Os09g0422500

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The Os09g0422500 gene can be called by another gene symbols like BC6 and OsCESA9 which similar to cellulose synthase .The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm and plays an important role in cell wall biosynthesis and plant growth.

Annotated Information

Introduction

A genomic fragment containing the mutant Bc6 gene (9374 bp), including 4.8 kbp of upstream sequence and 492 bp downstream, was digested with HindIII and subcloned into the binary vector pBIGRZ to yield the mutant construct, gBc6/pBIGRZ. The wild-type BC6 gene construct, gBC6/pBIGRZ, was generated by changing the mutated nucleotide at position 7112 from G to wild-type A by PCR mutagenesis with primers, PM-F1 (5′-GCAGTTCCCGCAGAGGTTCGACGGC-3′) and PM-R1 (5′-ATCGACGTCCACGACCGATACGCC-3′). These constructs were introduced into the wild-type plant, T65, by an Agrobacterium (Rhizobium radiobacter)-mediated method using strain EHA101. The T2 generation of transgenic plant was used for cell wall analysis.

Figure 1. rice from Brittle phenotype lines VS. Normal lines

1.Promoter

PromoterBC6:β-glucuronidase (pBC6:GUS) activity was assayed according to the method of Kosugi et al.The 4.8 kbp promoter region of BC6 was amplified by PCR using specific primers, gBc6-F1 and pBC6-R1 (5′-GAGGATCCATGGCCGCGCAACAACGGCCGG-3′), and fused with the GUS gene in the pBIGRZ vector, yielding pBC6:GUS. The nucleotide sequence of the construct was confirmed before transformation into the wild-type plant, T65, as described above. Culms, leaves, nodes, and seedlings of the transgenic plants harbouring the pBC6:GUS gene were cut into pieces, fixed in 5% (w/v) agar, and hand-sectioned. Sections from the transgenic plants were stained with a solution containing 0.5 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 50 mM phosphate buffer (pH 7.4) at 37 °C for 24 h, and observed under a microscope (Eclipse E400).

2.Quantitative analysis of BC1 and BC6 mRNAs

Relative amounts of BC1, BC3, BC6, OsCesA4, and OsCesA7 mRNA were estimated by quantitative RT-PCR. Single-stranded cDNA was synthesized from total RNA of the tissues or organs using oligo(dT)12–18 primer. The following specific primers were designed using the Primer3 program (http://frodo.wi.mit.edu/): for BC1 (Os03g0416200), BC1-RTP-F1 (5′-CGCATGAACTACACCCAGTG-3′) and BC1-RTP-R1 (5′-TCCATGAGCAGGTCGTTGTA-3′); for BC3 (Os02g0738900), BC3-RTP-F1 (5′-GGCCGAAACGATGAGATTTA-3′) and BC3-RTP-R1 (5′- AACATCAGCAGCTTGCATTG-3′); for BC6 (Os09g0422500), BC6-RTP-F1 (5′-TTAGCACGTTTGCGAGTTTG-3′) and BC6-RTP-R1 (5′-GAACTCGTCGTCCTCGTCTC-3′); for OsCesA4 (Os01g0750300), OsCesA4-RTP-F1 (5′-CTAATGCGACGAAGACGATG-3′) and OsCesA4-RTP-R1 (5′-GATTTAACGGTGCCCTCTCA-3′); for OsCesA7 (Os10g0467800), OsCesA7-RTP-F1 (5′-TCCATCTTCTCCCTCGTCTG-3′) and OsCesA7-RTP-R1 (5′-GAATCATCCATCCGGTCATC-3′); and for ACTIN1 (Os03g0718100), ACT1-RTP-F1 (5′-TTCCTACATCGCCCTGGACT-3′) and ACT1-RTP-R1 (5′-AGCCTTGGCAATCCACATCT-3′). The PCR was performed with a SYBR Premix Ex Taq kit under the following conditions: 10 s denaturing at 95 °C, 30 s annealing at 60 °C, and 20 s amplification at 72 C, 40 cycles. The PCR products were detected with Opticon 2 , and the mRNA amounts relative to ACTIN1 mRNA were calculated.

Function

1;This study characterizes a brittle culm (bc88) mutant of rice (Oryza sativa L.) obtained by ethylene methylsulfonate (EMS)-induced mutagenesis of Wuyunjing 7. The bc88 mutant exhibits a diversity of pleiotropic phenotypes, including brittle culm at the whole-plant growth stages, withered leaf tips at the seedling stage, and 18-d delay in heading date at the mature stage. Genetic analysis indicates that the bc88 mutant is controlled by a single recessive nuclear gene. The mutated bc88 gene isolated by map-based cloning contains only one point mutation in the 5th exon relative to its wild-type BC88 (LOC_Os09g25490 and Os09g0422500), leading to an amino acid change from P to L in bc88 plants. Alignment of the putative protein sequence with its homologs indicates that the mutation is located in the conserved region of the sequence. Detection of the transcription level of BC88 in rice plants shows that the expression level of BC88 is higher in spikes and culms than in leaves, roots, and leaf sheaths. These contribute to understanding of the molecular mechanism of cellulose synthesis. The target gene BC88 can be a useful tool in molecular marker-assisted selection for rice culm trait breeding.

2;Rice is a model organism in poaceae plants to study cell wall biosynthesis. In this study, a mutant S1-60 isolated from an EMS mutagenized japonica cultivar Nipponbare, is characterized by brittle culms that can be easily broken by bending. The reduction in mechanical strength was due to defect in thickening of the sclerenchyma cell wall. The amount of cellulose in S1-60 culms was reduced to 44.7% of that of wild-type plants. Besides, the mutant also exhibited pleiotropic phenotypes, such as dwarfism and partial sterility. Genetic analysis and map-based cloning showed that all the phenotype of S1-60 mutant was caused by a recessive point mutation in the OsCESA9 gene, which encodes the cellulose synthase A subunit 9. This yet uncharacterized missense mutation changed the highly conserved G905 to D at the beginning of the fifth transmembrane domain. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle phenotype in the culm. These results indicate that OsCESA9 plays an important role in cell wall biosynthesis and plant growth.

3;According to the MSU Rice Genome Annotation Release 7 (http://rice.plantbiology.msu.edu), there are 13 predicted open reading frames (ORFs) within the 118 kb fine mapping interval , including 7 ORFs with known biochemical functions, 5 ORFs encoding expressed hypothetical protein and 1 transposon . Among them, ORF3 (TIGR ID: LOC_Os09g25490) encoding cellulose synthase A catalytic subunit 9 (OsCESA9) was considered as the priority candidate gene, given that the amount of cellulose was reduced dramatically in S1-60 culm. Therefore, we sequenced and compared the mutant and wild type alleles of the OsCESA9 gene, which has 4643 bp in length and 11 exons and 10 introns. One base pair substitution was found in the last exon, changing GGC to GAC and the encoded amino acid from glycine to aspartic acid at the 905th position .The CESA9 protein has 1056 amino acids in length and eight putative transmembrane domains (TMDs), with two near the amino terminus and six clustered near the carboxyl terminus . There is a RING-type zinc finger in the N-terminal region, which might mediate the interaction between CESA9 and other CESA subunits. A large central domain containing two highly conserved motifs DXD and Q/RXXRW is required for the catalytic activity of the enzyme. The missense mutation in S1-60 occurs at the beginning of the fifth TMD, which might affect the plasma membrane localization of CESA9.

4;The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.

Expression

We examined the expression level of OsCESA9 in various rice organs by both semi-quantitative and quantitative RT-PCR. The OsCESA9 gene is predominantly expressed in the culms of mature stage plants, consistent with the brittle culm phenotype in the S1-60 mutant. The OsCESA9 gene is also expressed in panicle at mature stage. However, the expression level of OsCESA9 was relatively low at seedling stage, no matter in root, leaf blade or leaf sheath. This result showed the OsCESA9 gene mainly participates in the synthesis of secondary cell wall in mechanical tissue at late development stage.

The pattern of expression of BC6 was assessed by analysing T65 plants transformed with pBC6:GUS, a binary vector in which the 4.8 kbp region upstream of the BC6 gene was fused with the GUS reporter gene. BC6 promoter activity was detected in leaves, culms, and nodes, with relatively strong expression in culm vascular bundles 2 weeks after heading.Promoter activity was also observed in young tissues such as developing leaves. Although Bc6 mutation appeared to reduce cell wall thickness in sclerenchymal cells , promoter activity was not detected in developed sclerenchymal cells .It is possible that the reporter gene activity did not completely mirror the expression of the BC6 gene product, OsCesA9 protein. These patterns of BC6 promoter-driven gene expression were similar to the expression pattern of BC1 demonstrated by in situ hybridization .

In Arabidopsis, the COBL4 gene is co-expressed with the secondary cell wall-specific CesA genes and is presumed to play a role in the cellulose synthesis of secondary cell walls, although the precise molecular functions of COBRA-like proteins remain unclear .To examine the relationship between the secondary cell wall-specific CesA and COBRA-like proteins in rice, BC6 and BC1 mRNAs were quantitated in several tissues. Consistent with the results of the pBC6:GUS analysis, relatively high levels of BC6 mRNA were detected in culms and nodes.The level of Bc6 mRNA in roots was relatively low. Indeed, the brittle phenotype in roots was not clear compared with those in culms and leaves.Despite the apparent brittle phenotype in the leaves of Bc6 mutants, the level was low in both leaf blades and sheaths. Importantly, both BC6 and BC1 were highly expressed in culms, nodes, and flowers .These results indicated that BC6 and BC1 are co-expressed during development of secondary cell walls. On the other hand, the expression of BC6 was not related to that of BC3, suggesting that BC6 and BC3 are differently regulated.

The effect of the mutation on the expression of Bc6 was also examined in developing leaf blades. Bc6 mutants showed accumulation of BC6 mRNA comparable with T65.Furthermore, Bc6 mutation barely influenced the mRNA levels of other CesA genes, OsCesA4 and OsCesA7, which are expected to participate in cellulose synthesis in secondary cell walls, together with BC6 (OsCesA9).


Evolution

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Labs working on this gene

1. College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao 266109, China

2. National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China

3. Shandong Rice Research Institute, Jinan 250100, China

4. College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China

5. Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan

6. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China;

7. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

References

1.A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice. Wang D, et al. Plant Sci, 2012 Nov. PMID 23017906

2.Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. Kotake T, et al. J Exp Bot, 2011 Mar. PMID 21209026, Free PMC Article

3.The Rice Annotation Project Database (RAP-DB): 2008 update. Rice Annotation Project, et al. Nucleic Acids Res, 2008 Jan. PMID 18089549, Free PMC Article

4.Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana. Rice Annotation Project, et al. Genome Res, 2007 Feb. PMID 17210932, Free PMC Article

5.The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Ohyanagi H, et al. Nucleic Acids Res, 2006 Jan 1. PMID 16381971, Free PMC Article

Structured Information