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First published online October 2, 2003; 10.1104/pp.103.027714 Plant Physiology 133:1229-1239 (2003) © 2003 American Society of Plant Biologists Functional and Phylogenetic Analyses of a Conserved Regulatory Program in the Phloem of Minor Veins1,[w]Department of Plant Biology, Cornell University, Ithaca, New York 14853
The minor-vein phloem of mature leaves is developmentally and physiologically distinct from the phloem in the rest of the vascular system. Phloem loading of transport sugars occurs in the minor veins, and consistent with this, galactinol synthase is expressed in the minor veins of melon (Cucumis melo) as part of the symplastic-loading mechanism that operates in this species. A galactinol synthase promoter from melon drives gene expression in the minor-vein companion cells of both transgenic tobacco (Nicotiana tabacum) and Arabidopsis. Neither of these plants use galactinol in the phloem-loading process, implying that the promoter responds to a minor-vein-specific regulatory cascade that is highly conserved across a broad range of eudicotyledons. Detailed analysis of this promoter by truncation and mutagenesis identified three closely coupled sequences that unambiguously modulate tissue specificity. These sequences cooperate in a combinatorial fashion: two promote expression throughout the vascular system of the plant, whereas the third functions to repress expression in the larger bundles. In a complementary approach, phylogenetic footprinting was used to obtain single-nucleotide resolution of conserved sites in orthologous promoters from diverse members of the Cucurbitaceae. This comparative analysis confirmed the importance of the closely coupled sites but also revealed other highly conserved sequences that may modulate promoter strength or contribute to expression patterns outside of the phloem. The conservation of this regulatory design among species that phloem load by different mechanisms supports a model for organismal development in which tissues and cell types are controlled by relatively ancient and conserved paradigms but expression of genes influencing final form and function are relatively plastic.
Different vein classes in a leaf occupy distinct developmental and physiological niches. The leaf veins of tobacco (Nicotiana tabacum), for example, are subdivided into as many as six classes, with Class I consisting of the primary vein (mid-rib), and Classes V and VI representing the finest veins—referred to as minor veins—that enclose the areoles or terminate as blind endings in the areoles (Haritatos et al., 2000a  ). The smallest veins are not merely diminutive extensions of larger ones but are developmentally and functionally distinct. In growing leaves, the companion cells and sieve elements of the phloem are not fully developed in minor veins, and do not participate in nutrient transport. Rather, they mature during the sink-to-source transition when the productive capacity of the leaf exceeds its metabolic requirements and begins exporting photoassimilate to regions of net demand (Turgeon, 1989). Consistent with this, phloem loading, the energy-requiring process of accumulating photoassimilate in the phloem in preparation for export, occurs primarily, if not exclusively, in the minor veins.
Despite long-term interest in minor-vein structure and function, little is known at the molecular level. This is in part due to technical challenges. The minor veins are relatively inaccessible due to their position in the middle of the lamina. They are exceptionally small with an entire bundle being smaller in diameter than a mesophyll cell, and they are comprised of several distinct cell types (i.e. Haritatos et al., 2000b
An alternative approach to identifying minor-vein-specific genes may be through the presence of common sequence motifs in their promoters. Genes that are coordinately expressed, either during a developmental or an environmental cue, tend to respond to the same regulatory cascades and thus tend to share regulatory motifs in their promoters. This tendency has been exploited to identify unknown genes involved in a specific response by searching for the presence of regulatory elements similar to those found in well-characterized genes (Iyer et al., 2001
Because phloem loading is a prominent activity of minor veins, characterizing the regulation of a gene involved in this process is a reasonable starting point for identifying other minor-vein genes. Phloem loading occurs by one of two mechanisms: from the apoplast by cotransport with protons or from the symplast by a polymer-trapping mechanism (Grusak et al., 1996
However, the polymer trap mechanism of phloem loading is restricted to minor veins (Turgeon 1996
Consistent with its role in phloem loading, galactinol synthase immunolocalizes to the intermediary cells of minor veins in cucurbits (Beebe and Turgeon, 1992
This finding that the expression pattern conferred by the CmGAS1 promoter is conserved among species regardless of the requirement for galactinol indicates that the regulatory mechanisms conferring minor-vein expression are highly conserved irrespective of the loading mechanism employed. Such conservation in turn implies that the same regulatory system controls numerous other genes with disparate roles in the structure and function of minor-vein companion cells (Doebley and Lukens, 1998
To gain insight into the regulatory mechanisms that govern gene expression in the minor-vein phloem, functional domains of the CmGAS1 promoter were mapped by two complementary approaches: truncation/mutagenesis and comparative sequence analysis. The latter approach, referred to as phylogenetic footprinting, is based on the supposition that cis-acting sequences that interact with sequence-specific trans-acting factors are evolutionarily conserved, whereas neutral sequences undergo genetic drift (Fickett and Wasserman, 2000
Promoter Truncations
The CmGAS1 promoter (National Center for Biotechnology Information [NCBI] accession no. AF249912), isolated as a 3,081-bp fragment and fused to the uidA (
To further delimit the region of the promoter essential for the minor-vein expression pattern, additional truncations were made from both the 5' and 3' directions (Fig. 1B). The –60 region of the cauliflower mosaic virus (CaMV) 35S promoter (35S-60) was used to compensate for deletion of the CmGAS1 TATA box and transcriptional start site. The 35S-60 basal promoter provides sequences sufficient for transcription initiation, but does not itself result in gene expression unless coupled to transcriptional enhancer elements (Campisi et al., 1999 Promoter sequences from 1,816 to 123 bp upstream of the CmGAS1 ORF were fused to the 35S-60 basal promoter and the uidA reporter gene, and were analyzed for expression pattern and relative promoter strength in transgenic Arabidopsis. Systematic truncations of this element were made by PCR and site-directed mutagenesis and were similarly tested in transgenic Arabidopsis (Fig. 1B). On the basis of these truncations, we established that the smallest element necessary and sufficient for minor-vein expression was a 351-bp block located between nucleotides –1,333 and –983 (Fig. 1B). This element conferred the same pattern and approximate strength (within a factor of two) of uidA activity as all other constructs derived from the –1,816 to –123 element fused to the 35S-60 basal promoter. Sequences from –1,816 to –1,334, and from –982 to –123 are therefore dispensable for both tissue specificity and promoter strength. Further truncation of the minimal 351-bp element to –1,203 on the 5' side or –1,032 on the 3' side eliminated minor-vein activity; however, plants harboring the –1,333 to –1,032 fragment retained X-GlcA staining in the hydathodes (data not shown).
All constructs derived from the –1,816 to –123 element fused to the 35S-60 basal promoter demonstrated reduced activity relative to the construct pSGXho101. The promoter in pSGXho101 extends from –1,762 to +12 of the CmGAS1 sequence, and sequences from –122 to+12 may therefore contain enhancer elements that contribute significantly to promoter strength without affecting tissue specificity. This premise is supported by the presence of highly conserved sequences in the same region of CmGAS1 orthologs from other species in the Cucurbitaceae (see below). However, it is noted that the constructs are derived from different, albeit related, plasmid backbones (pGPTV-KAN versus pBi101.2; pGPTV-KAN is derived from pBi101.2; Becker et al., 1992
To pinpoint more precisely the cis-acting elements responsible for the minor-vein expression pattern, sequences within the –1,333 to –983 region were mutated as consecutive stretches of 10 nucleotides. Nucleotides were mutated in stretches of 10 to coincide roughly with the anticipated size of transcription factor-binding sites (six to 12 nucleotides). A SalI recognition site was incorporated in the center of each mutagenized sequence for convenient identification, and the remaining nucleotides were converted to noncomplementary transversions (i.e. A
As demonstrated by the histochemical assays, six mutations altered the tissue specificity of reporter gene expression from that observed with the parent construct, pGPTV-TT(–1,333–983; Fig. 2B), and clustered into three "blocks". Block I was defined by mutations 17 and 18, and includes nucleotides –1,173 to –1,154 (Fig. 2A). Mutation 17 abolished expression in minor veins, but expression was retained in hydathodes (Fig. 2C) and root apices (data not shown). Mutation 18 had minimal effect on staining intensity in the minor veins and hydathodes, but resulted in strong expression throughout the vascular network of the leaf, stem, and root, where it is otherwise not observed (Fig. 2D). Block II (Fig. 2A) was defined by mutations 21 and 22, and spans nucleotides –1,133 to –1,124. Both of these mutations abolished minor-vein expression. In addition to minor veins, mutation 21 abolished expression in all tissues (Fig. 2E), whereas mutation 22 retained expression in the hydathodes and root apices. Mutations flanking block II—particularly 20, 23, 24, and 25—exhibited reduced X-GlcA staining in the minor veins, however, the overall pattern was not altered from that observed with pGPTV-TT(–1,333–983). Block III (Fig. 2A) was defined by mutations 31 and 32, and consists of nucleotides –1,033 to –1,014. As with block II, mutation 31 abolished activity in all tissues and mutation 32 abolished activity only in the minor veins. Taken together, blocks I, II, and III are required for and work cooperatively to regulate expression from the CmGAS1 promoter in minor veins. One-half of block I, and both blocks II and III, are together required for expression in vascular tissues. The other half of block I is required to repress expression in the larger veins of the leaf as well as in the vascular bundles of stems and roots (Fig. 2D).
Arabidopsis, although a convenient heterologous host for functional analysis of a tissue-specific promoter, is not closely related to melon (Soltis et al., 1999
Sequence divergence among the species was initially unknown, and a broad sampling was therefore selected based on classifications derived from molecular and morphological data and on geographical distribution (Jeffrey, 1990
Local alignments in the GAS1 promoter regions of each representative specie were sought with the Gibbs sampler algorithm of the MACAW software package (Schuler et al., 1991
Three of the regions conserved among the species correlated with the three blocks determined by mutagenesis (Fig. 3, A and B). Sequence identity among the species was particularly high for blocks I and II and extended for several nucleotides beyond the block boundaries, as defined by site-directed mutagenesis (Fig. 3B). A T-rich stretch between blocks I and II was also conserved, but demonstrated considerable variation in length. The extent of sequence identity among the species in block III was not as pronounced, especially between the New and the Old World species (Fig. 3B). Upstream of block I, three regions of prominent sequence conservation were identified among melon, watermelon, L. cylindrica, T. kirilowii, and squash. These regions are immediately upstream of block I in L. cylindrica, T. kirilowii, and squash but are separated by roughly 1 kb in melon and watermelon (Fig. 3A). There is relatively little homology in these intervening regions. One of the upstream conserved regions (–2,366 to –2,345 of CmGAS1) was used as an anchor to obtain sequence information from M. charantia, S. humbertii, and S. edule. The upstream regions of homology are therefore not represented in these latter species. However, block I was within 100 bp of this conserved anchor, and these species therefore appear to have an upstream promoter architecture resembling that of L. cylindrica, T. kirilowii, and squash. Three stretches of near identity greater than 20 bp in length were found between block III and the GAS1 ORF, along with four closely clustered, shorter sequences (Fig. 3A and supplemental data). The distance between block III and the first of these sites varied greatly, from 176 bp in squash to 1,347 bp in S. edule. Within this region, there is some interspersed homology between melon and watermelon, and between L. cylindrica and T. kirilowii (data not shown). Further downstream are additional stretches of variable-length, non-conserved sequences interspersed among those that are conserved. Generally, sequence similarity between the species analyzed increases closer to the ORF. It is noteworthy that the blocks demonstrate substantial variation in spacing among the species, but that the order and orientation of individual blocks is constant, implying that over evolutionary time, the architecture of the promoter has been influenced substantially by insertion and deletion events.
The sequences identified as being functionally essential for minor-vein expression and conserved among the species analyzed were used to challenge the TRANSFAC database of characterized transcription factors (Hehl and Wingender, 2001
In the Cucurbitaceae, GAS1 catalyzes the synthesis of galactinol, which is the first committed step in the synthesis of RFO transport sugars and is an integral part of the polymer trap mechanism of phloem loading. Consistent with this, GAS1 activity localizes to the minor veins where RFOs are synthesized and loading occurs (Beebe and Turgeon, 1992 ). However, the CmGAS1 promoter confers gene expression to the minor veins in plants that do not use galactinol as an intermediate in the phloem-loading process, and where there is no a priori reason to expect this promoter to be recognized. This implies that the regulatory machinery governing minor-vein expression is conserved across a broad range of eudicotyledons, whereas the types of genes regulated by this machinery have changed. As an initial step in unraveling this genetic program, sequence elements within the galactinol synthase promoter that are necessary and sufficient for its precise tissue specificity were functionally characterized in transgenic Arabidopsis as a heterologous host. Promoter truncations revealed that sequences conferring minor-vein specificity were situated on a 351-bp fragment located 1,333 to 983 nucleotides upstream of the galactinol synthase ORF. The essential sequences were then mapped with 10-bp resolution by site-directed mutagenesis, and three blocks were characterized as being functionally significant: block I spans nucleotides –1,163 to –1,144 (mutations 17 and 18); block II spans nucleotides –1,133 to –1,114 (mutations 21 and 22); and block III spans nucleotides –1,033 to –1,014 (mutations 31 and 32). Promoter truncations also indicated that an essential element is located between nucleotides –1,333 and –1,203, however, this element was not identified by high-resolution mutagenesis or by comparative sequence analysis. The –1,333 to –1,203 element has an AT content of 84% that may be involved in chromatin structure and affect gene expression when deleted entirely, but may not be affected by smaller mutations.
The genera Arabidopsis and Cucumis are not closely related (Soltis et al., 1999
Representative species were selected based primarily on guide trees established by comparison of ribosomal internal transcribed spacer sequences with consideration of classifications based on morphological data (Jeffrey, 1990 Local alignment of sequences upstream of the CmGAS1 orthologs from the representative species identified regions of sequence conservation all along the 3,081 bp of the CmGAS1 promoter. However, based on functional analysis in Arabidopsis, sequences upstream of nucleotide –1,333 and downstream of nucleotide –983 are not essential for minor-vein expression. It is thus not clear why these other sequences are so strongly conserved. The two regions closest to the GAS1 ORF are either partially or fully downstream of CmGAS1 nucleotide –123, and may account for the difference in uidA expression observed between constructs derived from the –1,816 to –123 fragment, and pSGXho101 (Fig. 1). Similarly, regions upstream of nucleotide –1,762 may also be involved in promoter strength, because histochemical staining of transformed plants suggest that the promoter fragment contained on pSG3K101 is qualitatively stronger than that of pSGXho101 (data not shown).
In melon, CmGAS1 is expressed in maturing seeds (Volk et al., 2003
Due to our analysis of numerous species and our criterion for 90% similarity over at least eight consecutive nucleotides, the resolution at each site is higher than what is commonly reported in phylogenetic footprinting studies, in that each site is within the size range expected for the binding sites of individual transcription factors. By comparison, phylogentic footprinting studies among mammalian species frequently cite cis-acting elements that are 70% homologous over 100 nucleotides (Loots et al., 2000
It is worthy of note that there is no comparable homology with the promoter region of CmGAS2 (Volk et al., 2003 Three conserved regions were identified among the orthologs between nucleotides –1,333 and –983 of CmGAS1 that correlate with the three blocks identified by site-directed mutagenesis and functional analysis in Arabidopsis. Sequence identity was strong among the species analyzed in the vicinity of blocks I and II, and in both cases, this conservation extended beyond the boundaries of sequences that are functionally essential (Fig. 3B). Notwithstanding, sequence comparisons revealed nucleotides within each block that are not conserved among all species. These nucleotides are therefore not essential in the absolute sequence context, but may be essential for proper spacing within or between cis-acting promoter elements. In this important regard, phylogenetic footprinting improved the resolution of the promoter analysis beyond that obtained by site-directed mutagenesis alone. Sequence conservation was not as prominent in block III, particularly between the New World species, S. edule and squash, and the Old World species. Without the support of the mutagenesis data, the limited conservation in this region would have been dismissed as spurious background homology among the Old World species. This emphasizes the importance of selecting a range of species for analysis, as well as the requirement for a second, functional means to identify important sequences.
The first block does not display a clear sequence similarity to characterized transcription factor-binding sites, but does have remote homology to sequences recognized by a large class of transcription factors characterized by a homeodomain adjacent to a Leu zipper (HD-Zip). HD-Zip transcription factors form homo- and potentially heterodimers through the Leu zipper domain and tend to bind DNA at palindromic sequences (Sessa et al., 1998
In block II, mutation 21 disrupted sequences that correlate with the core recognition sequence of the dof family of transcription factors (CTTT; Yanagisawa and Schmidt, 1999 In block III, sequence conservation is strong among the Old World Cucurbitaceae, but is not as pronounced among the New World species. For this reason, a class of transcription factor that potentially interacts with this site cannot be assigned. As in blocks I and II, block III sequences were disrupted by two adjacent mutations, 31 and 32, each of which resulted in different expression patterns. Mutation 31 abolished activity in all tissues, and mutation 32 abolished activity only in the minor veins. Block III may therefore constitute the binding site for a single class of transcription factor, as proposed for block II, in which one mutation destroys the binding site core and the second disrupts those necessary only for binding minor-vein factors. Alternatively, block III may be a composite site that binds a complex, such as the dimer proposed for block I, and the different expression patterns may represent which constituents are able to bind.
Few other characterized promoters drive gene expression in mature minor veins, with exclusion from larger vascular bundles. One such promoter is from a stachyose synthase gene in Alonsoa meridionalis (AJ487031; Voitsekhovskaja, 2001 On the basis of these analyses, highly specific gene expression restricted to the companion cells of minor veins is attributable to the combinatorial activity of several trans-acting factors that function as either positive or negative regulators. The positive regulators must act together, because preventing any one of them from interacting with the promoter abolishes gene activity. Each one must therefore be present in, but not necessarily limited to, the minor veins where expression is ultimately observed. The negative regulator that interacts with block I to prevent expression in the major veins, is presumably absent from the minor veins, or is in some way prevented from interacting with the DNA in these tissues. This factor may instead be present in, and interact with, the CmGAS1 promoter in the major veins and other phloem tissues to repress activity. To directly test this model for regulating gene expression in the minor veins, DNA-binding proteins that interact with each sequence block are being sought. Knowledge of both the cis- and trans-acting factors that operate at the CmGAS1 promoter will provide a strong framework for identifying other genes that are coordinately regulated by the same pathway.
Promoter Truncations
All plasmid constructions were by standard procedures (Ausubel et al., 1995
Plasmid pSG3K101 was previously described (Haritatos et al., 2000a
Plasmid pGemTT was constructed to contain a multiple cloning site upstream of a basal promoter consisting of the first 60 bp of the CaMV 35S promoter and a translational enhancer from TEV (TT, transcription initiation/translational enhancer), both derived from pIBT210 (Haq et al., 1995
The CmGAS1 promoter fragments represented in Figure 1B were PCR-amplified with appropriate combinations of the following oligonucleotides using the Elongase polymerase preparation (Invitrogen) and pSG3K101 as template. The numeric in each oligonucleotide name indicates the first complementary sequence in the promoter. The forward oligonucleotides were GAS-1816F (tta agt aag ctt tgt gat cga tgg tat tag atg agt tcc), GAS-1333F (cat gtt aag ctt tag aac taa acc taa gtt atg tta tgt gtc), and GAS-678F (aga act aag ctt taa tca tta tca aaa aca cta cc). The reverse oligonucleotides were GAS-1149R (agg agg tct aga ttc tcc aca tta ttc gat tct tg), GAS-634R (cct aaa cat cta gat ttg att tca att ctg ttt taa ggt agt g), and GAS-123R (agc ttg tct aga gaa ggg atg gga agg agg c). The PCR products were extracted with chloroform and precipitated with ethanol, digested with HindIII and XbaI, gel purified, and sub-cloned into the same sites of pGemTT to create pGemTT(–1,816–123), pGemTT(–1,816–1,149), pGemTT(–1,333–634), and pGemTT(–678–123). The basal promoter (i.e. the –60 region of the 35S promoter with the translational enhancer element from TEV), and the promoter fusions were isolated as HindIII-SmaI fragments, and sub-cloned into the same sites of the binary vector pGPTV-KAN (Becker et al., 1992
The fragment corresponding to CmGAS1 sequences –1,333 to –634 contained sequences necessary and sufficient for uidA expression in minor veins of Arabidopsis, and pGemTT(–1,333–634) was therefore used for further truncations. Sequences within this fragment were altered to SalI recognition sites by site-directed mutagenesis (Kunkel et al., 1987
From the promoter truncation experiments, CmGAS1 promoter sequences between –1,333 and –983 were necessary and sufficient for uidA expression in minor veins of Arabidopsis, and therefore pGemTT(–1,334–983) was constructed for high resolution mutagenesis. pGemTT(–1,338–983) was created by digesting pGemTT-983 with SalI and XhoI and ligating the compatible cohesive ends. The sequences between positions –1,333 and –983 were mutagenized as consecutive blocks of 10 bp by site-directed mutagenesis. The sequences of the 35 mutagenic oligonucleotides used are available from the authors upon request. In general, each was 30 to 40 nucleotides in length, and introduced a SalI restriction site into the center of the target sequence along with two noncomplementary transversions on each side. The mutated sequences in the resulting 35 constructs are depicted in Figure 2A. Each of the mutagenized promoters were sub-cloned into pGPTV-KAN as HindIII-XmaI fragments.
pGPTV-KAN-derived binary vectors harboring promoter constructs were introduced to Agrobacterium tumefaciens strain GV3101 pMP90 by heat shock and selected on 2x YT medium supplemented with rifampicin and gentamycin, each at 50 mg L–1, and kanamycin at 100 mg L–1 (Koncz and Schell, 1986
Phylogenetic footprinting was employed to identify conserved sequences upstream of CmGAS1 orthologs in representative Cucurbitaceae species. Melon (Cucumis melo; Hales's Best Jumbo), watermelon (Citrullus lanatus; Burpee's Fordhook), and squash (Cucurbita pepo; Prolific Straight Neck) were obtained from W. Atlee Burpee & Co. (Warminster, PA). Cucumber (Cucumis sativum; Burpless Orient Express) was obtained from Germania Seed Company (Chicago). Momordica charantia and Seyrigia humbertii were obtained from the Cornell Conservatory. Luffa cylindrica and Trichosanthes kirilowii were obtained from Horizon Herbs, LLC (Williams, OR). Sechium edule was obtained from a local grocery. High Mr DNA was isolated by standard procedures (Doyle and Doyle, 1990
Galactinol synthase constitutes a small gene family, and steps were taken to ensure that sequences were obtained from CmGAS1 orthologs. Initially, GAS cDNA sequences were obtained from GenBank (rice [Oryza sativa], D26537; Vitis riparia, AF178569; common bugle [Ajuga reptans], AJ237693; pea [Pisum sativum], AJ243815; squash, AR014709; soybean [Glycine max], AR014710; canola [Brassica napus], AF106954; and Arabidopsis, AB062848) and aligned with ClustalX (Thompson et al., 1997
Sequence information for regions upstream of CmGAS1 orthologs in watermelon, squash, L. cylindrica, and T. kirilowii was obtained using the genome walking method (Siebert et al., 1995
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor. Novel sequences reported in this manuscript were submitted to NCBI GenBank: C. lanatus, AY37977; L. cylindrica, AY379778; T. kirilowii, AY379779; M. charantia, AY379780; S. humbertii, AY379781; S. edule, AY379782; and C. pepo, AY379783.
We thank Róisín C. McGarry and Ashlee R. McCaskill for reviewing the manuscript and Jian Hua for helpful discussion. Received May 28, 2003; returned for revision July 7, 2003; accepted July 18, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.027714.
1 This work was supported by the U.S. Department of Agriculture/Cooperative State Research, Education, and Extension Services/National Research Initiative Competitive Grants Program (proposal no. 2001–35318–10893 to R.T.).
[w] The online version of this article contains Web-only data.
2 Present address: Department of Biological Sciences, University of North Texas, Denton, TX 76203.
3 Present address: Department of Biology and Astrobiology Research Center, Pennsylvania State University, University Park, PA 16802. * Corresponding author; e-mail bga2{at}cornell.edu; fax 607–255–5547.
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-D galactopyranosyl-(1
1)-myo-inositol). Galactinol is the galactosyl donor for the synthesis of RFOs (Keller and Pharr, 1996
-glucuronidase [GUS]) reporter gene, confers prominent gene expression to the minor veins of both tobacco and Arabidopsis (Haritatos et al., 2000a
C, G
