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First published online October 13, 2006; 10.1104/pp.106.089169 Plant Physiology 142:1427-1441 (2006) © 2006 American Society of Plant Biologists Common Plantain. A Collection of Expressed Sequence Tags from Vascular Tissue and a Simple and Efficient Transformation Method1Molekulare Pflanzenphysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, D–91058 Erlangen, Germany (B.P., I.B., M.N., S.K., F.K., N.S.); and Syngenta Biotechnology, Inc., Research Triangle Park, Raleigh, North Carolina 27709 (J.S., R.A.D., R.J.M.)
The vascular tissue of higher plants consists of specialized cells that differ from all other cells with respect to their shape and size, their organellar composition, their extracellular matrix, the type of their plasmodesmata, and their physiological functions. Intact and pure vascular tissue can be isolated easily and rapidly from leaf blades of common plantain (Plantago major), a plant that has been used repeatedly for molecular studies of phloem transport. Here, we present a transcriptome analysis based on 5,900 expressed sequence tags (ESTs) and 3,247 independent mRNAs from the Plantago vasculature. The vascular specificity of these ESTs was confirmed by the identification of well-known phloem or xylem marker genes. Moreover, reverse transcription-polymerase chain reaction, macroarray, and northern analyses revealed genes and metabolic pathways that had previously not been described to be vascular specific. Moreover, common plantain transformation was established and used to confirm the vascular specificity of a Plantago promoter-  -glucuronidase construct in transgenic Plantago plants. Eventually, the applicability and usefulness of the obtained data were also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis (Arabidopsis thaliana) homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis as well. The presented vascular ESTs and the newly developed transformation system represent an important tool for future studies of functional genomics in the common plantain vasculature.
Certain cells or tissues are difficult to isolate from Arabidopsis (Arabidopsis thaliana) with classical techniques and, therefore, transcriptome or proteome analyses in these cells or tissues cannot be performed easily. One example of rather inaccessible tissue is the leaf vasculature. In an average Arabidopsis leaf blade, it represents less then 3% of the cell mass and its mechanical isolation is impossible because the strands are built by only few and, typically, tiny cells. The diameter of a companion cell (CC) or a sieve element (SE) can be 3 µm or less.
For several years, the laser microdissection and pressure-catapulting technique (Bonner et al., 1997
In the same article (Ivashikina et al., 2003
Several authors (Zhao et al., 2000
A different approach was used by Vilaine et al. (2003)
An entirely different and very elegant approach is the direct isolation of proteins from phloem sap. This technique was used by Balachandran et al. (1997)
Despite these different approaches, the total number of ESTs, especially from the phloem, is limited. Moreover, many of the plants that were used to collect phloem or vascular EST data (e.g. celery) are difficult or impossible to transform and EST information on these species can, therefore, only be used for other, heterologous plant systems. The largest dataset from a single plant is the collection of 989 ESTs from celery petioles (Vilaine et al., 2003
In our lab, common plantain (Plantago major) has been used repeatedly for analyses of SE or CC transporters (Gahrtz et al., 1994 Here, we describe a representative vascular transcript profile for Plantago, based on analysis of 5,900 EST sequences that represent 3,247 different mRNAs. The specificity of the library is confirmed by the identification of ESTs of genes previously identified in Plantago CCs, by the high expression levels of genes known to be vascular specific in other plants, by comparing expression levels in vascular versus nonvascular tissue using northern, reverse transcription (RT)-PCR and macroarray analyses, and in transgenic Plantago. Moreover, tissue specificity of promoters from Arabidopsis homologs of predicted vascular Plantago genes was analyzed and shown to confer vascular specificity in Arabidopsis. This result demonstrates that information obtained in Plantago may be transferred to other plant systems. All common plantain vascular EST sequence data are accessible via the Internet (http://www.plantain.de). Finally, a transformation technique is presented for common plantain. Together, these data provide the basis to perform functional genomics in Plantago, a species that differs significantly in vascular architecture and function from the model plant Arabidopsis.
Characterization of the Plantago Vascular EST Library
Vascular bundles (up to 10 cm and longer) are easily and rapidly pulled out from common plantain leaves and petioles due to the presence of an endodermis that surrounds the entire vascular tissue. The Casparian stripes within this endodermis rupture during extraction of the bundles (Gahrtz et al., 1994
A size-fractionated cDNA library from Plantago vascular tissue was generated in Of the obtained 5' sequences, 5,900 were used for further analyses; 40.1% of these sequences (2,347 ESTs) represented singlets and 59.9% (3,553 sequences) were found in two or more copies (maximum 150 copies). Overlapping and redundant sequences were assembled to 900 contigs with an average of 3.8 ESTs per contig, yielding a total of 3,247 independent Plantago vascular mRNAs. These sequences were characterized using BLASTX and BLASTN similarity searches in publicly available data libraries. With a threshold E value of 10–5, significant similarity matches were found for 55% of these sequences. Based on the predicted function of the encoded proteins, mRNAs with similarity to already known sequences were assigned to 11 functional groups. mRNAs for proteins with no functional predictions were divided into two groups that were named functionally uncharacterized proteins, for mRNAs giving similarity matches with functionally uncharacterized proteins in another organism, or novel genes, if no similarity matches were found. In Figure 1A , the percentage of independent mRNAs in each of the 13 groups is presented.
Of the 5,900 EST sequences, 26.4% (34.1% of the independent mRNAs) represent novel genes, making this the largest of the 13 groups shown in Figure 1A. The second largest group (18.9% of all ESTs and 20.9% of the independent mRNAs) is the functionally uncharacterized proteins group. The largest groups with known functions are the metabolism group, with 12.5% of all ESTs and the protein synthesis, turnover, and sorting group, with 10.9% of all ESTs. The smallest groups are the developmental and cell division control group, with 1.3% of all ESTs and the energy generation and photosynthesis group, with 1.5% of all ESTs. This distribution is expected because the corresponding genes are known to be either highly expressed (e.g. the protein synthesis, turnover, and sorting genes and many of the metabolism genes) or they are expected to have either low expression levels (developmental and cell division control genes) or expressed mainly in nonvascular tissue (e.g. the photosynthetic genes). The residual groups comprise between 2.6% (transport facilitators, channels, and pumps) and 8.8% (cellular response to hormones and stress) of all ESTs. The relatively high number of ESTs in the cellular response to hormones and stress group does not result from lengthy tissue sampling. The isolation of vascular tissue from Plantago leaves is too short (<5 s) to allow induction of stress-related genes. The other sequences group comprises 26 independent ESTs for a single mRNA with high homology to viral sequences (e.g. Japanese yam mosaic virus genomic RNA [accession no. AB027007] or the leek yellow stripe potyvirus [accession no. AJ307057]).
Figure 1A presents the percentage of ESTs (black numbers) and the percentage of different mRNAs (red numbers) for each of the 13 groups. The EST-to-mRNA ratios vary from 1.4 in the novel genes group to 4.8 in the cell structure group (Fig. 1B). Higher ratios indicate that one or more of the mRNAs in the respective group are encoded by highly expressed genes. This is expected for the cell structure group with mRNAs of genes, such as actins, tubulins, lamins, or matrix proteins. Interestingly, the mRNAs with the largest number of ESTs in this group encode a Gly/Pro-rich protein (GPRP1; Fig. 2
; Table I
). Expression of such genes is known to be associated with the vascular system (Keller et al., 1988
Figure 2 shows the distribution of ESTs per mRNA and the number of mRNAs for each EST count. The distribution is shown for all identified mRNAs (large image) and for the mRNAs of the novel genes and functionally uncharacterized proteins groups (Fig. 2, insets). As already mentioned, the large image demonstrates that MTs and GRPs are among the most highly expressed genes. It also shows the positions of the three mRNAs with the highest EST numbers of the metabolism group. These mRNAs encode a mannitol dehydrogenase, an  -amylase, and a Suc phosphate synthase (PmMTD1, PmAAMY1, PmSPS1; for accession numbers, see Table I).
Moreover, two mRNAs are marked that encode the CC-specific transporters PmSUC2 (for Suc; accession no. X75764; Gahrtz et al., 1994
In Table I, the mRNAs of the 10 most highly expressed genes from 12 groups plus the single mRNA from the other sequences group are listed together with their gene bank accession numbers. These 121 sequences, plus all other Plantago cDNA sequences, are deposited on a Web site (http://www.plantain.de). In addition to mRNAs from genes expressed in all tissues (e.g. histones, ubiquitins, tubulins, profilins), Table I shows numerous mRNAs that were previously shown to be specifically or preferentially expressed in the vascular tissue. These are, for example, mRNAs for thioredoxins (PmTRX1, PmTRX2, PmTRX3; Ishiwatari et al., 1998
For selected genes, the suggested vascular specificity or nonvascular specificity was tested by northern blots. The nonvascular RNA was isolated from the basal regions of leaves (blade and petiole) from which the vascular bundles had been extracted (the quality of these tissues has been demonstrated in figure 3
in Gahrtz et al., 1994
A larger number of genes was analyzed by RT-PCR (Fig. 3B). Again, the results confirmed the predicted vascular specificity. With the exception of PmUBQ1, which was used as a control gene and has similar expression levels in vascular and nonvascular tissue, and with the exception of PmPC1 (encoding a plastocyanine), which should be expressed more strongly outside the vasculature (similar to PmRBCS1 in Fig. 3A), all other genes showed varying degrees of vascular specificity. Some of them (e.g. THIOMETHYL-RIBOSE KINASE 1 [PmMTK1], POLYOL TRANSPORTER 1 [PmPLT1], XANTHINE/URACILE TRANSPORTER 1 [PmXUP1], AMINO ACID PERMEASE 1 [PmAAP1], two functionally uncharacterized genes [contigs P12.46.C1 and P12.40.C3], and a novel gene [contig P12.7.C65]) were expressed almost exclusively in the vasculature. For large-scale expression analyses, macroarrays with several hundred PCR-generated cDNA fragments (typical length: 250–300 bp) were hybridized to radiolabeled cDNA from vascular or nonvascular poly(A+) RNA. Figure 4 shows the results for 105 ESTs sorted by their vascular specificity (from left to right, increasing vascular specificity) and their relative expression intensity (high expression, Fig. 4A; medium expression, Fig. 4B; low expression, Fig. 4C). Genes expressed mainly in nonvascular tissue were identified only among the highly expressed genes (Fig. 4A). For example, the first EST in Figure 4A encodes a BURP domain-containing protein (PmBDC1) and the corresponding gene (accession no. AJ843872) has previously been shown to be expressed strongly and mainly in nonvascular tissue (M. Gahrtz and N. Sauer, unpublished data). Only three of the genes analyzed in Figure 4B (medium expression) and four of the genes analyzed in Figure 4C (low expression) have similar expression levels in vascular and nonvascular tissue. All other ESTs show higher or very high expression in the vasculature. ESTs 18 and 32 in Figure 4A represent the MT genes PmMT2 and PmMT1 that were also analyzed on northern blots (Fig. 3A) and that are listed among the top 10 genes in the cellular response to hormones and stress group (Table I). The relative expression intensities and the vascular specificity that is predicted for these genes from the macroarray in Figure 4A reflects the relative expression intensities predicted by the EST frequency listed in Table I and by the vascular specificity seen in the northern analyses in Figure 3A. This is also true for the PmAQP1 and PmPSP1 genes marked in Figure 4A or for the PmAAMY1 and PmSPS1 genes marked in Figure 4B (see also Table I for frequency of ESTs and/or Fig. 3B for vascular specificity). This demonstrates that the ratios (vascular to nonvascular) obtained by the analyses in Figure 4 are a useful measure for the vascular specificity of these mRNAs.
PCR-derived fragments from numerous mRNAs of the functionally uncharacterized proteins (Fig. 4, thin arrows) and the novel gene (Fig. 4, thick arrows) groups were included in these macroarrays. The vast majority of the corresponding genes turned out to be expressed preferentially or exclusively in the vascular tissue; some, for example, EST numbers 34 (contig P12.43.C2; accession no.AJ843979) or 35 (contig P12.635.C1; accession no. AM114423) in Figure 4A, even with higher specificity than PmMT1, a phloem marker protein.
An important question is, of course, whether such a set of expression data can be used to predict vascular specificity also for homologous genes from other plants (e.g. from Arabidopsis). Therefore, we isolated promoter sequences by PCR for Arabidopsis genes that showed significant homology to vascular-specific Plantago genes and used these promoters to drive expression of the GUS reporter gene in Arabidopsis.
The first gene we selected was AtMTK (At1g49820; 1,220-bp promoter sequence; Plantago homolog PmMTK1 [see Fig. 3B]). When the PmMTK1 sequence was first isolated from Plantago, BLAST searches found similarity only to a then functionally uncharacterized gene from Arabidopsis. Meanwhile, however, this Arabidopsis homolog was characterized as methylthio-Rib kinase (AtMTK; Sauter et al., 2004 The second gene we selected was PmTTPL1 (see Table I), which is homologous to three Arabidopsis genes (At2g16970, At2g16980, and At2g16990; highest identity values with At2g16970). These three genes encode so far uncharacterized tetracycline transporter-like proteins. We isolated the promoter of At2g16970 by PCR (1,104 bp) and used it to drive expression of the GUS reporter gene in Arabidopsis. The third gene we selected was PmXUP1, which codes for a putative xanthine/uracile transporter. There are six XUP homologs in Arabidopsis (xanthine/uracile permease-like family: At1g60030, At2g05760, At2g26510, At2g34190, At5g49990, and At5g62890), with At5g62890 sharing the highest degree of homology with PmXUP1. We isolated the promoter of At5g62890 by PCR (1,343 bp) and used it to drive expression of the GUS reporter gene in Arabidopsis. The fourth gene we selected was a Plantago member of the novel genes group (P12.0.C65; 1,428-bp 5'-flanking sequence; accession no. AM156930). We amplified a P12.0.C65 promoter fragment from Plantago genomic DNA by PCR based on sequence information that had been obtained by thermal asymmetric interlaced (TAIL)-PCR. This fragment was used to drive GUS expression in Arabidopsis. Vascular-specific expression of this gene in Plantago has previously been shown by northern analysis (N. Sauer and M. Gahrtz, unpublished data) and was confirmed by the RT-PCR shown in Figure 3B. Figure 5 demonstrates that source leaves of AtMTK promoter-GUS plants (seven of 10 analyzed transformants), of At2g16970 promoter-GUS plants (eight of 16 analyzed transformants), and of P12.0.C65 promoter-GUS plants (11 of 24 analyzed transformants) show GUS histochemical staining specifically in their vascular bundles. Only the GUS staining of At5g62890 promoter-GUS plants is not vascular specific and stains the trichomes of these plants with high specificity (11 of 12 analyzed plants). These data show that (1) Plantago-derived information on the vascular specificity of a unique gene is also valid in Arabidopsis (AtMTK); (2) vascular-specific Plantago promoters of genes (P12.0.C65) that do not have homologs in Arabidopsis confer also vascular-specific GUS expression in Arabidopsis; and (3) no prediction can be made on vascular specificity for members of larger Arabidopsis gene families (e.g. for the xanthine/uracile permease family). Nevertheless, the Plantago data suggest that at least one member of the Arabidopsis family may also be vasculature specific.
Establishing a Transformation System for Common Plantain A major prerequisite for analysis of expression patterns and gene functions in a given plant is the availability of a transformation system. So far, transformation had not been transcribed for common plantain. Based on Agrobacterium tumefaciens-mediated transformation of Arabidopsis, we developed an effective and simple system for producing common plantain transgenic plants.
For selection of possible transformants, we used the BASTA resistance gene in the vector pGPTV-bar (Becker et al., 1992
In a first approach, we tried to transform calli that had been obtained from leaves or roots of Plantago plants grown under sterile conditions and, in parallel, we tried to set up a regeneration system using untransformed calli. A similar approach has been published recently for Medicago truncatula (Crane et al., 2006
In a second approach, we tried to transform Plantago based on the floral-dip technique described for Arabidopsis (Clough and Bent, 1998 After dipping 24 Plantago plants into a suspension of Agrobacterium that harbored a PmPLT1 promoter-GUS construct, about 2,500 seeds from these plants (1.5 g) were put on soil and kept at 21°C in the growth chamber. Of the germinated seedlings, 32 survived repeated (3-fold) treatment with BASTA. Insertion of the GUS reporter gene into the genomes of these plants was checked by PCR on genomic DNA. In 15 of the analyzed plants, a PCR product could be identified (Fig. 6A ) that had the same size as the band obtained in a control reaction. In contrast, PCR with DNA from wild-type plants did not result in a PCR product with GUS-specific primers (Fig. 6A). A GUS assay with fully developed leaves of these 15 plants was performed and GUS histochemical staining was detected in the vascular tissue of 11 plants. GUS staining that was performed repeatedly with wild-type Plantago plants did not result in GUS-positive staining (data not shown).
These data demonstrate that (1) transgenic common plantain can be obtained using a modified floral-dip technique; (2) BASTA resistance is a suitable selection marker for Plantago; and (3) the used PmPLT1 promoter drives expression of the GUS reporter gene in Plantago vascular tissue.
This article presents data on the generation, characterization, and application of tools for the analysis of vasculature-specific gene expression in common plantain. More than 3,200 independent mRNAs were identified in an EST project that was based on a cDNA library constructed from vascular mRNA. In contrast to previously described vascular, phloem, or xylem ESTs that were obtained from petioles or hypocotyls, or that had been isolated from cells collected under conditions of mechanical stress, the mRNA used for the presented transcriptome analyses was derived from nonstressed vascular tissue of Plantago leaf blades. Vascular strands extracted from mature Plantago source leaves are branched and up to 15 cm long. The identified mRNAs are derived from genes expressed in the phloem and xylem of fully developed Plantago vasculature. Because most of the mature xylem vessels are no longer alive, the transcriptionally active cells in this tissue preparation represent mainly cells of the xylem parenchyma, phloem CCs, phloem SEs, and phloem parenchyma cells. Data presented in this article are useful for future studies of the complex and highly specialized long-distance transport system of higher plants. All of the identified genes are expressed within the vasculature and many of them were shown to be active preferentially or exclusively in this tissue. Due to the nature of the material that was used for the generation of the analyzed EST library (vascular bundles from fully developed source leaves), the obtained sequences represent primarily genes that encode proteins responsible for vascular structure and function (transport and signaling). In contrast, mRNAs from genes that regulate vascular development are expected only in smaller numbers because these are expressed mainly in the vasculature of sink leaves.
A comparison of the highly expressed genes of this Plantago EST library with vasculature marker genes previously identified in Plantago or in other plants underlines the vasculature specificity of this library. MTs (Vilaine et al., 2003 Moreover, the vasculature specificity of this library is supported by the identification of vasculature-typical metabolic pathways. For example, lignin biosynthesis is xylem specific and numerous mRNAs for enzymes of this pathway were identified. This includes the mRNAs for a Phe ammonia lyase (PmPAL1, AM159090), for three cinnamoyl-alcohol dehydrogenases (PmCAD1, AM11321; PmCAD2, AM159095; PmCAD3, AM159096),for three caffeoyl-CoA O-methyltransferases (PmCCoAOMT1, AM159088; PmCCoAOMT2, AM159089; PmCCoAOMT3, AM159091), for two 4-coumarate:CoA ligases (Pm4CL1, AM159092; Pm4CL2, AM159093), and for one sinapyl alcohol dehydrogenase (PmSAD1, AM159094).
We also identified almost complete sets of mRNAs for phloem-specific pathways. For example, Walz et al. (2002) We also identified the mRNAs for these three proteins (a Cu/Zn-superoxide dismutase [PmCSD1, AJ844003], an ascorbate peroxidase [PmAPX1, AJ843990], a monodehydroascorbate reductase [PmMDAR1, AM158910]), plus several other mRNAs for proteins potentially involved in ROS detoxification. Examples are a glutathione peroxidase (PmGPX1; AM159087), a thioredoxin-dependent peroxidase (PmTPX1; AJ843119), two glutaredoxins (PmGLX1, AJ844008; PmGLX2, AM111306), and three thioredoxins (PmTRX1, AJ844021; PmTRX2, AJ844022; PmTRX3, AJ844023).
Finally, numerous ESTs were identified for mRNAs encoding proteins of the ethylene biosynthetic pathway. We observed high expression levels of the gene for a 1-aminocyclopropane-1-carboxylate oxidase (PmACO1, AJ843131), the last enzyme in ethylene biosynthesis. Only recently, a regulatory function of ethylene in the induction of phloem defense responses has been postulated for different conifers (Hudgins and Franceschi, 2004 However, we also identified pathways that were so far not described to be vasculature specific or vasculature typical. For example, we identified several mRNAs for the polyamine biosynthetic pathway. Like ethylene biosynthesis (see above), polyamine biosynthesis starts with S-adenosyl-Met (SAM) as the primary substrate. The identified mRNAs for polyamine synthesis include two different SAM decarboxylases (PmSAMDC1, AM156953; PmSAMDC2, AM159097) that produce the aminopropyl groups for the polyamines spermine and spermidine. Moreover, we identified mRNAs for one spermidine synthase (PmSPDS1, AM158913) and for two different spermine synthases (PmSPMS1, AM158911; PmSPMS2, AM158912) that catalyze the formation of spermine or spermidine from decarboxylated SAM. This suggests that Plantago vasculature is an important site for polyamine biosynthesis.
A side product of ethylene and polyamine biosynthesis is 5' MTA, which is recycled to Met in the MTA cycle (or Yang cycle; Yung and Yang, 1982
Finally, we found vasculature-specific expression also for several other genes that were not previously described to be vascular specific. For example, vascular-specific expression has so far only been shown for a Our data also show vascular-specific expression for numerous genes encoding so far uncharacterized proteins or for novel genes. Analysis of their specific roles within the vasculature will be a major challenge for the next years.
Analyses of promoter activities from Arabidopsis genes that are homologs of identified Plantago vascular genes (Fig. 5) revealed vascular-specific expression for AtMTK1 and for the gene for the tetracycline transporter-like protein At2g16970. This demonstrates that careful predictions on the expression pattern of a gene of interest can be made from data obtained with homologous genes in other plant species. However, for numerous questions, it will be essential to modulate the expression of a gene directly in Plantago.
For example, in contrast to Arabidopsis, which translocates Suc and small amounts of raffinose in its phloem (Haritatos et al., 2000 Besides the mere fact that this promoter shows the expected GUS staining in the Plantago vasculature (Fig. 6), the successful and simple transformation of common plantain will provide the basis for further analyses. The physiological roles of the identified proteins and metabolic pathways can now be studied by overexpression of the respective genes or by down-regulation of their mRNA levels using RNAi or antisense RNA constructs.
Strains
Common plantain (Plantago major) plants were grown in a greenhouse on potting soil. Escherichia coli strain DH5
Total RNA from pure Plantago major vascular bundles (6 g) was isolated as described (Sauer et al., 1990
A directionally cloned (EcoRI/XhoI) cDNA library was generated from poly(A+) RNA using a cDNA inserts were PCR amplified with pUC-forward (5'-ACGACGTTGTAAAACGACGGCCAG-3') and pUC-reverse (5'-TTCACACAGGAAACAGCTATGACC-3') primers using whole cells as a template. PCR products were treated with exonuclease I and shrimp alkaline phosphatase for 30 min at 37°C and purified using 96-well purification plates (Edge Biosystems). Sequencing of the PCR products (Applied Biosystems 3700 automated DNA-sequencing system) was performed using the T3 forward primer. Vector and adaptor sequences were removed from the raw sequences. Resulting sequences were aligned to identify identical or overlapping ESTs for contig formation. Chimeric clones were identified by the presence of a cloning adaptor within the sequence.
Resulting contig and singlet sequences were compared against the Swissprot protein database using BLASTX (Altschul et al., 1997
For analysis of the vascular specificity of different Plantago genes, total RNA was extracted from isolated vascular bundles or from nonvascular tissue of Plantago using TrizolR reagent (Invitrogen). cDNA was synthesized from 5 µg of RNA in a total volume of 20 µL using the RevertAid H Minus first-strand cDNA synthesis kit (Fermentas). From these reactions, 0.5 µL were used as PCR templates with gene-specific 20-bp primers. Promoter sequences were PCR amplified from Arabidopsis genomic DNA using the following primers: MTK-fwd (5'-CAAATCATTTTTATACCTCGATGC-3') and MTK-rev (5'-GGCTTTTGGTACAAATTTTCAGA-3') for the amplification of a 1,220-bp promoter fragment of the Arabidopsis AtMTK gene, At5g62890-fwd (5'-CCCGACACTTAGAAATGTGTATCA-3') and At5g62890-rev (5'-CACAGAGAGAGAGAGAGGGAGAA-3') for the amplification of a 1,343-bp promoter fragment of the Arabidopsis At5g62890 gene, and At2g16970-fwd (5'-CTCTCTCTAAGCTTTCAAGGGTTATGTGAAATG GTA-3') and At2g16970-rev (5'-CAAGTCTATATTCCTCCATGGCT-3') for the amplification of a 1,104-bp promoter fragment of the Arabidopsis At2g16970 gene.
The promoter sequence of the gene encoding the Plantago contig P12.0.C65 was isolated by TAIL-PCR (Liu et al., 1995
For northern-blot analyses, 10 µg of total RNA were separated on denaturing agarose gels and transferred to nylon membranes as described (Maniatis et al., 1982 For macroarray analyses, 600 ng of PCR products of the cDNA fragments were spotted on nylon membranes and UV cross linked. Duplicates were spotted for each EST. PCR fragments for 216 different ESTs were spotted on the macroarrays used to study vascular specificity. PCR fragments for 108 different ESTs were spotted on those macroarrays that were used to study the salt responsiveness of gene expression. Hybridizations with 32P-labeled cDNAs were performed for 40 h at 42°C in 50% formamide, 5 x SSC, 5 x Denhardt's solution, 0.1% SDS, 50 µg mL–1 salmon sperm DNA, and 20 µg mL–1 polyuridylic acid. Filters were washed as described for the northern-blot analyses. Signals were detected as described for the northern blots.
The PmPLT1 promoter was obtained by two rounds of TAIL-PCR (Liu et al., 1995
Callus tissue was obtained from root sections of Plantago plants that were grown on Murashige and Skoog medium (Murashige and Skoog, 1962 Intact Plantago plants were regenerated from root-derived callus tissue in three steps. First, calli were transferred to shoot-inducing medium plates [containing Murashige and Skoog medium with 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (2 mg/L; Sigma-Aldrich)], where first leaves form after 3 weeks. For further growth, shoots were transferred in a second step to plastic boxes (5 cm high) with shoot-inducing medium. In the third and final step, shoots were transferred to boxes with root-inducing medium (Murashige and Skoog medium without hormones), where roots form after 2 to 3 weeks.
Transformation of common plantain plants was performed with Agrobacterium tumefaciens strain GV3101 (Holsters et al., 1980 Dipping was performed for 5 min under vacuum. Inflorescences with fully developed female flowers were used for the dipping procedure. Twenty-four and 48 h after the first dipping, dipping was repeated. Between dipping, plants were kept on the lab bench with no extra light. After the last dipping, plants were covered with plastic wrap to maintain sufficiently high humidity for Agrobacterium infection and transferred to the greenhouse (the wrap was removed after 2 d). After about 4 weeks, the dipped inflorescences had developed ripe seeds. At that stage, inflorescences were covered with a paper bag and seeds were harvested. BASTA-resistant offspring were identified by spraying soil-grown seedlings three times with BASTA (250 µL Liberty SL 200 g/L; ArgEvo).
Genomic DNA was isolated (Aitchitt et al., 1993 Sequence data from this article can be found in the GenBank/EMBL data libraries under the accession numbers listed in Table I. Received August 31, 2006; accepted October 2, 2006; published October 13, 2006.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. SA 382/15 to N.S.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Norbert Sauer (nsauer{at}biologie.uni-erlangen.de). www.plantphysiol.org/cgi/doi/10.1104/pp.106.089169 * Corresponding author; e-mail nsauer{at}biologie.uni-erlangen.de; fax 49–9131–8528751.
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-ZAP II (enriched in cDNAs longer than 500 bp; 2 x 106 plaque-forming units in the initial library). Inserts were excised to create a plasmid library in pBluescript SK–. According to the 
