The Plant Cell, Vol. 11, 1799–1810, September 1999, www.plantcell.org © 1999 American Society of Plant Physiologists

Identification of Two Essential Sequence Elements in the

Nonconsensus Type II

PatpB-290

Plastid Promoter by Using Plastid Transcription Extracts from Cultured TobaccoBY-2 Cells

Sanjay Kapoor

1

and Masahiro Sugiura

2

Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan

In higher plants, plastid genes are transcribed by at least two types of DNA-dependent RNA polymerases. One of them

is the well-known plastid-encoded prokaryotic type of polymerase that recognizes

s

70

-type promoters consisting of

2

35 and

2

10 consensus elements. The other recently recognized RNA polymerase has been found to be encoded en-tirely in the nucleus, and it recognizes a completely different set of promoters, designated previously as nonconsensustype II (NCII) promoters. Here, we report the development of an in vitro transcription system using nonphotosyntheticplastids of cultured tobacco BY-2 cells. This system preferentially and accurately initiates transcription from NCII pro-

moters. The conditions for in vitro transcription were optimized by using the tobacco

PatpB-290

promoter, which hasbeen found to be the most highly expressed NCII promoter in vivo. Analysis of in vitro transcription initiation in a seriesof

PatpB-290

5

9

deletion constructs revealed that sequences upstream of nucleotide

2

41 do not influence the tran-scriptional activity of this promoter. A 43-bp region (nucleotides

2

35 to

1

8) was further analyzed by introducing singleor multiple nucleotide substitutions into two regions (box I and box II) of high sequence conservation. We report herethat the ATAGAA sequence comprising box II and the

2

11 to

1

4 region (relative to transcription initiation) in box I sig-nificantly influence the activity of this NCII promoter.

INTRODUCTION

Plastids are plant-specific organelles that possess their owngenome and the capacity to express this genetic informa-tion. The transcription machinery of these organelles was

considered to be similar to that of

Escherichia coli

, becausehomologs of three of the bacterial RNA polymerase subunits(

a

,

b

, and

b9

) were found to be encoded on plastid ge-nomes. Incidentally, upstream regions of most of the initiallyidentified transcription initiation sites were found to resem-ble

2

10 and

2

35 consensus promoter elements (herein re-ferred to as consensus-type [CT] promoters) typical ofprokaryotic genes (reviewed in Igloi and Kössel, 1992;Gruissem and Tonkyn, 1993; Link, 1994; Hess and Börner,1999). Moreover, transcription factors bearing resemblanceto bacterial

s

factors were also identified as components ofthis transcription system (Lerbs et al., 1983; Bülow and Link,1988; Tiller et al., 1991; Link, 1994; Troxler et al., 1994; Tillerand Link, 1995; Tanaka et al., 1997; Kestermann et al., 1998;Tozawa et al., 1998).

In recent years, however, several reports have helped toestablish the existence of an entirely nucleus-encoded plas-tid RNA polymerase (NEP) in the plastids of higher plants(reviewed in Igloi and Kössel, 1992; Hess and Börner, 1999).Initial indications of completely nucleus-encoded plastidtranscriptional activity came from observations of RNA syn-thesis in the ribosome-deficient plastids (which were pre-sumably devoid of plastid-encoded proteins and thus of theplastid-encoded polymerase [PEP]) of heat-bleached ryeseedlings and

albostrian

mutants of barley (Bünger andFeierabend, 1980; Siemenroth et al., 1981). Later, it wasfound that only distinct sets of genes are transcribed in theribosome-deficient plastids of heat-bleached rye seedlings(Falk, et al., 1993), maize

iojap

mutants (Han et al., 1993),and barley

albostrian

mutants (Hess et al., 1993). As stron-ger evidence for the existence of NEP, the partial plastomeof

Epiphagus virginiana

, which lacks functional PEP subunitgenes, was found to be transcribed (dePamphilis andPalmer, 1990; Morden et al., 1991; Ems et al., 1995). Mean-while, a single subunit (110 kD) RNA polymerase activitywas detected in spinach chloroplast (Lerbs et al., 1985). Thisactivity was suggested to be the single subunit NEP or thenucleus-encoded catalytic core of a multimeric enzymebased on its functional similarity to T7 RNA polymerase(Lerbs-Mache, 1993). Recently, demonstration of transcription

1

Current address: Laboratory of Developmental Biology, NationalInstitute of Agrobiological Resources, Tsukuba Science City 305-8602, Japan.

2

To whom correspondence should be addressed. E-mail h44979b@nucc.cc.nagoya-u.ac.jp; fax 81-52-789-3081.

1800 The Plant Cell

initiation from a few plastid genes in

rpoB

(an essential sub-unit of PEP)-deleted tobacco seedlings (achieved by plastidtransformation and homologous recombination) finally andconclusively proved the existence of a plastidic transcrip-tional activity other than PEP (Allison et al., 1996). By use ofa similar experimental approach, it was confirmed that NEPdoes not share any of the core subunits of PEP (Serino andMaliga, 1998).

By analyzing the RNA species in

D

rpoB

tobacco seed-lings, translation-impaired plastids of wild-type tobacco,and nonphotosynthetic plastids of a cultured tobacco (BY-2)cell line, a number of NEP-dependent transcription initiationsites were identified and named nonconsensus type II (NCII)or NEP promoters (Allison et al., 1996; Kapoor et al., 1997).Sequence alignments of most of these promoters showedtwo regions of considerable similarity: an

z

15-bp regionsurrounding the transcription initiation site (box I) and a 6-bpregion near position

2

35 (box II), with reference to the site oftranscription initiation (Hajdukiewicz et al., 1997; Kapoor etal., 1997; Miyagi et al., 1998; Hess and Börner, 1999). Re-cently, similar promoters have also been identified in barleyand maize, suggesting that the nucleus-encoded transcrip-tion machinery is conserved between monocots and dicots(Hübschmann and Börner, 1998; Silhavy and Maliga, 1998).These observations have revealed that box II is more con-served in monocots than in tobacco (reviewed in Hess andBörner, 1999). In addition to box I– and box II–containingNCII promoters, there seem to be other types of NEP-recog-nized promoters that lack either one or both conservedboxes. For example, the maize

PrpoB

gene lacks box II(Silhavy and Maliga, 1998), whereas both of the conservedmotifs are lacking in tobacco

PclpP-53

(Sriraman et al., 1998).As a preliminary step toward understanding the biochemi-

cal properties and the promoter sequence requirements ofthe plastid-localized NEP transcription machinery, we reporthere the development of an in vitro transcription initiationsystem using the nonphotosynthetic plastids from BY-2cells. This system effects specific and accurate transcriptioninitiation from at least three tobacco NCII promoters. Fur-thermore, by using this system and a series of deletions andsubstitutions in the tobacco

PatpB-290

(NCII) promoter re-gion, we have identified two specific sequence elements re-quired for correct transcription initiation.

RESULTS

Development of an NCII Promoter–Specific Plastid in Vitro Transcription System

It is known that abolition of PEP (by blocking plastid transla-tion or deletion of

rpo

genes) results in a significant increasein the steady state levels of NCII transcripts, whereas abun-dance of transcripts resulting from CT promoters is greatly

reduced in such plastids (Hess et al., 1993; Allison et al.,1996; Kapoor et al., 1997; Miyagi et al., 1998; Serino andMaliga, 1998). Similar effects on the relative abundance oftwo transcript types were observed in developmentally dis-tinct (but genetically unaltered) nonphotosynthetic plastidsof cultured tobacco BY-2 cells and roots of wild-type to-bacco plants (Kapoor et al., 1997; Miyagi et al., 1998).

To ascertain whether the in vivo expression of NCII pro-moters in BY-2 cells also results from the activity of the NEPtranscription machinery, we analyzed the effects of plastidtranslation and transcription inhibitors on the respectivelevels of representative transcripts originating from NCII(

PatpB-290

and

Prrn16-64

) and CT (

PatpB-255

and

PpsbA-85

) promoters (Figure 1A). In light-grown wild-type tobaccoseedlings,

PatpB-255

(CT)–derived transcripts are

z

10times more abundant than those initiating from the NCII-type

PatpB-290

(Kapoor et al., 1997). On the other hand, inBY-2 cells, the

PatpB-255

–derived transcripts accumulateto only 40% of the levels derived from the

PatpB-290

pro-moter (Figure 1A, lane 1). A supplement of tagetitoxin (a PEPinhibitor) in the culture medium for 10 hr further reduced therelative amount of

atpB-255

transcripts by

z

15% (Figure1A, lane 2). In BY-2 cells, the tagetitoxin-influenced reduc-tion in the

atpB-255

transcript level was not as severe aspreviously observed (60% reduction) in the case of youngtobacco seedlings (Kapoor et al., 1997). At present, we donot have any suitable explanation for this observation ex-cept to suggest that there may be some differences in theuptake of tagetitoxin among BY-2 cells and young seed-lings.

The effect of supplementing the BY-2 culture mediumwith spectinomycin and streptomycin was more drastic be-cause addition of these supplements resulted in an almostcomplete elimination of the

PatpB-255

–derived transcript,with no inhibitory effect on the transcripts from

PatpB-290

(Figure 1A, lane 3). Similar results were obtained for

Prrn16-64

(NCII)– and

PpsbA-85

(CT)–derived transcript levels inBY-2 cells (Figure 1A, lanes 4 to 9). Taken together, thesedata show that analogous to the situation in wild-type to-bacco chloroplasts, in the plastids of BY-2 cells, the NCIIpromoters are utilized by the NEP transcription machinery.However, in contrast to chloroplasts, NEP transcription ac-tivity seemed to be much higher than the PEP activity in thenonphotosynthetic plastids of BY-2 cells.

Therefore, the cultured tobacco BY-2 cell line, whichmakes large quantities of developmentally homogenouscells easier to obtain, was selected as starting material forthe preparation of NEP-specific transcription extracts. Therelative abundance of NCII transcripts was not found to varysignificantly during the growth of the cell culture (Figure 1B).The mid-log phase, however, had previously been shown tobe high in RNA synthesis and low in RNase activities (Fanand Sugiura, 1995). Moreover, under our experimental con-ditions, the BY-2 cells harvested at the mid-log phaseyielded a higher percentage of intact plastids. Therefore, thisstage of cell growth was selected for preparation of the

In Vitro Transcription from NCII Promoters 1801

plastid in vitro transcription system. To minimize contamina-tion from other cell compartments, we used a gentle ap-proach involving protoplast preparation (Fan and Sugiura,1995). After physically disrupting the BY-2 protoplasts, in-tact nonphotosynthetic plastids were purified by differentialcentrifugation followed by sucrose step gradient centrifuga-tion. The transcription extracts were prepared after disrup-tion of purified plastids by sonication.

Optimization of in Vitro Transcription Reaction

The conditions for the in vitro transcription reaction were op-timized by using two adjacent

atpB

promoters,

PatpB-255

, aCT promoter, and

PatpB-290

, an NCII promoter, cloned inthe pBluescript II SK

1

vector (Figure 1C). The

PatpB-290

transcript is one of the most abundant among several NCIItranscripts that have been analyzed. Moreover, the inherentproximity of the

PatpB-255

and

PatpB-290

promoters pro-vided a unique opportunity to analyze the activity of twofunctionally distinct promoters in a single assay (Kapoor etal., 1997). The relative transcription activity of these two pro-moters was assayed by primer extension of in vitro tran-scripts using a 27-nucleotide primer complementary to the3

9

vector sequence. Selection of a primer complementary tovector sequences significantly reduces the background dueto in vivo transcripts as well as nonspecific transcriptionfrom endogenous DNA. The transcription initiation sites for thetobacco

atpB

/

E

promoters were mapped previously (Orozcoet al., 1990; Kapoor et al., 1994, 1997; Hajdukiewicz et al.,1997). Therefore, the dual promoter construct used in thisinvestigation was expected to yield two extension productsof 94 and 129 nucleotides for

PatpB-255

and

PatpB-290

, re-spectively (Figure 1C).

Figure 1.

Confirmation of NEP Activity in BY-2 Cells and SchematicRepresentation of the Dual Promotor Construct Used to Optimize inVitro Transcription Reactions.

(A)

Effect of plastid transcription and translation inhibitors on the invivo abundance of individual transcripts resulting from NCII and CTpromoters in BY-2 cells. Total RNA samples from BY-2 cells grownin the presence (

1

) or absence (

2

) of tagetitoxin (Tag) and spectino-mycin/streptomycin (Spc/Str) were subjected to primer extensionanalysis using gene-specific primers. The mean relative abundanceof individual transcripts, as estimated using a bioimage analyzer, isplotted in the form of a bar graph (the error bars represent the rangeof values from three different experiments).

(B)

Growth curve for BY-2 cells and the abundance of

atpB-

specifictranscripts. Total RNA isolated after several time periods was sub-jected to dot blot hybridization analysis by using a

32

P-labeled

atpB

gene–specific probe. The results were quantified and plotted as bars.

(C)

Schematic representation of the

atpB

promoter construct usedin this investigation. The in vitro transcript (bent arrows) resultingfrom an NCII (

PatpB-290

) and a CT (

PatpB-255

) promoter when sub-jected to primer extension analysis using the SKRP2 primer resultsin distinguishable extension products (left arrows) of 129 and 94 nu-cleotides (nt), respectively. Break in the

atpB

promoter represents

z

108 bp. Vector sequences are shown by hatched rectangles.

1802 The Plant Cell

Following the lead of earlier published results on PEP-specific plastid transcription extracts (Bottomley et al.,1971; Sun et al., 1986, 1989; Tiller and Link, 1993), prelimi-nary Mg

2

1

and K

1

concentrations for the in vitro transcrip-tion reaction were set at 7 and 50 mM, respectively, at pH8.0. Under these conditions, in vitro transcription followedby a primer extension assay yielded several nonspecific(

>

150 nucleotides) extension products in addition to thosecorresponding to

PatpB-255

and

PatpB-290

(see, e.g., Fig-ure 2A, lanes 3 to 5 and 10). However, after optimizing thereaction conditions for in vitro transcription to 20 mM Mg

2

1

,100 to 125 mM K

1

, and pH 7.8, specific transcription fromthe

PatpB-290

(NCII) promoter was observed (Figures 2Aand 2B). The peak of transcription activity was obtained byusing 84 nM template DNA, although this concentration alsoresulted in relatively higher background (Figure 2C). There-fore, for routine transcription assays, 42 nM template DNAwas used. Transcription activity was also dependent on totalprotein concentration (optimal at 1.2 mg mL

2

1

) as well as re-action time (optimal at 40 min; data not shown). Using thissystem, we could not detect any specific transcription activ-ity from linear DNA templates. The best results were ob-

tained with templates that were purified by using CsCldensity gradient centrifugation and that contained

,

5% ofDNA in linearized form (data not shown).

Effect of Transcription Inhibitors

The in vitro transcription reaction using the

atpB

promoterconstruct and the BY-2 extract under the optimized condi-tions produced two discrete bands of the predicted sizescorresponding to

PatpB-255

– and

PatpB-290

–derived tran-scripts (Figure 3A, lanes BEx). The site of in vitro transcrip-tion initiation was authenticated by electrophoresing theextended products simultaneously with the correspondingsequence ladder (Figure 3A). The relative levels of in vitro–transcribed products from

PatpB-290

and

PatpB-255

werefound to be similar to their respective in vivo levels in BY-2cells. It should be mentioned, however, that because the re-action conditions were optimized for NCII promoters, occa-sionally the activity of

PatpB-255

(CT promoter) was lowerthan expected. This template, when transcribed in vitro byusing tobacco chloroplast extracts, produced relatively high-

Figure 2. Optimization of in Vitro Transcription Reaction Conditions with Respect to K1 and Mg11 Concentrations, pH, and Template Amount.

The atpB promoter construct (Figure 1C) was in vitro transcribed as described in Methods. The relative transcription activity of PatpB-290 andPatpB-255 promoters (marked as 2290 and 2255, respectively) was assayed by primer extension of in vitro transcripts using the SKRP2 primerand analyzed on sequencing gels. The factors varied during in vitro transcription reactions are shown at the top of each panel. The solid trianglesrepresent relative increase or decrease in the units of the respective variable factor. Numbers at the left of each panel are the sizes (in nucle-otides) of marker DNA fragments.(A) Effect of K1 and Mg11 concentrations on in vitro transcription.(B) Effect of pH.(C) Effect of template amount.

In Vitro Transcription from NCII Promoters 1803

level transcription from the

PatpB-255

gene, but no in vitro–transcribed products corresponding to the

PatpB-290

(NCII)promoter were detected (Figure 3A, lanes CEx). The additionof tagetitoxin (100

m

M) had little or no effect on the abun-dance of the

PatpB-290

(NCII)–derived transcript. However,it caused

z

50% reduction in the levels of the

PatpB-255

–derived transcript (Figure 3B, lane Tag

1

). A few ratherlonger transcript species were detected with the chloroplastextract, which might have resulted from nonspecific tran-scription initiation and/or priming by the

SKRP2 primer thatwas used during the primer extension reactions (Figure 3A).

Sensitivity of the BY-2 plastid extract to other transcrip-tion inhibitors was also analyzed by supplementing the re-action mixture with 1 and 250 mg mL21 of rifampicin anda-amanitin, respectively (Figure 3B). No measurable effectsof either of the inhibitors were noted, suggesting that NCII-specific polymerase activity was due neither to contamina-tion by nuclear RNA polymerases nor to the prokaryotic-type RNA polymerase. Hence, finding an inhibitor(s) specificfor this newly characterized polymerase (NEP) activity wouldlead to a deeper understanding of its function.

In Vitro Transcription Initiation from Other NCII Promoters

Correct transcription initiation from two other NCII promot-ers, Prrn16-64 and Prpl32-1019 (Vera and Sugiura, 1995;Allison et al., 1996; Vera et al., 1996; Kapoor et al., 1997), wasalso achieved using this in vitro transcription system (Figure4). The construct containing both P1 and P2 promoters oftobacco rpl32 (see Methods) yielded only a single band (113bp) corresponding to the NCII Prpl32-1019 (P2) promoter. Inthe case of the rrn16 promoter construct, however, in vitrotranscripts initiating from both P1 (Prrn16-114 [CT]) andP2 (Prrn16-64 [NCII]) were detected, although the level ofthe Prrn16-114 transcript, which is the major in vivo 16Spre-rRNA transcript in photosynthetic plastids (Vera andSugiura, 1995), was <50% of that of the Prrn16-64 (NCII)–derived transcript. These results once again suggest thatNCII promoters are more efficiently utilized by this transcrip-tion system.

Identification of Sequences Required for Correct Transcription Initiation

Sequence alignments of the known NCII promoters revealedtwo regions of considerable similarity, hereafter referred toas box I (the sequence around the transcription start site)and box II (the sequence z35 bp upstream of the transcrip-tion start site; represented by a few examples in Figure 5).To analyze the importance of these two regions and to de-termine the sequences necessary for the nucleus-encodedmachinery to initiate transcription from NCII promoters, weconstructed a number of PatpB-290 promoter constructswith a series of deletion and substitution mutations (Figures6A and 7A) in the region upstream of the 2290 atpB tran-scription initiation site. Serial deletions from nucleotides2210 to 241 did not have any measurable adverse effect ontranscription from the PatpB-290 promoter; however, dele-tion of the region upstream of position 24 considerablyreduced its in vitro activity to negligible levels, thereby sug-gesting that the cis elements necessary for transcriptionfrom the PatpB-290 (NCII) promoter resided downstream ofthe 241 position (Figure 6B). Incidentally, this is the regionthat encompasses the conserved boxes I and II.

Figure 3. Determination of in Vitro Transcription Initiation Site andthe Effect of Transcription Inhibitors on Respective Transcription Ini-tiation from NCII (PatpB-290) and CT (PatpB-255) atpB Promoters.

(A) The atpB promoter construct (Figure 1C) was in vitro transcribedby using transcription extracts from BY-2 plastids (BEx) and leafchloroplasts of wild-type tobacco seedlings (CEx). The SKRP2primer extension products resulting from the relative transcriptionactivity of PatpB-290 and PatpB-255 promoters (marked as 2290and 2255, respectively) were analyzed on a sequencing gel along-side a sequence ladder (lanes T, G, C, and A), which was generatedby the same set of primer and template DNA. Tag, tagetitoxin.(B) The in vitro transcription reactions obtained by using BY-2 plas-tid extract and the atpB promoter construct were supplementedwith Tagetitoxin (Tag; 100 mM), rifampicin (Rif; 1 mg mL21), anda-amanitin (a-ama; 250 mg mL21). The bands corresponding to2290 and 2255 represent the relative amounts of in vitro–transcribedproducts resulting from PatpB-290 and PatpB-255, respectively.Numbers at left are the sizes (in nucleotides) of marker DNA fragments.

1804 The Plant Cell

To further analyze the specific sequences involved in NCIItranscription, we prepared seven (S1 to S7) 2290 atpBpromoter constructs with up to six A→T and C→G (exceptthe transcription initiation site at which T was substituted byC) base substitution mutations and analyzed them for invitro transcription activity (Figures 7A and 7B). Replacementof six of the most conserved base pairs (ATAGAA) in box II(Figure 7; construct S1 and lane S1) caused an z60% re-duction in the transcription activity of the PatpB-290 pro-moter. Similar severe losses of transcription activity wereobserved when changes in the transcription initiation site it-self (Figure 7, construct S5 and lane S5) or the adjacent up-stream region (nucleotides 29 to 211, included in box I)were made (Figure 7, construct S3 and lane S3). A 3-bpchange (nucleotides 12 to 14) immediately downstream ofthe transcription initiation site also affected transcription ef-ficiency, albeit slightly (z20%). Interestingly, substitution of

the 23 to 26 ATAG with TATC almost abolished transcrip-tion activity (Figure 7, construct S4 and lane S4). However,mutating three of the downstream AAT bases (Figure 7, con-struct S7 and lane S7) that were found to be fairly well con-served did not have any impact on the transcription activityfrom this promoter. The substitution S3 in the nonconservedregion (in between boxes I and II) also did not have any ef-fect on transcription activity (Figure 7B, lane S3). Taken to-gether, these results suggest that both conserved boxes Iand II are necessary components of the PatpB-290 promoterand that they are sufficient for accurate transcription.

As far as PatpB-255 is concerned, none of the mutationsin the upstream region of PatpB-290 affected its in vitrotranscription activity. However, a three-base change (AGA→TCT) corresponding to 12 to 14 of PatpB-290 consider-ably reduced the amount of in vitro–transcribed RNA fromPatpB-255. Incidentally, the PatpB-290 region between 11and 16 also represents the 235 region of PatpB-255 (a CTpromoter), which is known to be important for the efficiencyof CT promoters. Therefore, any disruption in this regionwould have affected the activity of the PatpB-255 promoter.

DISCUSSION

In this study, we characterize two important sequence ele-ments in PatpB-290 (an NCII promoter) by using an in vitrotranscription approach. To determine their significance, wefirst developed an in vitro transcription system using non-photosynthetic plastids of cultured tobacco BY-2 cells. Thissystem was tested for accuracy of transcription initiation byusing three NCII promoters, namely, PatpB-290, Prrn16-64,and Prpl32-1019. Analysis of a series of PatpB-290 59 dele-tion constructs suggested that sequences upstream of nu-cleotide 241 did not influence transcription initiation in vitro.Further characterization of this promoter by using base sub-stitutions in a 43-bp region (from nucleotides 235 to 18) re-vealed that two regions of high sequence similarity (box Iand box II), which have been observed in several NCII pro-moters (Kapoor et al., 1997; Miyagi et al., 1998; Hess andBörner, 1999), were in fact involved in the process of tran-scription initiation. Whereas most of the base changes inbox I and box II (S1, S3, and S5) caused an z40 to 60% de-crease, a 3-bp change from nucleotides 26 to 23 (S4) re-sulted in a severe reduction (z90%) in PatpB-290 promoteractivity. The latter region has been found to be fairly wellconserved among most of the NCII/NEP promoters identi-fied in tobacco (Hajdukiewicz et al., 1997; Kapoor et al.,1997; Hübschmann and Börner, 1998; Miyagi et al., 1998)and maize (Silhavy and Maliga, 1998).

Recently, in vitro characterization of the tobacco PrpoB-345 promoter element revealed that a CAT motif spanningnucleotides 28 to 26 is critical for transcription activity(Liere and Maliga, 1999). The CAT motif of the tobaccoPrpoB-345 promoter is well conserved in PatpB-290 (nucle-

Figure 4. In Vitro Transcription from Tobacco Prpl32-1019 (P2) andPrrn16-114 and Prrn16-64 (NCII) Promoters Along with That fromPatpB-290.

The genes are indicated at top; the respective in vivo transcript initi-ation sites are shown at right; sizes (in nucleotides) of marker DNAfragments are shown at left.

In Vitro Transcription from NCII Promoters 1805

otides 27 to 24). A severe reduction of PatpB-290 activitycaused by the replacement of three bases (ATA to TAT) inthis motif further validates its importance for NEP activity.This region also corresponds to a significant YRTA core se-quence motif that is found in most plant mitochondrial pro-moters (reviewed in Hess and Börner, 1999), suggesting thatmitochondrial RNA polymerase and the plastid NEP shareconsiderable similarity.

As far as minimal sequence requirements for transcriptioninitiation are concerned, PatpB-290 (described in this study)seems to differ from the recently characterized PrpoB-345promoter (Liere and Maliga, 1999). Our data suggest thatboth box I and box II are important for PatpB-290 activity,whereas, in the case of PrpoB-345, only a 15-bp sequence(nucleotides 214 to 11, equivalent to box I) was shown tobe sufficient for full promoter activity. Although there is agreat degree of similarity in the box I region of these twopromoters, PrpoB-345 completely lacks any region similarto box II. In addition to the tobacco PrpoB-345, PclpP-511(Hajdukiewicz et al., 1997) in tobacco and PrpoB in maize(Silhavy and Maliga, 1998) have also been found to lack se-quence conservation in the box II region. clpP-53 is an-other NEP-transcribed RNA that lacks both box I and box II(Sriraman et al., 1998). Most of the other NEP-utilized pro-moters identified to date, namely, tobacco Prrn16-64 (Veraand Sugiura, 1995; Allison et al., 1996), Prpl32-1019 (Vera etal., 1996), PatpI-208 (Hajdukiewicz et al., 1997; Miyagi et al.,1998), and PclpP-173 (Hajdukiewicz et al., 1997), containsequences similar to both box I and box II. Coincidentally,all of the genes containing box I– and box II–type NCII pro-moters are transcribed from multiple promoters, and at leastone of those promoters is a CT promoter.

When compared with nonphotosynthetic plastids, theabundance of transcript initiating from the box I– and box II–containing NCII promoters is greatly reduced in chloroplastsin which PEP takes over the major responsibility for plastidtranscription (Allison et al., 1996; Kapoor et al., 1997). Onthe other hand, promoters that lack either box II, namely,PrpoB-345, PclpP-511 (Silhavy and Maliga, 1998), andPaccD-129 (Hajdukiewicz et al., 1997), or both of the con-served boxes, for example, PclpP-53 (Sriraman et al., 1998),

have been found in the 59 regions of those genes for whicheither all or a major share of the respective transcript pool inchloroplasts is contributed by NEP-driven transcription. Thetobacco accD and rpoB genes have been shown to be tran-scribed from single promoters that are utilized by NEP(Hajdukiewicz et al., 1997; Liere and Maliga, 1999). In thecase of clpP, although one of the four promoters is a CT-uti-lized promoter (PclpP-95), a NEP-utilized promoter (PclpP-53) contributes significantly to the clpP transcript pool inchloroplasts (Hajdukiewicz et al., 1997). Therefore, to copewith the requirements of developing plastids, transcriptionfrom these promoters (which lack either one or both of theconserved boxes) might have to be regulated in a differentmanner than those sharing transcriptional responsibilitieswith CT promoters. In fact, rpoB transcription in barley hasbeen shown to increase severalfold before the remainder ofthe photosynthesis-related genes (Baumgartner et al., 1993).Based on the above-mentioned argument, we postulate thatthere might exist a correlation between the structure andregulation of NEP-utilized promoters.

The NCII-specific NEP activity described here has previ-ously been shown to be functionally (Allison et al., 1996;Kapoor et al., 1997) and structurally (Serino and Maliga,1998) distinct from the PEP. The first nonplastid-encodedRNA polymerase activity, however, was characterized fromspinach as a 110-kD single subunit activity (Lerbs-Mache,1993). This RNA polymerase was demonstrated to utilizephage T7 and a non-CT plastid (Prrn16PC) spinach pro-moter. We were, however, unable to detect any specifictranscription from either the T3 or the T7 promoter by usingtobacco BY-2 extracts (data not shown). Moreover, thespinach Prrn16PC promoter (utilized by the single subunitspinach RNA polymerase) does not show any significantsimilarity to the conserved regions of NCII promoters char-acterized thus far. In tobacco, clpP (encoding the catalyticsubunit of a Clp ATP-dependent protease) gene expressionalso has been characterized as NEP dependent. Becausethe clpP promoter (PclpP-53) does not exhibit any similarityto any other NEP-utilized (NCII) promoter, it has been cate-gorized separately and is believed to be transcribed by theNEP in conjunction with a specificity factor (Sriraman et al.,

Figure 5. Sequence Alignment of NCII Promoters.

Transcription initiation sites are marked with a bent arrow. Conserved motifs (box I and box II) are indicated. The sequence motif showing partial con-servation of the mitochondrial YRTA motif is underlined. The 39 A-rich region is shown in white letters on a black background. Dashes indicate noncon-served regions. Boldface letters represent the conserved nucleotides in box II. An AT-rich region in box I is shown in italic letters.

1806 The Plant Cell

1998). Paradoxically, the NEP machinery has been demon-strated to be fairly well conserved among dicots and mono-cots, exemplified by faithful transcription from maize rpoBand rice PclpP-111 promoters that was obtained by usingan in vitro (Liere and Maliga, 1999) and in vivo (Sriraman etal., 1998) approaches, respectively, with the tobacco NEP. It

Figure 6. In Vitro Transcription from a Series of Truncated atpBPromoter Constructs.

(A) Schematic representation of the deletion (D) mutations in theatpB promoter construct described in Figure 1C. The deleted por-tions are shown by broken lines. Two regions (box I and box II) ofhigh sequence conservation among known NCII promoter regionsare shown as I and II, respectively. The in vivo transcription initiationsites (2290 and 2255) are indicated with arrows; vector sequence(Seq.) is shown as black rectangles; open rectangles represent thetranscripts initiating from PatpB-290. WT, wild-type PatpB promoterwithout any modification.(B) Results of in vitro transcription reactions using deletion con-structs. The respective in vivo transcript initiation sites are displayedat right. The extract concentration (Conc.) control band in each lanerepresents a 192-bp 32P-labeled polymerase chain reaction (PCR)fragment that was mixed with the BY-2 in vitro transcription extractbefore its distribution to individual reactions, thereby creating an in-ternal standard for the amount of extract added to each reactionmixture. The bands corresponding to 2290 and 2255 represent therelative amounts of in vitro–transcribed products resulting fromPatpB-290 and PatpB-255, respectively. Numbers at left are thesizes (in nucleotides) of marker DNA fragments. An z130-nucleotideband (marked with an asterisk) that was detected occasionally mighthave resulted from nonspecific primer extension and/or transcrip-tion. WT, wild-type PatpB-290 promoter without any modification.

Figure 7. Significance of Sequences in the Conserved Regions ofthe PatpB-290 (NCII) Promoter for in Vitro Transcription.

(A) Seven constructs (S1 to S7) with base substitutions at severalpositions in the conserved (box I and box II) and nonconserved re-gions of the PatpB-290 promoter were prepared. The substitutionsin the PatpB-290 promoter sequence are marked by dashed lines.The conserved sequences are shown in boldface. The boxed se-quence in box I represents the 235 region of PatpB-255, a down-stream CT promoter.(B) Gel showing respective in vitro transcription initiation activity ofall the base substitution promoter constructs described in (A). Theextract concentration (Conc.) control band in each lane represents a192-bp 32P-labeled PCR fragment that was mixed with the BY-2 invitro transcription extract before its distribution to individual reac-tions, thereby creating an internal standard for the amount of extractadded into each reaction mixture. The bands corresponding to 2290and 2255 represent the relative amounts of in vitro–transcribedproducts resulting from PatpB-290 and PatpB-255, respectively.Numbers at left are the sizes (in nucleotides) of marker DNA frag-ments. WT, wild-type PatpB-290 promoter without any modification.

In Vitro Transcription from NCII Promoters 1807

would therefore be interesting to analyze whether the RNApolymerase utilizing spinach Prrn16PC and tobacco PclpP-53 promoters represents only a modified form of the NEP orconstitutes an altogether different polymerase.

A promising candidate for the gene encoding the plastidNEP has been recently identified from Arabidopsis (Hedtkeet al., 1997). This gene (RPOZ) has been reported to be fairlysimilar (55% identical residues) to its mitochondrial counter-part RPOY, and its 59 coding region resembles the se-quence of a plastid-targeting transit peptide. Most of theNCII/NEP promoters characterized thus far contain a coreYATA sequence that is very similar to the mitochondrial pro-moter core (YRTA) sequence, thus indicating that Arabidop-sis RPOZ could very well be the gene encoding the catalyticsubunit of NEP. This gene has been predicted to produce aprotein of 113 kD, which is very much in agreement with thesize ascribed to the spinach NEP activity (Lerbs-Mache,1993). To date, there is no experimental proof that RPOZ infact encodes plastid NEP. Once determined, it would be in-teresting to analyze how it relates to the NEP activities of to-bacco and spinach.

It certainly seems that the plastid transcription machineryis not as simple as it appeared to be a few years ago. Wehope that the in vitro transcription system described herewill help to answer at least some of the questions concern-ing the nucleus-encoded plastid-localized DNA-dependentRNA polymerase.

METHODS

Plant Materials

Tobacco (Nicotiana tabacum) BY-2 cells were grown in modifiedMurashige and Skoog medium (Wako Pure Chemicals, Osaka, Ja-pan), as described previously (Fan and Sugiura, 1995). The suspen-sion cultures were maintained by transferring aliquots to fresh medium(3:100) weekly and shaking at 130 rpm in the dark at 278C. Cells werecollected at mid-log phase (80 to 86 hr after inoculation) by passagethrough Miracloth (Calbiochem, San Diego, CA). For inhibition ofplastid protein synthesis, we supplemented the BY-2 culture mediumwith 200 mg mL21 (each) spectinomycin and streptomycin after 40 hrof growth in modified Murashige and Skoog medium alone. Tageti-toxin treatment was also given in a similar manner by adding thetoxin to a growing culture at a final concentration of 100 mM. Seed-lings of wild-type tobacco variety BY-4 were raised in a growthchamber (288C with continuous light) for 25 days.

Purification of BY-2 Plastids and Preparation of Plastid Transcription Extracts

Protoplast Preparation

Preparation of plastids from BY-2 cells, barring some minor modifi-cation, was essentially as described by Nemoto et al. (1988). BY-2

cells, which were collected from a 2-L suspension 80- to 84-hr cul-ture (z200 g fresh weight), were washed twice with 0.4 M mannitol,pH 5.0, and digested in 3 volumes of enzyme solution (1% Onozuka-RS cellulase [Yakult, Tokyo] and 0.1% pectolyase Y-23 [Kikkoman,Tokyo] in modified Murashige and Skoog medium containing 0.4 Mmannitol, pH 5.4) at 308C for 45 min (Nagata et al., 1981; Fan andSugiura, 1995). Protoplasts were harvested by centrifugation at 200gfor 5 min at 28C followed by three washes with 5 volumes each of ice-cold 0.4 M mannitol, pH 5.0. The pellet was suspended in plastid iso-lation buffer (0.4 M mannitol, 20 mM Tris-HCl, pH 7.6, 0.5 mM EDTA,1.2 mM spermidine, 7 mM b-mercaptoethanol, 0.6% [w/v] polyvi-nylpyrrolidone [PVP], and 0.1% [w/v] BSA).

Protoplast Lysis and Proplastid Purification

The protoplasts were broken by passing the suspension through twolayers of 20-mm nylon mesh under mild vacuum (changing the meshafter every 40 mL). The suspension of broken protoplasts was thencentrifuged at 250g for 5 min at 28C to pellet cell debris and nuclei.The supernatant was filtered through two layers of 10-mm nylonmesh under gravitational force. Percoll was added to the filtrate to afinal concentration of 15%. The proplastids were pelleted by centrif-ugation at 15,000g for 20 min at 28C and suspended in 4 volumes ofplastid isolation buffer. The proplastids were further purified from mi-tochondria and nuclear debris by sucrose density gradient centrifu-gation (30 to 50 to 70% sucrose in 20 mM Tris-HCl, pH 7.6, 0.5 mMEDTA, 1.2 mM spermidine, and 7 mM b-mercaptoethanol) at 1800gfor 40 min at 28C in a swinging bucket rotor. A yellow band of purifiedplastids that formed at the 50 to 70% sucrose interface was col-lected, diluted five times with plastid isolation buffer (minus PVP andBSA), and centrifuged at 4000g for 5 min at 28C.

Lysis of Proplastids

The resulting loose pellet was suspended in 1 volume of lysis buffer(50 mM Tricine-KOH, pH 8, 50 mM KCl, 0.5 mM EDTA, 0.5 mM DTT,0.5 mM phenylmethylsulphonyl fluoride, 1 mM benzamidine, 5 mMe-amino-n-caproic acid, and 5% [v/v] glycerol). The plastids were dis-rupted by sonication twice for 10 sec at power seven using a HandySonic sonicator (model UR-20P; Tomy, Tokyo, Japan) and centri-fuged at 50,000g for 30 min at 28C. The supernatant was dialyzedagainst 200 volumes of fresh lysis buffer for 3 hr at 28C. The resultingextract was divided into 30-mL aliquots, flash frozen in liquid nitro-gen, and stored at 2808C for up to 1 month. Typically, 500 mL ofplastid extract containing 20 to 25 mg mL21 protein was obtainedfrom 200 g of BY-2 cells. Chloroplast transcription extracts were pre-pared from 100 g of young expanded (3- to 6-cm) leaves. The chlo-roplasts were prepared according to Bartlett et al. (1982) and lysedas described by Orozco et al. (1986). The subsequent steps were thesame as described above for the BY-2 cells.

Determination of Mitochondrial Contamination

The procedure of Nemoto et al. (1988) for proplastid preparation wasexpected to yield extremely pure plastids from BY-2 cells. However,to be doubly sure of the extent of mitochondrial contamination, wesubjected aliquots (50-mg protein equivalent) from several stages ofplastid purification (protoplast preparation, protoplast lysis, pelletobtained after Percoll gradient centrifugation, and upper and lower

1808 The Plant Cell

bands obtained after sucrose density gradient centrifugation) to a cy-tochrome c oxidase assay (Orii and Okunuki, 1965). The final plastidpreparation was devoid of any detectable cytochrome c oxidase ac-tivity (data not shown).

Construction of DNA Templates

Unless indicated otherwise, all techniques used for manipulating nu-cleic acids were as described by Sambrook et al. (1989). The to-bacco atpB 59 upstream region from positions 2212 to 2479 relativeto the ATG codon of the ATP-synthase b subunit (positions 56,981 to57,248 of the tobacco chloroplast DNA) was polymerase chain reac-tion (PCR) amplified by using primers PA1 (GTATGGATCCATCTC-AAGTGGATGAATCAGAATCTTGAG) and PA2 (CAGATCTAGATA-CGGAATTCCTCTATGAATCTATGAAAGG), which contain BamHIand XbaI sites (underlined), respectively, using pTB27 from the to-bacco chloroplast clone bank as the template (Sugiura et al., 1986).The resulting fragment was digested with BamHI and XbaI andcloned at corresponding sites in pBluescript II SK1 (Stratagene, LaJolla, CA) and amplified in Escherichia coli XL-1 Blue cells (Strat-agene). The rrn16 and rpl32 promoter (corresponding to positions102,531 to 102,757 and 113,761 to 114,000 of tobacco chloroplastDNA, respectively) constructs were prepared in a similar manner, us-ing the same cloning sites as described above. The atpB promoter 59

serial deletion constructs were prepared by inverse PCR using ap-propriate primer pairs. The base substitution mutations in the pro-moter region were introduced by using the Transformer site-directedmutagenesis kit version 2 (Clontech, Palo Alto, CA). All of the con-structs were verified by sequence analysis. To be used as templates,all of the plasmids were isolated by using the alkaline lysis methodand purified by CsCl density gradient centrifugation.

In Vitro Transcription Reaction

A typical in vitro transcription reaction was performed at 308C for 40min in a 50-mL total volume, containing 50 mM Tris-HCl, pH 7.8, 100mM KCl, 20 mM MgCl2, 2 mM DTT, 10 units of RNase inhibitor, 0.5mM each nucleotide triphosphate (NTP), 42 nM template DNA, and60 mg of protein from the plastid extract. All of the components ex-cept for the NTPs were mixed and preincubated on ice for 10 min,and the reaction was initiated by adding 2 mL of a 12.5 mM solutionof each of the four NTPs. The reaction was stopped by extracting itwith an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1[v/v]). To the resulting aqueous phase, 0.1 pmol of a 59 32P-labeledprimer SKRP2 (59-TCGACGGTATCGATAAGCTTGATATC-39) com-plementary to the vector (pBluescript SK1) sequence (positions 675to 700) was added, and the nucleic acids were precipitated by add-ing 0.1 volume of 3 M sodium acetate and 2 volumes of ethanol. Afterwashing once with 70% ethanol, the pellet was air dried and dis-solved in 12.5 mL of reverse transcriptase buffer. Primer annealingwas performed by heating the template–primer mixture at 708C for 5min followed by a 5-min incubation at 658C and then gradually (38Cper min) cooling to 428C. The deoxynucleotide triphosphates (1 mMeach) and 10 units of avian myeloblastosis reverse transcriptasewere added while the reaction mixture was still at 428C, and primerextension was performed at the same temperature for 45 min. Forthe experiments shown in Figures 6 and 7, avian myeloblastosis re-verse transcriptase was replaced with ReverTra Ace (Moloney mu-rine leukemia virus reverse transcriptase without RNase H activity;

Toyobo Biochemicals, Osaka, Japan), and primer extension was per-formed at 508C for 45 min. After precipitating with 2 volumes of eth-anol, the DNA pellet was dissolved in 5 mL of loading buffer (25 mMTris-HCl, pH 8, 20 mM EDTA, pH 8, 0.5% bromophenol blue, 0.5%xylene cyanol, and 50% formamide). The samples were denatured at958C for 4 min and resolved on 6% denaturing polyacrylamide gels,and the results were analyzed by using a bioimage analyzer (modelBAS-2000; Fuji Photo Film Co., Tokyo).

During transcription analysis of deletion and substitution muta-tions (Figures 6 and 7), a 192-bp 32P-labeled PCR fragment wasmixed with the proplastid in vitro transcription extract before its dis-tribution to individual reactions, thereby creating an internal standardfor the amount of extract added to each reaction mixture. This frag-ment was prepared by PCR amplification of a pBluescript SK1 mul-tiple cloning site by using 32P-labeled T3 and M13 220 primersfollowed by gel purification (QIAquick gel purification kit; Qiagen,Chatsworth, CA).

ACKNOWLEDGMENTS

We thank Drs. Yoshinori Toyoshima, Mamoru Sugita, Hiroshi Takatsuji,Tetsuro Hirose, and Yasushi Yukawa for helpful discussions, Drs.Meenu Kapoor and Zbigniew Rybka for critical reading of the manu-script, and Dr. Naoki Takaya for help in experiments involvingcytochrome c oxidase estimation. This work was supported bygrants-in-aid from the Ministry of Education and Culture, Japan. S.K.was supported by a postdoctoral fellowship of the Japanese Societyfor the Promotion of Science.

Received April 12, 1999; accepted June 12, 1999.

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DOI 10.1105/tpc.11.9.1799 1999;11;1799-1810Plant Cell

Sanjay Kapoor and Masahiro SugiuraPromoter by Using Plastid Transcription Extracts from Cultured Tobacco BY-2 Cells

PlastidPatpB-290Identification of Two Essential Sequence Elements in the Nonconsensus Type II

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