A Novel FC116/ BC10 Mutation Distinctively Causes Alteration in the Expression of the Genes for Cell Wall Polymer Synthesis in Rice

Abstract

We report isolation and characterization of a fragile culm mutant fc116 that displays reduced mechanical strength caused by decreased cellulose content and altered cell wall structure in rice. Map-based cloning revealed that fc116 was a base substitution mutant (G to A) in a putative beta-1,6-N-acetylglucosaminyltransferase (C2GnT) gene (LOC_Os05g07790, allelic to BC10). This mutation resulted in one amino acid missing within a newly-identified protein motif "R, RXG, RA." The FC116/BC10 gene was lowly but ubiquitously expressed in the all tissues examined across the whole life cycle of rice, and slightly down-regulated during secondary growth. This mutant also exhibited a significant increase in the content of hemicelluloses and lignins, as well as the content of pentoses (xylose and arabinose). But the content of hexoses (glucose, mannose, and galactose) was decreased in both cellulosic and non-cellulosic (pectins and hemicelluloses) fractions of the mutant. Transcriptomic analysis indicated that the typical genes in the fc116 mutant were up-regulated corresponding to xylan biosynthesis, as well as lignin biosynthesis including p-hydroxyphenyl (H), syringyl (S), and guaiacyl (G). Our results indicate that FC116 has universal function in regulation of the cell wall polymers in rice.

Keywords: cell wall; cellulose; fragile culm; mechanical strength; rice.

Figure 1

Phenotypes and map-based cloning of the gene for fc116. (A) Three-week-old wild-type and fc116 plants. (B) Three-month-old wild-type and fc116 plant. (C,D) The mutant leaves and internodes are easily broken. (E) Plant height of wild-type and fc116. (F,G) Measurement of the force required for pulling and breaking wild-type and fcll6 mutant internodes. (H) The fc116 locus was mapped to a 142 kb region on chromosome 5. Black boxes represent exons and black lines represent introns. (I) Sequencing analysis revealed a point mutation in genomic DNA that results 3 bp deletion in fc116 CDS. Bar = 5 cm (A,B). ^**Significantly different (t = test at P < 0.01) compared with wild-type (E,F).

Figure 2

Analysis of FC116 protein. (A,B) Prediction of FC116 protein domain by the SMART and TMHMM2.0 program, showing a transmembrane helix domain between amino acid residues 20 and 42, a core-2/I-Branching enzyme between amino acid residues 86 and 345. Red star represents the mutation site of bc10; green star represents the mutation site of fc116. (C) Schematic diagram of the FC116 protein location in the Golgi membrane. (D) Three dimensional structure of FC116 protein. The black arrow was the deletion.

Figure 3

Phylogenetic tree and protein sequences alignment of FC116 protein family. (A) Unrooted tree of FC116 protein family and organization of exons and introns of the corresponding genes. (B) Black asterisk represents the deletion amino acid.

Figure 4

Co-expression profiling of the FC116 family in rice. (A) Expression patterns of the bc14 and fc116 in rice. The X-axis indicates the tissues at the developmental stages with 1, Calli (15 days after subculture); 2, Calli (5 days after regeneration); 3, Calli (Screening stage); 4, Calli (15 days after induction T2); 5, Calli (15 days after induction T3); 6, Seed imbibition; 7, Seed germination; 8, Plumule (48 h after emergence, Dark); 9, Plumule (48 h after emergence, Light); 10, Radicle (48 h after emergence, Dark); 11, Radicle (48 h after emergence, Light); 12, Seedling; 13, Young shoot; 14, Young root; 15, Mature leaf; 16, Old leaf; 17, Mature sheath; 18, Old sheath; 19, Young flag leaf; 20, Old flag leaf; 21, Young panicle stages 3 (secondary branch primordium differentiation stage); 22, Young panicle stages 4 (pistil/stamen primordium differentiation stage); 23, Young panicle stages 5 (pollen-mother cell formation stage); 24, Young panicle; 25, Old panicle; 26, Young stem; 27, Old stem; 28, Hull; 29, Spikelet; 30, Stamen; 31, Endosperm (7 days after pollination); 32, Endosperm (14 days after pollination); 33, Endosperm (21 days after pollination). The Y-axis represents the expression values obtained from the microarray analysis. (B) fc116 co-expression patterns in two rice varieties during the life cycles. The color scale representing the relative signal values is shown (green refers to low expression; black refers to medium expression and red refers to high expression). Black asterisk is the fc116.

Figure 5

Certain modules correlate with specific cell wall content in rice modified from Guo et al. (2014). Arrows represent the corresponding modules of the brittle culm (BCs), fragile clum (FCs) and CESA genes. Minus and plus represent the down and up regulation genes in fc116, respectively. Two minuses represent more down regulation genes in the module. Blue represent the modules secondary cell wall biosynthesis. Pink represent the modules of cellulose, xylose, arabinose, and guaiacyl lignin biosynthesis. Gray represents the modules of primary cell wall biosynthesis. Purple represent the modules of arabinose and galactose biosynthesis.

Figure 6

Staining of cellulose, lignin, and hemicellulose in wild-type and fc116 plants. (A) and (B) Calcofluor staining of the transverse culm sections of wild-type (A) and fc116 (B) plants, showing the decreased level of cellulose in the cell walls in the mutant culm. (C,D) Wiesner's staining of the transverse culm sections of wild-type (C) and fc116 (D), showing the increased level of lignin in the walls of sclerenchyma cells. (E,F) Immunohistochemical localization of xylan in leaf sections of wild-type and fc116 plants, showing the increased fluorescent signals in all cells visualized with xylan antibody CCRC-M148. Bar = 50 μm.

Figure 7

Expression of lignin monomers biosynthesis genes between wild-type and fc116. The y-axis represents the raw expression values obtained from the RNA-seq analysis. C3H, p-coumarate 3-hydroxylase; C4H, trans-cinnamate 4-hydroxylase; 4CL, 4-Coumarate-CoA ligase; CAD, Cinnamyl alcohol dehydrogenase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; CCR, cinnamoyl-CoA reductase; COMT, caffeic acid O-methyltransferase; COMTL, caffeic acid O-methyltransferase like; F5H, ferulate 5-hydroxylase; HCT, hydroxycinnamoyltransferase; PAL, phe ammonialyase.