Evolutionary expansion and functional diversification of oligopeptide transporter gene family in rice
© Liu et al.; licensee Springer. 2012
Received: 8 June 2012
Accepted: 22 June 2012
Published: 22 June 2012
Oligopeptide transporters (OPTs) play important roles in the mobilization of organic nitrogenous compounds and usually associate with tissues that show signs of rapid protein hydrolysis, such as germinating seeds and senescing leaves. This study is to investigate rice OPT genes.
A total of sixteen OsOPT genes (Os for Oryza sative L.) were identified in the rice genome, which were then classified into six sections that belong to two subfamilies (the PT and YSL subfamily). The major mechanisms for evolutionary expansion of the sixteen genes during the rice genome evolution include segmental and tandem duplication. Calculation of the duplication event dates indicated that the sixteen genes originated from nine original OsOPT genes, and the duplication events could be classified into three evolutionary stages. The first evolutionary stage occurred approximately 50 million years ago (Mya) and involved the evolution of four new genes. The second evolutionary stage was approximately 20 Mya and was marked by the appearance of two new genes, and the third evolutionary stage was approximately 9 Mya when two new genes evolved. Mining of the expression database and RT-PCR analysis indicated that the expression of most duplicated OsOPT genes showed high tissue specificities. Diverse expression patterns for the sixteen genes were evaluated using both semi-quantitative RT-PCR and the MPSS data. Expression levels of some OsOPT genes were regulated by abiotic and biotic stresses suggesting the potential involvement of these gene products in rice stress adaptation. Five OsOPT gene mutants showed abnormal development and growth, the primary analysis of five OsOPT gene mutants suggested that they may be necessary for rice development.
These results suggested that rice-specific OsOPT genes might be potentially useful in improving rice.
The acquisition and allocation of nitrogenous compounds are essential processes in plant growth and development (Williams and Miller ). Peptide transport has been demonstrated to play important roles in these processes and is a commonly observed physiological phenomenon in both prokaryotes and eukaryotes (Higgins and Payne ). Peptide transporters are characterized by the ability to transport a range in different types of peptides and their derivatives across membranes in an energy-dependent manner (Lubkowitz et al. ; Lubkowitz et al. ). The plant peptide transporters generally belong to three different gene families: the ABC-type transporter family, the peptide transporter family (PTR) and the oligopeptide transporter family (OPT) (Ouyang et al. ; Rentsch et al. ; Zhao et al. ), each of which recognizes peptides of a specific length. In this study, we focused on the rice OPT family. The OPT family was first characterized in yeast (Lubkowitz et al. ). Several other OPT genes have also been identified from other species, such as CaOPT1 from Candida albicans SpIsp4 from Schizosaccharomyces pombe, and ScOPT2 from Schizosaccharomyces cerevisiae (Lubkowitz et al. ). However, to date the OPT family has only been found in fungi, bacteria, plants and archaea, but not in animals (Lubkowitz ).
The OPT genes have been classified into two subfamilies by phylogenetic analysis: the PT and the YSL subfamily (Curie et al. ). Most of the information regarding the PT subfamily in plants comes from the study of Arabidopsis. Nine orthologs of the PT subfamily were identified in Arabidopsis and were named sequentially from AtOPT1 to AtOPT9. Five (AtOPT1, 4, 5, 6, and 7) of the orthologs were found to be functional tetra- and penta-peptide transporters by yeast complementation assay (Koh et al. ; Osawa et al. ). However, reduced AtOPT3 expression resulted in decreased accumulation of iron in seeds while high levels resulted in the accumulation of iron in other tissues, suggesting that AtOPT3 functions to maintain whole-plant iron homeostasis and iron nutrition of developing seeds (Stacey et al. ). AtOPT6 proteins have the ability to transport glutathione derivatives (Cagnac et al. ) and mammalian signaling peptides up to 10 amino acids in length (Pike et al. ). AtOPT promoter-GUS fusion analysis revealed differential expression of 8 AtOPT genes: 6 AtOPT genes (AtOPT1 AtOPT3 AtOPT4 AtOPT6 AtOPT7 and AtOPT8) were strongly expressed in vascular tissues, 3 (AtOPT1 AtOPT3 and AtOPT8) were uniquely expressed in pollens and one (AtOPT6) was expressed in ovules (Stacey et al. ). Several OPT genes were also identified from other plants. BjGT1 from B. juncea was found to transport glutathione and has been implicated in mediating heavy-metal toxicity (Bogs et al. ).
The YSL subfamily is usually considered to transport the metals (Inoue et al. ; Lee et al. ). Iron (Fe) is an important micronutrient for living organisms (Bashir et al. ). Maize yellow stripe1 (ZmYS1) from Zea mays was demonstrated as a phytosiderophore transporter (Curie et al. ; Roberts et al. ; Schaaf ). In rice, OsYSL2 (Koike et al. ), OsYSL15 (Inoue et al. ; Lee et al. ) and OsYSL18 (Aoyama et al. ) are similar to orthologs of ZmYSL1 in that they all have the ability to transport metal-nicotianamine. However, some members (OsOPT1 OsOPT3 OsOPT4 OsOPT5 and OsOPT7) of the PT subfamily in rice have been demonstrated to transport nicotianamine-bound iron (Vasconcelos et al. ).
The roles of the OPT genes in plants, especially in rice, are still under investigation. To the best of our knowledge, no biofunctional data regarding the OsOPT genes are currently available. In eukaryotes, functional diversification follows the evolutionary expansion of a gene family. The evolutionary expansion of a gene family follows duplication events of an individual gene, the chromosomal segment, or the whole genome (Lynch and Conery ; Moore and Purugganan ; Xue and Fu ). In this study, we identified the evolutionary expansion of the rice OsOPT family and showed function diversification of the duplicated genes by expression analysis and primary analysis of the mutants.
Identification of 16 OsOPT gene homologues in the rice genome
Basic information of sixteen rice OsOPT genes
Identity/similarity (E-value) to SpISP4
Phenotypes of the mutante
Dwarf, palegreen leaf, low tillerring, long clum, late heading
Dwarf, short panicle, lesion leaf, low tillering
Dwarf, glabrous leaf, semi-short clum, white belly rice kernel
Yellow-green plant, late heading, semi-dwarf, low tillerring
Semi-dwarf, low tillering, semi-sterile pollen
Phylogenetic relationship, chromosomal location and gene structures of the OsOPT genes
We also determined the intron-exon boundaries for the 16 OsOPT genes to obtain more information on the genomic organization of rice OsOPT genes (Figure 2b). According to the annotation of the rice genome, three of the six sections (A2, B1 and B2) had similar intron-exon boundaries. All three genes found in section A2 (OsOPT2, OsOPT3 and OsOPT4) contain six introns and seven exons, but the length from the fourth to the sixth exon were identical. The genes in section B1 (OsYSL10, OsYSL12 and OsYSL13) had four identical exons. All four genes found in section B2 (OsYSL9, OsYSL16, OsYSL2 and OsYSL15) contain six introns and seven exons, but the length from the third to the fifth exon were identical. The identical exons were connected by slashes (Figure 2b). In addition, a 98 bp-exon was found in most of the 13 OsOPT genes except OsOPT1, OsOPT3 and OsOPT8.
Duplication and evolution analysis of the OsOPT genes
To reconstruct the expansion history of the 16 OsOPT genes in the rice genome, duplication and evolution analysis was performed. According to the duplication regions of rice chromosomes, we found that seven genes (OsOPT2, OsOPT3 and OsOPT4 in section A2, OsYSL12 and OsYSL13 in section B1, OsYSL2 and OsYSL15 in section B2) came from gene tandem duplication, which were shown in the box in Figure 3. However, three pairs (OsYSL9/OsYSL16, OsYSL2/OsYSL15 and OsOPT8/OsOPT6) were located in duplicated chromosomal regions, suggesting that these six genes came from segmental duplication.
Ka/Ks analysis and estimate of the absolute dates for the duplication events between the duplicated OsOPT genes
Date (million years)
We also calculated the dates of the duplication events (Table 2) by molecular clock theory (Yu et al. ) using the nucleotide substitution rate (Ks) and the neutral evolutionary rate (λ) of 6.5 × 10−9 substitutions per silent site per year for rice genome (Yu et al. ). According to the first whole-genome duplication event of grass genomes (Gaut ; Salse et al. ), duplication events for the three pairs (OsYSL15/OsYSL2, OsYSL16/OsYSL15 and OsYSL9/OsYSL2) occurred within the last 70 to 50 million years, after the origin of grasses (Gaut ; Kellogg ) and before the divergence of rice and maize (Gaut ; Stebbins ). Duplication events for the two pairs (OsOPT4/OsOPT3 and OsOPT4/OsOPT2) occurred within the last 50 to 20 million years, after the divergence of rice and maize, but before Zizaniinae and Oryzinae were diverged from one another (Guo and Ge ). Duplication events for the other two pairs (OsYSL12/OsYSL13 and OsOPT6/OsOPT8) occurred within the last 20 to 9 million years, after Zizaniinae and Oryzinae were separated and before the Oryza genus branched off from the remaining genera of Oryzeae (Guo and Ge ). Therefore, the evolutionary origin of the 16 OsOPT genes may have occurred in three evolutionary stages. The genes that came from the three evolutionary stages were shown in three different colors (Figure 3).
Differential expression of the OsOPT genes under normal growth conditions
Data from the MPSS database suggested that some genes showed differential expression profiles in the developmental organs (Figure 4A). For the vegetative organs, four genes (OsOPT4, OsOPT7, OsYSL13 and OsYSL12) were differentially expressed among the roots of 14-day-old seedlings and 60-day-old seedlings; however, three genes (OsOPT4, OsOPT7, OsYSL13) were expressed at a higher level and one gene (OsYSL12) was expressed at a lower level in the 60-day-old roots than the 14-day-old roots. Three genes (OsOPT2, OsOPT7 and OsOPT9) had lower expression in the 14-day-old leaves than the 60-day-old leaves. For the reproductive organs, only five genes (OsOPT5, OsOPT8, OsOPT9, OsOPT12 and OsYSL16) were expressed in the male organs (mature pollen); whereas, six genes (OsOPT4, OsOPT8, OsOPT9, OsYSL12, OsYSL13 and OsYSL16) were expressed in the female organs (ovary and mature stigma). In addition, seven genes were expressed in the 35-day-old callus.
Expression of the OsOPT genes under abiotic and biotic stresses
In response to salt (250 mM NaCl) treatment, four genes (OsOPT4, OsOPT7, OsYSL13 and OsYSL16) were upregulated in the roots; however, five genes (OsOPT2, OsOPT7, OsOPT9, OsYSL13 and OsYSL12) were downregulated and one gene (OsOPT3) was upregulated in the leaves. In response to drought treatment, seven genes (OsOPT2, OsOPT4, OsOPT7, OsOPT8, OsYSL13 OsYSL15 and OsYSL16) were upregulated and one gene (OsYSL9) was downregulated in the roots; however, only two genes (OsYSL12 and OsYSL13) were upregulated and six genes (OsOPT2, OsOPT3, OsOPT4, OsOPT7, OsOPT9 and OsYSL16) were downregulated in the leaves. In response to cold (4oC) treatment, three genes (OsOPT8, OsYSL9 and OsYSL12) in the roots and seven genes (OsOPT2, OsOPT3, OsOPT4, OsOPT7, OsOPT9, OsYSL13 and OsYSL16) were downregulated in the leaves, and only one gene (OsYSL9) was upregulated in the leaves.
We also analyzed the responses of the OsOPT genes to infection with a fungus, Magnaporthe grisea, or a bacteria, Xanthomonas oryzae (Figure 6), which are known to cause rice blast and rice bacterial streak, two main rice diseases that cause severe decreases in rice yield. The expression changes of OsPTRs were determined after the inoculation. The microarray data and the infection with M. grisea and X. oryzae were published previously (Nobuta et al. ). We found that four genes (OsOPT8 OsYSL12 OsYSL13 and OsYSL16) were upregulated and three genes (OsOPT3 OsOPT5 and OsYSL9) were downregulated by M. grisea treatment (Figure 6A), and there was no response difference among these seven genes between the resistance reaction and the susceptible reaction of the rice variety Nipponbare. In response to X. oryzae treatment (Figure 6B), upregulated expression of two genes (OsOPT8 and OsYSL12) were observed only during the early inoculation, and two genes (OsYSL13 and OsYSL16) were upregulated during the whole inoculation; and two genes (OsOPT2 and OsOPT5) showed downregulation.
Expression response of the OsOPT genes to hormone treatments
Primary phenotypes of the inserted mutants for five OsOPT genes
To acquire more clues regarding the biological function of OsOPT genes, we searched main rice inserted mutant databases. Inserted mutants of all sixteen OsOPT genes were found in different mutant databases (Additional file 1: Table S1). However, the phenotype records of mutants of only five OsOPT genes were found in two mutant databases and their observed phenotypes were integrated into Table 1. The segregation rate and the plant numbers recorded in the mutant database can be found in Additional file 1: Table S1. From the records of the database, we found that some plants of the five OsOPT mutants displayed abnormal phenotypes under normal growth conditions (Table 1, Additional file 1: Table S1). Of course, it should be further investigated whether the phenotypes of the mutants from the database are linked with the five OsOPT genes.
Evolutionary expansion of the OsOPT gene family in rice
Gene duplication plays an important role in the expansion of a gene family (Adams and Wendel ; Hughes ). Several mechanisms have been suggested to drive the expansion of a gene family, including segmental duplication, tandem duplication and retroposition (Cannon et al. ; Kong et al. ). In this study, we identified sixteen members of the OsOPT gene family from the rice genome. We found that both the segmental duplication and the tandem duplication contributed to the evolutionary expansion of the OsOPT gene family in rice.
To speculate on the evolutionary orders of the rice OsOPT family, we considered protein sequence similarities with SpISP4 (Table 1), dates of the gene appearance (Table 2), rice genome evolutionary events (Krom and Ramakrishna ), etc. The rice OsOPT members, which are more similar to SpISP4 from S. pombe, might have evolved earlier in the rice genome (Krom and Ramakrishna ). The molecular clock was also used to compute the date of duplication by calculating the number of substitutions per silent site (Ks). Yu et al. () identified an evolutionary rate of 6.5 × 10−9 substitutions per silent site per year by analyzing the duplication history of the rice genome, dating the duplication event to 53 million years ago (Mya). Based on the dates of the duplication events (Table 2) and similar E-value to SpISP4 (Table 1), we speculated that the evolutionary expansion of the OsOPT gene family in the rice genome underwent three stages. The three evolutionary stages of the OsOPT genes were shown with three colors in Figure 3. Before the evolutionary expansion of the OsOPT gene family in the rice genome, nine OsOPT genes (gene name in black) were distributed on six chromosomes (Figure 3). The first evolutionary stage was from the origin of the grasses (70 million years ago) to the divergence of rice and maize (approximately 50 million years ago). The original nine OsOPT gene homologues were also found in the maize genome (data not shown). During the first stage, OsYSL15 arose from OsYSL2 located on chromosome 2 after one tandem duplication event. Next, through one segmental duplication event between chromosome 2 and chromosome 4, OsYSL9 and OsYSL16 arose from OsYSL2 and OsYSL15 on chromosome 4. The four newly evolved genes were shown in red (Figure 3). The second evolutionary stage occurred at the point of divergence between rice and maize (approximately 50 million years ago) to the separation of Zizaniinae and Oryzinae (approximately 20 million years ago). In the second stage, OsOPT4 and OsOPT3 arose from OsOPT2 on chromosome 6 by two tandem duplication events. The two newly evolved genes were shown in green (Figure 3). The third evolutionary stage was from the separation of Zizaniinae and Oryzinae (approximately 20 million years ago) to the point where Oryza branched-off from the remaining Oryzeae genera (approximately 9 million years ago). In the third stage, OsYSL13 arose from OsYSL12 on chromosome 4 after one tandem duplication event; OsOPT6 arose from OsOPT8 on chromosome 4 following one segmental duplication event. The two newly evolved genes were shown in blue (Figure 3). After the three evolutionary stages, the number of the rice OsOPT gene family increased to sixteen genes. Notably, purifying selection occurred at stage 1 and stage 2, but positive selection occurred at stage 3.
Different functions and multiple roles of the OsOPT genes in rice development
Some plant OPT genes have been characterized in embryo development (Stacey et al. ), glutathione transport (Zhang et al. ), heavy metal detoxification (Cagnac et al. ), seed germination (Muntz ), and long distance nitrogen allocation (Williams and Miller ). The genome-wide expression analysis of the OsOPT gene family showed that eleven genes (OsOPT2 OsOPT3 OsOPT 4 OsOPT7 OsOPT8 OsOPT9 OsYSL2 OsYSL9 OsYSL12 OsYSL13 and OsYSL16) had transcript accumulation in all tissues, and some in germinating seeds, roots, panicles and callus (Figure 4). These data suggested multiple functions for these oligopeptide transporters in rice and they might be involved in housekeeping functions during rice growth and development. On the other hand, some genes may play roles in specific tissues. The expression of OsOPT6 was not detectable in most of the tested tissues (Figure 4), but it was specifically expressed in the 1.2-2.0 mm portion of the anthers (Data from RiceXpro expression database).
AtOPT6 (Pike et al. ; Cagnac et al. ) and OsOPT3 (OsGT1) (Zhang et al. ) have been characterized GSH transporters and have been suspected of having a role in mediating heavy metal phytotoxicity. However, little is known about the role of the OsOPT genes in response to stress. We obtained some clues regarding the role of OsOPT genes in response to stresses by examining their expression level. Nine of the 16 OsOPT genes (OsOPT2 OsOPT4 OsOPT7 OsOPT7 OsOPT9 OsYSL12 OsYSL13 OsYSL15 and OsYSL16) showed a response to at least one abiotic stress treatment (Figure 5). Of the two main rice disease treatments, the expression of seven genes (OsOPT3 OsOPT5 OsOPT8, and OsYSL9 OsYSL12 OsYSL13 and OsYSL16) changed after M. grisea treatment and the expression of six genes (OsOPT2 OsOPT5 OsOPT8 OsYSL12 OsYSL13 and OsYSL16) changed after X. oryzae treatment (Figure 6).
Functional diversification of OsOPT genes
Functional diversification is a result of the evolutionary expansion of a gene family by gene duplication and often accompanies changes in the expression profile of the gene family member (Hughes ; Lynch and Conery ). The OPT family has been divided into two subfamilies (the PT and YSL subfamily) (Curie et al. ), where the YSL subfamily was believed to be able to transport iron compounds and the PT subfamily was believed to be able to transport oligopeptides with four-five amino acid residues (Lubkowitz ). However, several members of the PT subfamily have also been identified as transporters of iron compounds, such as AtOPT3 (Stacey et al. ), OsOPT1 OsOPT4 and OsOPT7 (Vasconcelos et al. ). These data may support the idea that the two subfamilies originated from a common ancestor.
Expression diversification between the duplicated OsOPT genes
The transport functions and the expression patterns of the sixteen OsOPT genes have been diversified following their evolution. In the PT subfamily, three members (OsOPT1 OsOPT4, and OsOPT7) have been shown to be able to transport iron-chelates in yeast (Vasconcelos et al. ). OsOPT3 (OsGT1) could also transport glutathione, some amino acids and peptides (Zhang et al. ). In the YSL subfamily, OsYSL2 (Koike et al. ) and OsYSL15 (Inoue et al. ; Lee et al. ) have been shown to be able to transport metal-nicotianamine. OsOPT6 arose from OsOPT8 by one segmental duplication event approximately 22.58 million years ago, but their expression profiles were different. OsOPT8 was expressed in all developing organs (Figure 4) and it was also induced by M. grisea; however, OsOPT6 was not expressed under normal growth conditions and during testing with biotic and abiotic stresses. OsOPT6 is not a pseudogene because it has a corresponding full-length mRNA (AK070801) that was found in the RAD-DB database (Table 1) and it might be specifically expressed in the 1.2-2.0 mm portion of the anthers.
To our knowledge there are few biofunctional data about rice OsOPT genes. Some offspring of the five inserted OsOPT mutants showed abnormal symptoms which were recorded in the mutant database and the symptoms of the five OsOPT mutants were distinct (Table 1). Mutants of OsYSL2 OsYSL12 and OsOPT3 were Tos17 insertion and mutant of OsOPT4 was T-DNA insertion. Of course, these phenotypes from the mutant database might not link with the OsPTR genes, because the mutant lines may carry multiple Tos17 or T-DNA insertions and the tissue-culture induced mutations (Hirochika et al. ). By observing the phenotypes of two homozygote T-DNA inserted mutant osopt3 (osgt1) and osopt9, we found osptr3 showed a lesion mimic phenotype when the osptr3 mutant plant was grown under light of normal growth field (Figure 8A), but no this symptom under shade (data now shown). In addition, OsOPT3 (OsGT1) has been found to be a glutathione transporter (Zhang et al. ). Glutathione is the major endogenous antioxidant produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen compounds (Szalai et al. ). These suggested that OsOPT3 (OsGT1) may play a role in tolerance to oxidative burst. The homozygote osptr9 mutant showed semi-sterile pollens (Figure 8B), which suggested that OsPTR9 may play a role in pollen development. These preliminary data could suggest that the rice OPT family may be necessary for rice growth and they may play diverse roles in rice.
The OsOPT gene family has been researched for more than ten years. In plants, it seems to have the ability to transport oligopeptide or metals, but due to the large number of potential substrates, the real roles of the OsOPT gene family are not fully understood. In this study, we described the evolutionary expansion and expression profiling of the rice OsOPT gene family and provided useful information for further study on the function of the OsOPT gene family in rice.
Rice OPT family contains 16 genes, and is classified into the PT and YSL subfamily with 9 and 7 genes, respectively. The 16 OsOPT genes evolved from 9 original OsOPT genes after 3 evolutionary stages. Their expression patterns of 16 OsOPT genes and phenotypes of five OsOPT mutants are different.
Identification of OsOPT gene homologues
To identify putative OPT genes in the rice genome, a reference OPT protein (SpISP4) (GI:19112445) from S. pombe was chosen as a query sequence to search in the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) by BLASTP. The protein sequences satisfied E < 0.1 were selected as the candidate proteins, which were compared with the OPT records in GenBank. Then, the physical and chemical parameters of the putative OsOPT proteins were computed by ProtParam in the ExPASy Proteomics Server (http://www.expasy.ch/tools/protparam.html). The prediction of transmembrane helices was performed by TMHMM Server v. 2.0 (http://genome.cbs.dtu.dk/services/TMHMM-2.0/). To confirm above OsOPT genes as the OPT gene homologue,we also analyzed the predicated OsOPT proteins in the Pfam database (http://www.sanger.ac.uk/Software/Pfam/search.shtml).
Phylogenetic analysis and gene structure
Multiple sequence alignments of amino acid sequences were performed using ClustalX (version 2.0.9) (Thompson et al. ). Unrooted phylogenetic and molecular evolutionary analyses were constructed using MEGA4.0 by the neighbor-joining method with a Poisson correction model, using 1000 replicates for bootstrap analysis (Tamura et al. ). Exon-intron organization was determined using the genome browser in RAP-DB (http://rapdb.dna.affrc.go.jp/). Gene structures were displayed using Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/chinese.php).
Chromosomal localization and gene duplication
The chromosomal localization of the OsOPT genes was determined by mapview in NCBI (http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid=4530). The OsOPT genes present on the duplicated chromosomal segments were identified by segmental genome duplication of rice available at MSU-RGA (http://rice.plantbiology.msu.edu/analyses_search_blast.shtml) with the maximum distance permitted between collinear gene pairs of 100 kb. The OsOPT genes separated by a maximum of twenty genes were identified as tandem duplicated genes (Lynch and Conery ; Moore and Purugganan ).
Ka/Ks analysis and calculation of the date of duplication events
The number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) of duplicated genes were calculated with DnaSP Version 5. The duplication date could be computed with the number of substitutions per silent site (Ks) (Comeron , Shiu et al. ; Yang et al. ). Yu et al. () calculated a neutral evolutionary rate (λ) of 6.5 × 10−9 substitutions per silent site per year to analyze the duplication history of the rice genome. Here, we calculated the dates of the duplication events by the equation T = Ks/2λ, for rice, the λ = 6.5 × 10−9 (Yang et al. ).
Rice materials and treatments
The rice cultivar Nipponbare (O. sativa L. japonica) was used in this study. For expression analyzes of the OsOPT genes under normal conditions, total RNA was extracted from different tissues of the rice plants grown in the natural field of South China Botanical Garden, at the booting stage.
For phytohormone treatments, seeds were soaked in water and germinated at 28 °C for 2 days and then grown in Hoagland's Solution (Baba and Takahashi ) under natural conditions for 3 weeks. Next, the seedlings were cultured in Hoagland's solution containing 5 μM gibberellic acid (GA3), 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D, an auxin), 5 μM kinetin (KT, a cytokinin), or 25 μM abscisic acid (ABA), respectively, for 24 h.
For various treatments, published data were used as a reference for the growth conditions, treatments and experiments on the plant materials in the digital expression database (Nobuta et al. ).
RNA extraction and semi-quantitative RT-PCR analysis
Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions(Invitrogen, http://www.invitrogen.com). First-strand cDNA was synthesized from 3 μg of total RNA treated with DNase I using M-MLV reverse transcriptase (Promega, http//http://www.promega.com). The first-strand cDNA was used as a template for semi-quantitative PCR after normalizing to rice Actin1 (AB047313). Semi-quantitative RT-PCR was performed in a 20 μL reaction volume containing 1 μL cDNA solution, 1 × PCR buffer, 0.25 μM dNTPs, 1.0 μM gene-specific primers and 0.5 U Taq polymerase (Takara, Japan) using the following conditions: 94 °C for 3 min (1 cycle), 94 °C for 30 s, 60 °C for 30 s, 72 °C for 2 min (30-35 cycles) and 72 °C for 10 min (1 cycle). All primer sequences used in this study are listed in Additional file 2: Table S2. To ensure the primers were specific for the correct OsOPT genes, primers were designed using software Primer Primier v5.00 (PREMIER Biosoft international). The amplification products obtained with the different primer pairs were purified using gel electrophoresis and sequenced. Sequences were then aligned on the RAP-DB website to determine their specificity.
Digital expression analysis using the MPSS database
The massively parallel signature sequencing (MPSS) database (http://mpss.udel.edu/rice/) was searched for 20-base signatures from 22 mRNA libraries representing 18 different tissues/organs in rice expression evidence analysis (Nakano et al. ). Only the signatures uniquely identified for an individual gene and had a perfect match (100 % identity over 100 % of the length of the tag) were used for analysis. The normalized abundance (tags per million, tpm) of these signatures for a given gene in a given library represents the quantitative estimate of the expression of that gene. The 22 mRNA libraries of MPSS have been group into four groups to analyze the expression profile of the OsOPT family in various developmental stages, organs and various stress treatments.
Biofunctional analysis of OsOPTs
Two approaches were used to obtain biofunctional clues on OsOPTs. First, different rice inserted mutant databases were searched. The information on the inserted mutants of OsOPTs was retrieved from OryGenesDB (an interactive tool for rice reverse genetics, http://orygenesdb.cirad.fr/index.html), searched by their gene locus ID, and the results were listed in Additional file 1: Table S1. Then, two mutant databases (OTL: http://urgi.versailles.inra.fr/OryzaTagLine/cgi-bin/focus_mutant.pl and NIAS: http://tos.nias.affrc.go.jp/index.html.en) were further searched using the plant names of the inserted mutants in Additional file 1: Table S1 to obtain the observed phenotypes of these mutants. Five OsOPTs mutants had their observed phenotypes in two mutant databases. The second approach was analysis of two OsOPTs (OsOPT3 and OsOPT9) inserted mutants, which were from NIAS, and their main phenotypes were obtained from the homozygote osopt3 and osopt9 mutant plants.
We thank to National Institute of Agrobiological Sciences (NIAS), Japan, for rice mutants (ND1017 and NG0095). This research was partly supported by NSFC grants (30900116/31071352), grant (KSCX3-EW-N-07) of the Chinese Academy of Science, Technology Innovation Program of Functional Development of Instrument and Equipment (YG2011081) of the Chinese Academy of Sciences, and grants (2011A020201003/2007A0201000001/S2011040000236) from Guangdong Province, China.
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