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First published online June 18, 2004; 10.1105/tpc.022574 © 2004 American Society of Plant Biologists A Multidrug Resistance–Associated Protein Involved in Anthocyanin Transport in Zea maysDepartment of Biological Sciences, Stanford University, Stanford, California 94305-5020 2 To whom correspondence should be addressed. E-mail deang{at}unimelb.edu.au; fax 650-725-8221.
Anthocyanin biosynthesis is one of the most thoroughly studied enzymatic pathways in biology, but little is known about the molecular mechanisms of its final stage: the transport of the anthocyanin pigment into the vacuole. We have identified a multidrug resistance–associated protein (MRP), ZmMrp3, that is required for this transport process in maize (Zea mays). ZmMrp3 expression is controlled by the regulators of anthocyanin biosynthesis and mirrors the expression of other anthocyanin structural genes. Localization of ZmMRP3 in vivo shows its presence in the tonoplast, the site at which anthocyanin transport occurs. Mutants generated using antisense constructs have a distinct pigmentation phenotype in the adult plant that results from a mislocalization of the pigment as well as significant reduction in anthocyanin content, with no alteration in the anthocyanin species produced. Surprisingly, mutant plants did not show a phenotype in the aleurone. This appears to reflect the presence of a second, highly homologous gene, ZmMrp4, that is also coregulated with the anthocyanin pathway but is expressed exclusively in aleurone tissue. This description of a plant MRP with a role in the transport of a known endogenous substrate provides a new model system for examining the biological and biochemical mechanisms involved in the MRP-mediated transport of plant secondary metabolites.
Sequestration of reactive metabolites is crucial for cell survival. Plant cells in particular produce many secondary metabolites that must be excluded from the cytoplasm to prevent cellular damage. Anthocyanin pigments are a visible example of this type of plant product. Compartmentalization of anthocyanin in the vacuole is required both to limit the mutagenic and oxidative effects of synthesis pathway intermediates (Ahmed et al., 1994  ; Rueff et al., 1995) and for proper biological function of the final product. The biosynthesis of anthocyanin is one of the most thoroughly studied biochemical pathways in biology. Easily identified phenotypes and complete mutant viability have made anthocyanin production an ideal model for analysis of gene regulation, paramutation, and transposon biology. Recent work on the last genetically defined step in this pathway (Marrs et al., 1995; Alfenito et al., 1998) has also identified anthocyanin biosynthesis as an important model for studying the mechanisms for the detoxification and vacuolar sequestration of heterocyclic organic anions. In addition to anthocyanins, this class of compounds includes a vast array of endogenous and xenobiotic chemicals involved in pest and disease resistance, pigmentation, UV protection, intracellular and extracellular signaling, and weed control (Abell et al., 1993; Shirley, 1996; Hammerschmidt, 1999). Understanding the molecular mechanisms involved in transporting these molecules to the correct cellular locations represents a fundamental, yet poorly understood, problem in biology.
The synthesis of anthocyanin bears a striking similarity to the detoxification of heterocyclic organic xenobiotics. Both processes occur in the cytoplasm and begin with a relatively nonreactive hydrophobic compound that is modified by a series of enzymes to introduce reactive centers and add hydrophilic molecules such as sugars or amino acids. Such modifications not only reduce hydrophobicity but can also act as tags for recognition by the proteins that transport the modified product out of the cytoplasm (Ishikawa et al., 1997
Also termed GS-X pumps because of their association with the transport of glutathionated compounds, the
Structurally, MRPs are large, modular proteins made up of three transmembrane domains (TMDs) and two ATP binding cassette domains (nucleotide binding domains [NBDs]) (Borst et al., 1999
Several lines of indirect evidence implicate an MRP in the anthocyanin biosynthetic pathway of Zea mays. Genetic analysis demonstrates that the actions of the known anthocyanin biosynthetic genes are not cell autonomous. In tissues from mutant plants, all of the anthocyanin pathway genes are present and active, with the exception of the mutated structural gene. Revertant sectors generated by unstable mutants of individual structural genes all have indistinct boundaries resulting from the sequestration of a small amount of anthocyanin in vacuoles of nonrevertant cells surrounding the revertant sector (Figure 1). This indicates that some component produced by the intact anthocyanin pathway in the revertant sector can move to the adjacent cells, partially complementing the mutation in those cells. In tissues from plants with unstable mutations in the anthocyanin regulators, in our lines a complex of the myc and myb-type proteins B and Pl in the plant body and R and C1 in the aleurone, none of the anthocyanin structural genes are active outside of the revertant sectors. If the known structural genes represent all of the anthocyanin biosynthetic genes, we would expect that the components that can diffuse from structural gene revertant sectors would also be able to diffuse out of revertant sectors generated by unstable mutants of the transcription factors and that these revertant sectors would also have diffuse boundaries. However, revertant sectors of the anthocyanin transcription factors have distinct boundaries (Figure 1; Kermicle and Alleman, 1990
Further evidence suggests the presence of such a transporter and offers clues to its identity. The ability of vanadate, a specific inhibitor of the ABC family of transmembrane transporters, to block pigment sequestration in maize cells (Marrs et al., 1995 ) strongly suggests that anthocyanin transport requires the activity of an ABC transporter. Similarities between the anthocyanin biosynthesis and xenobiotic detoxification pathways, particularly the requirement for the activity of a GST to ensure proper anthocyanin localization, suggest that the ABC transporter responsible for anthocyanin sequestration is an MRP. Indeed, in a study of anthocyanin production in maize cell culture, two ABC transporters, one of which is an MRP, were identified as being upregulated in cells overexpressing the anthocyanin transcription factors R and C1 (Bruce et al., 2000). In this article, we identify that MRP, ZmMRP3, as coregulated with the anthocyanin pathway in planta, characterize the ZmMRP3 gene, and demonstrate the involvement of the corresponding protein in the transport of anthocyanin into the vacuole.
MRP Expression and Anthocyanin Biosynthesis Screening of 30,000 Pioneer Hi-Bred maize EST sequences for those with homology to human, Arabidopsis, and yeast MRPs identified nine candidate MRPs. Full sequencing of the inserts yielded 1.0 to 3.0 kb (20 to 60% of estimated cDNA size) of the 3' end of seven unique genes. One of these was found to be an ABC transporter of a different class and was not characterized further. Subsequent searches of the publicly available EST database yielded 3' sequence for four additional MRP-like genes. Two of the genes identified in this screen were recently cloned and named ZmMrp1 and ZmMrp2 (Swarbreck et al., 2003 ). Fragments of the 3' end from each of these 10 genes were used as probes on RNA gel blots to compare expression patterns in eight tissue types using samples from plants with either high (B and Pl) or no (b and pl) anthocyanin (Figures 2A and 2B). Six of the ten MRPs were expressed in at least one tissue (Table 1), but only ZmMrp3 showed an expression pattern that correlated with anthocyanin levels and with the expression of Anthocyaninless2 (A2), the gene encoding anthocyanidin synthase (Figure 2C). DNA gel blot analysis using the same ZmMRP3 probe indicates that it hybridizes to a single gene (data not shown).
It was surprising to find that neither ZmMrp3 nor A2 were expressed at detectable levels in fully pigmented, mature adult leaves. To determine if this was a consequence of leaf age, we screened adult leaves of the same anthocyanin genotypes shortly after they emerged and before the development of full pigmentation. Both ZmMrp3 and A2 were highly expressed in expanding adult leaves from pigmented plants (Table 1; data not shown). This indicates that as adult leaves age and accumulate the indelible dark pigmentation, they downregulate expression of the anthocyanin pathway genes. This downregulation appears to eliminate detectable anthocyanin gene expression by the 15th to 20th day after leaf emergence. In addition to anthocyanin-regulated expression, ZmMRP3 transcript was also found in the unpigmented developing tassel and in the developing ear, where anthocyanin is also absent (Figure 2C, Table 1). This suggests functions for this MRP in addition to anthocyanin transport. Both the developing ear and immature tassels are composed of several tissue types; therefore, the expression data offer few clues to ZmMrp3 functions. The lack of expression of ZmMrp3 in mature pollen and the scarcity of pollen-specific enhancer elements in the ZmMrp3 promoter (Figure 3C) suggest that ZmMrp3 does not have a function within pollen.
Previously, Bruce et al. (2000) reported that the expression of a second ABC protein, originally identified as an MRP, was coregulated with anthocyanin biosynthesis in Black Mexican Sweet culture cells after coexpression of the R and C1 transcription factors. However, in differentiated maize tissues, the expression of this gene is regulated by light and is not affected by the expression of the anthocyanin transcription factors (Figure 2D, Table 1). Sequencing of a full-length cDNA clone of this gene allowed us to determine that it is not an MRP but is instead a member of the ABCF family of ABC proteins (Dean and Allikmets, 2001). Based on its structure, we have named this gene ZmAbcf1. Like the other ABCF genes described to date, the predicted ZmABCF1 protein lacks TMDs and is therefore assumed to be soluble. The other members of the ABCF family described to date are not involved in transmembrane transport. Rather, they appear to play a role in protein translation (Tyzack et al., 2000). A complete survey of the expression of the available maize MRPs (Table 1) showed that five maize MRPs are expressed in multiple tissues. Of these, ZmMrp3, ZmMrp5, ZmMrp6, and ZmMrp7 are expressed in all of the tissues that synthesize anthocyanin. As noted above, however, only ZmMrp3 shows any change in expression in response to anthocyanin content. ZmMrp7 shows the most consistent levels of expression throughout the plant but is not expressed in silks or in mature pollen. One of the most striking findings was that none of the ZmMrps tested showed detectable expression in mature pollen.
In our RNA gel blot analysis, we found that ZmMrp1 is expressed primarily in seedling tissues, whereas ZmMrp2 expression is only detectable in adult leaves. Within the limits of the differing genetic backgrounds and tissue types examined in these two studies, our results for the expression of ZmMrp1 and ZmMrp2 agree with an RT-PCR based expression analysis reported by Swarbreck et al. (2003) Four of the ZmMrps are not expressed at detectable levels in the tissues tested but may be active in tissue types that were not included in this survey or, as noted above, may be present at levels below the detection limits of RNA gel blots. To shed light on the expression pattern of these MRPs, we examined the tissue of origin for their corresponding ESTs. Three of the ZmMrps were identified in tissues not included in the blot. ZmMrp4 ESTs were recovered from libraries of stressed seedlings and Lesion mimic mutant leaves, ZmMrp9 was found in a tassel primordium library, and ZmMrp10 ESTs were recovered from both the immature embryo and tassel primordia cDNA collections. The ZmMrp8 ESTs were recovered from a library derived from a mixture of adult tassel, silk, kernel, root, and leaf RNA, making it impossible to determine if this MRP is present at levels below the limit of RNA gel blot detection or was derived from one of the library tissues not included in our survey.
Structure of ZmMrp3
BLAST searches using the 1480–amino acid predicted protein sequence identified significant homology between ZmMrp3 and MRPs from other organisms, particularly A. thaliana and O. sativa. BLAST CD motif searches identified two ABC TMDs and two ABC transporter ATPase motifs (NBDs) arranged in the TMD-NBD-TMD-NBD sequence characteristic of many full-sized eukaryotic ABC transporters (Theodoulou, 2000
A genomic library was screened by PCR to recover 0.69 kb of sequence upstream of the putative transcription start site. Consistent with this being a promoter sequence, the cloned region contains both TATA and CAAT motifs directly upstream of the proposed transcription start site (Figure 3C). The promoter also contains both published types of anthocyanin-responsive element (ARE) (Tuerck and Fromm, 1994
To further test the regulation of ZmMrp3 by the anthocyanin transcription factors, we fused 687 bp of the ZmMrp3 promoter, including the putative ARE site, to a luciferase reporter gene (Bodeau and Walbot, 1996
Sequence Relationship between ZmMrp3 and Other MRPs
The most striking finding in the sequence analysis was the similarity between ZmMRP3 and OsMRP13. Over the entire protein, these two genes share >79% amino acid identity. This similarity increases to 85% if the highly variable 5' TMD is excluded. When compared with the relationships between all MRPs identified from A. thaliana (Kolukisaoglu et al., 2002 Although the lack of full-length sequence from most of the maize MRPs precluded useful phylogenetic analysis, ClustalW comparisons of the available EST sequences did identify an extremely close homology between ZmMrp3 and ZmMrp4. The alignment of the entire 1.7-kb EST sequence of ZmMrp4 with the corresponding region of ZmMrp3 shows a nucleotide identity of 77%, which rises to 88% if only the coding regions of the genes are compared. Comparison of the predicted protein sequence shows 90% amino acid identity over the available open reading frame. Additional genomic sequencing of ZmMrp4 identified several differences in intron/exon structure compared with ZmMrp3, confirming that ZmMrp4 is a unique gene (data not shown). As noted in Table 1, ZmMrp4 expression was not detectable by RNA gel blot analysis in the tissues tested.
ZmMRP3 Is Localized to the Tonoplast
Antisense Mutants Alter Anthocyanin Biosynthesis To test the in vivo role of ZmMrp3, we generated transgenic plants expressing an antisense construct of the 3'end of ZmMrp3 driven by the anthocyanin-regulated promoter of the Bronze1 gene (Figure 5A). This promoter was chosen to ensure that the antisense construct would be expressed coordinately and specifically with the anthocyanin pathway; if ZmMrp3 performs essential roles in other cells, expression should be unaffected. Twelve independent transformants were recovered after particle bombardment. The initial transgenic lines contain nonfunctional alleles of the required anthocyanin transcription factors. Consequently, each independent transformant was introgressed into two different stocks in which deep pigmentation is expected in leaves, tassel, anthers, and/or aleurone. In one of the twelve independent transformants screened, plants carrying the transgene and the transcription factors necessary for full pigmentation showed a marked reduction in pigmentation. The observed phenotype varied with genetic background, ranging from a burnished color to an almost complete absence of pigment in anthocyanin containing tissues of the adult plant, particularly the leaves and tassel (Figure 5B). These phenotypes bear a striking resemblance to that seen in the leaves and tassel of plants carrying a mutation in the Bz2 gene (Neuffer et al., 1997 ).
To define the cellular nature of this phenotype, we examined the localization of anthocyanin in the face of the glume, a single layer tissue in which pigmentation is regulated by B/Pl expression. Consistent with this regulation, glumes from plants without anthocyanin (b/pl) lacked pigmentation, whereas glumes from plants with high levels of anthocyanin expression (B/Pl) showed high anthocyanin content with the pigment localized to the vacuole (Figure 5C). Glume tissue from the  ZmMRP3 plants showed the bronze color characteristic of anthocyanin that cannot enter the vacuole (Marrs et al., 1995). This phenotype is strikingly similar to that seen in glumes from bronze2 mutant plants (Figure 5C). Because of the predominance of the vacuole in glume cells, it is impossible to determine if the mislocalized anthocyanin is accumulating in the thin layer of cytoplasm or is being deposited in the cell wall. In all cases, reduced pigmentation was correlated with the presence of the transgene and elimination of the full-length ZmMrp3 transcript as tested by RNA gel blot analysis (Figure 5D). To confirm that reduction in ZmMRP3 expression resulted from antisense suppression and was not a product of the insertion of the transgene into an anthocyanin-related gene, we took advantage of the tight genetic linkage between the herbicide resistance marker and the transgene (data not shown) and the natural tendency of transgenes to be transcriptionally silenced over several generations of backcrossing. Silenced plants were identified by the absence of herbicide resistance combined with the presence of the transgene (Figure 5E). This indicates silencing of the entire transgene array, including both the herbicide resistance gene and the antisense construct. In silenced plants, the levels of both native ZmMrp3 expression and anthocyanin content were restored to wild-type levels (Figure 5E), confirming that transgene expression is required to produce the observed phenotype.
To characterize the biochemical nature of the color phenotype in comparison to mutants in both synthesis and localization, flavonoids were isolated from the adult leaves of sibling plants in families in which the transgene was segregating; siblings expressing the transgene are referred to as
HPLC analysis of the flavonoids in maize shows that the pigment profile in the bronze1 mutant differs from that seen in the ZmMRP1 and bronze2 plants in terms of anthocyanin species present. Purple maize contains three main anthocyanin pigments: cyanidin 3-glucoside (C3G), cyanidin 3-malonyl glucoside, and cyanidin 3-dimalonyl glucoside as well as a small amount of the C3G precursor cyanidin (Figure 6B; Fossen et al., 2001). The absence of the cyanidin specific UDP:glucosyl transferase activity in the bronze1 mutant (Larson and Coe, 1977) is reflected in the almost complete absence of C3G in bronze1 mutants as compared with wild-type plants (Figures 6B and 6C). By contrast, anthocyanins produced by the ZmMrp3 and bronze2 plants are found in ratios indistinguishable from wild-type plants (Figures 6B and 6C), indicating that all of the enzymatic steps of anthocyanin biosynthesis remain fully functional in both the ZmMrp3 and bronze2 plants. Therefore, the reduction in anthocyanin content in the ZmMRP3 and bronze2 plants appears to stem from the inability of C3G to be transported into the vacuole.
The Lack of an Aleurone Phenotype Suggests Redundancy in Anthocyanin Transport
To investigate this possibility, we used the suite of MRP-specific probes described earlier to screen blots containing aleurone RNA. As expected, ZmMrp3 was expressed strongly in pigmented aleurone but was not expressed in near-isogenic unpigmented tissue (Figure 7A). This confirms that ZmMrp3 is coregulated with the anthocyanin structural genes in the aleurone. Surprisingly, ZmMrp4, which is not expressed in the plant body (Table 1), is highly expressed in pigmented aleurone (Figure 7A). ZmMrp4 was the only other MRP to show anthocyanin-regulated activity in the aleurone. It was curious to note that the ZmMRP4 transcript at
Given the extensive sequence similarity between ZmMrp3 and ZmMrp4, there was a significant possibility that the ZmMrp3 construct could suppress the expression of both genes. To test the effect that the presence of the ZmMrp3 transgene has on ZmMrp4 expression, we use quantitative RT-PCR to measure the expression of ZmMrp3 and ZmMrp4 in individual aleurones taken from an ear that was segregating for transgene expression. In aleurones that expressed the transgene, there is a fivefold reduction in ZmMrp3 expression (Figure 7B). However, there is no statistically significant change in ZmMrp4 expression in the presence of the transgene (Figure 7B).
The MRPs are the most thoroughly studied of the plant ABC transporters, with many in vitro studies detailing the transport kinetics and offering suggestions of possible biological roles. This characterization of ZmMrp3 is however the first to describe the role of a plant MRP in the in vivo transport of a specific endogenous substrate. Our work demonstrates that ZmMrp3 is strongly expressed in tissues actively synthesizing anthocyanin (Figure 2C), and its promoter is regulated by the maize anthocyanin transcription factors (Figure 3E). As well as being expressed in the expected tissue types, we also show that the ZmMRP3 protein is localized to the tonoplast, the site of transport of the anthocyanin pigment into the vacuole (Figure 4). Transgenic antisense plants in which ZmMrp3 expression is reduced below detectable levels are defective in anthocyanin accumulation, but in the presence of a silenced transgene, expression and pigmentation are restored (Figures 5 and 6). Together, these data confirm the role the encoded protein plays in the transport of anthocyanin. Although ZmMrp3 plays a role in the proper sequestration of anthocyanin, it does not appear to be the only transporter involved in this process. The antisense knockout of ZmMrp3 produces a strong phenotype in the leaves and tassels of pigmented plants, even when it is present as a single transgene array. However, this same construct fails to produce a phenotype in the aleurone when one, two, or three copies of the transgene array are present. In addition, the large number of genetic screens done by our lab and others have failed to identify a transporter gene as a component of the anthocyanin pathway in maize aleurone. This suggests the presence of a redundant transporter and/or an alternative and essential biological role for ZmMrp3.
Expression evidence argues strongly for the presence of an alternative transporter in the aleurone. ZmMrp4, a close homolog of ZmMrp3, is expressed in pigmented aleurone in a manner consistent with regulation by R and C1 (Figure 7A) and is unaffected by the expression of the antisense transgene (Figure 7B). This pattern of regulation is consistent with ZmMrp4 playing a role in anthocyanin sequestration in the aleurone and with genetic evidence suggesting that the transport step confers cell autonomy to the anthocyanin pathway. It is interesting to note that the transcript size of ZmMrp4 is significantly shorter than ZmMrp3, possibly reflecting the absence of the 5'-most TMD as seen in MRPs identified in maize (Swarbreck et al., 2003
Redundancy in anthocyanin structural genes is not uncommon. Both the chalcone synthase and chalcone isomerase genes are present in multiple copies in maize (Coe et al., 1981
The existence of another possible mechanism of anthocyanin transport has been inferred by data from A. thaliana, indicating that flavonoid transport is not limited to the MRPs. The tt12 mutant described by Debeaujon et al. (2001)
The tt12 mutant has no impact on the anthocyanin content in the Arabidopsis plant body, indicating that another transporter is involved in anthocyanin sequestration in these tissues. Indeed, maize, Arabidopsis, and petunia (Petunia hybrida) all require the activity of a specific GST for correct anthocyanin sequestration (Marrs et al., 1995
The presence of a MATE-type flavonoid transporter needs to be reconciled with the GST-mediated transport of anthocyanin in maize, petunia, and Arabidopsis. The finding that the tt19 gene in Arabidopsis encodes a GST that is required for anthocyanin pigmentation in the plant body (Kitamura et al., 2004
Another possibility is that the MRP is not a transporter but acts to modify the activity of MATE permeases. In mammals, a subclass of the MRPs termed sulfonylurea receptors (SUR) form a multimeric complex with a potassium channel, with the SUR proteins regulating the activity of the channel proteins (Babenko et al., 1998
Indirect evidence suggests that ZmMrp3 may play other, possibly essential roles, in maize. The expression of ZmMrp3 in the developing ear and unpigmented developing tassel (Figure 2C, Table 1) strongly argues for ZmMrp3 functions beyond anthocyanin transport. This would not be surprising given the extensive range of substrates and pleiotropic mutant phenotypes found for the A. thaliana MRPs (Lu et al., 1998 Our discovery of an anthocyanin-specific MRP provides an important model system for investigating the biology and biochemistry of both GST and MRP activity. Anthocyanin sequestration, with a known substrate and specific GST and MRP requirements, provides an excellent system for investigating several additional questions relating to the molecular mechanism of GST-MRP transport. Is the GST only involved in binding the substrate or, once bound, does it actively direct the substrate to the MRP? Does transport involve a direct GST–MRP interaction, and is this process selective? Does the GSH bound to the GST act to enhance the rate of transport? In light of the size of the GST and MRP families and the rate at which endogenous GST and MRP substrates are being identified, discovering the role of each component of this transport mechanism will be key to understanding how these molecules are efficiently moved from the cytoplasm to the correct subcellular location.
Plant Material The R-r C1 B Pl and r-g c1 b pl anthocyanin tester lines are in A188/W23 genetic background. Transgenic lines were generated in A188xB73 (Hi Type II) and crossed into the anthocyanin tester lines listed. Inbred line A188 (r-r c1 b pl) seedlings were grown as recommended by Sheen (1993) for protoplast isolation. Genomic and cDNA sequences were generated from RNA and DNA extracted from A188 or W23 inbred lines.
Reagents
Gene Expression Analysis
Quantitative RT-PCR was done using an Opticon 2 (MJ Bioworks, South San Francisco, CA). Primers used for amplification were as follows: ZmMrp3, 5'-CATCTCTGTGGAAAGGGTTC-3' and 5'-CTGTTCGCCCAACTATACCG-3'; ZmMrp4, 5'-TTCCGTGACCTCGTCAAAG-3' and 5'-TAGTTCTTCGGATGTGCAAG-3'; ZmMrp3, 5'-CGGAATAAAGCGGACACGTT-3' and 5'-TCTAGTGGAAGGCGCATCAA-3'; and actin, 5'-CGATTGAGCATGGCATTGTCA-3' and 5'-CCCACTAGCGTACAACGAA-3'. All primer pairs were determined to have equal amplification efficiency, and the PCR products were resolved by electrophoresis and sequenced to confirm the identity of each amplicon. The rate of amplification was monitored using SyberGreen fluorescence at 83°C. The amplification of all samples was normalized to actin amplification to account for variation in RNA levels, and all biological samples were tested in triplicate. Results are reported as 2– Ct, where Ct is the number of PCR cycles required to the log phase of amplification for the experimental gene minus the same measure for actin (Livak and Schmittgen, 2001), or as 2–Ct normalized to wild-type 2–Ct. Aleurone samples giving a positive signal for the ZmMrp3 were classified as transgenic. Standard error of the mean was calculated using standard statistical methods.
Cloning and Characterization of ZmMrp3 and ZmAbcf1 Full-length cDNA clones were amplified by PCR using Taq HiFi (Invitrogen) with the following primers. For ZmMrp3: D109F, 5'-CCATCGATATCCGCACGCGCCTTTAGCTACTA-3', and D95R, 5'-TACCGCGGATTAGATGTGTATGACCAGTACTC-3'; for ZmABCF1I: D88F, 5'-GCGGATCCACCCAACCCAACCAAGAAACAGAGG-3', and D89R, 5'-GGAATTCATAGCATACGGCACAGCACGCAGAGT-3'. ZmMrp3 is toxic in standard Escherichia coli cloning strains so it was cloned into the ClaI/SstII sites of Phagescript M13 (Stratagene, La Jolla, CA). ZmABCF1 was cloned into the EcoR1/BamH1 sites of pBluescript SK– (Stratagene). Both clones were fully sequenced. Genomic sequence of ZmMrp3 was recovered by PCR with Taq HiFi. Primers designed to sequences throughout the gene were used to ensure 3x coverage of the entire gene. Promoter sequence was recovered by digesting genomic DNA with HindIII and ligating it into pBluescript KS+. The resulting library was amplified by nested PCR using Taq HiFi. Amplification was done in two rounds with M13 reverse and T3 sequencing primers and the gene-specific primers D110R, 5'-GCAGCTGGAGGAGTCGAGTATGTC-3', and D129R, 5'-TCCCCGCGGATGATTCGTGCTTCAACTCCA-3'. The product was TA cloned into the pGEM-T vector (Promega, Madison, WI). Three independent clones were sequenced.
ARE motifs in ZmMRP3 were identified by manual sequence analysis and using the MEME system (Bailey and Elkan, 1994
Phylogenetic and Sequence Analysis
GFP Constructs and Confocal Scanning Microscopy
ZmMrp3:GFP was electroporated into etiolated maize protoplasts as described (Sheen, 1993 Scanning confocal imaging was done using a Nikon Diaphot inverted fluorescence microscope (Tokyo, Japan) with a Nikon 60 x 1.2 numerical aperture water immersion objective and a Bio-Rad MRC 1024 confocal head. Images were analyzed with LASERSHARP software (Bio-Rad, Hercules, CA).
Transgenic Plants
Both constructs
Pigment Extraction, Quantification, and HPLC Anthocyanin extractions for HPLC analysis were done as above except the tissue was ground in 0.1 N HCl/methanol for 1 min and immediately spun at full speed in a microcentrifuge for 5 min. The supernatant was removed and centrifuged for a further 5 min. The resulting supernatant was diluted with 5% acetic acid in ratios ranging from 1:1 to 20:1, depending on tissue type, and immediately loaded onto the HPLC. HPLC was done using a Dionex GP40 gradient pump (Dionex, Sunnyvale, CA) using a Microsorb 100-5 C18 column (Varian, Palo Alto, CA). Data were collected and analyzed using PEAKNET software (Dionex, Sunnyvale, CA). Pigment separation was by gradient elution with a flow rate of 0.75 mL/min. Solvent A, 5% acetic acid; solvent B, acetonitrile, 1 min at 90% A, 10% B; from 90% A, 10% B to 55% A, 45% B in 17.5 min; to 100% B in 2.5 min, at 100% B for 1 min; to 90% A, 10% B in 3 min; at 90% A, 10% B for 3 min. Absorbance was detected at 512 nm using a Dionex AD20 detector. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AP005828.1, AY609318 (ZmMrp3), and AY615521 (ZmAbcf1).
We would like to thank R. Meeley and Pioneer Hi-Bred for access to their EST database and the provision of materials, F. Theodoulou and J.V. Dean for providing data before publication, and R. Kurtz and MJ Bioworks for providing the Opticon 2 and for technical advice on quantitative real-time PCR. We would also like to thank K. Brewer, M. Abreu, J. Fernandes, T. DeHoog, and other members of the Walbot Lab for laboratory and field assistance. We would like to acknowledge D. Ehrhardt for the gift of the 35SGFP constructs and assistance with confocal microscopy. We would like to thank S. Long for providing materials and HPLC equipment. L. Mueller, G. Rudenko, C. Schmid, and S. Shah provided helpful suggestions and discussion. This work was supported by a grant from the National Science Foundation (IBN 9603927). C.D.G. was supported in part by a Natural Sciences and Engineering Research Council of Canada PGS-B fellowship.
1 Current address: School of Botany, University of Melbourne, Victoria, Australia, 3052.  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.plantcell.org) is: Virginia Walbot (walbot{at}stanford.edu). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.022574. Received April 1, 2004; accepted April 19, 2004.
Abell, L.M., Schloss, J.V., and Rendina, A.R. (1993). Target-site directed herbicide design. ACS Symp. Ser. 524, 16–37.
Ahmed, M.S., Ainley, K., Parish, J.H., and Hadi, S.M. (1994). Free radical-induced fragmentation of proteins by quercetin. Carcinogenesis 15, 1627–1630.
Alfenito, M.R., Souer, E., Goodman, C.D., Buell, R., Mol, J., Koes, R., and Walbot, V. (1998). Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases. Plant Cell 10, 1135–1149. Babenko, A.P., Aguilar-Bryan, L., and Bryan, J. (1998). A view of sur/KIR6.X, KATP channels. Annu. Rev. Physiol. 60, 667–687.[CrossRef][Web of Science][Medline] Bailey, T.L., and Elkan, C. (1994). Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2, 28–36.[Medline]
Bakos, E., Evers, R., Szakács, G., Tusnády, G.E., Welker, E., Szabó, K., de Haas, M., van Deemter, L., Borst, P., Varádi, A., and Sarkadi, B. (1998). Functional multidrug resistance protein (MRP) lacking the N-terminal transmembrane domain. J. Biol. Chem. 273, 32167–32175. Bodeau, J.P., and Walbot, V. (1996). Structure and regulation of the maize Bronze2 promoter. Plant Mol. Biol. 32, 599–609.[CrossRef][Web of Science][Medline] Borst, P., Evers, R., Kool, M., and Wijnholds, J. (1999). The multidrug resistance protein family. Biochim. Biophys. Acta 1461, 347–357.[Medline]
Bruce, W., Folkerts, O., Garnaat, C., Crasta, O., Roth, B., and Bowen, B. (2000). Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P. Plant Cell 12, 65–79.
Coe, E.H., McCormick, S.M., and Modena, S.A. (1981). White pollen in maize. J. Hered. 72, 318–320. Cormack, B.P., Valdivia, R.H., and Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.[CrossRef][Web of Science][Medline] Dean, M., and Allikmets, R. (2001). Complete characterization of the human ABC gene family. J. Bioenerg. Biomembr. 6, 475–479.
Debeaujon, I., Peeters, A.J.M., Léon-Kloosterziel, K.M., and Koornneef, M. (2001). The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell 13, 853–871. Edwards, R., and Dixon, D.P. (2000). The role of glutathione transferases in herbicide metabolism. In Herbicides and Their Mechanisms of Action, A.H. Cobb and A.C. Kirkwood, eds (Sheffield: Sheffield Academic Press), pp. 33–71. Felsenstein, J. (1989). PHYLIP—Phylogeny inference package (version 3.2). Cladistics 5, 164–166. Fossen, T., Slimestad, R., and Andersen, Ø.M. (2001). Anthocyanins from maize (Zea mays) and reed canarygrass (Phalaris arundinacea). J. Agric. Food Chem. 49, 2318–2321.[Medline] Gaedeke, N., Klein, M., Kolukisaoglu, U., Forestier, C., Müller, A., Ansorge, M., Becker, D., Mamnun, Y., Kuchler, K., Schulz, B., Mueller-Roeber, B., and Martinoia, E. (2001). The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement. EMBO J. 20, 1875–1887.[CrossRef][Web of Science][Medline] Grotewold, E., and Peterson, T. (1994). Isolation and characterization of a maize gene encoding chalcone flavonone isomerase. Mol. Gen. Genet. 242, 1–8.[Web of Science][Medline] Hammerschmidt, R. (1999). Phytoalexins: What have we learned after 60 years? Annu. Rev. Phytopathol. 37, 285–306. Higgins, C.F., Hiles, I.D., Salmond, G.P.C., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W., and Hermodson, M.A. (1986). A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature 323, 448–450.[CrossRef][Medline]
Hinder, B., Schellenberg, M., Rodoni, S., Ginsburg, S., Vogt, E., Martinoia, E., Matile, P., and Hörtensteiner, S. (1996). How plants dispose of chlorophyll catabolites. Directly energized uptake of tetrapyrrolic breakdown products into isolated vacuoles. J. Biol. Chem. 271, 27233–27236.
Hipfner, D.R., Almquist, K.C., Leslie, E.M., Gerlach, J.H., Grant, C.E., Deeley, R.G., and Cole, S.P.C. (1997). Membrane topology of the multidrug resistance protein (MRP). A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus. J. Biol. Chem. 272, 23623–23630. Ishikawa, T., Li, Z.S., Lu, Y.P., and Rea, P.A. (1997). The GS-X pump in plant, yeast, and animal cells: Structure, function, and gene expression. Biosci. Rep. 17, 189–207.[CrossRef][Web of Science][Medline]
Jasinski, M., Ducos, E., Martinoia, E., and Boutry, M. (2003). The ATP-binding cassette transporters: Structure, function, and gene family comparison between rice and Arabidopsis. Plant Physiol. 131, 1169–1177. Kermicle, J.L., and Alleman, M. (1990). Gametic imprinting in maize in relation to the angiosperm life cycle. Dev. Suppl. 9–14. Kitamura, S., Shikazono, N., and Tanaka, A. (2004). TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J. 37, 104–114.[CrossRef][Web of Science][Medline] Klein, M., Martinoia, E., Hoffmann-Thoma, G., and Weissenböck, G. (2000). A membrane-potential dependent ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides: Regulation of glucuronide uptake by glutathione and its conjugates. Plant J. 21, 289–304.[CrossRef][Web of Science][Medline] Klein, M., Perfus-Barbeoch, L., Frelet, A., Gaedeke, N., Reinhardt, D., Mueller-Roeber, B., Martinoia, E., and Forestier, C. (2003). The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use. Plant J. 33, 119–129.[CrossRef][Web of Science][Medline]
Klein, T.M., Roth, B.A., and Fromm, M.E. (1989). Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles. Proc. Natl. Acad. Sci. USA 86, 6681–6685. Kolukisaoglu, H.Ü., Bovet, L., Klein, M., Eggmann, T., Geisler, M., Wanke, D., Martinoia, E., and Schulz, B. (2002). Family business: The multidrug-resistance related protein (MRP) ABC transporter genes in Arabidopsis thaliana. Planta 216, 107–119.[CrossRef][Web of Science][Medline] Larson, R.L., and Coe, E.H. (1977). Gene dependent flavonoid glucosyltransferase in maize. Biochem. Genet. 15, 153–156.[CrossRef][Medline]
Lesnick, M.L., and Chandler, V.L. (1998). Activation of the maize anthocyanin gene a2 is mediated by an element conserved in many anthocyanin promoters. Plant Physiol. 117, 437–445.
Liu, G.S., Sánchez-Fernández, R., Li, Z.S., and Rea, P.A. (2001). Enhanced multispecificity of Arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2. J. Biol. Chem. 276, 8648–8656. Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408.[CrossRef][Web of Science][Medline]
Lu, Y.-P., Li, Z.-S., Drozdowicz, Y.M., Hörtensteiner, S., Martinoia, E., and Rea, P.A. (1998). AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites: Functional comparisons with AtMRP1. Plant Cell 10, 267–282.
Lu, Y.P., Li, Z.S., and Rea, P.A. (1997). AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: Isolation and functional definition of a plant ATP-binding cassette transporter gene. Proc. Natl. Acad. Sci. USA 94, 8243–8248. Marrs, K.A., Alfenito, M.R., Lloyd, A.M., and Walbot, V. (1995). A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature 375, 397–400.[CrossRef][Medline] Martinoia, E., Grill, E., Tommasini, R., Kreuz, K., and Amrhein, N. (1993). ATP-dependent glutathione S-conjugate export pump in the vacuolar membrane of plants. Nature 364, 247–249.[CrossRef]
Matthews, H., Clendennen, S.K., Caldwell, C.G., Liu, X.L., Connors, K., Matheis, N., Schuster, D.K., Menasco, D.J., Wagoner, W., Lightner, J., and Wagner, D.R. (2003). Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15, 1689–1703.
Morita, Y., Kataoka, A., Shiota, S., Mizushima, T., and Tsuchiya, T. (2000). NorM of vibrio parahaemolyticus is an Na(+)-driven multidrug efflux pump. J. Bacteriol. 182, 6694–6697. Neuffer, M.G., Coe, E.H., and Wessler, S.R. (1997). Mutants of Maize. (Plainview, NY: Cold Spring Harbor Laboratory Press), p. 77. Pollak, P.E., Hansen, K., Astwood, J.D., and Taylor, L.P. (1995). Conditional male-fertility in maize. Sex. Plant Reprod. 8, 231–241. Raizada, M.N., Benito, M.I., and Walbot, V. (2001). The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs. Plant J. 25, 79–91.[CrossRef][Web of Science][Medline]
Raizada, M.N., and Walbot, V. (2000). The late developmental pattern of Mu transposon excision is conferred by a cauliflower mosaic virus 35S-driven MURA cDNA in transgenic maize. Plant Cell 12, 5–21. Rea, P.A., Li, Z.S., Lu, Y.P., Drozdowicz, Y.M., and Martinoia, E. (1998). From vacuolar GS-X pumps to multispecific ABC transporters. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 727–760.[CrossRef][Web of Science]
Roth, B.A., Goff, S.A., Klein, T.M., and Fromm, M.E. (1991). C1- and R-dependent expression of the maize Bz1 gene requires sequences with homology to mammalian myb and myc binding sites. Plant Cell 3, 317–325.
Rudenko, G.N., and Walbot, V. (2001). Expression and post-transcriptional regulation of maize transposable element MuDR and its derivatives. Plant Cell 13, 553–570.
Rueff, J., Gaspar, J., and Laires, A. (1995). Structural requirements for mutagenicity of flavonoids upon nitrosation. A structure-activity study. Mutagenesis 10, 325–328. Sainz, M.B., Grotewold, E., and Chandler, V.L. (1997). Evidence for direct activation of an anthocyanin promoter by the maize C1 protein and comparison of DNA binding by related Myb domain proteins. Plant Cell 9, 611–625.[Abstract]
Sánchez-Fernández, R., Davies, T.G.E., Coleman, J.O.D., and Rea, P.A. (2001). The Arabidopsis thaliana ABC protein superfamily, a complete inventory. J. Biol. Chem. 276, 30231–30244. Sheen, J. (1993). Protein phosphatase activity is required for light-inducible gene expression in maize. EMBO J. 12, 3497–3505.[Web of Science][Medline] Shirley, B.W. (1996). Flavonoid biosynthesis: ‘New’ functions for an ‘old’ pathway. Trends Plant Sci. 1, 377–382.
Swanson, S.J., Bethke, P.C., and Jones, R.L. (1998). Barley aleurone cells contain two types of vacuoles. Characterization of lytic organelles by use of fluorescent probes. Plant Cell 10, 685–698. Swarbreck, D., Ripoll, P.-J., Brown, D.A., Edwards, K.J., and Theodoulou, F. (2003). Isolation and characterisation of two multidrug resistance associated protein genes from maize. Gene 315, 153–164.[Medline] Theodoulou, F.L. (2000). Plant ABC transporters. Biochim. Biophys. Acta 1465, 79–103.[Medline] Tommasini, R., Vogt, E., Fromenteau, M., Hörtensteiner, S., Matile, P., Amrhein, N., and Martinoia, E. (1998). An ABC-transporter of Arabidopsis thaliana has both glutathione-conjugate and chlorophyll catabolite transport activity. Plant J. 13, 773–780.[CrossRef][Web of Science][Medline] Tuerck, J.A., and Fromm, M.E. (1994). Elements of the maize A1 promoter required for transactivation by the anthocyanin B/C1 or phlobaphene P regulatory genes. Plant Cell 6, 1655–1663.[Abstract]
Tyzack, J.K., Wang, X., Belsham, G.J., and Proud, C.G. (2000). ABC50 interacts with eukaryotic initiation factor 2 and associates with the ribosome in an ATP-dependent manner. J. Biol. Chem. 275, 34131–34139. Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951.[Web of Science][Medline] This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
190-kD MRPs constitute large protein families in many eukaryotes. There are nine MRPs described in Homo sapiens (Dean and Allikmets, 2001
