Os06g0660200

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Annotated Information

Function

OsPIN2was proposed as an auxin efflux transporter and a member of the PIN family (Wanget al., 2009). The cDNA sequence of OsPIN2(AK101191) comprises 2276 nucleotides and alignment between the cDNA, and the genomic sequence revealed that the gene has seven exons and six introns (Figure S1A). The ORF encodes a protein of 629 amino acid residues and shows 56.3% sequence identity with Arabidopsis AtPIN2 .Hydropathy and transmembrane (TM) motif analyses of the amino acid sequence revealed that the OsPIN2 protein contains three characteristic regions, including a hydrophobic region with five TM domains, a predominantly hydrophilic core, followed by another hydrophobic region with four TM segments .

The gain of function ofOsPIN2in rice plants

It was previously reported that knockdown of OsPIN1bexpression very significantly increased the number of tillers and tiller angle, while over-expression did not alter the shoot architecture of rice (Xuet al., 2005). In the rice genome, OsLazy1 has been identified as a novel grass-specific protein playing a negative role in polar auxin transport; its mutation resulted in an increase in the rice tiller angle (Abe et al., 1996; Li et al., 2007; Yoshihara and Iino, 2007 ). It is interesting to speculate whether enhancingOsLazy1expression could compact rice architecture further. In addition, OsTAC1was defined as a tiller angle control gene in rice, its expression level positively correlated with tiller angle and numbers (Yuet al., 2007)). Transgenic plants over-expressingOsPIN2showed larger tiller angles, lower plant height, more tillers than WT (Figure 2c–e), which was similar to theOsPIN1bknockdown mutants (Xu et al., 2005). The change of phenotype caused by alteration of OsPIN2andOsPIN1bexpression supported the common observation that rice plant height was negatively correlated with tiller number (Wang and Li, 2005). Therefore, auxin might play a key role in the crosstalk between the plant height and branching, an aspect of canopy architecture which is poorly understood.

Expression

Generation of transgenic rice showingOsPIN2 over-expression and phenotypes of transformants Ubiquitin promoter (pUbi) has been long reported as a useful strong promoter in a variety of applications in gene transfer studies and drives the gene expression most actively in rapidly dividing cells (Cornejoet al., 1993) . To investigate the function ofOsPIN2in rice, we made transgenic rice plants over-expressing the full length of cDNA driven by pUbi. Southern blotting analysis revealed that they came from two transgenic lines (O1 and O2, respectively) distinct from each other and both had one copy of the transgene (Figure 1a). Real-time RT-PCR analyses showed that transcriptional expression ofOsPIN2was moderate in the root–shoot junctions and roots but very faint in the leaves of WT (the untransformed plants as control) (Figure 1b). In both the transgenic lines, OsPIN2had the most abundant transcripts in the root–shoot junction, and similar levels in the leaves and roots (Figure 1b). The increases in OsPIN2transcriptional expression driven by pUbi in these tissues reached by 3–15 times.

Over-expression ofOsPIN2significantly increased tiller angle and numbers and decreased height of the rice in comparison with WT (Figure 1i). At 80 days after germination, O1 and O2 plants had a wider tiller angle (the angle between the outermost tillers in the left and right side) (44° and 48°, respectively) than the WT (22°). In addition, the two transgenic lines had many more tillers per plant (21 and 23, respectively) than the WT (17 on the average). In contrast, the whole plant height was very significantly decreased byOsPIN2over-expression. The transgenic plant phenotypes were inheritable from T0 (Figure 2f–h) to T2 generations. At the ripening stage, theOsPIN2-over-expressing plants showed shorter panicle length, less number of grains per panicle and lower grain weight per panicle in comparison with the control. Even though the seed setting rate was similar between O1, O2 and WT, O1 plants had a 35% increase in the effective tiller number, leading to a 16% grain yield increase per plant relative to control plants. O2 plants showed the same tendency but not significant increase in panicle number and grain yield. OsPIN2over-expression also decreased the grain length and breadth by 4%–7% and 12%–16%, respectively. The changes in the grain size lead to 10%–12.5% decreases of 1000-grain weight compared to WT. Moreover, over-expression of the OsPIN2significantly decreased the number of adventitious roots and the total root length by 22%–28%.

There was no putative gene in the insertion site for both O1 and O2. This direct genetic evidence further supported the OsPIN2 contribution to the altered phenotype of the transgenic rice in Figure 1

Auxin transport altered byOsPIN2over-expression in rice To assess whether the over-expression ofOsPIN2affected the free auxin levels, the concentration of endogenous free IAA in various organs of the transgenic and WT plants was quantified by high-performance liquid chromatography (HPLC). In the first and second leaves from the top, O1 and O2 transgenic plants contained free IAA concentrations that were 11%–35% lower than that in the control plants (Figure 3a). In the sheath of the first leaf, the transgenic plants and WT had nearly the same concentration of free IAA, while in the sheath of the second leaf, O1 and O2 plants had 38%–53% lower free IAA concentration than that in WT (Figure 3b). In contrast, in the root– shoot junction (shoot base), the two transgenic lines contained 65%–128% more free IAA than that in WT (Figure 3c). To detect the effect ofOsPIN2over-expression on auxin distribution, we separated individual roots into three segments: 0–4 cm including the root apex and elongation zones; 4–8 cm, the lateral root area; and 8–12 cm which was adjacent to the root–shoot junction. Both transgenic and WT roots showed the same patterns of free IAA concentration with an abrupt decrease from the tip to the root base (Figure 3d).

Evolution

Structure and evolution of the PIN super family

Genes homologous to the Arabidopsis PIN sequences are present in genomes throughout the plant kingdom. These include representatives in non-vascular plants such as Physcomitrella; in vascular plants, PIN sequences appear to be ubiquitous. Both before and since the divergence of monocot and dicot plants there have been significant changes in the number and the structure of PINs. However, in dicot plants, the phylogenetic structure of the family has been broadly conserved ( Figure 2a). Both Medicago and potato contain at least five PIN sequences (these data were collected from EST sequencing projects and, accordingly, have the propensity to exclude sequences from mRNA expressed either at a low level or exclusively in certain tissue types). Each of these genes shows similarity to one of the eight PINs of Arabidopsis. Both Medicago and potato contain two genes homologous to AtPIN1 and two genes homologous to the AtPIN3, AtPIN4, AtPIN7 group. Medicago has one gene homologous to AtPIN2. Potato has one gene homologous to AtPIN6. There is additional evidence for such a broadly conserved phylogenetic structure in the PIN genes of soybean (Glycine max). Here, there exist single homologous genes to AtPIN1, AtPIN2, AtPIN5, AtPIN8, and two genes homologous to the AtPIN3, AtPIN4, AtPIN7 cluster of Arabidopsis.

To confirm that the changed phenotype of O1 and O2 was caused by over-expression of OsPIN2in the transgenic plants, we detected the location of one copy T-DNA insertion in these two lines. The T-DNA containing ubi-promoter was inserted in the chromosome 3 and 10 in the genome of O1 and O2, respectively (Figure 2).

Labs working on this gene

1.State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China

2.Institut für Biologie II, Universität Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.

Structured Information

Gene Name	Os06g0660200
Description	Similar to Auxin efflux carrier protein
Version	NM_001064803.1 GI:115469337 GeneID:4341736
Length	3804 bp
Definition	Oryza sativa Japonica Group Os06g0660200, complete gene.
Source	Oryza sativa Japonica Group ORGANISM Oryza sativa Japonica Group Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP clade; Ehrhartoideae; Oryzeae; Oryza.
Chromosome	Chromosome 6
Location	Chromosome 6:28077811..28081614
Sequence Coding Region	28077886..28078901,28078996..28079180,28079269..28079572,28080611..28080696,28080796..28080953 ,28081075..28081151,28081241..28081307
Expression	GEO Profiles:Os06g0660200
Genome Context	<gbrowseImage1> name=NC_008399:28077811..28081614 source=RiceChromosome06 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008399:28077811..28081614 source=RiceChromosome06 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atgatcaccggacgcgacatctacgacgtgctggcggcgatcgtgccgctgtacgtggcgatgttcctggcgtacgggtcggtgcggtggtgggggatattcacgccggaccagtgctccggcatcaaccgcttcgtcgccgtcttcgccgtgccgctcctctccttccacttcatctccaccaacgacccctactccatgaactaccgcttcctcgccgccgactcgctccagaagctcgtcatcctcgccgcgctcgccgtctggcacaacctgctctcccgctaccgccgcaatggcggcgccgccgcgtcgctcgactggaccatcaccctcttctcgctgtccacgctgcccaacacgctggtcatgggcatcccgctgctccgcgccatgtacggcgacttctccggctcgctcatggtgcagatcgtcgtgctccagagcgtcatctggtacaccctcatgctcttcctcttcgagtaccgcggcgccaaggcgctcatctccgagcagttcccgccggacgtcggcgccagcatcgcctcgttccgcgtcgactccgacgtcgtctcgctcaacgggagggaggcgctgcaggcggacgccgaggtggggcgcgacggccgcgtccacgtcgtcatccgccgctccgcctcggcctccaccacgggcggcggcggcggcgcggcgcgctccggcgtgtcccgggcgtacggcgcgtccaacgccatgacgccgcgcgcctccaacctcaccggcgtggagatctactcgctgcagacgtcgcgcgagcccacgccgcgggcgtccagcttcaaccaggccgacttctacgccatgttctccggcagcaagatggccagccagatggctagccccatggcgcagcacggcggcgccggcggccgcgcccagggcctcgacgagcaggtcaccaacaagttcgcctccggcaaggccgccgacccgccgtcgtatcccgccccgaaccccggcatgatgccggcgccaaggaagaaggagctcgggggctcaaactccaactccaacaaggagctacacatgttcgtgtggagctctagcgcgtcgccggtgtcggaggccaacctccgcaacgccgtcaaccacgccgcctccaccgacttcgcctccgcgccgccgccggcagccgttcccgtcggcggcgccactcccaaaggggtgagtggcagtgtcacgccggcggcgaagaacggcggcggcgagttggagatcgaggacgggctgaagagcccggcggcggggctggcggcgaagttcccggtgtcggggtcgccgtacgtggcgccgaggaagaagggcggcggcgccgacgtgcccgggctggcggaggcggcgcacccgatgccgccgacgagcgtgatgacgcggctcatcctcatcatggtgtggcgcaagctcatcagaaaccccaacacctactccagcctcatcggcctcgtctggtccctcgtctccttcaggtggaatatccaaatgccttcaataataaagggctcaatatcaatattgtcagatgcagggctaggaatggctatgttcagcttaggcttgttcatggctctgcaaccaaagatcatttcttgtggcaagaccgttgcgacatttgcaatggcagtgaggttcttgactggtccagctgttattgcagctacttccattgccattgggctcaggggagtactcttgcatgttgccattgttcaggcagcacttccacaaggcattgtcccgtttgtgtttgccaaggagtacaattgccatcctcaaatacttagcacagcggttatttttgggatgctcatcgcgcttccgatcacgatactctactatgtgcttcttgggatatag</cdnaseq>
Protein Sequence	<aaseq>MITGRDIYDVLAAIVPLYVAMFLAYGSVRWWGIFTPDQCSGINR FVAVFAVPLLSFHFISTNDPYSMNYRFLAADSLQKLVILAALAVWHNLLSRYRRNGGA AASLDWTITLFSLSTLPNTLVMGIPLLRAMYGDFSGSLMVQIVVLQSVIWYTLMLFLF EYRGAKALISEQFPPDVGASIASFRVDSDVVSLNGREALQADAEVGRDGRVHVVIRRS ASASTTGGGGGAARSGVSRAYGASNAMTPRASNLTGVEIYSLQTSREPTPRASSFNQA DFYAMFSGSKMASQMASPMAQHGGAGGRAQGLDEQVTNKFASGKAADPPSYPAPNPGM MPAPRKKELGGSNSNSNKELHMFVWSSSASPVSEANLRNAVNHAASTDFASAPPPAAV PVGGATPKGVSGSVTPAAKNGGGELEIEDGLKSPAAGLAAKFPVSGSPYVAPRKKGGG ADVPGLAEAAHPMPPTSVMTRLILIMVWRKLIRNPNTYSSLIGLVWSLVSFRWNIQMP SIIKGSISILSDAGLGMAMFSLGLFMALQPKIISCGKTVATFAMAVRFLTGPAVIAAT SIAIGLRGVLLHVAIVQAALPQGIVPFVFAKEYNCHPQILSTAVIFGMLIALPITILY YVLLGI</aaseq>
Gene Sequence	<dnaseqindica>76..1091#1186..1370#1459..1762#2801..2886#2986..3143#3265..3341#3431..3497#aattagctcgttctcttgtgtcaagaaaaaaaaagaaagaaaaagctcgccgccgccgccaccgtcgccggcgcgatgatcaccggacgcgacatctacgacgtgctggcggcgatcgtgccgctgtacgtggcgatgttcctggcgtacgggtcggtgcggtggtgggggatattcacgccggaccagtgctccggcatcaaccgcttcgtcgccgtcttcgccgtgccgctcctctccttccacttcatctccaccaacgacccctactccatgaactaccgcttcctcgccgccgactcgctccagaagctcgtcatcctcgccgcgctcgccgtctggcacaacctgctctcccgctaccgccgcaatggcggcgccgccgcgtcgctcgactggaccatcaccctcttctcgctgtccacgctgcccaacacgctggtcatgggcatcccgctgctccgcgccatgtacggcgacttctccggctcgctcatggtgcagatcgtcgtgctccagagcgtcatctggtacaccctcatgctcttcctcttcgagtaccgcggcgccaaggcgctcatctccgagcagttcccgccggacgtcggcgccagcatcgcctcgttccgcgtcgactccgacgtcgtctcgctcaacgggagggaggcgctgcaggcggacgccgaggtggggcgcgacggccgcgtccacgtcgtcatccgccgctccgcctcggcctccaccacgggcggcggcggcggcgcggcgcgctccggcgtgtcccgggcgtacggcgcgtccaacgccatgacgccgcgcgcctccaacctcaccggcgtggagatctactcgctgcagacgtcgcgcgagcccacgccgcgggcgtccagcttcaaccaggccgacttctacgccatgttctccggcagcaagatggccagccagatggctagccccatggcgcagcacggcggcgccggcggccgcgcccagggcctcgacgagcaggtcaccaacaagttcgcctccggcaaggccgccgacccgccgtcgtatcccgccccgaaccccggcatgatgccggcgccaaggtaaaatgaaactgattattaacacctcaaaatttctgttcatcgatcgtgttttgatcgagttggatttttgattttgtcgccattgctacaggaagaaggagctcgggggctcaaactccaactccaacaaggagctacacatgttcgtgtggagctctagcgcgtcgccggtgtcggaggccaacctccgcaacgccgtcaaccacgccgcctccaccgacttcgcctccgcgccgccgccggcagccgttcccgtcggcggcgccactcccaaaggtactctctcatccatccattgacgcacacgacgacgatcacaccacatgtgttcaaggcttcgtctaatggtgcgtgcatgccacaggggtgagtggcagtgtcacgccggcggcgaagaacggcggcggcgagttggagatcgaggacgggctgaagagcccggcggcggggctggcggcgaagttcccggtgtcggggtcgccgtacgtggcgccgaggaagaagggcggcggcgccgacgtgcccgggctggcggaggcggcgcacccgatgccgccgacgagcgtgatgacgcggctcatcctcatcatggtgtggcgcaagctcatcagaaaccccaacacctactccagcctcatcggcctcgtctggtccctcgtctccttcaggtattggattctcaccgatgttttcggtactcaagctgacgttctcgcttccacagttccacttcatcgacgcccgctttcccggtcgcttccctctaagatggaacactcttctgccaatacattttgacatcccacagctatgacagatcgcttatcccggttcatcttcagggcaagggcgctttttttcaagttataccgaattatcaaaacagattatttactctccatttcaaaaaagtattttataaaaaaaaatattcttctactacattacttagattacttgttttaagctagttgttaagtttttttttaaattgtttaatttatttgtataatgaaatcgataatctgcatagtaatctaaaccaaataagtaatatacacttgagttcacgatctgatcaatttatgttgttcgaagctacaaaaaggatatatttttgctatgctattcgttgctagattgcttgaaattttaatctaggaagaggcaagatcgatgagaacttacttaacttcacaactggattagtgttttctgaacgcatgtgagagcacatgataattacgttggtctcggatctggtccagcacatgcaaagaacatacacacgtatgcagatgcaggtgcaggtgcaggtcttatgtgagccctggagtttagcagcagagaccaaatctaccgaatcagctgcgattgccgtgtttgtccccatccaagacagacacggcgaattcggctcaactagctagatgatccatcatgtgtacaagaagaagagcaaaaaatgtacaatgatatggatgcatcattgattcattgtactactatcttaccattagatggcacatctagagggagggttgcaagaacagggcatttttagtggggaggagcacatgcctgcaagcatctaaaattgcatacatgcatgatacatctagcattgattctcttgtcatacaatttttttttacattgtgaaaatatatgcttaattgcttattgactttgtgaatttggatcaggtggaatatccaaatgccttcaataataaagggctcaatatcaatattgtcagatgcagggctaggaatggctatgttcagcttaggtacagagttgacctttttgatacatatctatagccataactagtgagttttttctttgctttgaccaagaaattctgacaatagtatatgtaacccaggcttgttcatggctctgcaaccaaagatcatttcttgtggcaagaccgttgcgacatttgcaatggcagtgaggttcttgactggtccagctgttattgcagctacttccattgccattgggctcaggggagtactcttgcatgttgccattgttcaggtaagcagtagattctttatttcttgcaatcatcttaaaagaaaaaaaatattaaattttgtaattatggtgaattggaatgtattgtggtgatcttatagattaattgattgtcaaacaggcagcacttccacaaggcattgtcccgtttgtgtttgccaaggagtacaattgccatcctcaaatacttagcacagcgtaagaaatgcatgcttaaactttctatttgttcgtccatacggttgctgaagtatatagcctaaaatatataaatgtgaaattttcagggttatttttgggatgctcatcgcgcttccgatcacgatactctactatgtgcttcttgggatatagtgttcttgaagaaggcaaaaaagaaagagtagggaaaaaaataggattctaggtttctagaggaaaatgcaaaagaaatatgatatgggctttcttgaagacctgaagaactaccagagctgaagaatagggaaatgagatcaagtaggatcctagctagagagaaatgcaaaggaaagacaccccttgattacaattttttaattttttctgcaactgttttggcatcaaagtaaaggttagggccttgagtatgaagagttcagccgttaatttgacaagttgggttggcggtactaaagattcc</dnaseqindica>
External Link(s)	NCBI Gene:Os06g0660200, RefSeq:Os06g0660200