analysis of the rolc promoter region lnvolved in somatic ... · tained the rolc promoter internal...

Plant Physiol. (1994) 104: 1151-1157

Analysis of the rolC Promoter Region lnvolved in Somatic Embryogenesis-Related Activation in Carrot Cell Cultures'

Nobuharu Fujii*, Ryusuke Yokoyama, and Hirofumi Uchimiya

lnstitute of Molecular and Cellular BioSciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (N.F., H.U.); and Department of Botany, Hokkaido University, Sapporo 060, Japan (R.Y.)

In cell cultures of carrot (Daucus carota l.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' non- coding sequence of the Ri plasmid rolC and a structural gene for bacterial 0-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low 0- glucuronidase (CUS) activity in calli and proembryogenic masses (PEM) and high CUS activity in heart- and torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Interna1 deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35s core region (-90 to +6 bp), it conferred relatively low CUS activity in calli and PEM but high CUS activity in heart and torpedo embryos. When -848- to -255-bp or -255- to -94- bp fragments of the rolC promoter were fused to the same CaMV 35s core region, CUS activity patterns were not related to somatic embryogenesis. These results suggest that the combination of sev- era1 regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures.

Dicotyledonous plants infected by the plant-pathogenic bacterium Agrobacterium rhizogenes produce adventitious roots (Riker et al., 1930; Moore et al., 1979; White et al., 1985). The induction of hairy roots is due to integration of a part of the Ri plasmid into the plant genome and subsequent expression of T-DNA-linked genes in plant cells (Chilton et al., 1982; White et al., 1982; Willmitzer et al., 1982; SpanÒ et al., 1982). Severa1 genes of the T-DNA are expressed in a cell-specific or developmentally regulated manner (Schmiill- ing et al., 1989; Sugaya et al., 1989; Capone et al., 1991; Chichiriccò et al., 1992). For instance, the rolC gene promoter is expressed in vascular tissues (Schmiilling et al., 1989; Sugaya et al., 1989) and seed embryos (Sugaya and Uchimiya, 1992).

This research was supported by Fellowships from the Japan Society for the Promotion of Science for Japanese Junior Scientists, by the Ministry of Education, Science and Culture of Japan, and by a grant from the Torey Science Foundation.

* Corresponding author; fax 81-3-3812-2910. 1151

Somatic embryogenesis in carrot (Daucus carota L.) cell- suspension cultures has become an attractive model system for the study of plant development (Steward et al., 1958). Carrot cells cultured in a medium supplemented with 2,4-D proliferate as unorganized cell clusters. Upon remova1 of 2,4- D from the culture medium, these cells undergo somatic embryogenesis through globular, heart, and torpedo stages. Numerous studies have been made to identify genes associated with somatic embryogenesis ( e g Borkird et al., 1988; Aleith and Richter, 1990; Hata et al., 1991; Kawahara et al., 1992). Nevertheless, little is known about the regulation of gene expression in which the activation of promoters is associated with somatic embryogenesis. We previously found that expression of the uidA gene under the control of the rolC promoter was increased during somatic embryogenesis in transgenic carrot cell cultures (Fujii and Uchimiya, 1991). In the present paper we report the findings of our investigation of the precise association between rolC-uidA expression and the developmental stages of somatic embryos. Additionally, we report on our evaluation of the cis-acting regions of the rolC promoter that are required for SERA.

MATERIALS AND METHODS

Plasmid Construction

The methods used to construct the plasmid pBI101-012- GUS and 5' deletions of the rolC promoter have been described previously (Sugaya et al., 1989; Sugaya and Uchi- miya, 1992). Interna1 deletions of the rolC promoter were produced as follows. A fragment (HindIII/BamHI) containing the 5' upstream region of rolC in pBR328 (Oono et al., 1987) was inserted into the BamHIIHindIII site of pUC12. Using this plasmid pUC12-012-HB as a template, the DNA fragment containing the rolC promoter (-848 to -255 bp) was amplified using M13 primer and -255/-271 primer by PCR. The amplified DNA fragment was digested with HindIII, followed by successive treatments with T4 DNA polymerase and T4 polynucleotide kinase. This DNA fragment was inserted into a HincII site of pUC19. The presence of the expected DNA sequences was confirmed by dideoxy sequencing (Sanger et al., 1977). DNA fragments (blunt end/EcoRI) containing deleted upstream-region series (-255, -230,

Abbreviations: CaMV, cauliflower mosaic virus; GUS, 0-glucuronidase encoded by the uidA gene; MS, Murashige-Skoog; 4-MU, 4- methyl-umbelliferyl-P-D-glucuronide; PEM, proembryogenic masses; SEM, somatic-embryogenesis-related activation.

https://plantphysiol.orgDownloaded on January 21, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://plantphysiol.org

1152 Fujii et al. Plant Physiol. Vol. 104, 1994

-192, -134, -94, and +2 bp) of the rolC gene and the codingregion of the uidA gene (Jefferson, 1987; Jefferson et al.,1987) were inserted into the Smal/EcoRl site of the plasmid,pUC-848/-255. DNA fragments (Hmdlll/EcoRI) that con-tained the rolC promoter internal deletions (A—255/—255,A-255/-230, A-255/-192, A-255/-135, A-255/-94, andA—25S/+2) and the uidA gene were inserted into the Hindlll/EcoRI site of pBIN19.

An EcoRV/BomHI fragment that contained the CaMV 35Score region (—90 bp) and the BamHI/EcoRI fragment thatcontained the uidA gene and the nos terminator of pBI221(Jefferson, 1987) were inserted into the HmcII/EcoRI site ofpIBISl, thus creating a 35S core region-uidA gene. Thisplasmid, pUC12-O12-pl, and the deleted -255-bp upstreamregion of rolC-uidA in pUC12 were digested with Avail andtreated with Klenow polymerase, followed by digestion withHindlll, which created rolC upstream fragments, the —848/-94 region, and the -2S5/-94 region, respectively. A frag-ment of the rolC upstream region (—848/—255) was obtainedby Hmdlll and Smal digestion of pUC-848/-255. Each ofthese fragments of the rolC upstream region (Hindlll/bluntend) was cloned in the H;'ndIII/filled-in Xhol site of thepIBI31-35S core region-MidA gene. Subsequently, each het-erologous promoter-widA gene (Hi'rtdlll/EcoRI) was insertedinto the Hindlll/EcoRI site of pBIN19.

Establishment of Transformed Cells

Constructed vectors were transferred into Agrobacteriumtumefaciens strain LBA4404 by a freeze-thaw method (Hols-ters et al., 1978). One-week-old hypocotyl segments of carrot(Daucus carata L. cv US-Harumakigosun) were transformedwith A. tumefaciens as described previously (Fujii and Uchi-miya, 1991). Independently obtained transgenic callus fromeach hypocotyl was cultured separately in MS liquid medium(Murashige and Skoog, 1962) containing 1 mg/L 2,4-D, 100mg/L kamamycin sulfate, and 500 mg/L Claforan and usedas the different transgenic cell group.

Cell Cultures

Cell cultures selected for kanamycin resistance were main-tained in MS liquid medium containing 0.1 mg/L of 2,4-D.Somatic embryos were separated into different stages accord-ing to the method of Giuliano et al. (1983) with minormodifications. Three-week-old cells that passed through a106-/im mesh, but that were retained on a 37-^m mesh, werewashed with MS hormone-free liquid medium. To inducesomatic embryogenesis, cells suspended in the same mediumwere transferred into MS hormone-free medium (1 mLpacked cell volume/L medium). After 1 week, cells that

Call!(MS plus 2,4-D)

PEM fraction(106-38nm)

1 week(MS minus 2,4-D)

IFraction180-63nm

Globular fraction(150-63nm)

Subculture to freshMS minus 2,4-D medium

2 days Heart fraction•(250-180nm)

6 days

. Torpedo fraction(355-300nm)

Figure 1. Illustration of the fractionation of morphologically different cell types of transgenic carrot cells possessing therolC-uidA fusion gene. The different stages (i.e. globular, heart, and torpedo) can be separated as described in "Materialsand Methods." a, Globular embryos in the 63- to 150-^m fraction; b, heart embryos in the 180- to 250-^m fraction; c,torpedo embryos in the 300- to 355-ium fraction. Cells treated with 5-bromo-4-chloro-3-indoyl-/3-D-glucuronic acid areshown on the right.



rolC Promoter in Somatic Embryogenesis 1153

passed through a 150-pm mesh, but that were collected on a 63-pm mesh, were obtained. After sedimentation in the medium, the supematant was discarded. This fraction consisted primarily of the globular stage. To obtain heart and torpedo embryos, the fraction that passed through a 180-pm mesh, but that was retained on a 63-pm mesh, was allowed to settle, then was washed and transferred to fresh MS medium. Two days later, the fraction that passed through a 250-pm mesh, but that was retained on a 180-gm mesh (heart-stage embryos), was collected. Six days later, the fraction that passed through a 355-pm mesh, but that was retained on a 300-pm mesh (torpedo-stage embryos), was also collected. These fractions were allowed to settle and were washed twice. The globular, heart, and torpedo embryo fractions that we' obtained are shown in Figure 1. For 5'-deletion analysis of the rolC promoter, cells that passed through a 63-pm mesh, but not through a 37-pm mesh, were resuspended and washed with MS hormone-free liquid medium. The washed cells were maintained in medium (1 mL packed cell volume/L medium) with or without 2,4-D for 12 d.

CUS Assay

GUS enzyme activity of the transformants was assayed as described by Jefferson (1987). Briefly, cultured cells were homogenized in a lysis buffer containing 50 m~ NaP04 (pH 7.0), 1 mM EDTA, 0.1% Sarkosyl, 0.1% Triton X-100, and 10 m~ P-mercaptoethanol. After centrifugation (7500g, 5 min), the supernatant was assayed for GUS activity. Protein con- centrations were determined by the method of Bradford (1976) with a kit supplied by Bio-Rad Laboratories. Samples were incubated in lysis buffer containing 1 mM 4-MU at 37OC for 30 min. Reactions were stopped with 0.2 M Na2C03. The fluorescence of the 4-methylumbelliferone product was measured with excitation at 365 nm and emission at 455 nm. For histochemical staining, cultured cells were fixed in a solution of 0.3% formaldehyde, 10 mM Mes, pH 5.6, and 0.3 M mannitol for 20 min at room temperature, followed by incubation overnight with 1 mM 5-bromo-4-chloro-3-indolyl- P-glucuronide in 50 mM NaH2P04 (pH 7.0) at 37OC.

RESULTS Activation of the rolC Promoter-uidA Cene Expression during Somatic Embryogenesis

We previously showed that the expression of the rolC-uidA fusion gene was activated during somatic embryogenesis of transgenic carrot cell cultures (Fujii and Uchimiya, 1991). However, we did not determine GUS activity in fractionated cells corresponding to the embryo stages. The different stages ( e g globular, heart, and torpedo embryos) can be separated using an appropriate sieving method (Fig. 1). As shown in Figure 2A, significantly higher GUS activity was found in heart and torpedo embryos than in calli and PEM. The GUS activity was increased with the progress of somatic embryogenesis from PEM to globular embryos and from globular to heart embryos (each GUS activity was significantly different; F > 0.01). When harvesting heart embryos (collected with 250- and 180-pm meshes), we also obtained globular embryos from a smaller fraction (collected with 150- and 63-pm meshes). Similarly, when harvesting torpedo embryos (col-

lected with 355- and 300-pm meshes), globular and heart embryos were obtained. To investigate whether activation of the rolC promoter depends on the duration of culture in somatic embryo induction medium (-2,4-D) or on the proc- esses of somatic embryo development, we compared the GUS activities in the embryos described above. Figure 2B shows that the GUS activity in globular embryos was fairly constant at each harvest time. In contrast, GUS activities in heart and torpedo embryos were higher than those in globular embryos at each harvest time. These results indicate that the activation of the rolC promoter is dependent on the stage of somatic embryo development.

5' Deletion Analysis of rolC Promoter

To determine the regulatory region in the rolC promoter that is responsible for SERA, chimeric genes consisting of deleted fragments of the rolC 5' flanking region and the uidA structural gene were introduced into carrot cells. Using transformed cells, GUS activities in calli (+2,4-D) and somatic embryos (-2,4-D) were measured. In both cases, 12-d-old samples were analyzed. When the rolC 5' upstream region

A

4 'C .- 200 A T

B

T i

Globular Heart Torpedo

Hsrvest Time

Figure 2. A, Comparison of GUS activities in different cell types of transgenic carrot cells possessing the ro/C-uidA fusion gene. Exper- iments were repeated at least three times using two independently obtained transgenic cell groups. GUS activity in the heart embryos (2161 & 1163 [mean & SE] pmol 4-MU min-' mg-' protein) was defined as 100%. Lines at t h e end of each bar represent the SE. B, Comparison of GUS activities at various harvest times. GUS activities at different embryonic stages (globular, heart, and torpedo) were measured at various harvest times for globular, heart, and torpedo embryos (details can been found in the text). Experiments were repeated at least three times using one transgenic cell group. GUS activity in the heart embryos (2266 & 1487 pmol 4-MU min-' mg-' protein) was defined as 100%. Lines at the end of each bar represent the SE.




Figure 3. Analysis of CUS activities of 5' deletions of the rolC promoter in transgenic carrot cells. The numbers indicate deletion end points in nucleotides from the transcriptional start site (Kanaya et al., 1990). CUS activity (pmol 4-MU min-' mg-' protein) was measured in calli and somatic embryos cultured for 12 d as described in "Materials and Methods."

was deleted from -848 to -255 bp, GUS activities decreased gradually in somatic embryos (Fig. 3). At each deletion, GUS activities were much lower in calli than in somatic embryos. Further deletions from -230 bp resulted in significantly low GUS activities.

lnternal Deletion Analysis

The 5' deletion analysis suggested that the region downstream from -255 bp is involved in the regulation of SERA. It was unclear whether the deletion up to -230 bp involved deletion of the regulatory region of SERA or loss of the enhancer element. Therefore, we canied out intemal deletion analysis, in which the upstream region (-848 to -255 bp) was ligated to respective deletions in the -255- to +2-bp region (Fig. 4A). Using transgenic calli that expressed GUS activity and had the potential for somatic embryo formation, GUS activity was measured at the various developmental stages (as shown jn Fig. 2A). The results indicate that GUS activity begins to increase at either the globular stage or the heart stage (Fig. 4, B-G). A11 intemal deletions resulted in

relatively low GUS activity in calli and PEM, whereas high activity was detected in heart and torpedo embryos.

Heterologous Promoter Fusion Analysis

The results of the deletion analysis prompted 11s to investigate whether the regulatory elements associated with somatic ernbryogenesis exist from +2 bp to the 3' region or whether they are present in at least two regions, namely from -255 bp to the 5' region and from -255 bp to the 3' region. To answer this question, we constructed fusion promoters consisting of the CaMV 35s core region and severa1 DNA fragments of the rolC promoter (Fig. 5A). The CaMV 35s core region (-90 to +6 bp) contains the as-2 site and a TATA box (Larn et al., 1989). Chimeric genes shown in Figure 5A were tramferred into carrot cells, and GUS activities were analyzed at different stages of somatic embryogenesis. When the 35s core region alone was fused to the uidA gene, GUS activities in calli and PEM were not significantly different in embryos (Fig. 5E). In contrast, when the rolC promoter fragment (-848 to -94 bp) was fused to the 35s core region, it conferred relatively low GUS activity in calli and PEM but high GUS activities in heart and torpedo embryos (Fig. 5B). Thus, the regulatory region for SERA is not distrilmted solely in the 3' region from -94 bp in the rolC promoter. When -848- to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the 355 core region, thc pattem of GUS acíivity was changed. Namely, GUS activtties in calli and PEld were higher than (or equal to) those Iobserved in embryos (Fig. 5, C and D).

DISCUSSION

We previously demonstrated that the rolC prornoter (-848 to +23 bp) fused to the uidA reporter gene confer:j an expression pattem parallel to somatic embryogenesis in the carrot (Fujii and Uchimiya, 1991). In the present report, using fractionated embryo cells, we showed that the highest GUS activity .was associated with the heart and torpedo stages. To

Figure 4. Comparison of CUS activities in different cell types of transgenic carrot cells pos- sessina internal deletions of the rolC promoter. A A, Schkmatic diagram illustrating t h e series of internal deletions of the rolC promoter. The numbers indicate the distance of the end points of the rolC promoter fragments in nucleotides from the transcriptional start site. A-Control indicates athe control construct containing a polylinker site at -255 bp. B to C, The relative GUS activity obtained from each construct. lnternal deletions of the rolC promoter are as follows: A-Control (B), A-255/-230 (C),

(F), and A-255/+2 (C). Experiments were repeated at least three times. CUS activity in the heart embryos was defined as 100'70, which corresponded to 705 f 68 (B), 731 f 100 (C) , 2246 f 228 (D), 1548 -+. 60 (E), 7548 f 1354 (F), and 2930 f 1130 (C) pmol 4-MU min-' mg-' protein.

A-255/-192 (D), A-255/-134 (E), A-255/-94



ro/C Promoter in Somatic Embryogenesis 1155

Figure 5. Comparison of GUS activities in different cell types of transgenic carrot cells possessing the ro/C/35S fusion promoter. A, Schematic diagram illustrating the series of constructs of the ro/C/35S fusion promoter. The ro/C promoter fragments are represented by black lihes, and the numbers indicate the distance of the end points of the ro/C promoter in nucleotides from the transcriptional start site. The CaMV 35s core region (-90/+6) i s shown as a hatched box. B to E, The relative GUS activity obtained from each of the constructs. The ro/C/35S fusion promoters are as follows: -848 to -94/35S core (B), -848 to -255/35S core (C), -255 to -94/35S core (D), and 35s core region (E). Experiments were repeated at least three times. GUS activity in the heart embryos was defined as 100%, which corresponded to 376 & 52 (B), 452 f 37 (C), 294 * 39 (D), and 258 2 13 (E) pmol 4-MU min-’ mg-’ protein.

determine which cis elements are required for gene activation in somatic embryogenesis, we performed a deletion analysis of the rolC promoter and performed heterologous promoter fusion experiments.

Figure 6 summarizes the rolC promoter constructs and the activation of GUS associated with somatic embryogenesis. The rolC promoter can be divided into three regions containing putative cis regulatory elements. These proposed regions can be referred to as region I (-848 to -255 bp), region I1 (-255 to -94 bp), and region I11 (-94 to +23 bp) (Fig. 6). The replacement of region I11 in the -848-bp rolC promoter by the 35s core region did not diminish SERA (Fig. 5B), indicating that region 111 alone does not control gene activation. However, with the -255-bp 5‘ deletion and the intemal deletion (A-848 to -94 bp), substitction of the 355 core region for region I11 changed the expression pattem (Fig. 5, C and D). This phenomenon may be interpreted to mean that the combination of either region I or I1 with the 355 core region conferred a new expression pattem or that such constructs removed callus/PEM repressors.

On the other hand, retention of region I11 and delction of either region I or I1 retained SERA, which suggests that regions I and I1 may be functionally compensatory with regard to SERA. If so, then the combination of regions I and I1 may synergistically confer SERA on the 35s core region. Interaction and synergism between regulatory elements have been observed in severa1 plant promoters; for example, in the developmental and tissue-specific expression pattem of the 35s promoter (Benfey et al., 1990a, 1990b) and in the virus and salicylate inducibility of the PR-lu promoter (van de Rhee and Bol, 1993).

The intemal deletion of the rolC promoter from -255 to +2 bp, which lacks CAAT- and TATA-box sequences, conferred uidA gene expression (Fig. 4G). Similarly, van de Rhee and Bol (1993) showed that fusion of the double-enhancer sequence of the 35s promoter directly to the uidA gene can

+

+ I

+ I

+ I

Figure 6. Schematic presentation of the ro/C promoter constructs and activation of uidA gene expression associated with somatic embryogenesis in carrot transformed with these constructs. Num- bers represent regions containing putative regulatory elements in- dicated by circles and the square. SERA: +, GUS activity in somatic embryos was higher than that in calli; -, GUS activity in somatic embryos was equal to or less than that in calli.




confer high-leve1 expression in the absence of CAAT- and TATA-box sequences.

In the region from -94 to +23 bp o f the rolC gene, as well as in typical eukaryotic regulatory sequences such as the CAAT and TATA boxes, AT-1-box-like sequences can be found (-77AATATTTTTATT-66). The AT-1 box, AATATTT- TATT, is a series of consensus sequences within the upstream region of light-regulated genes (Datta and Cashmore, 1989). This sequence is homologous to an A/T-rich motif, AA- TATTTTAAT, that is found in the 5' upstream region of the ,f3-phaseolin gene that binds to nuclear extracts from imma- ture bean cotyledons (Bustos e t al., 1989). We previously demonstrated that a n AT-1-like sequence in the rolC promoter is capable of binding to nuclear proteins from tobacco hairy root and wheat germ (Kanaya et al., 1990, 1991).

When the upstream region and the downstream region from -255 bp are compared, severa1 AT-rich regions are present. In addition, both regions contain G-box-like sequences (-429TACGTG-424 and -222CACGTG-217). G-box motifs, or related motifs with a n ACGT tetranucleotide core, have been identified in the promoters of various genes, including those that respond to visible and UV light (Schulze- Lefert et al., 1989), ABA (Guiltinan et al., 1990), wounding (Rosahl e t al., 1986), and anaerobiosis (DeLisle and Ferl, 1990). Salinas et al. (1992) showed that tetramers of the G- box-related motif GTACGTGGCG conferred regulated expression in developing seeds. Studies of the function of the G-box-related motif during somatic embryogenesis are cur- rently in progress.

ACKNOWLEDCMENT

We thank E.T. Aspuria for providing critica1 reading of the manuscript .

Received October 1, 1993; accepted January 4, 1994. Copyright Clearance Center: 0032-0889/94/104/1151/07.

LITERATURE ClTED

Aleith F, Richter G (1990) Gene expression during induction of somatic embryogenesis in carrot cell suspensions. Planta 183

Benfey PN, Ren L, Chua NH (1990a) Tissue-specific expression from CaMV 35s enhancer subdomains in early stages of plant development. EMBO J 9 1677-1684

Benfey PN, Ren L, Chua NH (1990b) Combinatorial and synergistic properties of CaMV 35s enhancer subdomains. EMBO J 9

Borkird C, Choi JH, Jin ZH, Franz G; Hatzopoulos P, Chorneau R, Bonas U, Pelegri F, Sung ZR (1988) Developmental regulation of embryonic genes in plants. Proc Natl Acad Sd USA 8 5 6399-6403

Bradford MM (1976) A rapid and sensitive method for the quanti- tation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana1 Biochem 7 2 248-254

Bustos MM, Guiltinan MJ, Jordano J, Begum D, Kalkan FA, Hall TC (1989) Regulation of 8-glucuronidase expression in transgenic tobacco plants by A/T-rich, cis-acting sequence found upstream of a French bean P-phaseolin gene. Plant Cell 1: 839-853

Capone I, Cardarelli M, Mariotti D, Pomponi M, De Paolis A, Costantino P (1991) Different promoter regions control leve1 and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants. Plant Mo1 Biol 1 6 427-436

Chichiriccò G, Costantino P, SpanÒ L (1992) Expression of the rolB

17-24

1685-1696

oncogene from Agrobacterium rhizogenes during zygotic embryogenesis, in tobacco. Plant Cell Physiol 3 3 827-832

Chilton MD, Tepfer DA, Petit A, David C, Cassse-Delbart F, Tempé, J (1982) Agrobacterium rhizogenes inserts T-DNA into the genome of host plant root cells. Nature 295 432-434

Datta N, Cashmore AR (1989) Binding of a pea nuclear protein to promoíiers of certain photoregulated genes is modula.ted by phos- phoryliation. Plant Cell 1: 1069-1077

DeLisle AJ, Ferl RJ (1990) Characterization of the Anzbidopsis Adh G-box binding factor. Plant Cell2 547-557

Fujii N, Uchimiya H (1991) Conditions favorable foi: the somatic embryogenesis in carrot cell culture enhance expression of the rolC promoiter-GUS fusion gene. Plant Physiol95 238-241

Giulianol G, Rosellini D, Terzi M (1983) A new method for the purification of the different stages of carrot embryoids. Plant Cell Rep 2 216-218

Guiltinan MJ, Marcotte WR, Quatrano RS (1990) A plant leucine zipper protein that recognizes an abscisic acid respcinse element. Sdence 2 5 0 267-271

Hata S, Kouchi H, Suzuka I, Ishii T (1991) Isolation and characterization of cDNAclones for plant cyclins. EMBO J 1 0 2681-2688

Holsters M, de Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978) Transfection and transformation of A. tumefaciens. EAol Gen Genet 163 181-187

Jeffersoni RA (1987) Assaying chimeric genes in plaiits: the GUS gene fusion system. Plant Mo1 Biol Rep 5 387-405

Jeffersoni RA, Kavanagh TA, Bevan MW (1987) GLS fusions: p- glucuronidase as a sensitive and versatile gene marker in higher plants. EMBO J 6: 3901-3907

Kanaya IK, Hayakawa K, Uchimiya H (1991) In vitro binding of wheat-germ proteins to the 5'-upstream region of thc rolC gene of Ri plasmid. Plant Cell Physiol32 295-297

Kanaya K, Tabata T, Iwabuti M, Uchimiya H (1'990) Specific binding of nuclear protein from tobacco hairy roots cultured in vitro to a 5'-upstream region of the rolC gene of th2 Ri plasmid. Plant Cell Physiol 31: 941-946

Kawahara R, Sunabori S, Fukuda H, Komamine A ('1992) A gene expressed preferentially in the globular stage of somatic embryogenesis, encodes elongation-factor 1 alpha in carrot. Eiur J Biochem

Lam E, Benfey PN, Gilmartin PM, Fang RX, Chua NII (1989) Site- specific mutations alter in vitro factor binding and change promoter expreseion pattem in transgenic plants. Proc Natl A.cad Sci USA

Moore L,, Warren G, Strobel G (1979) Involvement of plasmid in the hairy root disease of plants caused by Agrobacteriiim rhizogenes. Plasmid 2 617-626

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 1 5 473-497

Oono Y, Handa T, Kanaya K, Uchimiya H (1987) 'fie TL-DNA gene of Ri plasmids responsible for dwarfness of tobacco plants. Jpn J Genet 6 2 501-505

Riker A], Banfield WM, Wright WH, Keitt GW, Sagen HE (1930) Studies on infection of hairy root on nursery apple tree. J Agric Res 41: 507-540

Rosahl S, Schmidt R, Schell J, Willmitzer L (1986) Isolation and characterization of a gene from Solanum tuberosum encoding pa- tatin, the major storage protein of potato tubers. Mo1 Gen Genet

Salinas J, Oedo K, Chua NH (1992) Two G-box-related sequences confer different expression pattems in transgenic tobacco. Plant Cell4 1485-1493

Sanger E:, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Cci USA 7 4

Schmidling T, Schell J, Spena A (1989) Promoters of the rolA, B, and C genes of Agrobacterium rhizogenes are differentially regulated in tramgenic plants. Plant Cell 1: 665-670

Schulze-Lefert P, Dangl JL, Becker-Andre M, Hanhbrock K, Schulz: W (1989) Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene. EMBO J 8: 651-656

SpanÒ L, Pomponi M, Costantino P, Van Slogteren GMS, Tempé

209 157-162

86: 789'0-7894

203 214-220

5463-5467



rolC Promoter in Somatic Embryogenesis 1157

J (1982) Identification of T-DNA in the root inducing plasmid of the agropine type Agrobacterium rhizogenes 186. Plant Mo1 Biol 1:

Steward FC, Mapes MO, Mears K (1958) Growth and organized development of cultured cells. 11. Organization in cultures grown from freely suspended cells. Am J Bot 4 5 705-708

Sugaya S, Hayakawa K, Handa T, Uchimiya H (1989) Cell-specific expression of the rolC gene of the TL-DNA of Ri plasmid in transgenic tobacco plants. Plant Cell Physiol 3 0 649-653

Sugaya S, Uchimiya H (1992) Deletion analysis of the 5’-upstream region of the Agrobacterium rhizogenes Ri plasmid rolC gene required for tissue-specific expression. Plant Physiol 9 9 464-467

van de Rhee MD, Bol JF (1993) Induction of the tobacco PR-Za gene

291-300

by virus infection and salicylate treatment involves an interaction between multiple regulatory elements. Plant J 3 71-82

White FF, Ghidossi G, Gordon MP, Nester EW (1982) Tumor induction by Agrobacterium rhizogenes involves the transfer of plasmid DNA in the plant genome. Proc Natl Acad Sa USA 7 9

White FF, Talor BH, Huffman GA, Gordon MP, Nester EW (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol

Willmitzer L, Sanchez-Serrano J, Buschfeld E, Schell J (1982) DNA from Agrobacterium rhizogenes is transferred to and expressed in axenic hairy root plant tissues. Mo1 Gen Genet 186 16-22

3193-3197

164: 33-44



analysis of the rolc promoter region lnvolved in somatic ... · tained the rolc promoter internal...

Documents