In Silico Analysis of a MRP Transporter Gene Reveals Its Possible Role in Anthocyanins or Flavonoids Transport in Oryze sativa

There are many studies on enzymatic pathways of anthocyanin biosynthesis, but little is known about the anthocyanins transport in Oryze sativa. In silico analysis, the OsMRP15 (LOC_Os06g06440), an orthologous gene of mazie anthocyanin transporter ZmMRP3, has been identified in rice. The OsMRP15 contained a 4425bp open reading frame (ORF) encoding a 1475 amino acid protein, belonging to a MRP subfamily of ABC transporters, and has a high sequence identity, very similar protein structure, and the same arrangement of domains to ZmMRP3, but the genomic structure of OsMRP15 was significant difference with ZmMRP3. The prediction promoter of OsMRP15 has many presumed anthocyanin regulatory sites. The phylogenetic analysis of MRPs in rice, mazie and Arabidopsis showed that OsMRP15 and ZmMRP3 belonged to the same subbranch. The expression pattern indicated that OsMRP15 was co-expression with two anthocyanin transcription factors. These analysis results implied that as an ortholog of ZmMRP3, the function of OsMRP15 was possibly as a membrane-bound transporter required for vacuolar uptake of anthocyanins in rice.


Introduction
The multidrug resistance-associated proteins (MRPs) belong to ATP-dependent, proton-gradient-independent transporters (ATP-Binding Cassette, ABC) superfamily [1], and the primary function of plant MRPs are involved in the vacuolar sequestration of potentially toxic metabolites, for example flavonoids and other secondary metabolites [2].The full-size or typic MRP transporters consist of five core domains [3]: three hydrophobic transmembrane domains (TMDs) that each contains multiple transmembrane α-helices; and two soluble nucleotide binding domains (NBDs) that each is composed of the Walker A and B motives linked by an ABC signature motif.These domains are arranged with the topology: TMD0-TMD1-NBD1-TMD2-NBD2, in which the TMD0 is required for correct targeting and two (TMD-NBD)s compose a transmembrane transport channel.Now, many MRPs have been identified in some different plants, but only several genes involved in flavon-oids transport have been studied.There are 15 putative MRP genes in Arabidopsis genome.AtMRP1 and AtMRP2 transport anthocyanin-glutathione conjugates in vacuolar yeast [4,5].Moreover, AtMRP2 is involved in chlorophyll degradation in vivo [6].AtMRP4 and AtMRP5 are involved in guard cell regulation [7].Maybe due to the function redundance in vacuolar uptake of anthocyanins, flavonoidless phenotypes have not been obatained by knockout mutants in some AtMRPs [2].In mazie, ZmMRP3 is a membrane-bound anthocyanin transporter.The expression pattern of ZmMRP3 correlated with the anthocyanin accumulation pattern and is controlled by anthocyanin regulators [8].ZmMRP4, the other related MRP, acts mainly as phytic acid transporter in the seed and its other possible function is partially redundant with ZmMRP3 in anthocyanin transport in the scutellum [9].
In rice, we identified 16 MRP genes and excluded the LOC_Os11g05700 that is a half transporter and consists of a single TMD and NBD, and originally called OsMRP16 in [3], using the latest RGAP V6.1 (Rice Genome Annotation Project) database, and only one gene was studied.OsMRP5 (LOC_Os03g04920, now-called OsMRP13 in this paper), an orthologus gene of ZmMRP4, is a phytic acid transporter in seeds [10].
Anthocyanin is an important biologically active substance in black, purple and red rice [11].Although the regulators and key enzymes have been studied, little is known about anthocyanin transport in the final phase of anthocyanin biosynthesis in rice.In order to improve the content of anthocyanin in rice seeds, in this paper we identified a MRP gene, OsMRP15, and analyzed its possible role in anthocyanins or flavonoids transport.

Plant Materials
The Green rice varieties (ZH11, Nipponbare and T65) and red leaf variety ZY were planted in our lab.

RNA Isolation, cDNA Synthesis and RT-PCR Analysis
The total RNA was isolated by Trizol reagent kit (Invitrogen, USA) and two micrograms of total RNA were reverse-transcribed using M-MLV kit (Promega, USA), according to the manufacturer's instructions.Using Os-Actin1 as a reference gene, two primers of OsMRP15, F15 (5'-agatgggtcgaattggagcatgggtc-3') and R15 (5'-cgtaggtttgtcatac tccaccac-3') were designed to RT-PCR analysis, according to the procedure: predenaturation at 94˚C for 4 min, 35 cycles at 94˚C for 0.5 min, 58˚C for 0.5 min and 72˚C for 0.5 min, followed by the final extension at 72˚C for 5 min.

Bioinformatics Analysis
The latest RGAP V6.1 data was used to assay the rice MRP gene family.Gene prediction and structure analysis used a FGENESH web software.Molecular characterization and multiple sequence alignment of OsMRP15 was analyzed using Vector NTI 10.0 program (Invitrogen, USA).The promoter prediction and cis-acting regulatory elements analysis utilized the latest PromPredict program [12] and PLACE program [13], respectively.The conserved protein domain analysis used the SMART web tool, and protein subcellular localization prediction adopted WolF PSORT program.The peptide sequences of MRPs were aligned with Clustal W and subsequently a phylogenetic tree was constructed by the neighbor-joining (NJ) method with MEGA 5.0 by bootstrap with 1000 replicates [14].Our Rice GeneChip Database was used to analyze gene expression patterns.

Molecular Characteristics of OsMRP15
Using ZmMRP3 as a probe, the orthologus gene Os-MRP15 was identified by BLAST search in RGAP V6.1 database.The OsMRP15 was located in Chr.6, and the TIGR number is LOC_Os06g06400.The genome length of OsMRP15 was 8445bp including 299bp 5'-UTR, 4425bp ORF and 389bp 3'-UTR.Although the gene CDS structure of OsMRP15 including 11 exons and 10 introns is very different with ZmMRP3 CDS structure that only contained 4 exons and 3 introns, the two genes have very similar phase of introns which implied that the introns loss maybe happened in ZmMRP3 (Figure 1).In addition, there were two introns in 5'-UTR of ZmMRP3, but no introns in prediction The subcellular localization prediction of OsMRP15 showed its presence in the plasma membrane and was an integral membrane protein, which is similar with Zm-MRP3 that located in the tonoplast [8].And 48 conserved phosphorylation sites were predicted by NetPhos 2.0.These sites suggested that posttranslational modification may play an important role for OsMRP15 normal function.The protein structure analysis displayed that OsMRP15 and ZmMRP3 have similar second structure and almost exactly same of conserved structures except for lacking two transmembrane-spanning alpha-helices of TMD2 in ZmMRP3 (Figure 3).

The Analysis of Prediction Promoter Region of OsMRP15
In the 1.5 kb upstream of 5'-UTR of OsMRP15, a prediction core promoter region from −443bp to −503bp, many  cis-active elements and many myb or myc cis-regulatory elements were found by using PromPredict and PLACE program, respectively (Figure 4).Moreover, a cis-sequence of OsMRP15 promoter similar to an anthocyanin regulatory element (ARE) [8], is present at −570 position (Figure 4).These promoter characteristics of OsMRP15 conformed to the ZmMRP3 promoter and implied that OsMRP15 expression was controlled by similar antho-cyanin regulatory genes.

The Phylogenetic Tree Analysis of OsMRP15
Using the latest RGAP V6.1 data, all 16 rice MRP genes were identified by BlASTP searches.The nomenclature for OsMRP1 to OsMRP7 and OsMRP9 to OsMRP15 were in accordance with [3].The OsMRP8 was only one new locus not two in [3], and old OsMRP16 in [3] is a single TMD and NBD structure which was excluded in the rice MRP family.So, the new named OsMRP16 in this paper was the old OsMRP17.
In the phylogenetic tree (Figure 5), there were two clades: the clade I including two rice MRPs in which the possible function of OsMRP1 maybe was related with transport of anthocyanin-glutathione conjugates like AtMRP1 and AtMRP2 in yeast [4,5]; the clade II containing many subcludes in which OsMRP15 and ZmMRP3, and OsMRP13 and ZmMRP4 all belonged to the same subbranch with 100 bootstrap value, respectively.Therefore, it can be postulated that OsMRP15 was an anthocyanin transport similar with ZmMRP3, and OsMRP13 has possible redundant function in anthocyanin transport like ZmMRP4.

The Expression Pattern of OsMRP15
In our Rice GeneChip Database, OsMRP15 expression was higher in stigma than embryo, and very low in other tissues and organs.Furthermore, OsMRP15 was co-expression with two anthocyanin transcription factors, OsC1 and OsB1, in all tissues and organs, except for embryo (Figure 6).These expression patterns resembled Zm-MRP3 [3] that expressed in all anthocyanin assembled and colored tissues and organs, and was controlled by the anthocyanin regulators B (myc type) and Pl (myb type).
In red leaf rice, the anthocyanin biosyntheses is controlled by OsC1 and OsB (OsB1 and OsB2) [15].But in green leaf, the OsC1 is induced by UV-B and very low expression, and the OsB2 is non-function.Moreover, the anthocyanin key structure gene DFR is mutant and nonfunction in many japonica rice varieties, for example Nipponbare [15].
In order to detect the co-expression pattern of OsMRP15, the RT-PCR was analyzed in a red leaf variety ZY and 3 green leaf varieties ZH11, T65, and Nipponbare.The RT-PCR result indicated that OsMRP15  expression was co-expression with anthocyanin regulators in red leaf of ZY, but no expression in green leaf varieties (Figure 7).The integrative expression pattern suggested that OsMRP15 played a possibly important role in rice anthocyanin synthesis.

Discussion
As an ortholog of ZmMRP3 [8], a mazie anthocyanin transport, OsMRP15 was identified in silicon analysis using latest rice genome data.At the level of genome structure (Figure 1), it was very different that OsMRP15 has many exons in CDS and no introns in prediction 5'-UTR, but ZmMRP3 was only 4 exons in CDS and two introns in 5'-UTR.Compared with ZmMRP3, other orthologus structure were very similar with OsMRP15, which implied that introns have lost in ZmMRP3.On the other hand, at the level of protein, OsMRP15 has highest identity, the same protein structure, conserved domains and similar sublocation with ZmMRP3 (Figures 2 and 3).In prediction promoter of OsMRP15, many cis-active elements and cis-regulatory elements were very similar with ZmMRP3 promoter, specially the similar ARE [8].
The result of OsMRP15 and ZmMRP3 with high bootstrap value in the same subbranch of phylogenetic tree strongly suggested that the two genes have similar function.Interestingly, OsMRP16, the same subclade with OsMRP15, was no or very low expression in all tissue and organs (Figure 6), which implied that OsMRP16 maybe was a non-function gene in rice.The expression pattern of OsMRP15 co-expression with OsC1 and OsB1 was strongly supported by RT-PCR between red and green leaf rice varieties.
In green stigma, the function of OsMRP15 maybe was a flavonoids transport, in which transported some flavonoids, e.g.flavonols.Maybe because some key structure genes in anthocyanin biosynthesis were non-function or mutant, the pathway was incomplete though OsC1 and OsB1 were high expression.In addition, in embryo, Os-MRP15 was expression but OsC1 and OsB1 were no expression.These results suggested that maybe OsMRP15 has other regulatory factors and played an unknown role in embryo, which was different with ZmMRP3.Based on in silicon analysis and RT-PCR result, it can be deduced that OsMRP15 was a membrane-bound transporter that is required for vacuolar uptake of anthocyanins or flavonoids in rice, like ZmMRP3 in maize.Moreover, there was a possible GST gene presumptively forming anthocyanin-glutathione conjugates by OsMRP15 transporting into vacuolar in rice.

Figure 1 .Figure 2 .
Figure 1.The genome structure of OsMRP15 compared with ZmMRP3.The number of 0 and 1 indicated the phase of introns: phase-0 introns (intron located between two codons), phase-1 introns (intron located between first and second nucleotide of a codon), respectively.

Figure 6 .
Figure 6.The expression patterns of OsMRP15 in different tissues and organs of rice.The relative level is the ratio of the signal intensity values between genes and OsACTIN1.

Figure 7 .
Figure 7.The RT-PCR analysis of OsMRP15 in green and red leaf rice cultivars.
In Silico Analysis of a MRP Transporter Gene Reveals Its Possible Role in Anthocyanins or Flavonoids Transport in Oryze sativa 556