Os11g0235200

SP1 encodes a putative transporter that belongs to the peptide transporter (PTR) family,and regulates panicle development and seed size in rice.

Annotated Information

Function

SP1 encodes a putative transporter that belongs to the peptide transporter (PTR) family. SP1 is preferentially expressed in young panicle，especially in the phloem of the branches of young panicle.SP1 regulates panicle development and seed size in rice.

Mutation

Figure1. Phenotype of the sp1-2 mutant. (from reference)

sp1-2 is one TOS17 insertion mutant.
(1) Phenotypic comparison between wild-type and sp1-2 plants after heading.
The sp1-2 mutant plant showed a slight reduction in plant height, owing to the shorted panicle (Figure 1a).
(2) Comparison of panicle sizes between the wild-type and sp1-2 plants at the grain filling stage.
In comparison with the wild type, the mature panicle length of sp1-2 was strikingly reduced (Figure 1b).
(3) Comparison of mature grains size between the wild type and sp1-2.
The seed size of the sp1-2 mutant was smaller than the wild type (Figure 1c).
(4) Comparison of mature grains weight between the wild type and sp1-2.
In consisted with the smaller seeds, the weight of 1000 seeds was lower of the sp1-2 mutant compared to the wild type (Figure 1d).
These results indicated that the SP1 affects not only the panicle size, but also the grain size.

Cloning of the SP1 gene

250pxFigure 2. Map-based cloning and confirmation of the SP1 gene. (from reference)

Map-based approach was used to clone the SP1 gene. Primary gene mapping showed that the SP1locus is located between the molecular markers S21074 and RM3701 on chromosome 11 (Figure 2a), and then fine mapping narrowed the locus to an 8-kb region between markers M7 and M8 (Figure 2b). Annotation of the 8-kb sequence identified an open reading frame (ORF), LOC_Os11g12740, which is composed of five exons and four introns (Figure 2c). DNA sequence comparison revealed a 31-bp deletion in the third exon in sp1-2. The identity of SP1 is further confirmed by genetic complementation. The plasmid pSPC containing the entire LOC_Os11g12740 ORF and pSPCT containing the partial coding region of the ORF were introduced into the sp1-2 mutant (Figure 2d). All the 10 transgenic lines of pSPC showed a complementation of the sp1-2 phenotype, whereas all of the eight lines of pSPCT failed to rescue the sp1-2 phenotype (Figure 2e,f). RT-PCR analysis showed that the transcription level of the SP1 gene is dramatically decreased in young panicles of sp1-2 (Figure 2g). These results presented above showed that the rice SP1 gene and its mutation is responsible for the altered phenotype of sp1.

Expression

Figure 3. Expression pattern of SP1.(from reference)

The spatial and temporal expression pattern of SP1 is analyzed by GUS reporter system and RT-PCR.
As shown in figure 3a-m, weak GUS signals could be detected in panicle axes upon the formation of spikelets (Figure 3a), subsequently, the GUS signals become stronger in rachis, branches, stigmas, ovules, and the surfaces of palea and lemma during the elongation stage of the young panicle (Figure 3b-g). The GUS signals are barely detectable in mature panicle (Figure 3h). And the GUS signals are hardly detected in root, culm and leaf (Figure 3i-k). Interesting, the SP1-GUS fusion protein is specially located in the phloem of vascular bundles of panicle rachis (Figure 3l-m). RT-PCR analysis results indicated that SP1 highly expressed in young panicle (Figure 3n).

Evolution

Figure 4. Phylogentic analysis of the SP1 amino acid sequence with plant peptide transporter (PTR) proteins. (from reference)

Bioinformatic analysis revealed that the deduced amino acid sequence of SP1 contains a conserved PTR2 domain, implying that SP1 may encode a PTR family protein. Phylogenetic analysis showed that the SP1 gene is classified into a distinct clade for nitrate transporters, and is closest to AtNRT1:2, an Arabidopsis nitrate transporter (Figure 4).
(Footnote: In Figure 4,the amino acid sequences of the entire proteins were aligned by CLUSTALX, and the phylogenetic tree was constructed using the neighbor-joining algorithm. Bootstrap values are shown at each node. The circle and the triangle indicate peptide and nitrate transporters, respectively. The square indicates a dicarboxylate transporter.)

Knowledge Expansion

Figure 5. SP1 sequences and the predicted hydropathy profile of SP1.(from reference)

Figure 6. Subcellular localization of the SP1-GFP fusion protein. .(from reference)

Most members of the PTR family contain 12 a-helical transmembrane domains, and are classified into four groups according to their structure . Hydropathy analysis shows that SP1 protein indeed contained 12 transmembrane domains (TMDs), harboring a long hydrophilic loop between TM6 and TM7 (Figure 5b,c). Three consensus sequences, GxxIADxWLGxFx TIxxxxxVxxxG, LGTGGIKPxV and FxxFYLxINxGSL, have been characterized for PTR proteins. However, these motifs were not strictly conserved in all PTR proteins . This is also the case in SP1, in which there are several alternatives within the conserved motifs (GxxLSDxYLGxFxTMxxxxxVxxxG, LGSGCLKPxI and FxxAYFxFCxGEL). In consistent with the 12 TMDs, the SP1 is located at the plasma membrane ,which is correlates with the SP1 action ,as one nitrate transporter(figure 6). In contrast with the control, signal is observed ubiquitously in onion epidermal cells, in which the SP1-GFP fusion protein is exclusively localized in the plasma membrane (Figure 6a–d). And analysis of ACTIN:SP1-GFP transgenic rice also indicates that SP1 localizes at the plasma membrane in rice (Figure 6e–f).
(Footnote: In Figure 5,(a) The nucleotide sequence and the deduced amino acid sequences of SP1. Numbers at left refer to the positions of nucleic acids. The box highlights the 31-bp deletion in sp1-2. The asterisk indicates the stop codon introduced by the 31-bp deletion. The shaded letters refer to the conserved PTR2 domains, and the underlined letters indicate the 12 transmembrane domains. (b) Prediction of membrane-spanning regions and their orientation of SP1 made by the TMPRED program. (c) A prediction model for the transmembrane topology of SP1.)

Labs working on this gene

• State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China,
• State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences,Hangzhou 310006, China,
• Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8652, Japan,
• Research Institute for Bioresources, Okayama University, 2-20-1, Chuo, Kurashiki, Okayama 710-0046, Japan