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DNA Research Advance Access originally published online on December 12, 2006
DNA Research 2006 13(5):205-228; doi:10.1093/dnares/dsl013
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© The Author 2006. Kazusa DNA Research Institute.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Genome-wide analysis of ATP-binding cassette (ABC) proteins in a model legume plant, Lotus japonicus: comparison with Arabidopsis ABC protein family

Akifumi Sugiyama1, Nobukazu Shitan1, Shusei Sato2, Yasukazu Nakamura2, Satoshi Tabata2 and Kazufumi Yazaki1,*

1 Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere Kyoto University, Gokasho, Uji 611–0011, Japan
2 Kazusa DNA Research Institute, 2–6–7 Kazusa-Kamatari, Kisarazu, Chiba, 292–0812, Japan

Received 3 August 2006; revised 30 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Acknowledgements
 REFERENCES
 
ATP-binding cassette (ABC) proteins constitute a large family in plants with more than 120 members each in Arabidopsis and rice, and have various functions including the transport of auxin and alkaloid, as well as the regulation of stomata movement. In this report, we carried out genome-wide analysis of ABC protein genes in a model legume plant, Lotus japonicus. For analysis of the Lotus genome sequence, we devised a new method ‘domain-based clustering analysis’, where domain structures like the nucleotide-binding domain (NBD) and transmembrane domain (TMD), instead of full-length amino acid sequences, are used to compare phylogenetically each other. This method enabled us to characterize fragments of ABC proteins, which frequently appear in a draft sequence of the Lotus genome. We identified 91 putative ABC proteins in L. japonicus, i.e. 43 ‘full-size’, 40 ‘half-size’ and 18 ‘soluble’ putative ABC proteins. The characteristic feature of the composition is that Lotus has extraordinarily many paralogs similar to AtMRP14 and AtPDR12, which are at least six and five members, respectively. Expression analysis of the latter genes performed with real-time quantitative reverse transcription–PCR revealed their putative involvement in the nodulation process.

Key words: ABC protein; domain-based clustering analysis; genome-wide analysis; Lotus japonicus; SMC subfamily


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and Methods
 3. Results and Discussion
 Acknowledgements
 REFERENCES
 
ATP-binding cassette (ABC) proteins constitute one of the largest families in plants, with more than 120 members each in Arabidopsis and rice, which function as transporters, channel regulators and molecular switches. ABC proteins share highly conserved amino acid sequence domains designated nucleotide binding domains (NBDs). Each NBD contains three characteristic motifs of Walker A [GX4GK(ST)], Walker B boxes [(RK)X3GX3L (hydrophobic)3]1Go and an ABC signature [(LIVMFY)S(SG)GX3(RKA)(LIVMYA)X(LIVFM) (AG)],2Go the last of which is situated between two Walker boxes. Most ABC proteins contain one or two transmembrane domains (TMDs), which generally consist of 4–6 transmembrane {alpha}-helices, while several members lacking TMD appear to be ‘soluble’ proteins. The majority of eukaryotic members characterized so far are ‘full-size’ ABC proteins, which contain two NBDs and two TMDs in a single polypeptide, either forming ‘forward’ TMD1-NBD1-TMD2-NBD2 or ‘reverse’ NBD1-TMD1-NBD2-TMD2 orientation. Those with one NBD and one TMD are referred to as ‘half-size’ ABC proteins.

Inventories of plant ABC proteins are available for Arabidopsis and rice,3Go–5Go whereas little is known about ABC proteins in an important family, Fabaceae. Fabaceae, which is composed of 700 genera and 20 000 species,6Go represents the third largest plant family next to Orchidaceae and Asteraceae and has significant agricultural importance as dicots. A hallmark feature of legumes is their ability to obtain nitrogen-containing nutrients via symbiosis with soil microbes. This ability is important not only for agriculture but also for the environment as it replaces synthetic nitrogen fertilizers. Thus, studies of the mechanism of symbiotic nitrogen fixation (SNF) between fabaceous plants and rhizobia are of particular importance from agricultural and environmental viewpoints as well as for basic science on plant-microbe interactions.

In this report we provide an inventory of ABC proteins in Lotus japonicus, a model legume, whose genome sequence information is available from the Lotus genome project of Kazusa DNA Research Institute. As an informatics approach, we have applied a new method ‘domain-based clustering analysis’ for the phylogenetic analyses of NBDs and TMDs of ABC proteins. By comparing Lotus ABC proteins with those of Arabidopsis and rice, we have identified the features of Lotus ABC proteins, and demonstrated expression analysis for those characteristic genes of Lotus in their relevance to nodulation and SNF.


   2. Materials and Methods
Top
 Abstract
 1. Introduction
 2. Materials and Methods
3. Results and Discussion
 Acknowledgements
 REFERENCES
 
2.1. Domain-based clustering analysis of ABC proteins
The amino acid sequences of all ABC proteins of Arabidopsis were divided into fragments of NBDs and TMDs. For NBD fragments, we used 220 amino acid sequences starting from 10 residues ahead of the consensus glycine of Walker A. TMDs of Arabidopsis ABC proteins were extracted from the web page ARAMEMNON (http://aramemnon.botanik.uni-koeln.de).7Go We used the predicted TMD regions of AtATH1, AtMDR1, AtMRP1, AtWBC1 and AtPDR1 as representatives of each subfamily ABCA', B, C and G, respectively, to define the TMD regions of the other members in respective subfamilies, where multiple alignments were made with the ClustalW program.8Go AtMRP6, 11, 15 are ABCC members that lack in the N-terminal extension T0, and these are not found in the T0 cluster in Fig. 1.


Figure 1
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  		Figure 1. Phylogenetic relationship of NBDs and TMDs of Arabidopsis ABC proteins. The amino acid sequences of NBDs of all Arabidopsis ABC proteins and those of TMDs of membrane-localized ABC proteins in Arabidopsis were aligned using the ClustalW program as described in Materials and Methods. The nomenclature of Sanchez-Fernandez et al.3Go was used, and the abbreviations of ABC proteins are as follows: ATH, ABC-two-homolog; ATM, ABC transporter of mitochondria; GCN, general control non-repressible; MDR, multi-drug resistance; MRP, multi-drug resistance-associated protein; NAP, non-intrinsic ABC protein; PDR, pleiotropic drug resistance; PMP, peroxisomal membrane protein; RLI, RNase L inhibitor; SMC, structural maintenance of chromosome; TAP, transporter associated with antigen processing; WBC, white-brown complex. N1 and N2 were referred to as NBD1 and NBD2, respectively, and T0, T1 and T2 represent TMD0, TMD1 and TMD2, respectively. ATH is ABCA', MDR, TAP and ATM are ABCB, MRP is ABCC, PMP is ABCD, RLI is ABCE, GCN is ABCF and WBC is ABCG in the systemic name used in the text.

 
NBDs of L. japonicus were extracted from Lotus fragments with ABC signature(s) as described above for the phylogenetic analyses of NBDs. Fragments containing <220 amino acids were also used if they have an ABC signature. The shortest fragment was 75 amino acids of Ljwgs_041501.2 in ABCC subfamily, but most of the fragments contained a large part of NBD. For the phylogenetic analyses of TMDs, Lotus fragments that show amino acid similarity to >50% of the TMD region of Arabidopsis counterparts were used. Multiple alignments were performed using ClustalW program with the default setting, and phylogenetic trees were viewed with TreeView.9Go

2.2. Identification of ABC proteins in L. japonicus genome
All putative amino acid sequences of L. japonicus available from the genome sequencing project of Kazusa DNA Research Institute were used as query sequences for BLASTP searches10Go against the proteome of A. thaliana. We employed L. japonicus proteins whose top hits in the BLAST search were ABC proteins for further analysis. Each Lotus protein was aligned with Arabidopsis counterparts using the ClustalW program to find consensus sequences of Walker A, Walker B and the ABC signature.

2.3. Identification of SMC proteins in the rice genome
Arabidopsis SMC proteins were used as query sequences for BLASTP searches.10Go Putative SMC proteins of rice were aligned with Arabidopsis SMC proteins, and the presence of the Walker A box, Walker B box and the ABC signature was confirmed.

2.4. Plant materials
Seeds of L. japonicus cv. MG-20 were scarified with sandpaper (no. 120), and surface-sterilized with 1% sodium hypochlorite for 10 min. Surface-sterilized seeds were sown on autoclaved vermiculite supplemented with NF medium11Go and germinated at 25°C under illumination with a 16/8 h (light/dark) photoperiod (120 µmol m–2 s–1) and 1-week-old seedlings were used for inoculation with symbiotic bacteria, Mesorhizobium loti strain Tono12Go, which had been cultured in YEM medium 13Go for 2 days at 25°C in the dark. Uninoculated plants were used as a negative control. Both inoculated and uninoculated plants were grown in a growth chamber at 25°C with 16/8 h (light/dark) photoperiod (120 µmol m–2 s–1); nodule formation was only observed in inoculated plants. After 3 weeks of inoculation, leaves, stems, roots and nodulated roots were harvested. These organs were immediately frozen in liquid nitrogen and kept at –80°C until the extraction of total RNA.

2.5. RNA isolation and real-time reverse transcription (RT)–PCR
Total RNA was isolated with the RNeasy Plant Mini-Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. Reverse transcription was done with SuperScript III reverse transcriptase (Invitrogen, CA), followed by incubation with RNase H (Invitrogen, CA). Real-time PCR reactions were performed with the Roter-Gene 3000A (Corbett Research, Australia), using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, CA) according to the manufacturers' instructions. Briefly, the PCR reaction mixture consisted of 10 ng of cDNA template, 5 pmol of primers, 1 µl of fluorescent probe provided by the above kit and 12.5 µl of Platinum Quantitative PCR SuperMix-UDG in a total volume of 25 µl, and the standard reaction condition was as follows: 95°C for 10 min, 40 cycles of 95°C 15 s, 55°C for 30 s, 72°C 30 s. The primers used to detect each mRNA species are listed in Table 1.


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  		Table 1. Oligonucleotide primers used in this work

 

   3. Results and Discussion
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
Acknowledgements
 REFERENCES
 
3.1. Domain-based clustering analysis of ABC proteins
The genome sequences of L. japonicus, available from the genome sequencing project of Kazusa DNA Research Institute, consist of sequences derived from transformation-competent artificial chromosome (TAC) clones, bacterial artificial chromosome (BAC) clones and those from whole genome shotgun sequencing. The estimated coverage is 70% (331 Mb) of the whole genome (470 Mb),14Go and these sequences will be publicly available soon. Since these sequences cover ~90% of the expressed-sequence tags registered in the database (S. Sato et al., unpublished data), i.e. a large part of euchromatin region was sequenced, we can use these sequence data to analyze ABC protein family in a genome-wide manner. Our analysis in L. jaoponicus provides a valuable in silico tool for other researchers to obtain information from the data of other genome projects, which mainly aim to sequence the euchromatin region.

As full-size ABC proteins are large, typically composed of >1200 amino acids, most of the putative open reading frames (ORFs) estimated in the genome sequence are fragmental, i.e. the full-length sequence was obtained in only limited members. Thus, we have devised a new method ‘domain-based clustering analysis’ to classify the member composition of ABC proteins, in which amino acid sequence of NBDs and TMDs instead of the entire proteins are employed to cluster them. First, we applied this method to Arabidopsis ABC proteins to assess the validity of this method.

Sanchez-Fernandez et al.3Go reported that Arabidopsis contains 129 ABC proteins, and van den Brule and Smart15Go later added two more pleiotropic drug resistance (PDR) members by detailed search of this subfamily in the Arabidopsis genome, resulting in 131 members of ABC proteins in Arabidopsis. In our analysis of their NBDs, however, it was revealed that 8 of the 131 members, i.e. AtATH8 (ABC-two-homolog 8) (At2g39190), AtATH9 (At2g40090), AtATH10 (At4g01660), AtATH13 (At5g64940), AtNAP1 (non-intrinsic ABC protein 1) (At4g04770), AtNAP4 (A1g03900), AtNAP5 (A1g71330) and AtNAP6 (At1g32500), do not have consensus NBD sequences, including an ABC signature, despite being annotated as ABC proteins. Thus, these sequences were excluded from the domain-based clustering analysis of NBD in this study, and members of the structural maintenance of chromosome (SMC) subfamily, which have a largely separated NBD, were also not analyzed. We then extracted fragments of NBD and TMD from 119 sequences as described in Materials and Methods, which were named to reflect the position of the full-length polypeptide sequence; for example, NBD1, NBD2, TMD1 and TMD2 of AtMDR1 were designated as AtMDR1-N1, AtMDR1-N2, AtMDR1-T1 and AtMDR1-T2, respectively. The first TMD of the members of the multi-drug-resistance-associated protein (MRP) subfamily was indicated as T0.

A phylogenetic tree was constructed from all amino acid sequences of NBD (Fig. 1). In Arabidopsis, NBD1 and NBD2 of full-size ABC proteins as well as soluble ABC proteins having two NBDs like GCN (general control non-repressible) subfamily were separately clustered except for MDR and NAP subfamilies. Rice ABC proteins5Go also gave nearly identical relationship of NBD sequences. This suggests that in the MDR subfamily similarity between NBD1 and NBD2 is clearly higher than in other subfamilies, whereas NAP members are very divergent in their amino acid sequence. This phylogenetic relationship within NBD is nearly identical to that of full-length proteins.3Go We then applied this method to all amino acid sequences of TMDs to construct the phylogenetic tree (Fig. 1). It was clearly shown that each TMD1 and TMD2 of all full-size ABC protein subfamilies was clustered in an individual manner, including the MDR subfamily, indicating that the similarity among TMD1 of each full-size ABC protein is higher than the similarity between TMD1 and TMD2 in the same molecule. TMDs of half-size ABC transporters such as TAP (transporter associated with antigen processing) and ATM (ABC transporter of mitochondria) were also likely to be clustered together. These findings indicate that TMD1 and TMD2 sequences are also conserved individually enough to distinguish TMDs of ABC proteins from those of other membrane proteins in sequence-based homology searches of Lotus genome. Comparing these phylogenetic trees to those of full-length polypeptides,3Go domain-based clustering analysis can be reasonably applied to fragments of L. japonicus ABC proteins. In MRPs, TMD0 was divided into two clusters, whereas TMD1 and TMD2 form each group. It is interesting that the split TMD0 clusters appear to reflect the predicted subcellular localization of either the vacuole or plasma membrane, indicating that TMD0 may be the determinant of the targeting membrane. These results indicate that each NBD or TMD represents the whole protein sequence, and therefore, domain-based clustering analysis can be used to classify an anonymous fragment of ABC proteins either with NBD or TMD obtained from the genome sequence into an appropriate subfamily, and can also be utilized to identify that the NBD or TMD belong to either N- or C-terminal flanking region. These results also strongly suggest that NBD1 of full-size ABC proteins are not equivalent to NBD2 except for MDR subfamily members, and that TMD1 of full-size ABC proteins are not equivalent to TMD2 either, even for the MDR subfamily.

Frequent gene duplication of NBDs has been suggested from the phylogenetic analysis of NBDs of human ABC protein genes.16Go The phylogenetic analysis of Arabidopsis TMDs also provides an evidence for high duplication of TMDs in a similar manner as NBDs, i.e. several independent gene duplication events, because the diversity of two TMDs in a single polypeptide is higher than that within TMD1s or TMD2s in the subfamily.

3.2. Identification of ABC proteins in L. japonicus
In the genome of L. japonicus, we found 394 ORFs that gave higher similarity to ABC proteins than to any other proteins of Arabidopsis. Among those 394 putative polypeptides, 112 ORFs contained at least one ABC signature (Table 2). TM, BM, CM and Ljwgs in Table 2 refer to TAC clone, BAC clone, contig of TAC and BAC clones and whole genome shotgun, respectively. The nomenclature of Sanchez-Fernandez et al.3Go has been widely used for plant ABC proteins by other plant researchers,15Go,17Go,18Go while Garcia et al.5Go used another nomenclature for rice ABC proteins. We employed in this report the nomenclature of human ABC proteins16Go that is generally accepted by most mammalian ABC protein researchers and more common for eukaryotic ABC proteins. Most subfamilies of plant ABC proteins have corresponding counterparts in the human genome, which are classified into eight subfamilies ABCA–ABCG, whereas plant-specific subfamilies like PDR are named with conventional designation.


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  		Table 2. Inventory of Lotus japonicus putative ABC proteins

 
3.3. ABCA and ABCA' subfamily
The plant ABCA subfamily has been considered to consist of full-size and half-size proteins, and the latter are also called the ATH subfamily, which stands for ABC-two-homolog. As all human ABCA proteins are full-size, it may be confusing to use the conventional name ‘ABCA’ for half-size members, and the name ‘ABC two’ has not been commonly used in recent years. We would like to propose ABCA' subfamily for half-size members ATHs. By this classification, we can ordinary distinguish full-size ABCA members and sequentially similar half-size members from other subfamilies. Only one full-size ABCA member (AtABCA1) is present in the Arabidopsis genome, whereas no homolog has been found in the rice genome.3Go,18Go In the genome sequences of L. japonicus, we found nine fragments similar to AtABCA1, eight of which showed high amino acid similarity to AtABCA1 (Table 2). Ljwgs_011569.2, Ljwgs_024406.1, Ljwgs_026106.1.1, Ljwgs_031377.1, Ljwgs_039697.1, Ljwgs_047252.1, Ljwgs_058746.1 and Ljwgs_058851.1 show similarity to the following regions: 742–1037, 1–115, 1034–1105, 520–578, 412–518, 1159–1253, 689–740 and 1268–1329 amino acid position of AtABCA1, respectively (see Supplementary Figure 1). As each of those fragments showed similarity to different parts of AtABCA1 and no overlapped fragment having different DNA sequence was seen, it can be concluded that these eight fragments are derived from one ABCA protein of L. japonicus.

Arabidopsis and rice contain 16 and 7 members of half-size ABCA' proteins, respectively,3Go,5Go whereas in L. japonicus we found 34 fragments that show striking amino acid similarity to ABCA' proteins of both model plants (Table 2). Among them two fragments have an ABC signature, indicating that at least two members of ABCA' protein (TM1130.25 and TM1130.29) exist in the L. japonicus genome (Table 3).


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  		Table 3. Comparison of the ABC protein superfamily in plants and humans

 
Some members of this subfamily have been studied intensively in humans due to their clinical importance. ABCA1, ABCA2 and ABCA4 have been reported to be involved in cholesterol transport, drug resistance and Rod photoreceptor retinoid transport, respectively.19Go In plants, however, the physiological functions of these subfamily members are not clear, except for AtABCA1, which is preferentially expressed in the pollen of Arabidopsis (K. Yazaki; unpublished data) and seems to regulate pollen germination (personal communication from Dr C. Forestier of Cadarache Institute). Since all plant AtABCA1 orthologs found in EST databases are limited in dicots, e.g. soybean (Glycine max), common bean (Phaseolus vulgaris), potato (Solanum tuberosum) and tomato (Lycopersicon esculentum), while only half-size members are observed in monocots, ABCA proteins may have a function specific to dicots, or ABCA' members may complement the function of ABCA member.

3.4. ABCB subfamily
The Arabidopsis ABCB subfamily consists of 22 full-size members, which are conventionally named MDR or PGP, five half-size members that comprise 2 TAPs and 3 ATMs,3Go whereas 24 full-size (MDR) and 4 half-size (3 TAPs and 1 ATM) proteins are present in the rice genome.5Go In Lotus, we found 60 fragments that showed similarity to Arabidopsis full-size proteins, 23 of which had at least one ABC signature (Table 2). Since NBD1 and NBD2 of the ABCB subfamily of Arabisopsis are not obviously distinguishable as shown in Fig. 1, domain-based clustering analysis with NBDs of Lotus and Arabidopsis did not show clear separation between NBD1 and NBD2 (Fig. 2A). We then employed TMDs for domain-based clustering analysis to clarify the phylogenetic relationship of this subfamily in Lotus as a more reliable method for this subfamily. A phylogenetic tree was constructed with 22 TMDs obtained from 18 fragments of Lotus and TMDs of all Arabidopsis full-size ABCB proteins (Fig. 2B). From this result it is predicted that at least 12 full-size proteins of the ABCB subfamily are present in the Lotus genome, because 9 fragments contain TMD1, 7 fragments contain TMD2 and 3 fragments contain both TMD1 and TMD2.


Figure 2
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  		Figure 2. Domain-based clustering analysis. (A) The amino acid sequences of putative NBDs of Lotus fragments similar to ABCB proteins and Arabidopsis ABCB subfamily members were aligned using the ClustalW program. (B) The amino acid sequences of putative TMDs of Lotus fragments similar to ABCB proteins and Arabidopsis ABCB subfamily members were aligned using the ClustalW program. (C) The amino-acid sequences of NBDs of Lotus fragments showing highest similarity to ABCC proteins and those of Arabidopsis NBDs of ABCC members were aligned using the ClustalW program. (D) The amino acid sequences of NBDs of Lotus fragments similar to ABCF proteins with at least one ABC signature and those of Arabidopsis NBDs of ABCF members were aligned using the ClustalW program. (E) The amino acid sequences of NBDs of Lotus fragments similar to PDR proteins with at least one ABC signature and those of Arabidopsis NBDs of PDR members were aligned using the ClustalW program. N1 and N2 represent NBD1 and NBD2, respectively, and T1 and T2 represent TMD1 and TMD2, respectively. Each number before the hyphen corresponds to the nomenclature of the ABCB member of Arabidopsis according to Sanchez-Fernandez et al.3Go Ljwgs is referred to as sequences assembled from the whole genome shotgun, and TM, CM are referred to as sequences from TAC and BAC clones. Lotus genes are shown in red.

 
We also found four fragments showing striking similarity to Arabidopsis TAP and one fragment of ATM half-size protein (Table 2). Two of the TAP-like fragments have an ABC signature, suggesting that two TAP proteins are in Lotus as in other plant species analyzed. In total, the number of Lotus ABCB proteins is estimated as 15, which implies 12 full-size MDR-type, 2 TAP-like members and 1 ATM-like protein (Table 3).

In humans this subfamily is comprised of 11 members, including 7 half-size proteins. Most intensive studies of this subfamily have been done with ABCB1, also called MDR1, whose gene product was designated to be PGP.20Go This was the first eukaryotic ABC protein identified as the drug efflux pump responsible for multi-drug resistance in cancer cells.21Go Only one member of this subfamily in Arabidopsis, AtMDR1, also known as AtPGP1, was reported to confer herbicide tolerance when over-expressed in plants.22Go Our trials to over-express other MDR-type ABC transporters derived from various plants including Arabidopsis failed to confer a multi-drug resistance phenotype, suggesting that the name MDR is actually inappropriate as the general name of full-sized ABCB subfamily members. It is noteworthy that this subfamily has attracted the particular attention of plant hormone researchers, because some full-size members of this subfamily have been involved in polar auxin transport in plants. Geislar et al.23Go showed the AtPGP1-mediated efflux of auxin in yeast and HeLa cells, and counterparts of this ABC protein in maize and sorghum were reportedly also responsible for auxin transport in monocots.24Go Another member, AtPGP4, was recently reported as an auxin transporter regulating basipetal transport in the root.25Go,26Go Although not all full-size ABCB proteins may be involved in auxin transport, the ABCB subfamily probably plays a central role in auxin transport together with the PIN family, also in Lotus. Plant full-size ABCB proteins contain members functioning as inward transporters, contrary to other eukaryotic ABC proteins that mediate the efflux of substrate from cytosol.27Go The Lotus ABCB subfamily will provide other members for study of the mechanism to determine transport direction.

Half-size proteins in humans, ABCB2 and ABCB3, are also called TAP1 and TAP2, respectively, which form a heterodimer to transport peptides into the ER lumen for peptide processing, resulting in presentation on the cell surface by class I major histocompatibility complex molecules.28Go A barley half-size TAP-like protein, IDI7, was identified as an iron-deficiency-induced gene,29Go but its physiological functions, including the transport substrate, remain to be clarified. Another half-size protein, ABCB7 of humans, also known as ABC7, is an ortholog of yeast ATM1 and has been reported to be a mitochondrial protein that functions in the biogenesis of iron–sulfur proteins.30Go Arabidopsis ATM homolog AtATM3, also called STA1, was identified from the starik mutant, which showed dwarfism and chlorosis, and was reported to function in the biogenesis of iron–sulfur clusters.31Go Recently, AtATM3 was reported to be involved in heavy metal resistance.32Go

3.5. ABCC subfamily
The ABCC subfamily consists of full-size ABC proteins conventionally designated as MRP, which often have N-terminal extension of the TMD. There are 15 and 17 members in the ABCC subfamily in Arabidopsis and rice, respectively,3Go,5Go including a pseudogene AtMRP15 in Arabidopsis.17Go In Lotus, we found 71 fragments with strong similarity to Arabidopsis MRP proteins (Table 2). Among them, 23 fragments have at least one ABC signature. Domain-based clustering analysis was performed with the putative NBD regions of those fragments and those of Arabidopsis ABCC proteins (Fig. 2C). Analysis showed that 7 fragments contain NBD1, 15 fragments contain NBD2 and 2 fragments contain both NBD1 and NBD2. Taken together, it is estimated that 17 ABCC proteins exist in Lotus (Table 3). Domain-based clustering analysis with TMDs also gave a similar result (data not shown).

As is the case for human ABCC proteins like ABCC1, ABCC2 and ABCC3,16Go some plant ABCC proteins also recognize glutathione conjugates as their transport substrate. For instance, AtMRP1 shows substrate specificity for a wide variety of glutathione (GSH) conjugates of cadmium, dinitrophenol and metolachlor, as well as oxidized GSH, while both AtMRP2 and AtMRP3 transport chlorophyll catabolite adding to several GSH conjugates.33Go–35Go In addition, an ABCC protein of maize has been reported to be involved in the transport of anthocyanin, probably in a GSH-dependent manner.36Go Human ABCC subfamily members also function as ion channel and/or channel regulators, e.g. ABCC7, also known as cystic fibrosis transmembrane conductance regulator (CFTR), is a chloride ion channel,37Go whereas ABCC8 and ABCC9, conventionally named sulfonylurea receptor 1 (SUR1) and SUR2, respectively, act as regulatory subunits of the potassium channel regulating insulin secretion38Go in an ATP-sensitive manner. Despite detailed analysis, plants do not seem to have bona fide counterparts of CFTR and SUR, whilst AtMRP4 and AtMRP5 are involved in stomata movement,39Go–41Go where ion channels play a central role, and the latter could bind sulfonylurea,42Go suggesting that these are, at least, functional counterparts of SUR proteins in Arabidopsis.

3.6. ABCD subfamily
ABC proteins localized at the peroxisome have characteristic sequences highly conserved only among this subfamily, which is classified as the ABCD subfamily. In humans, this subfamily consists of four members, which are all half-size ABC proteins. In contrast, Arabidopsis and rice ABCD contain one and two full-size ABCD proteins, respectively, in addition to one half-size protein for each plant.3Go,5Go In Lotus, four and three fragments have similarity to half-size and full-size ABCD proteins, respectively (Table 2). Although ABC signatures are not observed in those fragments, it is presumed that the number of half-size and full-size ABCD protein in the Lotus genome sequence is at least one each (Table 3).

Three independent research groups reported full-size Arabidopsis ABCD protein nearly at the same time, named PXA1 (Peroxisomal ABC transporter 1),43Go PED3 (Peroxisome defective 3)44Go and CTS (Comatose) meaning ‘forever dormant’.45Go They demonstrated that this ABCD member has the function of transporting acyl-CoA esters from cytosol to peroxisome, resulting in energy generation from storage lipid in seeds, which promote seed germination. Other phenotypes of this mutant were high resistance to indolebutyric acid and 2,4-dichlorophenoxybutyric acid, growth retardation and fewer lateral roots.43Go,44Go In contrast to full-size ABCD protein, there is no report on half-size ABCD proteins in plants, although they are close counterparts of human ABCD protein (PMP70), which has been intensively studied as the transporter responsible for the import of fatty acids and/or fatty acyl-CoAs into peroxisome.16Go

3.7. ABCE subfamily
The ABCE subfamily is a group of soluble ABC proteins having two NBDs in a molecule but no TMD. In the human genome, only one member was found in this ABCE subfamily, which is known as RNase L inhibitor (RLI).46Go Each Arabidopsis and rice genome has two members in this subfamily,3Go,5Go whereas we found seven fragments in Lotus that have similarity to Arabidopsis ABCE proteins (Table 2). One of the fragments contained two ABC signatures, suggesting that Lotus has at least one ABCE proteins (Table 3). One of the Arabisposis ABCE proteins, AtRLI2, was recently reported to be highly expressed in transgenic plants showing RNA interference, irrespective of the target genes.47Go If plant ABCE proteins also function as RLI, these members may be involved in physiological functions relevant to viral resistance in plants.

3.8. ABCF subfamily
Another soluble ABC protein group having two NBDs and no TMD, i.e. the ABCF subfamily, is also called the GCN subfamily. Three members are known in humans, and human ABCF1 reportedly functioned in the enhancement of protein synthesis and in the inflammation process.48Go In Lotus we found 19 fragments with significant similarity to ABCF proteins of Arabidopsis and, 8 have at least one ABC signature (Table 2). Domain-based clustering analysis of NBDs of both Arabidopsis ABCF proteins and eight fragments of Lotus suggests that two fragments have NBD1, two fragments have NBD2 and four fragment have both NBD1 and NBD2 (Fig. 2D). From this analysis, we estimated the number of Lotus ABCF proteins as at least six, whereas both Arabidopsis and rice have five ABCF proteins (Table 3).3Go,5Go There has been no report on GCN proteins in plants so far, but most members are highly expressed in the root tissues of Arabidopsis (personal communication with Dr T. Kato of Oji-paper Company). In fact, EST analysis also suggests all Arabidopsis ABCF proteins except AtGCN2 are expressed, although their functions are still unknown.

3.9. ABCG subfamily
The ABCG subfamily consists of half-size proteins with ‘reverse’ orientation, which was conventionally designated as the white–brown complex (WBC) subfamily. Arabidopsis and rice contain 29 and 30 members of this subfamily, respectively,3Go,5Go and this represents the largest subfamily in both plants. The large subfamily size of the ABCG is one of the characteristics of plant ABC proteins, as the human genome has only six ABCG members. In Lotus we found 64 fragments that can be classified into ABCG proteins (Table 2) due to their similarity to Arabidopsis orthologs. Among them 24 fragments each possess an independent ABC signature, which indicates that Lotus has at least 24 ABCG proteins (Table 3). Domain-based clustering analysis was performed with the putative NBD regions of those fragments and those of Arabidopsis ABCG proteins, but we could not find Lotus-specific clade (Supplementary Figure 2).

Intensive studies on ABCG proteins have been performed for Drosophila ABCG proteins. Members designated ‘white, brown and scarlet proteins’ are presumed to be involved in the transport of pigment precursors in the eyes of Drosophila.49Go In humans, ABCG1 was reported to play a role in the regulation of cholesterol transport,50Go whereas other members, ABCG5 and ABCG8, were reported to form a heterodimer and transport cholesterol in epithelial cells of the intestine.51Go In plants, cloning of these subfamily members has been only recently reported, e.g. GhWBC1 of cotton (Gossypium hirsutum), which is highly expressed in developing fiber cells,52Go NtWBC1 of tobacco (Nicotiana tabacum) expressed specifically in reproductive organs,53Go and AtWBC12 proposed to be relevant to transport cuticular wax at the stem epidermis.54Go Adding to them, AtWBC19 shows kanamycin resistance in Arabidopsis;55Go however, no member with a corresponding function to human ABCGs has been reported so far.

3.10. PDR subfamily
The PDR subfamily is comprised of full-size proteins in the ‘reverse’ orientation, NBD1-TMD1-NBD2-TMD2. This subfamily is recognized as being specific to plants, including yeast, and is not found in the human genome. In Arabidopsis and rice, 15 and 21 members have been identified, respectively,3Go,5Go while in yeast (Saccharomyces cerevisiae), 9 PDRs exist out of 30 ABC protein genes.56Go,57Go A genome-wide search in Lotus resulted in 71 fragments that showed the highest similarity to Arabidopsis PDR proteins (Table 2). Of these we found 18 fragments with at least one ABC signature. Domain-based clustering analysis was performed with the putative NBD sequences of those fragments and those of Arabidopsis PDR proteins (Fig. 2E). Analysis showed that 6 fragments contained NBD1, 10 fragments contained NBD2, and 2 fragments contained both NBD1 and NBD2. Based on these data, we presumed that the Lotus genome possesses at least 12 PDR proteins (Table 3). In our analysis, NBDs of PDR showed the most clear separation between NBD1 and NBD2, which suggests that the tandem repeat of this subfamily is composed of two relatively divergent structure units.

The name PDR is derived from yeast PDR5 that was identified as a transporter conferring multi-drug resistance,58Go and its functions have been elucidated in detail. PDR5 is a plasmamembrane-localized transporter that can export a broad range of drugs, such as anti-fungals, anti-cancer drugs, detergents, ionophores and steroids.59Go–62Go The first characterized plant PDR gene, SpTUR2, was cloned from Spirodela polyrrhiza63Go as a gene highly induced in turion formation, and NpPDR1, formally called NpABC1 of Nicotiana plumbaginifolia, was reported to function in the plant defense pathway by excreting an endogenous anti-fungal diterpene, sclareol.64Go,65Go It has been reported very recently that AtPDR8 contributes non-host resistance in Arabidopsis by two independent groups.66Go,67Go All these reports indicate that plant PDR proteins appear to play important roles in plant defense responses, while it was also suggested that AtPDR12 is involved in lead resistance68Go and that NtPDR3 is induced by iron deficiency.69Go

3.11. SMC subfamily
SMC proteins are found in both prokaryotes and eukaryotes, and their functions are proposed to be chromosome condensation, sister chromatid cohesion, sex-chromosome gene dosage compensation, DNA recombination and DNA repair pathways.70Go These proteins are not ordinarily considered to be ABC proteins due to the lack of an ABC signature between the Walker A and Walker B motifs, although plant SMC proteins uniquely have an ABC signature in addition to Walker A and Walker B motifs. In SMC proteins, the Walker A sequence and ABC signature are largely separated by an insertion of 1000–1100 amino acids, which makes domain-based clustering analysis difficult. Arabidopsis contains four members in this subfamily.3Go Since Garcia and co-workers5Go did not report SMC proteins in rice, we searched for rice SMC proteins as described in Materials and Methods, resulting in the identification of four SMC proteins in the rice genome (Table 4). In Lotus we found 24 fragments similar to Arabidopsis SMC proteins, and one fragment has an ABC signature (Table 2). This analysis suggests that Lotus has at least one SMC proteins (Table 3), although the probable number of these subfamily members was hard to predict due to their unique structure. There has been no report on the physiological or biochemical functions of SMC proteins in plants so far.


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  		Table 4. SMC subfamily of rice

 
3.12. NAP subfamily
This subfamily consists of ‘bacterial-type’ ‘soluble’ ABC proteins with a single NBD. Prokaryotic ABC proteins are encoded by four different genes, each corresponding to either NBD or TMD, which are assembled at the membrane to form a transporter complex. It can be implied that plant NAP proteins also form a heterotetramer complex to function as a transporter. Arabidopsis has 15 members, but 4 of themlack an ABC signature, whereas rice has 10 members.3Go,5Go In the Lotus genome sequence we found 23 fragments similar to Arabidopsis NAP proteins, 10 of which had an ABC signature (Table 2), indicating that Lotus has at least 10 NAP members (Table 3).

There have been several reports on Arabidopsis NAP members. AtNAP1, reported as AtABC1, appeared to function in the transport of protoporphyrin IX at the envelope of chloroplast because mutant strains accumulated this chlorophyll precursor.71Go AtNAP1 was also suggested to represent an atypical SufB protein involved in iron–sulfur cluster assembly,72Go whereas another member AtNAP7 was suggested to represent SufC protein.73Go These authors also demonstrated the interaction between AtNAP6 and AtNAP7 as well as between AtNAP1 and AtNAP7, which was indicative of an AtNAP1-AtNAP6-AtNAP7 complex formed in plastids.

3.13. Features of the Lotus ABC protein family
According to the analysis described above, the total number of ABC proteins in L. japonicus genome is presumed to be at least 91 (Table 3). When calculated with the coverage of ESTs (90%) and undetermined sequence of the genome (~32.4%), the total number of Lotus ABC proteins in the whole genome is suggested to be 100–130. This number is consistent with those of Arabidopsis and rice ABC proteins. When analyzing in more detail, we found that 6 of 17 fragments with the ABC signature that belong to Arabidopsis ABCC proteins are highly similar to one member AtMRP14, and this number is extraordinarily high compared with other ABCC homologs. Since all six AtMRP14-like fragments contain NBD2 of ABCC proteins, it is presumed that Lotus has six AtMRP14-like genes. The high redundancy of this particular molecular species is one of the characteristic features of Lotus ABC proteins. AtMRP14 was reported to be expressed in most organs of Arabidopsis,17Go although its function is still unknown.

In other subfamilies, we also found that nine of 23 fragments with the ABC signature are exclusively similar to AtPDR12. One fragment (chr3. CM0026.74) contained both NBD1 and NBD2, four (Ljwgs 020627.1, Ljwgs 060957.1, Ljwgs 077747.1 and chr3. CM0026.70.1) contained NBD1, and four (Ljwgs 036170.1, Ljwgs 080010.1, Ljwgs 085739.1 and chr3. CM0026.70) contained NBD2, which did not overlap in their sequences, suggesting that at least five AtPDR12-like genes exist in Lotus. AtPDR12 and its homologs in various plants, SpTUR2, NpPDR1 and NtPDR1, are presumed to be involved in the plant defense reaction.64Go,65Go,74Go–76Go The high redundancy of this particular homolog in Lotus may be relevant to the symbiosis of this plant, for example, highly sophisticated responses are involved in distinguishing symbiotic and pathogenic bacteria.

Our real-time quantitative reverse transcription (RT)–PCR analysis showed that the expressions of those AtMRP14-like and AtPDR12-like members in Lotus were regulated in an organ-specific manner, and some members were inducible upon inoculation with Mesorhizobium loti in root tissues. As shown in Fig. 3, the expression of three AtMRP14-like genes, Ljwgs 051480.1, Ljwgs 147765.1 and chr5. CM0456.6.2, was >2-fold higher in leaves compared with other organs, whereas that of Ljwgs 008083.1 and Ljwgs 041501 was slightly higher in inoculated roots than in uninoculated samples. Comparison of the expression level and pattern of those five genes similar to AtMRP14 suggested that they are not functionally redundant but have distinct physiological roles. In contrast to these moderate responses to symbiotic bacteria, most AtPDR12-like genes were strongly inducible in inoculated roots with symbiotic bacteria, as shown in Fig. 3, suggesting that at least some AtPDR12-like genes indeed have an important role relevant to SNF by responding to either symbiotic or non-symbiotic bacteria. One of those fragments (chr3. CM0026.74) was actually reported to be up-regulated during the early stage of nodulation in cDNA microarray analysis.77Go The expression of Ljwgs 087804.1 and Ljwgs 036170.1 was not detected with two different primer sets in our experiments.


Figure 3
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  		Figure 3. Real-time RT–PCR to compare the expression profiles of five AtMRP14-like genes and eight AtPDR12-like genes of L. japonicus. Transcript levels are shown as relative values to those of actin used as the internal control. Primer sets to specifically amplify each gene are designated in Table 1. Data represent the means ± SD of three replicates. L, leaves; S, stems; UR, roots of uninoculated plants; IR, roots of inoculated (nodulated) plants.

 
In conclusion, in this report we devised a ‘domain-based clustering analysis’ method, in which both NBD and TMD fragments were excised and independently compared to characterize the ABC protein family in L. japonicus. This method was found to be very useful to characterize ABC proteins, i.e. it is applicable even when their entire amino acid sequences are not available, and this method provides detailed information about whether a particular fragment is either from the N-terminal or C-terminal region. It seems also useful to predict to whether ABCC (MRP) subfamily members are to be localized to either tonoplast or plasma membrane. In fact, AtMRP1 and AtMRP2 have been reported to be localized at tonoplast,78Go,79Go whereas AtMRP4 has been to be at plasma membrane.41Go By this method, we analyzed the genome sequence of a model legume plant L. japonicus, and found that the Lotus genome has at least 91 ABC proteins and probably 100–130 members exist in the whole genome sequence. An interesting finding about Lotus ABC proteins is that they show high redundancy in two particular members, i.e. six AtMRP14-like ABCC genes and at least five AtPDR12-like genes. This high redundancy in only two characteristic members of each two different subfamilies was unique in Lotus. Expression analysis using real-time RT–PCR suggests that at least some AtPDR12-like genes may have functions related to SNF in inoculated roots. Further experiments to characterize the functions of individual members in L. japonicus are necessary to elucidate the detailed roles of those genes in plant–microbe interaction, but those genes will be good candidates to understand such legume-specific biological events as nodulation and SNF. Many transport processes, e.g. signal secretion and perception of these molecules, as well as exchanging nutrients, are important for the initial nodulation process and the development of SNF, and ABC transporters of symbiotic rhizobia have been known to function in the exchange of metabolites in nodules. 80Go–82Go It would be of great importance to identify ABC transporters in plant side that function in those processes, which will provide clearer insights in the molecular mechanism of symbiotic processes.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and Methods
 3. Results and Discussion
 Acknowledgements
REFERENCES
 
We thank Dr Shingo Hata of Kyoto University for invaluable advice and the gift of M. loti, the National Bioresource Project (Lotus japonicus, Glycine max) for seeds of L. japonicus, and Mr Masafumi Hamamoto of Kyoto University for searching for ABCA proteins for in silico analysis of the EST data of ABCA. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 17051018 and 17027016 to K.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists (No. 183051 to A.S.). We also thank the Uehara Memorial Foundation for an additional grant in support of this study.

Supplementary data: Supplementary data are available at www.dnares.oxfordjournals.org

Note added in proof: After the acceptance of this article, plant ABC protein community including the corresponding author has organized a new unified nomenclature for plant ABC proteins, which is basically the same as appeared in this paper with some exceptions. In the new unified nomenclature, ABCA and ABCA' are classified in one group as ABCA, PDR is included in the group of ABCG, and NAP is called ABCH.


   Footnotes
 
*To whom correspondence should be addressed. Tel. +81-774-38-3617, Fax +81-774-38-3623, E-mail: yazaki{at}rish.kyoto-u.ac.jp

Communicated by Mikio Nishimura


   REFERENCES
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