Os09g0494200

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Annotated Information

Function

The rice Os09g0494200 was reported as Brittle Culm15 (BC15) which encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice. Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. BC15 was designated as Chitinase-Like Protein1(OsCTL1) and will henceforth be referred to as BC15/OsCTL1.


BC15/OsCTL1 is an N-glycosylated membrane protein. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth, showed in Figure 1. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. And it is localized in the Golgi apparatus with its C-terminus facing the Golgi lumen. [1]

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Expression

To date, at least 12 bc mutants (bc1, bc2, bc3, bc4, bc5, bc6, bc7, bc10, bc11, bc12, bc14 and bc15) have been characterized in rice. BC15 encodes a membrane-associated chitinase-like protein.[2] Quantitative real-time polymerase chain reaction and β-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. BC15/OsCTL1 is ubiquitously expressed in many organs. The expression pattern of BC15/OsCTL1 was analyzed by quantitative real-time (qRT)-PCR using RNAs isolated from various organs from wild-type plants. As shown in Figure 2A, BC15/OsCTL1 is expressed in several organs, with relatively high levels in the internodes and nodes. This expression pattern fits with the brittle culm phenotype of bc15.


To further examine its expression at the tissue level, the promoter of BC15/OsCTL1 was fused to the GUS reporter gene and the construct was expressed in the wild-type plants. We detected GUS activity in the coleoptiles, roots, shoots, and lamina joints of transgenic seedlings (Fig. 2, B–E). Careful observation revealed stronger GUS-derived signal in the elongation zone of the roots than in the root tips (Fig. 2C) and strong GUS-derived signal in the vascular bundles of the root mature zone (Fig. 2D). GUS activity was also observed in the panicle branches and glumes of mature plants (Fig. 2F). Fresh hand-cut sectioning of the stained internodes clearly showed strong GUS activity in the vascular bundles (Fig. 2G).Therefore, BC15/OsCTL1 is ubiquitously expressed, especially in the tissues that confer mechanical strength in fully developed rice plants.

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Structure

BC15 Encodes a Chitinase-Like Protein. The coding sequence of BC15 (981 nucleotides) encodes a protein comprising 326 amino acids. Pfam analysis showed that BC15 belongs to the GH19 family (PF00182) and is a class II chitinase (EC 3.2.1.14). Plaza 2.5 software further identified 27 orthologs (ORTHO004207) and 306 homologs (HOM000272) of BC15 in different plant species. Because all of them are plant chitinases, BC15 thus encodes a plant chitinase. To check whether BC15 preserves the catalytic and substrate-binding domains of chitinases, we aligned the sequences of BC15 and its orthologs. BC15 possesses the conserved signatures of GH19. One common signature of GH19 proteins is devoid of the N-terminal Cys-rich region that is considered for chitinase binding. Moreover, the H-E-T-T motif required for catalytic activity is replaced by the S-K-T-S motif. Construction of a phylogenetic tree that includes all of the BC15 orthologs and its homologs from rice and Arabidopsis indicated that the closest ortholog of BC15 is AtCTLl. Therefore, BC15 was designated as Chitinase-Like Protein1 (OsCTL1) and will henceforth be referred to as BC15/OsCTL1 in this paper.


BC15/OsCTL1 is an N-Glycosylated Membrane Protein. Plant chitinases often target the extra cellular matrix or various organelles. To investigate the locations where in BC15/OsCTL1 exists, we used the TMHMM 2.0 and SignalP 4.0 software to predict whether BC15/OsCTL1 has any localization motifs. These analyses indicated that BC15/OsCTL1 is a classical type II membrane protein and possesses an N-terminal transmembrane domain (Fig. 3A). To verify this prediction in vivo, we generated transgenic bc15 mutant plants that express GFP-fused BC15/OsCTL1 (pBC15GFP; Fig. 2B). The resulting construct fully rescued the mutant phenotypes, indicating that the tagging with GFP does not affect BC15/OsCTL1 function. We extracted the total proteins from pBC15GFP transgenic plants, fractionated them into soluble, total membrane, and cell wall fractions, and probed them using anti-GFP antibody; BC15/OsCTL1 was mainly detected in the membrane fraction (Fig. 3B), suggesting that BC15/OsCTL1 is membrane associated. This result prompted us to treat the membrane fraction with several chemicals. As shown in Figure 3C, BC15/OsCTL1 was partially solubilized following Triton X-100 treatment. BC15/O sCTL1 is thus an integral membrane protein. In addition, the Prositescan algorithm predicts that BC15/OsCTL1 has three N-glycosylation sites. Changes in the extent of migration of BC15/CTL1-GFP on a SDS-PAGE gel after treatment with PNGase F, an amidase widely used to remove N-linked glycans from glycoproteins, suggested that BC15/OsCTL1 is N-glycosylated (Fig. 3D).

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Localization

To determine whether BC15/OsCTL1 resides on the plasma membrane or in intracellular compartments, we further separated the total membrane fraction isolated from pBC15GFP transgenic plants into the plasma membrane and endomembrane fractions. Protein gel blotting showed that BC15/OsCTL1 is predominantly present in the cellular endomembrane systems (Fig. 4A). We then investigated whether BC15/OsCTL1 targets a particular subcellular organelle. Signal peptide (SP) sequences provide valuable clues regarding the subcellular localization of chitinases. In contrast to AtCTL1, which has a secretion SP at the N terminus (D score = 0.865 in the SP prediction by SignalP 4. 0), BC15/OsCTL1 lacks this motif (D score = 0 .2 77), suggesting that BC15/OsCTL1 and AtCTL1 have different subcellular localization patterns. We viewed the root epidermal and coleoptiles cells of plants expressing the pBC15GFP transgene, and the dot-like fluorescent signals were observed in those living cells (Fig. 4B). The dot-like signals were almost identical to those labeled by a Golgi marker, while we coexpressed CTL1-GFP and Man49-m Cherry in rice protoplast cells (Fig .4C). Therefore, OsCTL1 is localized in the Golgi apparatus. As a type II membrane protein, the C terminus of BC15/OsCTL1 that contains a catalytic domain is proposed to face the Golgi lumen. To verify this topological structure, we treated the membrane extracts from plants expressing BC15/OsCTL1-GFP with proteinase K in the presence or absence of the detergent Triton X-100. Immunoblotting assays revealed that GFP fused to the C terminus of BC15/OsCTL1 was sensitive to proteinase K treatment in the presence of Triton X-100 (Fig. 4D), indicating that the C terminus of BC15/OsCTL1 faces the Golgi lumen.

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Labs working on this gene

  • Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

References

  1. Wu B, et al. Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice[J]. Plant Physiol, 2012, 159(4):1440-52.
  2. Rao Y C, et al. Characterization and cloning of a brittle culm mutant (bc88) in rice ( Oryza sativa L.)[J]. Chin Sci Bull, 2013, 58: 3000-3006.

Structured Information

Gene Name

Os09g0494200

Description

Similar to Chitinase-like protein (EC 3.2.1.14)

Version

NM_001070084.1 GI:115479910 GeneID:4347451

Length

1807 bp

Definition

Oryza sativa Japonica Group Os09g0494200, 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 9

Location

Chromosome 9:19801754..19803560

Sequence Coding Region

19802078..19802480,19802568..19802724,19802797..19803217

Expression

GEO Profiles:Os09g0494200

Genome Context

<gbrowseImage1> name=NC_008402:19801754..19803560 source=RiceChromosome09 preset=GeneLocation </gbrowseImage1>

Gene Structure

<gbrowseImage2> name=NC_008402:19801754..19803560 source=RiceChromosome09 preset=GeneLocation </gbrowseImage2>

Coding Sequence

<cdnaseq>atgaagaggaagactcggaacaagataatcctgacgacgctgctggtgtcggcggcggcgatcctgatcggcgggacggtggcgctgatcctgacggcggggacatggaaggtgaagatgaaggagtcgcgcgagaagatctgcgacaaggggtgggagtgctcgggtagcaagtactgctgcaacgacaccatcaccgacttcttcaaggtgtaccagttcgagaacctcttctccaagcgcaacagccccgtcgcccacgccgtcggcttctgggactaccagtccttcatcaccgccgccgccctcttcgagcccctcggcttctgcaccaccggcggcaagcagatgcagatgatggagctctgcgccttcctcggccatgtcggctccaagacctcatgtggatttggtgtggcgaccggcgggccgacggcgtgggggctctgctacaaccacgagatgagccctaaggaggattactgcgacaagacgaacctgcagtatccctgcgtcgagggcgccgagtactacggccgcggcgccatccccgtcttctggaactacaactatggcgcggcgggcgacgggatacacgaggacctgctccaccacccggagtacctggagcagaacgcgacgatggcgttcatggcggcgatgtggcggtggatgacgccgatgaagaagaagcagccgtcggcgcacgacgtgttcgtgggcaactggaagcccaccaagaacgacacgctcgccaagcgcctccccgggttcggcgccaccatgaacgtgctctacggcgaccagatctgcggcaagggctacatcgacgacatgaacgtcatcatctcgcactaccagtactacctcgacctcatgggcgtcggccgcgagcactccggcgacaaccgcgactgcgccgagcaggccgccttcaacccctcctacaagaagcccgacgatcagcagcaacaaagctag</cdnaseq>

Protein Sequence

<aaseq>MKRKTRNKIILTTLLVSAAAILIGGTVALILTAGTWKVKMKESR EKICDKGWECSGSKYCCNDTITDFFKVYQFENLFSKRNSPVAHAVGFWDYQSFITAAA LFEPLGFCTTGGKQMQMMELCAFLGHVGSKTSCGFGVATGGPTAWGLCYNHEMSPKED YCDKTNLQYPCVEGAEYYGRGAIPVFWNYNYGAAGDGIHEDLLHHPEYLEQNATMAFM AAMWRWMTPMKKKQPSAHDVFVGNWKPTKNDTLAKRLPGFGATMNVLYGDQICGKGYI DDMNVIISHYQYYLDLMGVGREHSGDNRDCAEQAAFNPSYKKPDDQQQQS</aaseq>

Gene Sequence

<dnaseqindica>325..727#815..971#1044..1464#acacacaacggcaacgtcttaccagctcggagctctctcgcttgctgcctcttctcttcttcctcccgccaccgacaccacctccaccagcagcttcgccttcgccgcgtcggcatctgcagttgccacttcgctttcttccactccctcctcctcctcgcttacctcacactcctcccccccaatttcatcccccacccaccaccagatcccccaccacctgccgcattctcccccccaagatccagatcgccggtcacccccacgaactcgctcgagatccagagagagagagagaggcagtttcttggttgattttcgaggatgaagaggaagactcggaacaagataatcctgacgacgctgctggtgtcggcggcggcgatcctgatcggcgggacggtggcgctgatcctgacggcggggacatggaaggtgaagatgaaggagtcgcgcgagaagatctgcgacaaggggtgggagtgctcgggtagcaagtactgctgcaacgacaccatcaccgacttcttcaaggtgtaccagttcgagaacctcttctccaagcgcaacagccccgtcgcccacgccgtcggcttctgggactaccagtccttcatcaccgccgccgccctcttcgagcccctcggcttctgcaccaccggcggcaagcagatgcagatgatggagctctgcgccttcctcggccatgtcggctccaagacctcatgtgtgtagcttcttcgttttcgctatggtttgatttggatttgaggttttaagctcgccattgatggggatttgtgtgtgtgtgcaggtggatttggtgtggcgaccggcgggccgacggcgtgggggctctgctacaaccacgagatgagccctaaggaggattactgcgacaagacgaacctgcagtatccctgcgtcgagggcgccgagtactacggccgcggcgccatccccgtcttctggtaagcttcgcttcgtcgccgccatctcgatcgcttgatttgatgattgattcggattcggattcggaataggaactacaactatggcgcggcgggcgacgggatacacgaggacctgctccaccacccggagtacctggagcagaacgcgacgatggcgttcatggcggcgatgtggcggtggatgacgccgatgaagaagaagcagccgtcggcgcacgacgtgttcgtgggcaactggaagcccaccaagaacgacacgctcgccaagcgcctccccgggttcggcgccaccatgaacgtgctctacggcgaccagatctgcggcaagggctacatcgacgacatgaacgtcatcatctcgcactaccagtactacctcgacctcatgggcgtcggccgcgagcactccggcgacaaccgcgactgcgccgagcaggccgccttcaacccctcctacaagaagcccgacgatcagcagcaacaaagctagaacagatcacccccaattccccccaattccacggtaaagaattcacccaatgtgttgttgtatacatcttcttgctactgcaatcgctattgccaatgcaaaccaaccattgccattgctgttgcctcgtgagctatacagattaacggattcttcatcagtgtgaggagctgtggtttgcattggtgtatttctgagtagcggattttgagggaagtgtcaactgtgtgtgtgatgcctatgggatttgggaaatttgttgtagcccttgttgtaccatagtgtacattttcattccatttgatttttgtcacatatacatgtttaaagaggaggattcagg</dnaseqindica>

External Link(s)

NCBI Gene:Os09g0494200, RefSeq:Os09g0494200