requirement for Pfr. In sharp contrast, severa1 other transcripts, including ribulose bisphosphate carboxylase small subunit (RbcS) and chlorophyll a/b binding (Cab)mRNAs, increased in abundance much more gradually and lost far-red light sensitivity more rapidly. The relative rapidity of the Fed-7 response in pea buds and its more prolonged requirement for Pfr indicate that one or more biochemical steps in phytochrome regulation of Fed-7 mRNA abundance differ from those regulating the RbcS and Cab transcript levels in this species. To gain a further understanding of the mechanisms governing Fed-7 light responses, the pea Fed-7 gene was isolated and the expression of various chimeric derivatives was characterized in transgenic tobacco (Elliott et al., 1989a, 1989b). Dark adaptation experiments showed that light is required for high-leve1expression in green leaf tissue. However, it is not known whether peaFed-7 expression is regulated by phytochrome in transgenic tobacco. Current address: Texas Agricultura1 Experiment Station, Weslaco,
TX 78596. To whom correspondence should be addressed.
In this study, we examined the light responsivenessof Fed-7 in etiolated transgenic tobacco seedlings. We monitored changes in transcript levels in response to continuous illumination with white light and to treatment with short pulses of red and far-red light designed to stimulate the phytochrome system. We showed that the white light response of Fed-7 mRNA in tobacco seedlings closely parallels that in pea seedlings. However, red light alone is less effective in tobacco than it is in pea. Thus, photoreceptors other than phytochrome appear to play a larger role in the tobacco response.
RESULTS White Light lnduces Rapid and Extensive Fed-7 mRNA Accumulation in Both Pea and Transgenic Tobacco Seedlings We first compared a time course for white light induction of Fed-7 transcripts in tobacco seedlings containing the intact Fed-7 gene construct (Elliott et al., 1989a) to a similar time course for the native Fed-7 gene in pea. Six-day-oldetiolated seedlings were transferred to light and harvested after 2, 4, or 24 hr. Controls were harvested after 6 and 7 days in complete darkness. Total RNA from pea buds and whole tobacco seedlings was analyzed on RNA gel blots. Figure 1 shows the result of this time course for seedlings of pea and two independent tobacco transformants containing the intact Fed-7 gene. The etiolated seedlings of both pea
384
The Plant Cell
Tobacco Pea Co 2
4
24C24
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104
CD 2
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and Cab mRNAs in response to white light. Transgenic tobacco seedlings carrying the intact pea Fed-1 gene were treated as in Figure 1, and total RNA was isolated from whole seedlings. Figure 2 shows that generally similar patterns of accumulation were seen for the Fed-1 and RbcS mRNAs. Both transcripts continued to increase up to 24 hr, although the Fed-1 mRNA showed a more rapid response at the early time points. Tobacco Cab transcripts showed the fastest rate of accumulation, reaching maximum levels after only 4 hr of continuous white light. Overall, each set of transcripts had a unique pattern of accumulation, with the pea Fed-1 response more closely resembling that of tobacco flbcS transcripts.
Phytochrome Does Not Induce High Levels of Fed-1 mRNA in Etiolated Tobacco Seedlings
2
4
24
C24
Hours of Light
Figure 1. Time Course of Pea Fed-1 mRNA Accumulation in Etiolated Pea and Transgenic Tobacco Seedlings. Total RNA was isolated from the buds of etiolated pea seedlings and from whole tobacco seedlings derived from two different transformants (plants 104 and 120) containing the intact pea Fed-1 gene. In each case, seedlings were grown in continuous darkness for 6 days and then exposed to continuous white light for 0, 2, 4, or 24 hr. C0, 6-day-old etiolated seedlings; C24, 7-day-old etiolated seedlings. (A) Fed-1 mRNA detected on gel blots of total cellular RNA (5 ng per lane) hybridized with a 32P-labeled RNA probe as described in Methods. (B) Histogram depicting the net counts of ^P-label hybridized to Fed-1 RNA in each lane of the gel blot shown in (A) measured from a radioanalytical scan as described in Methods.
and the transgenic tobacco contained low amounts of Fed-1 mRNA on day 6 (Figure 1, CQ). However, Fed-1 mRNA increased dramatically after only 2 hr of continuous white light, and by the 4-hr time point, the level was already more than half of that accumulated by 24 hr. The extent of Fed-1 mRNA accumulation over this time course was quite similar in both pea and tobacco, with both species showing an approximately 25-fold increase in Fed-1 mRNA abundance relative to the starting point (C0) and a sixfold increase over the 7-day dark control (C24). Thus, the accumulation of Fed-1 mRNA in etiolated tobacco seedlings containing the intact Fed-1 gene was a rapid, strongly light-dependent phenomenon similar to that found in pea seedlings. To compare the response of the Fed-1 transgene to those of other light-regulated genes in the tobacco system, we also measured the accumulation of the endogenous tobacco FlbcS
In etiolated pea seedlings, phytochrome has a major influence on Fed-1 mRNA accumulation. A single red light pulse induces a large increase in the amount of transcript, and the importance of phytochrome as the predominant photoreceptor in this response is clearly established by its far-red reversibility (Kaufman et al., 1985a, 1986). However, Figure 3 shows that a red light pulse that is able to induce a strong response in pea buds gives only marginal induction in tobacco seedlings. In pea, Fed-1 transcripts reached levels nearly half as high as those of the white light controls, whereas transgene mRNA
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