Os07g0102300
SEMI-ROLLED LEAF 1 (SRL1), a gene involved in the regulation of leaf rolling.
Leaf rolling is an important agronomic trait in rice (Oryza sativa) breeding and moderate leaf rolling maintains the erectness of leaves and minimizes shadowing between leaves, leading to improved photosynthetic efficiency and grain yields. Although a few rolled-leaf mutants have been identified and some genes controlling leaf rolling have been isolated, the molecular mechanisms of leaf rolling still need to be elucidated.
Contents
Annotated Information
Function
SRL1 is expressed in various tissues and is expressed at low levels in bulliform cells. SRL1 protein is located at the plasma membrane and predicted to be a putative glycosylphosphatidylinositol (GPI)-anchored protein.
Xiang et al. have demonstrated that SRL1 regulates leaf rolling through inhibiting the formation of bulliform cells by negatively regulating the expression of genes encoding vacuolar H+-ATPase subunits and H+-pyrophosphatase, which will help to understand the mechanism regulating leaf rolling[1]. Xiang et al. thought that SRL1 Negatively Regulates the Formation of Bulliform Cells and Modulates Leaf Rolling.SRL1 Is a Putative GAP.SRL1 May Inhibit the Formation of Bulliform Cells by Suppressing the Expression of Genes Encoding Vacuolar H+-ATPase Subunits and H+-Pyrophosphatase.
Additional srl1 mutant alleles were searched against the T-DNA flanking sequences in Shanghai T-DNA insertion population. A candidate mutant with T-DNA located at the fifth intron of SRL1 was identified and named as srl1-2. Analysis using SRL1-specific primer F4805-R and T-DNA border primers (NTL1, NTL2, and NTL3), respectively, confirmed the insertion of T-DNA in SRL1 and Southern-blot analysis of srl1-2 genomic DNA using a fragment of hygromycin phosphotransferaseII gene as probe indicated the single insertion of T-DNA . Segregation analysis using the progeny of heterozygous srl1-2 revealed that of the 480 individuals, 132 showed leaf-rolling phenotype (similar to srl1-1) that are homozygous for T-DNA insertion, while 348 displayed flat leaves similar to wild type that are heterozygous for T-DNA insertion or do not harbor T-DNA insertion. The segregation ratio (348:132; χ2 = 1.6, degrees of freedom = 1, P = 0.21) was consistent with classical Mendelian ratio (3:1), indicating the genetic linkage of SRL1 and leaf rolling. Further quantitative (q)RT-PCR analysis revealed the significantly decreased expression of SRL1 in the srl1-2 homozygous mutants, and phenotypic observation, cross-section analysis, toluidine blue O staining of the homozygous srl1-2 plants confirmed the rolled leaf resulting from the increased bulliform cells on the adaxial cell layers, which resembles the srl1-1 mutant and verifies the crucial role of SRL1 in leaf-rolling control.
Mutation
Mutants srl1-1 (point mutation) and srl1-2 (T-DNA insertion) exhibit adaxially rolled leaves due to the increased numbers of bulliform cells at the adaxial cell layers, which could be rescued by complementary expression of SRL1.Phenotypic analysis revealed that srl1-1 exhibited incurved leaves from the seedling stage to reproductive stage(Figure 1).
In grass species such as rice, leaf rolling is induced by water loss from bulliform cells on the leaf upper epidermis, therefore the number and density of bulliform cells may affect the extent of leaf rolling. However, there has been controversy over the roles of bulliform cells in leaf rolling . Studies by Shield demonstrated that water loss from the adaxial subepidermal sclerenchyma and mesophyll also contributed to leaf rolling, and rolling could occur in leaves lacking bulliform cells. In our study, srl1-1 and srl1-2 mutants displayed adaxially rolled leaves resulted from increased number of bulliform cells on the adaxial side of leaf blades, which may substantiate the role of bulliform cells in controlling leaf rolling.
Overproliferation of bulliform cells resulted in the increased number of bulliform cells, while the total number of cells on the adaxial side of leaf blades did not change in srl1-1 and srl1-2, suggesting that more bulliform cells and less epidermal cells were formed from the early undifferentiated epidermis. Therefore, SRL1 may control leaf rolling by negatively regulating the formation of bulliform cells. In rice, the young leaf is adaxially rolled after differentiation from the leaf primordium, and unrolling occurs when the leaf blade emerges from the leaf sheath. The leaves of srl1-1 and srl1-2 continuously exhibit adaxial rolling after the emergence from leaf sheath, indicating the defects in regulation of unrolling. Compared with the thick and cutinized cell walls of epidermal cells, bulliform cells are large, thin walled, and highly vacuolated, and the increase of bulliform cells in srl1-1 and srl1-2 mutants may result in altered mechanical properties of the leaf adaxial surface, leading to adaxial rolling of leaves.
Expression
SRL1 is expressed in various tissues and is expressed at low levels in bulliform cells.SRL1 is located in the plasma membrane and predicted to be a putative glycosylphosphatidylinositol(GPI)-anchored protein.Moreover,analysis of gene expression profile of laser microdissection and quantitative RT-PCR revealed that the expression of gene encoding vacuolar H+-ATPase(subunit A,B,C and D)and H+-pyrophosphatase were increased in srl-1 compared with wild type.These results together demonstrate that SRL1 may negatively regulate the expression of genes encoding vacuolar H+-ATPase subunits and H+-pyrophosphatase,thereby inhibiting the formation of bulliform cells and modulating leaf rolling.
SRL1 was mapped primarily with SSR and STS markers using 186 F2 mutant plants. Of 10,000 F2 plants, 2,342 segregants showing thesrl1-1 mutant phenotype were used for fine mapping and the SRL1 gene was localized within a 21.8-kb region between two SSR markers S5869-3 and S5869-4. New molecular markers were developed by comparing original or cleaved amplified polymorphic sequences between indica var 9311 and Nipponbare according to data published at the National Center for Biotechnology Information The PCR procedure for mapping was as follows: 94°C for 4 min, followed by 35 cycles of 94°C, 30 s; annealing temperature for 30 s; 72°C, 40 s; and a final elongation step at 72°C for 10 min. The PCR products were analyzed on 5% to 6% agarose gels. To define molecular lesions, 21.8-kb genomic DNA from the srl1-1 and relevant wild-type variety (Nipponbare) were amplified by PCR. All PCR products were sequenced and the candidate gene was amplified from both srl1-1 and Nipponbare genomic DNAs using different primers. Obtained sequences were analyzed by DNAMAN software (Version 5.2.2, Lynnon Biosoft).
To express the SRL1-GFP fusion protein, the whole coding sequence of SRL1 were divided into two fragments amplified by primers 5′-GGGATGGCCCCCGCCGGT-3′ and 5′-TCCGAGGGCATACTTGGA-3′, 5′-TGCATCTTTTGACCCAGCTAC-3′ and 5′-CTAGACTTGTAGCCAAATAGC-3′, respectively, and subcloned into the pA7 vector, resulting in the insertion of GFP at 364 amino acid of SRL1, upstream of the predicted C-terminal GPI-modification site (ω-site). The resultant construct was introduced into Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts , rice protoplasts of leaf sheath , and onion (Allium cepa) epidermal cells (Yang et al., 2011), respectively, and the green fluorescence was observed by confocal laser-scanning microscopy (Zeiss LSM 510 META) with an argon laser excitation wavelength of 488 nm.
Evolution
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Labs working on this gene
- National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032 Shanghai, China
- State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, Zhejiang, China
- Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
- US Department of Agriculture/Agricultural Research Service, Plant Gene Expression Center, 800 Buchanan St, Albany, CA 94710, USA
- National Institute of Agrobiological Sciences, Department of Molecular Genetics, Head of Laboratory of Gene Expression; 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan
References
[1] Xiang J J, Zhang G H, Qian Q, et al. SEMI-ROLLED LEAF1 Encodes a Putative Glycosylphosphatidylinositol-Anchored Protein and Modulates Rice Leaf Rolling by Regulating the Formation of Bulliform Cells[J]. Plant Physiology, 2012,159(4): 1488-1500.
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[4] Bowman JL, Eshed Y, Baum SF. Establishment of polarity in angiosperm lateral organs[J]. Trends Genet(2002),18: 134–141.
[5] Capron A, Gourgues M, Neiva LS, Faure JE, Berger F, Pagnussat G, Krishnan A, Alvarez-Mejia C, Vielle-Calzada JP, Lee YR, et al. Maternal control of male-gamete delivery in Arabidopsisinvolves a putative GPI-anchored protein encoded by the LORELEI gene[J]. Plant Cell(2008),20: 3038–3049.
[6] Coppinger P, Repetti PP, Day B, Dahlbeck D, Mehlert A, Staskawicz BJ.Overexpression of the plasma membrane-localized NDR1 protein results in enhanced bacterial disease resistance inArabidopsis thaliana. Plant J(2004),40: 225–237.
