both interna1 and externa1 regulatory elements control expression of the pea gene … · the plant...

The Plant Cell, Vol. 4, 389-395, April 1992 O 1992 American Society of Plant Physiologists

Both Interna1 and Externa1 Regulatory Elements Control Expression of the Pea Fed-7 Gene in Transgenic Tobacco Seedlings

Maria Gallo-Meagher,ai’ Dolores A. Sowinski,b Robert C. Elliott,bi2 and William F. T h o m p s ~ n ~ ~ ~ ~ ~ a Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695

Department of Botany, North Carolina State University, Raleigh, North Carolina 27695

In previous studies using leaves of light-grown transgenic tobacco plants, we have shown that sequences located within the transcribed region of the pea Fed-l gene (encoding ferredoxin I) are major cis-acting determinants of light-regulated mRNA accumulation. However, we show here that these internal sequences are less important for the Fed-l light response in etiolated tobacco seedlings than they are in green leaves and that upstream elements confer organ specificity and contribute significantly to Fed-7 light responses in etiolated material. Light effects mediated by upstream response elements are thus most pronounced during the initial induction of gene activity, whereas internal elements play a more prominent role in modulating Fed-l expression once the gene is already active.

INTRODUCTION

Fed-7, a singlecopy nuclear gene encoding ferredoxin I (Dobres et al., 1987; Elliott et al., 1989b), exhibits a number of unusual light responses in the buds of etiolated pea seedlings (Kaufman et al., 1985, 1986). We have argued that these differences indicate that Fed-7 regulatory mechanisms probably differ from those of other light-responsive genes (Thompson, 1988). Con- sistent with this expectation, we have recently used chimeric gene constructs in transgenic tobacco plants to show that important cis-acting light-regulatory element(s) are located within the transcription unit of the pea Fed-7 gene (Elliott et al., 1989a). This observation contrasts sharply with the upstream location of regulatory elements in other plant genes and raises the possibility that Fed-1 light responses may be controlled by mechanisms affecting processes other than transcriptional initiation. Additional experiments have further localized the internal control region and shown that it can confer light- responsive expression when fused to reporter gene sequences (L.F. Dickey, M. Gallo-Meagher, and W.F. Thompson, unpublished data).

However, these experiments used green leaves from light- grown primary transformants (Elliott et al., 1989a), and it is known that many features of light-regulated gene expression are dramatically different in etiolated and green tissues (for reviews, see Jenkins, 1991; Thompson and White, 1991). For example, mRNA for a pea glutamine synthase gene, GS2, is more rapidly induced by white light in green plants than in

Current address: Texas Agricultura1 Experiment Station, Weslaco,

Current address: John lnnes Institute, Norwich N R 4 7UH, UK. To whom correspondence should be addressed.

TX 78596.

etiolated seedlings (Edwards and Coruzzi, 1989), and ribulose bisphosphate carboxylase small subunit (RbcS) gene transcripts that accumulate in response to a red light pulse in etiolated pea seedlings do not do so in dark-adapted green leaves of the same species (Fluhr and Chua, 1986). Perhaps the most dramatic example comes from studies of the det-7 mutant in Arabidopsis (Chory et al., 1989b). Photoregulated genes are expressed constitutively in etiolated seedlings of this mutant, but the same genes show normal responses when green plants are subjected to dark treatments followed by reillumination.

Given the differences between green plants and etiolated seedlings, we wanted to extend our previous work on chimeric Fed-1 genes by measuring the response of the same gene constructs in etiolated seedlings. In this study, we show that the relative contributions of upstream and internal cis-acting light- regulatory elements in the expression of Fed-7 differ between etiolated cotyledons and green leaves, with internal elements contributing a smaller portion of the total response in etiolated cotyledons. We also show that sequences 5’to the transcription start site determine organ-specific expression of Fed-7 in both seedlings and mature plants.

RESULTS

Transgenic Tobacco Seedlings

Seedlings were derived from transgenic tobacco plants containing severa1 of the chimeric genes described by Elliott et

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390 The Plant Cell

al. (1989a). Figure 1 illustrates these constructs, including anintact Fed-1 gene, long and short Fed-1 promoter fusions, andthe Fed-1 "message" construct in which the Fed-1 transcrip-tion unit is transcribed from the cauliflower mosaic virus (CaMV)35S promoter. (For simplicity, we use the term "promoter" torefer collectively to all c/s-acting regulatory sequences 5' tothe start of transcription.) The control construct was pBI121(Jefferson et al., 1987), in which the p-glucuronidase (gusA)reporter is driven by the CaMV 35S promoter.

35S-GUS (Control)

CaMVSSS GUS Nos

Fed-Fed (Intact Fed-1 Gene)

h . 5' Flanking Fed 3° Flanking |

Hind III Alul Alul Avail EcoRI

Fed-GUS (Long Promoter)

5' Flanking GUS Nos

Hind III Alul Alul

FedtSl-GUS (Short Promoter)

Promoter GUS Nos

Alul Alul

35S-Fad (Fed-1 "Message")

CaMV 35S ;<:fc;Fed

Alul Avail

Figure 1. Constructs Used.

Various Fed-1 constructs along with the control construct pBI121(CaMV 35S promoter driving expression of gusA with a nopaline syn-thase [nos] terminator; Jefferson et al., 1967) were inserted into thebinary vector pBIN19 (Bevan, 1984) and introduced into tobacco byway of Agrobacterium-mediated leaf disc transformation (Elliott et al.,1989a). The intact Fed-1 gene construct (Fed::Fed) contains the tran-scribed region plus flanking sequences from -2000 to +1600. Thelong promoter construct (Fed::GUS) contains sequences from -2000to +5 driving the expression of GUS with a nos terminator. The shortpromoter construct (Fed(S)::GUS) contains sequences from -495 to+5 of the 5' flanking region driving the expression of gusA with a nosterminator. The Fed-1 message construct (35S::Fed) contains the CaMV35S promoter driving the expression of the Fed-1 transcribed regionfrom +5 to +824 with the Fed-1 polyadenylation site.

________Tobacco________ PeaPromoter 35S Fed Fed(S) Fed 35S FedMessage GUS Fed GUS .GUS. Fed Fed

R t L f R t L f R t L f R t L f R t L f R t L f

GUS

Fed It m f^̂ ^

Figure 2. Organ Specificity in Pea Seedlings and Mature TransgenicPlants.

Total RNA (5 ng per lane) was isolated from leaves or roots of 1-week-old pea plants, as well as from leaves or roots of 6-week-old transgenictobacco plants containing the Fed-1 constructs. The RNA gel blot washybridized with a mixture of 32P-labeled RNA probes to detect Fed-1and gusA. Lf, leaf; Rt, root.

Each chimeric gene was carried in the binary vector pBIN19(Bevan, 1984), which contains a bacterial neomycin phos-photransferase (nptll) gene conferring kanamycin resistance.Kanamycin cannot be used to select transgenic individualsfrom populations of etiolated seedlings because discrimina-tion between sensitive and resistant phenotypes requiresgrowth in the light. However, the number of T-DNA loci ineach primary transformant can be determined genetically byanalyzing segregation ratios for kanamycin resistance. Threerepresentative transformants were analyzed in this way for eachgene construct in Figure 1, and seeds were taken from trans-formants with two or more transgenic loci for most subsequentstudies.

Organ-Specific Expression Is Mediated by theFed-1 Promoter

Organ specificity of Fed-1 expression was assessed by com-paring leaves or roots of 6-week-old transgenic tobacco plantscontaining the chimeric constructs illustrated in Figure 1, aswell as leaves and roots of 1-week-old pea plants. Plantsrepresenting at least four different primary transformants weretested for each construct, and representative results are shownin Figure 2. When a Fed-1 promoter was driving transcription,regardless of the gene to which it was attached, mRNA wasdetected only in leaves. The expression of the native Fed-1 genein pea was also restricted to leaves. In contrast, Fed-1 mRNAtranscribed from the CaMV 35S promoter accumulated to read-ily detectable levels in both leaves and roots of transformedtobacco, indicating that pea Fed-1 mRNA can accumulate inroot cells when it is synthesized from a constitutive promoter.

If the regulatory mechanisms involved in organ specificityare independent of the stage of development, we might ex-pect the Fed-1 promoter to confer cotyledon-specific expressionin tobacco seedlings because in this species the cotyledonsbecome green upon illumination. To test this prediction, seed-lings from transformants containing Fed::GUS or 35S::GUS

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Fed-1 Control Elements 391

constructs were grown in the dark for 6 days and then exposedto light for 24 hr. Cotyledons were separated from the remainderof the seedling (hypocotyl and root), and GUS activity was mea-sured in soluble protein extracts. Figure 3 shows that GUSactivity in seedlings containing the long or short Fed-1 pro-moter constructs was essentially restricted to the cotyledons,which had >90% of the total seedling activity. In contrast, the35S::GUS construct was more strongly expressed in hypocotylsand roots than in cotyledons. The organ specificity of the Fed-1promoter in transgenic tobacco seedlings was confirmed usinga GUS histochemical assay as described by Jefferson (1987).The blue precipitate indicative of GUS activity was evident inthe cotyledons, vascular tissue, and roots of seedlings con-taining the 35S::GUS construct, but restricted to the cotyledonsin seedlings containing the Fed-1 promoter fusions (data notshown).

The Fed-1 Promoter Is Light Responsive in EtiolatedSeedlings

To determine whether the Fed-1 promoter confers light respon-siveness in transgenic tobacco seedlings, RNA gel blot analysiswas performed on seedlings containing the Fed::GUS and35S::GUS constructs. Seedlings representing six independentprimary transformants for each construct were either grownin complete darkness or given 24 hr of light prior to harvest.Representative results are shown in Figure 4. Seedlings con-taining the 35S::GUS construct did not display a significantlight response. In most cases, however, a clear increase in

35S • GUS

51 80 82LD LD LD

Fed • GUS

202 216 218LDLD LD

Figure 4. Comparison of Fed::GUS and 35S::GUS Expression in Light-and Dark-Treated Transgenic Seedlings.Total RNA (5 ng per lane) was isolated from 7-day-old transgenic seed-lings that were grown either in complete darkness (D) or given 24 hrof light (L) before harvest. Gel blots were hybridized with a ̂ P-labeledRNA probe to detect gusA. mRNA data are shown for representativetransformants containing the 35S::GUS construct (51, 80, and 82) orthe Fed::GUS construct (202, 216, and 218).

mRNA abundance could be seen when the gusA gene wasdriven by the Fed-1 promoter. Table 1 shows that light-treatedFed:-.GUS seedlings produced an average of threefold to four-fold more gusA transcripts than the dark controls. Similar resultswere obtained when samples of the same seedling popula-tion were also assayed for extractable GUS activity using afluorescence assay (Jefferson, 1987).

80 -

60 -

40 -

20 -

0

i-i

,152 168 202 216Fed(S)-GUS Fed-GUS

I51 8235S-GUS

Transformants

Figure 3. Organ Specificity in Transgenic Tobacco Seedlings.Seedlings from independent primary transformants containing the Fed-1promoter (transformants 152,168, 202, and 216) or the CaMV 35S pro-moter (transformants 51 and 82) fused to the gusA gene were grownin liquid culture in the dark for 6 days and then exposed to light for24 hr. GUS activity was determined fluorometrically in extracts fromcotyledons (open bars) or from hypocotyls and roots (filled bars). TotalGUS activity for each construct is set to 100%.

Contribution of Internal Regulatory Elements to theLight Response in Seedlings

Although clearly significant, the threefold to fourfold responseof the Fed::GUS fusion is smaller than the sixfold to ninefoldincrease in Fed-1 mRNA in seedlings containing the intact Fed-1gene (see Figure 5; Gallo-Meagher et al., 1992). Thus, itseemed likely that internal c/'s elements were also essentialfor normal light regulation in seedlings. We tested the activityof internal elements using seedlings derived from transformantscontaining the 35S::Fed "message" construct (Figure 1), inwhich Fed-1 transcription is driven by the CaMV 35S promoter.Initial experiments were carried out as before, with RNA be-ing extracted from whole seedlings. In these experiments,transformants containing the intact Fed-1 gene displayed theexpected sixfold to ninefold increase in mRNA abundance, buta survey of seedlings derived from several independent trans-formants containing the message construct showed onlyapproximately a twofold response (data not shown). This re-sponse is considerably smaller than the fourfold to sixfoldinduction seen for the same construct in mature plants (seeElliott et al., 1989a). However, whole seedlings were used forthese experiments. Because transcription of the message con-struct is driven by the CaMV 35S promoter rather than by the

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392 The Plant Cell

Table 1. Light Induction Ratios for Transcript Abundance andGUS Activity of Independent Transformants Containing 35S::GUSand Fed::GUS Promoter Fusion Constructs.

Light/Dark Ratio

35S::GUS

515468788082

Mean± so

RNA

1.0ND

3.11.60.71.0

1.6± 1.1

Activity

0.61.50.80.31.01.5

1.0± 0.5

Fed::GUS

202216218222224228

Mean± so

Light/Dark Ratio

RNA

1.34.72.06.62.44.3

3.6± 2.0

Activity

4.23.63.04.13.72.7

3.6± 0.6

Total RNA was isolated from 7-day-old transgenic seedlings containingthe indicated constructs. Seedlings were grown either in completedarkness or given 24 hr of white light immediately before harvest.For transcript analysis, gel blots were hybridized with a 32P-labeledantisense RNA probe complementary to gusA mRNA. Light/dark ra-tios were calculated from autoradiographs analyzed by scanning den-sitometry. GUS activity was determined by fluorometry using solubleprotein extracts from separate groups of seedlings given the samelight treatments.

organ-specific Fed-1 promoter, these transformants might syn-thesize Fed-1 mRNA in hypocotyl and root cells that normallydo not do so and that may therefore lack the ability to regulateits abundance. If a large amount of unregulated Fed-1 mRNAwere to accumulate in hypocotyls or roots, it might mask lightresponses occurring only in the cotyledons. This possibilitywas seemingly supported by the observation that histochemi-cal staining was very strong in the roots of seedlings containing35S::GUS constructs, as reported previously by others (e.g.,Benfey et al., 1989).

The experiment was therefore repeated with RNA extractedfrom excised cotyledons, using a novel microscale extractionprocedure to isolate RNA from 20 to 60 pairs of cotyledons(see Methods). Because crude RNA extracts were used, it wasimpossible to measure the amount of RNA loaded on the gel.Therefore, Fed-1 transcript levels in each lane of the gel blotswere normalized to the signal produced by hybridizing withan excess of an end-labeled oligonucleotide complementaryto the 18S ribosomal RNA (Barbu and Dautry, 1989; de Leeuwet al., 1989).

Figure 5 showsthat strong hybridization signals were ob-tained with RNA from light-treated cotyledons and from intactseedlings derived from a transformant containing the 35S::Fed

Fed-1Probe

CPM

EntireSeedling

L D

18

35S : Fed

CotyledonsL D

26 10

Hypocotyls +Roots

L D

4 3

Fed : Fed

EntireSeedling

L D

I97 13

rRNAProbe • t

CPM 174 118 306 223 39 34 10 12

Figure 5. Light Response of 35S::Fed Construct in Whole Seedlings and Excised Cotyledons.

Total RNA extracts were prepared from 60 pairs of excised cotyledons, sets of roots and hypocotyls, or intact tobacco seedlings containing theFed-1 message construct (35S::Fed, transformant 312), using a microscale procedure as described in Methods. For comparison, another setof seedlings containing the intact gene construct (Fed::Fed, transformant 124) was treated and extracted similarly. Seedlings grown in liquid cul-ture were given the light treatments described in Figure 4. Fed-1 mRNA was detected on RNA gel blots by hybridization with a 32P-labeled antisenseRNA probe. Phosphorus-32 labeling in each band was measured in counts per minute by radioanalytical scanning, and values are listed beloweach lane. L, light; D, darkness. To correct for variation in the yield of the microscale RNA procedure, the same gel blot was reprobed with amolar excess of a 32P-end-labeled oligonucleotide complementary to the 18S rRNA (see Methods). Normalized light induction ratios were calcu-lated by dividing the ratio of the two Fed-1 signals by the corresponding 18S rRNA ratio. Normalized light induction ratios for the 35S::Fed chimericgene were 2.4, 1.9, and 1.2 for whole seedlings, excised cotyledons, and hypocotyls plus roots, respectively, whereas a normalized ratio of 9.0was calculated for the Fed::Fed construct.

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message construct, but that relatively high levels of Fed-7 RNA were also observed in the dark. In this and severa1 similar experiments (data not shown), an approximately twofold increase (after correcting for loading differences using the rRNA signal) was observed in cotyledons of illuminated seedlings. Because less RNA was obtained from hypocotyls and roots than from cotyledons, Fed-7 hybridization signals were cor- respondingly weak. Thus, it is unlikely that RNA from other parts of the seedling would obscure a light response in cotyledons, and the twofold response seen for the transformant used in these experiments can be compared to approximately twofold responses obtained with whole seedlings in the survey mentioned above.

Taken together, our results indicate that both the upstream and internal cis-acting elements can independently regulate Fed-7 transcript levels in response to light. However, the inter- na1 cis element(s) mediate significantly smaller light effects in etiolated seedlings than they do in green leaves of mature plants, and neither region by itself can convey the full response of the intact Fed-7 gene.

DISCUSSION

Role of the Fed-l Promoter

Previous work with dark-adapted transgenic tobacco leaves indicated that the Fed-7 promoter was unable to confer light responsiveness when fused to the gusA gene sequence (Elliott et al., 1989a), whereas a construct in which the CaMV 35s promoter was fused to the Fed-7 gene sequence exhibited strong light induction. These results were unambiguous with respect to the effect of the Fed-7 message sequence, but in- terpretation of the data from Fed-7 promoter fusions was complicated because a 35S::GUS control construct exhibited an unanticipated negative light effect, with significantly lower gusA mRNA levels in the light than in the dark (Elliott et al., 1989a, Figure 4). More recent data obtained from chloramphen- icol acetyltransferase (CAT) and luciferase (LUC) gene fusions indicate that this negative light effect was mediated by gusA sequences rather than by the CaMV 35s promoter (L.F. Dickey, M. Gallo-Meagher, and W.F. Thompson, unpublished data) and thus that it probably masked a positive light effect on the Fed-7 promoter fusions in our original experiments. Consistent with this conclusion, we show here that in seedlings, where light does not affect expression of the 35S::GUS construct, Fed-7 promoter fusions exhibited light responses averaging threefold to fourfold.

In addition to its role in light-regulated expression, the Fed-7 promoter also confers organ specificity in both seedlings and green plants. Regardless of the mRNA sequence to which it was attached, the Fed-7 promoter was able to direct expression in leaves or cotyledons, but never in roots (Figures 2 and 3). These results are consistent with those of Vorst et al.

(1990), who showed that the promoter of an Arabidopsis ferredoxin I gene mediates organ-specific expression in transgenic tobacco.

Variation in the Role of Interna1 Elements

Together with other data, the results presented here document differences in the mechanisms controlling Fed-7 expression in green leaves and etiolated seedlings. In our previous work with green leaves (Elliott et al., 1989a; L.F. Dickey, M. Gallo- Meagher, and W.F. Thompson, unpublished data), we observed light effects averaging fourfold to sixfold for the message construct containing only the internal cis element(s). These responses are two to three times larger than those of seedlings containing the same construct, indicating that internal elements mediate a larger portion of the total light response in green leaves than they do in etiolated seedlings.

The many morphological, cellular, and biochemical differences between etiolated cotyledons and green leaves are likely to have profound effects on the photoperception and signal transduction mechanisms controlling gene expression (e.g., Choryet al., 1989a, 1989b; reviewed by Thompson and White, 1991). However, the differences between Fed-7 responses in etiolated and green material may be even more fundamental because in etiolated seedlings we are measuring induction of a previously inactive gene, whereas in leaves we are measuring the ability of light to modulate mRNA levels after maximal expression has already been achieved. Molecular mechanisms might be expected to differ in such different developmental circumstances, and it is logical to expect transcriptional induction to play a more important role in initial expression than in subsequent modulation. Our working hypothesis is that upstream elements in the pea Fed-7 promoter are responsible for the initial expression of Fed-7 mRNA in appropriate cells of potentially photosynthetic organs. Subsequently, Fed-7 mRNA levels are modulated by mechanisms involving both promoter sequences and internal elements, with the internal elements assuming a dominant role in mature green tissue.

Relation to Other Systems

Thus far, the parsley 4CL-7 gene is the only other example of a plant gene in which internal elements have been shown to play a role in photoregulated expression. A recent report by Douglas et al. (1991) demonstrated that neither the 4CL-7 promoter region fused to the gusA reporter gene nor the 4CL-7 exon sequences fused to the CaMV 35s promoter was sensitive to induction by elicitor or light treatment in transgenic tobacco or parsley suspension cells. However, gene fusions combining promoter and transcribed sequences showed normal inducible expression, indicating that sequences internal to the transcription unit are required for inducibility.

A major difference between the 4CL-7 and Fed-7 systems is that the promoter and transcribed sequences of Fed-7 can

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394 The Plant Cell

each act independently to confer a light response, whereas in the 4CL-7 system both segments of the gene must appar- ently be present together. Although there is as yet no direct evidence, this difference suggests that the interna1 cis elements in the two systems may exert their effects by different mechanisms. The 4CL-7 element may be analogous to the downstream portions of certain animal and vira1 promoters that require specific sequences on both sides of the initiation start site (e.g., Jarrell and Meselson, 1991), whereas the Fed-7 in- terna1 element(s) might act by affecting processes such as transcriptional elongation or mRNA stability, or perhaps by a mechanism similar to that of the TAR element in the human immunodeficiencyvirus (e.g., Braddock et al., 1991; Gatignol et al., 1991).

METHODS

Plant Growth Conditions

Transgenic tobacco (Nicotiana tabacum SRl cv Petite Havana) seed derived from primary transformants (Elliott et al., 1989a) were surface- sterilized by immersion in 0.5% hypochlorite for 5 min followed by two 15-min washes with sterile deionized water. For most experiments, seeds were transferred to 150-mm-diameter Petri plates containing two layers of gel blotting paper(No. GB004; Schleicher and Schuell, Keene, NH) saturated with sterile half-strength Hoagland solution modified according to Downs and Thomas (1991). For the experiments shown in Figures 3 and 5, seeds were germinated and grown on a rotary shaker in 125- mL flasks containing 10 mL of the same half-strength Hoagland s o b tion. To promote germination, seeds were exposed to fluorescent light (40 pmol m+ sec -I) for 1 hr. lmmediately after this treatment, seedlings were transferred to darkness, where they were grown for 6 days prior to the inductive light treatments. For dark growth, flasks were wrapped in two layers of aluminum foi1 and placed on a rotary shaker (1 15 rpm) in a growth chamber (27%); plates were transferred to a dark room and placed in a loosely covered plastic box wrapped with two layers of black cloth. After growth in darkness for 6 days, etiolated seedlings were transferred to a 27% growth chamber containing a mixture of fluorescent and incandescent light (60 pmol m+ sec-I, derived from two 100-W incandescent and four 40-W fluorescent bulbs) or kept in the dark for an additional 24 hr.

For RNA gel blot analysis of root and leaf RNA, transgenic tobacco plants were grown for 5 to 7 weeks in soil under continuous light at 22OC, as described previously (Elliott et al., 1989a). When the plants were 50 to 80 cm tall, the largest leaf still expanding (usually 10 cm long) was harvested. Roots from the same plants were rinsed with dis- tilled water to remove soil, and one-half to three-quarters of the entire root system was used for RNA extraction. Peas were grown for 7 days in continuous light on Kimpac (Industrial Paper Products, Inc., Burling- ton, NC) moistened with deionized water, and buds and roots were used for RNA isolation.

Kanamycin Segregation Ratios

Seeds obtained from self-fertilized primary transformants were surface- sterilized as described above and germinated on 1% agar containing

hormone-free MS medium (Murashige and Skoog, 1962), pH 5.5, sup- plemented with 300 pglmL kanamycin. After 2 weeks of growth in the light (60 pmol m-2 sec -l, nOC), kanamycin-sensitive seedlings were bleached and could be easily distinguished from the green kanamycin- resistant seedlings (Potrykus et al., 1985).

RNA Extraction and Gel Blots

For most experiments, RNA was extracted as described previously (Gallo-Meagher et al., 1992). For excised cotyledon and rooffhypocotyl RNA extractions, we developed a nove1 procedure to accommodate much smaller amounts of tissue. Cotyledons and roots/hypocotyls from 60 seedlings were separated, frozen in liquid Nz, and ground with a plastic pestle (No. 749520; Kontes, Vineland, NJ) in a 15" microcentrifuge tube containing 60 pL of RNA extraction buffer (1% [whr] SDS, 10 mM Tris, pH 7.5, 1 mM EDTA, 1% [whr] triisopropylnapthalenesul- fonic acid, 4% [whr] paminosalicylic acid, 1 mM aurintricarboxylic acid, 2% [vlv] P-mercaptoethanol) and 60 pL of phenol reagent (phenol sta- bilized with 0.8% hydroxyquinoline, equilibrated with Tris-HCI at pH 8.0, and then mixed with an equal volume of 24:l chloroformlisoamyl alcohol). Aftercentrifuging for 2 min at 13,000 rpm, 60 pL of the super- natant was transferred to a new microcentrifuge tube containing 200 pL of denaturation buffer (6.5% formaldehyde, 50% formamide, and 1 x Mops buffer; 1 x Mops buffer is 20 mM 3[N-morpholino]propane- sulfonic acid, pH 7.0,5 mM sodium acetate, 1 mM EDTA, and 250 pM aurintricarboxylic acid). After denaturing for 15 min at 55OC, 40 pL of 0.2% bromophenol blue and 50% glycerol in 1 mM EDTA, pH 8.0, was added, and 40 pL of sample was loaded onto a 1% agarose minigel containing 1.1% formaldehyde in 1 x Mops buffer. Gels were electro- phoresed at 3 Vlcm for 1 to 3 hr. Although removing cotyledons from 60 seedlings took up to 30 min, control experiments showed no effect of wounding on Fed-7 mRNA levels during this time. Procedures for blotting and hybridization with 32P-labeled RNA probes were as described previously (Galio-Meagher et al., 1992). Radioactive bands on the RNA blots were quantitated directly (AMBIS Radioanalytic Im- aging System; AMBIS Systems, Inc., San Diego, CA).

Following radioanalytical scanning, the blots were stripped by wash- ing for 2 hr at 65OC in 10 mM sodium phosphate buffer, pH 7.0, containing 50% formamide. After rinsing with 2 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate), each blot was rehybridized with a 17-base oligonucleotide (5'-CGACGGGCGGTGTGTAC-3') complementary to the 18s rRNA. The oligonucleotide was labeled according to Maniatis et al. (1982) by phosphorylation with T4 polynucleotide kinase. A molar excess of labeled oligonucleotide was added to blots that had been prehybridized overnight at 37% in a buffer containing 5 x SSC, 50 mM Tris-HCI, pH 7.5, 5 mM EDTA, 0.1% (whr) sodium pyrophosphate, 1% SDS, 0.2% polyvinylpyrrolidone (WP 40,000), 0.2% Ficoll, 0.2% BSA, and 400 pglmL calf liver RNA. Hybridizations were carried out in the same buffer overnight at V C , and then the blots were given three 10-min washes at 42OC in 6 x SSC, 0.1% SDS before autoradi- ography and radioanalytical scanning.

Fluorometric and Histochemical Analysis of GUS Actlvity

To measure extractable GUS activity, cotyledons were separated from the hypocotyls and roots of 80 tobacco seedlings grown from the seed of two individual plants for each construct. Extraction and fluorometric assay procedures were carried out as described by Jefferson (1987). For histochemical staining, tobacco seedlings were incubated at 37%

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overnight (-46 hr) in 1.5-mL microcentrifuge tubes containing 200 WL of a 5-bromo-4-chloro-3-indolyl glucuronide solution (1 mglmL in 50 mM sodium phosphate buffer, pH 7.0, 1% dimethylformamide). Stained seedlings were cleared in 70% ethanol at rwm temperature and stored in the same solution until analysis.

ACKNOWLEDGMENTS

This work was supported in part by the National lnstitutes of Health (Grant No. GM-43108) and the National Science Foundation (Grant No. DCB-8817276). We thank Lynn Dickey for her advice and encourage- ment and Keith Everett for his expert photographic assistance.

Received November 14, 1991; accepted January 27, 1992.

REFERENCES

Barbu, V., and Dautry, F. (1989). Northern blot normalization with a 28s rRNA oligonucleotide probe. Nucl. Acids Res. 17, 7115.

Benfey, P.N., Ren, L., and Chua, N.-H. (1989). The CaMV 35s en- hancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J.

Bevan, M.W. (1984). Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12, 5711-5721.

Braddock, M., Thorburn, A.M., Kingsman, A.J., and Kingsman, S.M. (1991). Blocking of Tat-dependent HIV-1 RNA modification by an inhibitor of RNA polymerase II processivity. Nature 350,439-441.

Chory, J., Peto, C.A., Ashbaugh, M., Saganich, R., Pratt, L., and Ausubel, F. (1989a). Different roles for phytochrome in etiolated and green plants deduced from characterization of Arabidopsis thaliana mutants. Plant Cell 1, 867-880.

Chory, J., Peto, CA., Feinbaum, R., Pratt, L., and Ausubel, F. (1989b). Arabidopsis thaliana mutant that develops as a light-grown plant in the absence of light. Cell 58, 991-999.

de Leeuw, W.J.F., Slagboom, P.E., and Vijg, J. (1989). Quantitative comparison of mRNA levels in mammalian tissues: 28s ribosomal RNA leve1 as an accurate interna1 standard. Nucl. Acids Res. 17,

Dobres, M.S., Elliott, R.C, Watson, J.C, and Thompson, W.F. (1987). A phytochrome regulated transcript encodes ferredoxin I. Plant MOI. Biol. 8, 53-59.

Douglas, C.J., Hauffe, K.D., Ites-Morales, M.E., Ellard, M., Paszkowski, U., Hahlbrock, H., and Dangl, J.L. (1991). Exonic sequences are required for elicitor and light activation of a plant defense gene, but promoter sequences are sufficient for tissue specific expression. EMBO J. 10, 1767-1775.

Downs, R.J., and Thomas, J.F. (1991). Phytotron Procedural Manual for Controlled-Environment Research at the Southeastern Plant En- vironment Laboratory. NCARS Technical Bulletin No. 244 (Revised). (Raleigh, NC: North Carolina Agricultura1 Research Service).

Edwards, J.W., and Coruui, G.M. (1989). Photorespiration and light act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase. Plant Cell 1, 241-248.

8, 2195-2202.

10137-10138.

Elliott, R.C., Dickey, L.F., White, M.J., and Thompson, W.F. (1989a). cis-Acting elements for light regulation of peaferredoxin I gene expression are located within transcribed sequences. Plant Cell 1,

Elliott, R.C., Pedersen, T.J., Fristensky, B.A., White, M.J., Dicky, L.F., and Thompson, W.F. (1989b). Characterization of asingle copy gene encoding ferredoxin I from pea. Plant Cell 1, 681-690.

Fluhr, R., and Chua, N.-H. (1986). Developmental regulation of two genes encoding ribulose bisphosphate carboxylase small subunit in pea and transgenic petunia plants: Phytochrome response and blue light induction. Proc. Natl. Acad. Sci. USA 83, 2358-2362.

Gallo-Meagher, M., Sowinski, D.A., and Thompson, W.F. (1992). The pea ferredoxin I gene exhibits different light responses in pea and tobacco. Plant Cell 4, 383-388.

Gatignol, A., Buckler-White, A., Berkhout, B., and Jeang, K.T. (1991). Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science 251, 1597-1600.

Jarrell, K.A., and Meselson, M. (1991). Drosophila retrotransposon promoter includes an essential sequence at the initiation site and requires a downstream sequence for full activity. Proc. Natl. Acad. Sci. USA 88, 102-104.

Jefferson, R.A. (1987). Assaying chimeric genes in plants: The GUS gene fusion system. Plant MOI. Biol. Rep. 5, 387-405.

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: P-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907.

Jenkins, G.I. (1991). Photoregulation of plant gene expression. In Plant Biotechnology, Vol. 2, Developmental Regulation of Plant Gene Ex- pression, D. Grierson, ed (Glasgow/London: Blackie), pp. 1-41.

Kaufman, L.S., Briggs, W.R., and Thompson, W.F. (1985). Phyto- chrome control of specific mRNA levels in developing pea buds: The presence of both very low fluence and low fluence responses. Plant Physiol. 78, 388-393.

Kaufman, L.S., Roberts, L.L., Briggs, W.R., and Thompson, W.F. (1986). Phytochrome control of specific mRNA levels in developing pea buds: Kinetics of accumulation, reciprocity, and escape kinetics of the low fluence response. Plant Physiol. 81, 1033-1038.

Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982). Molecular Clon- ing: A Laboratory Manual. (Cold Spring Harbor, NY Cold Spring Harbor Laboratory).

Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497.

Potrykus, I., Paszkowski, J., Saul, M.W., Petruska, J., and Shillito, R.D. (1985). Molecular and general genetics of hybrid foreign gene introduced into tobacco by direct gene transfer. MOI. Gen. Genet. 199, 169-177.

Thompson, W.F. (1988). Photoregulation: Diverse gene responses in greening seedlings. Plant Cell Environ. 11, 319-328.

Thompson, W.F., and White, M.J. (1991). Physiological and molecular studies of light-regulated nuclear genes in higher plants. Annu. Rev. Plant Physiol. Plant MOI. Biol. 42, 423-466.

Vorst, O., Dam, F.V., Oosterhoff-Teerstra, R., Smeekens, S., and Weisbeek, P. (1990). Tissue-specific expression directed by an Arabidopsis thaliana pre-ferredoxin promoter in transgenic tobacco plants. Plant MOI. Biol. 14, 491-499.

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both interna1 and externa1 regulatory elements control expression of the pea gene … · the plant...

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