Genome-wide analysis of the cellulose synthase-like (Csl) gene family in bread wheat (Triticum aestivum L.).

BACKGROUND: Hemicelluloses are a diverse group of complex, non-cellulosic polysaccharides, which constitute approximately one-third of the plant cell wall and find use as dietary fibres, food additives and raw materials for biofuels. Genes involved in hemicellulose synthesis have not been extensively studied in small grain cereals.
RESULTS: In efforts to isolate the sequences for the cellulose synthase-like (Csl) gene family from wheat, we identified 108 genes (hereafter referred to as TaCsl). Each gene was represented by two to three homeoalleles, which are named as TaCslXY_ZA, TaCslXY_ZB, or TaCslXY_ZD, where X denotes the Csl subfamily, Y the gene number and Z the wheat chromosome where it is located. A quarter of these genes were predicted to have 2 to 3 splice variants, resulting in a total of 137 putative translated products. Approximately 45% of TaCsl genes were located on chromosomes 2 and 3. Sequences from the subfamilies C and D were interspersed between the dicots and grasses but those from subfamily A clustered within each group of plants. Proximity of the dicot-specific subfamilies B and G, to the grass-specific subfamilies H and J, respectively, points to their common origin. In silico expression analysis in different tissues revealed that most of the genes were expressed ubiquitously and some were tissue-specific. More than half of the genes had introns in phase 0, one-third in phase 2, and a few in phase 1.
CONCLUSION: Detailed characterization of the wheat Csl genes has enhanced the understanding of their structural, functional, and evolutionary features. This information will be helpful in designing experiments for genetic manipulation of hemicellulose synthesis with the goal of developing improved cultivars for biofuel production and increased tolerance against various stresses.

Background

Plant cell wall consists of three main polysaccharide fractions: cellulose, hemicellulose, and pectin, with lignin and proteins being the other two constituents. Grass walls contain mainly two of the three polysaccharide fractions with pectin being a rather minor constituent. Hemicelluloses are plant cell wall matrix polysaccharides that possess diverse linear or branched structures [1, 2]. These mainly encompass 1–4-β-glucan, 1,3;1,4-β-glucan, galactan, and glucomannan in grasses [3]. In addition, glucuronoarabinoxylan is a major grass wall constituent. Because of the presence of heterogeneous substituents or other linkages on their polymer backbones, hemicelluloses are non-crystalline and can be readily hydrolysed in comparison to cellulose. These polysaccharides can interact with cellulose microfibrils through hydrogen bonds [4].

Hemicellulosic polysaccharide backbones in plants are made by the cellulose synthase-like (Csl) enzymes, which are members of a much larger superfamily of genes referred to as glycosyltransferase 2 (GT2) [5]. Several other GTs, i.e., xyloglucan α-1,6-xylosyltransferases (GT34), xyloglucan fucosyltransferases (GT37), and xyloglucan galactosyltransferases (GT47) have been reported to be involved in the biosynthesis of xyloglucans [6]. Genes encoding Csl enzymes share sequence similarity with the cellulose synthase A (CesA) gene family known to form cellulose throughout the plant kingdom [7]. A variable number of Csl genes ranging from 30 to 50 have been reported from different plant species and are classified into nine subfamilies (CslA–CslH and CslJ) [8, 9]. Cereals generally lack CslB and CslG families. Among the remaining families, CslA, CslC, and CslD are conserved in all land plants, whereas CslF, CslH are restricted to grasses [10, 11]. A poorly understood subfamily, CslJ, has been reported in grasses as well as dicots, which contrasts with the previous claims of its occurrence only in grasses [12, 13]. Similarly, the subfamilies CslB and CslG were previously reported to be specific to dicots [14]. However, a recent report established the presence of the CslB subfamily in monocots as well [12]. Several of the Csl subfamilies have been reported to be involved in the biosynthesis of different cell wall polysaccharides. For example, subfamily CslA was shown to form β-1,4-mannan backbone of galactomannan and glucomannan [15, 16]. Similarly, CslF and CslH subfamilies were shown to make 1–3;1–4-β-glucan in grasses [17, 18], whereas CslC genes were associated with the formation of the 1–4-β-glucan backbone of a xyloglucan and some other polysaccharides [19].

Wheat is a major cereal crop grown on the largest area of arable land in the world, is second only to maize in grain production, and feeds approximately 40% of the world population [20]. It has a large genome size (~17 Gb), of which ~80–90% is repetitive [21]. Even after the complete genome sequence became available [22], Csl genes remain unidentified and uncharacterized in bread wheat. In general, homeologous copies of most of the genes are located on each of the three chromosomes belonging to each of the subgenomes (A, B, and D), suggesting that the number of Csl genes is expected to approximately three-times that of a diploid species like rice. We used publicly available resources to retrieve wheat genome sequence. Large-scale data mining was performed using the Pfam domain models for the identification of Csl gene family members, which are reported in this study.

Methods

Data sources and sequence retrieval

Wheat genome data were downloaded from the Ensembl Plants FTP server (ftp://ftp.ensemblgenomes.org/pub/current/plants/fasta/triticum_aestivum/), generated by the International Wheat Genome Sequencing Consortium (IWGSC) and converted into a local BLAST database using the UNIX pipeline. BLAST analyses (BLASTN as well as BLASTP) were performed using the stand-alone command line version of NCBI (National Center for Biotechnology Information) blast 2.2.28+ (ftp://ftp.ncbi.nih.gov/blast/executables/LATEST/), released March 19, 2013. A query file was generated from Pfam domain models; PF00535 (GT2) domain and PF03552 (Cellulose_synt) downloaded from Pfam 30.0 June 2016 release [23]. The sequences of splice variants were also retrieved from Ensembl Plants browser (http://plants.ensembl.org/Triticum_aestivum/Info/Index). Analysis of splice variants was conducted as described by Kim et al. (2007) [24]. Previously known Csl sequences from Arabidopsis thaliana, Oryza sativa, and Zea mays were downloaded from the Cell Wall Navigator database [25]. For Brachypodium, sequences were retrieved from phytomine (https://phytozome.jgi.doe.gov). Amino acid sequences of the aforementioned CSL proteins are given in Additional file 1: Figure S1.

Blast searches for wheat homologs

All query files containing the two Pfam domain models (PF00535 and PF03552) were used to perform the BLASTn searches against the local blast database of bread wheat. All blast hits with E-value >1.0 were removed. Using cut-off E- value <1.0, all previously known CesA genes were retrieved. After the compilation of all the sequences below the cut-off value, CD-hit program was used to obtain non-redundant sequences. Higher cut-off E- value was used to ascertain the identification of all the genes that possessed the Pfam domains PF00535 and PF03552. These genes were further filtered through phylogenetic analysis alongwith previously known CSL proteins from Arabidopsis, Brachypodium, maize, and rice, which reflected some non-targeted genes that were removed from further analysis [26]. Phylogenetic analysis was also implemented to categorize different Csl sub-families. CesA genes were distinguished from the Csl genes with the CXC motif, which is diagnostic of the CesA but absent from the Csl proteins [7, 27]. Presence of the conserved domains Cellulose_synt/GT2 was confirmed using a batch blast search at the CDD (conserved domain database) of NCBI. Homeologous genes from each of the three genomes were named TaCslXY_ZA, TaCslXY_ZB, or TaCslXY_ZD, where X denotes the Csl subfamily, Y the gene number and Z the wheat chromosome where it is located. Alignment of the sequences of all newly identified wheat Csl genes is given in Additional file 2: Figure S2.

Protein structure and motif/domain identification

Protein sequences were downloaded from the Ensembl Plants FTP server (ftp://ftp.ensemblgenomes.org/pub/current/plants/fasta/triticum_aestivum/), developed by the International Wheat Genome Sequencing Consortium (IWGSC) [22]. Multiple protein sequence alignments were performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) [28]. The resulting alignments were analysed for the presence of conserved motifs (D, D, DXD, QXXRW) of the GT2 superfamily. Conserved patterns of aligned sequences were highlighted using the sequence manipulation suite: Color align conservation (http://www.bioinformatics.org/sms2/color_align_cons.html) [29]. The conserved domains were predicted using CCD database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [22, 30, 31]. Wheat Csl genes were named based on their sequence identity, coverage, presence of conserved domains and motifs similar to those of the previously identified rice Csl genes. The number of genes in in a subfamily exceeded that of rice, the additional genes were given new names. Because of the resemblance of CslD genes with CesA genes and their probable role in cellulose synthesis, we specifically focused on the TaCslD subfamily. Gene structures and intron evolution of TaCslD members were predicted using the gene structure display server 2.0 (http://gsds.cbi.pku.edu.cn/) using the genomic and cDNA sequences.

Evolutionary relationships of Csl genes

A total of 215 CSL proteins from Arabidopsis, maize, rice and wheat were aligned using MAAFT (v1.3.6) [32]. Sequences that did not extend over the conserved core region were removed. Positions where more than 40% of the sequences contained a gap were also removed. The phylogeny and 1000 bootstrap replications of these sequences was inferred using Seqboot (v3.696) [33] and FastTree (v2.1.10) implemented on the Guillimin cluster [34].

The phylogeny of the CslD subfamily was also determined separately from Arabidopsis, Brachypodium, maize, rice and wheat. For phylogenetic analysis, the amino acid sequences of CSL proteins were aligned using MUSCLE and their evolutionary history was inferred using Neighbor-Joining methods [35]. The tree was drawn to scale, with branch lengths being equivalent to the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed with a Poisson correction and are given as the number of amino acid substitutions per site. The rate of variation among sites was modeled with a gamma distribution (shape parameter = 1) and all positions containing gaps and missing data were removed. Evolutionary analyses were conducted in MEGA6 [36].

RNA-seq expression analysis

Publicly available RNA-seq data generated from bread wheat (var. Chinese Spring) was used to study the expression of newly identified wheat Csl genes. The data were compiled from five different wheat tissues (spike, leaf, stem, root, and grain) collected at seedling, vegetative and reproductive stages of development [37]. The relative expression of each TaCsl subfamily was presented as a heat map generated from the relative abudnace of transcripts (per 10 million reads) for each gene using wheat expression browser powered by expVIP (http://www.wheat-expression.com).

Results

Identification and classification of Csl gene family members in bread wheat

Database searches for bread wheat using conserved pfam motifs PF00535 and PF03552, which are specific to the GT2 superfamily, resulted in the identification of 108 cellulose synthase-like (TaCsl) genes (Table 1). Two to three homeologous copies of each gene from the A, B and D genomes were common. The identified genes were named following the nomenclature of rice, which shares synteny with wheat. To avoid the complexity of the nomenclature, a suffix corresponding to the chromosome number and the specific wheat genome identifier (A, B, or D) has been used for each gene name [7]. For example, the first gene of subfamily CslA; CslA1 on the long arm of chromosome 1 of genomes A, B, and D is named as TaCslA1_1AL, TaCslA1_1BL, and TaCslA1_1DL, respectively.

Table 1

Homeologous copies of the bread wheat Csl genes

No.	Ensembl ID	Gene name	Corresponding gene in rice
1	TRIAE_CS42_6BS_TGACv1_513375_AA1639370.1	TaCslA1_6BS	CslA1
2	TRIAE_CS42_6AS_TGACv1_485966_AA1554960.1	TaCslA1_6AS	CslA1
3	TRIAE_CS42_2AL_TGACv1_093375_AA0278800.1	TaCslA2_2AL	CslOS09G39920
4	TRIAE_CS42_2BL_TGACv1_129747_AA0394630.1	TaCslA2_2BL	CslOS09G39920
5	TRIAE_CS42_2DL_TGACv1_160461_AA0550770.1	TaCslA2_2DL	CslOS09G39920
6	TRIAE_CS42_1AS_TGACv1_019142_AA0061550.1	TaCslA2_1AS	CslOS09G39920
7	TRIAE_CS42_7BS_TGACv1_592860_AA1945380.1	TaCslA3_7BS	CslA3
8	TRIAE_CS42_7DS_TGACv1_623146_AA2050070.1	TaCslA3_7DS	CslA3
9	TRIAE_CS42_7AS_TGACv1_569190_AA1809650.1	TaCslA3_7AS	CslA3
10	TRIAE_CS42_6DS_TGACv1_543811_AA1744360.1	TaCslA4_6DS	CslA10/4/2
11	TRIAE_CS42_6AS_TGACv1_487286_AA1569690.1	TaCslA4_6AS	CslA10/4/2
12	TRIAE_CS42_6BS_TGACv1_513376_AA1639390.1	TaCslA4_6BS	CslA10/4/2
13	TRIAE_CS42_2BS_TGACv1_146583_AA0468630.1	TaCslA5_2BS	CslA5/7
14	TRIAE_CS42_2AS_TGACv1_113418_AA0355820.1	TaCslA5_2AS	CslA5/7
15	TRIAE_CS42_2DS_TGACv1_177473_AA0578070.1	TaCslA5_2DS	CslA5/7
16	TRIAE_CS42_3DL_TGACv1_249033_AA0835410.1	TaCslA6_3DL	CslA11
17	TRIAE_CS42_3B_TGACv1_221079_AA0729630.1	TaCslA6_3B	CslA11
18	TRIAE_CS42_3AL_TGACv1_197519_AA0666560.1	TaCslA6_3AL	CslA11
19	TRIAE_CS42_2AS_TGACv1_113300_AA0354190.1	TaCslA7_2AS	CslA5/7
20	TRIAE_CS42_2DS_TGACv1_177798_AA0584795.1	TaCslA7_2DS	CslA5/7
21	TRIAE_CS42_3B_TGACv1_220828_AA0720500.1	TaCslA8_3B	CslA11
22	TRIAE_CS42_3DS_TGACv1_273022_AA0927600.1	TaCslA8_3DS	CslA11
23	TRIAE_CS42_U_TGACv1_642146_AA2112270.1	TaCslA9	CslA9
24	TRIAE_CS42_7BL_TGACv1_579090_AA1903960.1	TaCslA9_7BL	CslA9
25	TRIAE_CS42_7AL_TGACv1_558725_AA1795700.1	TaCslA9_7AL	CslA9
26	TRIAE_CS42_U_TGACv1_642146_AA2112290.1	TaCslA10	CslA9
27	TRIAE_CS42_7DL_TGACv1_602617_AA1962870.1	TaCslA10_7DL	CslA9
28	TRIAE_CS42_7AL_TGACv1_557254_AA1778850.1	TaCslA10_7AL	CslA9
29	TRIAE_CS42_7BL_TGACv1_578444_AA1895100.1	TaCslA10_7BL	CslA9
30	TRIAE_CS42_3AS_TGACv1_210508_AA0674280.1	TaCslA11_3AS	CslA11
31	TRIAE_CS42_3DS_TGACv1_272005_AA0912960.1	TaCslA11_3DS	CslA11
32	TRIAE_CS42_3B_TGACv1_223332_AA0780350.1	TaCslA11_3B	CslA11
33	TRIAE_CS42_3DL_TGACv1_251593_AA0882850.1	TaCslC1_3DL	CslC1
34	TRIAE_CS42_3AL_TGACv1_197197_AA0665370.1	TaCslC1_3AL	CslC1
35	TRIAE_CS42_3DS_TGACv1_271926_AA0910940.1	TaCslC3_3DS	CslC3
36	TRIAE_CS42_3B_TGACv1_220758_AA0718310.1	TaCslC3_3B	CslC3
37	TRIAE_CS42_3AS_TGACv1_211225_AA0686890.1	TaCslC3_3AS	CslC3
38	TRIAE_CS42_1DL_TGACv1_061928_AA0205730.1	TaCslC7_1DL	CslC7
39	TRIAE_CS42_1BL_TGACv1_030750_AA0099830.1	TaCslC7_1BL	CslC7
40	TRIAE_CS42_1AL_TGACv1_001272_AA0028090.1	TaCslC7_1AL	CslC7
41	TRIAE_CS42_1DL_TGACv1_062162_AA0209740.1	TaCslC9_1DL	CslC10/9
42	TRIAE_CS42_1BL_TGACv1_030501_AA0092480.1	TaCslC9_1BL	CslC10/9
43	TRIAE_CS42_5BL_TGACv1_404820_AA1311790.1	TaCslC10_5BL	CslC10/9
44	TRIAE_CS42_5DL_TGACv1_435778_AA1454840.1	TaCslC10_5DL	CslC10/9
45	TRIAE_CS42_5AL_TGACv1_374268_AA1195590.1	TaCslC10_5AL	CslC10/9
46	TRIAE_CS42_1BL_TGACv1_030586_AA0094860.1	TaCslD1_1BL	CslD1
47	TRIAE_CS42_1AL_TGACv1_001700_AA0034150.1	TaCslD1_1AL	CslD1
48	TRIAE_CS42_1DL_TGACv1_063091_AA0223780.1	TaCslD1_1DL	CslD1
49	TRIAE_CS42_2BS_TGACv1_148683_AA0494520.1	TaCslD3_2BS	CslD3
50	TRIAE_CS42_2DS_TGACv1_177279_AA0572180.1	TaCslD3_2DS	CslD3
51	TRIAE_CS42_2AS_TGACv1_114244_AA0365360.1	TaCslD3_2AS	CslD3
52	TRIAE_CS42_1BS_TGACv1_049706_AA0160220.1	TaCslD4_1BS	CslD4
53	TRIAE_CS42_5BS_TGACv1_425241_AA1392650.1	TaCslD4_5BS	CslD4
54	TRIAE_CS42_5DS_TGACv1_457675_AA1488780.1	TaCslD4_5DS	CslD4
55	TRIAE_CS42_7BL_TGACv1_577301_AA1871610.1	TaCslD5_7BL	CslD5
56	TRIAE_CS42_7AL_TGACv1_559436_AA1799630.1	TaCslD5_7AL	CslD5
57	TRIAE_CS42_7DL_TGACv1_603510_AA1985050.1	TaCslD5_7DL	CslD5
58	TRIAE_CS42_5DL_TGACv1_433536_AA1415830.1	TaCslE1_5DL	CslE6/1
59	TRIAE_CS42_5BL_TGACv1_406235_AA1342600.1	TaCslE1_5BL	CslE6/1
60	TRIAE_CS42_6DL_TGACv1_526558_AA1687090.1	TaCslE2_6DL	CslE2
61	TRIAE_CS42_6AL_TGACv1_471004_AA1500600.1	TaCslE2_6AL	CslE2
62	TRIAE_CS42_6BL_TGACv1_499967_AA1596110.1	TaCslE2_6BL	CslE2
63	TRIAE_CS42_U_TGACv1_683314_AA2158770.1	TaCslE3	CslE6/1
64	TRIAE_CS42_6DS_TGACv1_543277_AA1737920.1	TaCslE4_6DS	CslE6/1
65	TRIAE_CS42_5DL_TGACv1_433536_AA1415840.1	TaCslE6_5DL	CslE6/1
66	TRIAE_CS42_5BL_TGACv1_406235_AA1342610.1	TaCslE6_5BL	CslE6/1
67	TRIAE_CS42_5AL_TGACv1_376126_AA1232370.1	TaCslE6_5AL	CslE6/1
68	TRIAE_CS42_2DL_TGACv1_159781_AA0542640.1	TaCslF1_2DL	CslF1/2/4
69	TRIAE_CS42_2AL_TGACv1_094713_AA0301960.1	TaCslF1_2AL	CslF1/2/4
70	TRIAE_CS42_2DL_TGACv1_160109_AA0546890.1	TaCslF1_2DL	CslF1/2/4
71	TRIAE_CS42_2BL_TGACv1_130934_AA0420130.1	TaCslF1_2BL	CslF1/2/4
72	TRIAE_CS42_7BL_TGACv1_580651_AA1914920.1	TaCslF2_7BL	CslF1/2/4
73	TRIAE_CS42_7AL_TGACv1_557532_AA1782680.1	TaCslF2_7AL	CslF1/2/4
74	TRIAE_CS42_7DL_TGACv1_602590_AA1961740.1	TaCslF2_7DL	CslF1/2/4
75	TRIAE_CS42_2AS_TGACv1_113659_AA0359050.1	TaCslF3_2AS	CslF3
76	TRIAE_CS42_2DS_TGACv1_177641_AA0581710.1	TaCslF3_2DS	CslF3
77	TRIAE_CS42_2BS_TGACv1_148608_AA0494060.1	TaCslF3_2BS	CslF3
78	TRIAE_CS42_2BS_TGACv1_146146_AA0456710.1	TaCslF4_2BS	CslF1/2/4
79	TRIAE_CS42_2DS_TGACv1_179076_AA0604160.1	TaCslF4_2DS	CslF1/2/4
80	TRIAE_CS42_2DS_TGACv1_178985_AA0603230.1	TaCslF5_2DS	CslF3
81	TRIAE_CS42_2AS_TGACv1_112790_AA0345230.1	TaCslF5_2AS	CslF3
82	TRIAE_CS42_2BS_TGACv1_148027_AA0489970.1	TaCslF5_2BS	CslF3
83	TRIAE_CS42_7BL_TGACv1_577473_AA1876170.1	TaCslF6_7BL	CslF6
84	TRIAE_CS42_7AL_TGACv1_555973_AA1751470.1	TaCslF6_7AL	CslF6
85	TRIAE_CS42_7DL_TGACv1_607937_AA2011180.1	TaCslF6_7DL	CslF6
86	TRIAE_CS42_5BL_TGACv1_409916_AA1366600.1	TaCslF7_5BL	CslF7
87	TRIAE_CS42_5DL_TGACv1_433902_AA1424880.1	TaCslF7_5DL	CslF7
88	TRIAE_CS42_5AL_TGACv1_374191_AA1193100.1	TaCslF7_5AL	CslF7
89	TRIAE_CS42_2BS_TGACv1_148916_AA0495580.1	TaCslF8_2BS	CslF8
90	TRIAE_CS42_2DS_TGACv1_178471_AA0596060.1	TaCslF8_2DS	CslF8
91	TRIAE_CS42_2AS_TGACv1_112322_AA0335280.1	TaCslF8_2AS	CslF8
92	TRIAE_CS42_2AS_TGACv1_112322_AA0335290.1	TaCslF9_2AS	CslF9
93	TRIAE_CS42_2BS_TGACv1_147667_AA0486240.1	TaCslF9_2BS	CslF9
94	TRIAE_CS42_2DS_TGACv1_177329_AA0573830.1	TaCslF9_2DS	CslF9
95	TRIAE_CS42_U_TGACv1_641498_AA2096480.1	TaCslF10	CslF9
96	TRIAE_CS42_1BS_TGACv1_049866_AA0163180.1	TaCslF10_1BS	CslF9
97	TRIAE_CS42_2AL_TGACv1_094351_AA0296300.1	TaCslH1_2AL	Cslh1/2
98	TRIAE_CS42_2DL_TGACv1_158387_AA0517170.1	TaCslH1_2DL	CslH1/2
99	TRIAE_CS42_2BL_TGACv1_129372_AA0380770.1	TaCslH1_2BL	CslH1/2
100	TRIAE_CS42_3B_TGACv1_221049_AA0728260.1	TaCslH2_3B	Csl
101	TRIAE_CS42_3DS_TGACv1_273502_AA0931770.1	TaCslH2_3DS	Csl
102	TRIAE_CS42_3DS_TGACv1_271739_AA0907200.1	TaCslH3_3DS	Csl
103	TRIAE_CS42_3AS_TGACv1_212952_AA0704280.1	TaCslH3_3AS	CslH3
104	TRIAE_CS42_3B_TGACv1_222234_AA0760340.1	TaCslH3_3B	Csl
105	TRIAE_CS42_3DS_TGACv1_272297_AA0918580.1	TaCslJ1_3DS	Csl
106	TRIAE_CS42_3AS_TGACv1_210908_AA0681280.1	TaCslJ1_3AS	Csl
107	TRIAE_CS42_3B_TGACv1_221705_AA0747940.1	TaCslJ2_3B	Csl
108	TRIAE_CS42_3DS_TGACv1_272756_AA0924850.1	TaCslJ2_3DS	Csl

An unrooted neighbor-joining (NJ) tree for the 215 derived Csl proteins from Arabidopsis, maize, rice and wheat is shown in Fig. 1. TaCsl proteins grouped into seven subfamilies: TaCslA (32 proteins), TaCslC (13 proteins), TaCslD (12 proteins), TaCslE (10 proteins), TaCslF (29 proteins), TaCslH (8 proteins), and TaCslJ (4 proteins) (Fig. 2). The TaCslA and TaCslC subfamilies were closely related as shown by their taxonomic distribution and phylogenies. As expected, these subfamilies were conserved across the plant species. Although TaCslD is present in all the plant species whereas TaCslF is specific to grasses, their proximity to each other suggests a common origin [12]. Among the sequences common to both dicots and grasses, subfamily CslA appeared to be the most divergent between these two groups of plants. Whereas the sequences within the subfamilies CslC and CslD were interspersed between Arabidopsis and grasses, all the subfamily CslA sequences of Arabidopsis clustered together, separately from the grass CslA sequences. Proximity of the CslB and CslH subfamilies points to their common origin before the separation of grasses from dicots. Similarly, CslG and CslJ apparently had a common origin.

Fig. 1

An unrooted maximum likelihood phylogenetic tree of the Cellulose synthase-like (Csl) gene family from Arabidopsis, maize, rice and wheat using FastTree (v2.1.10) according to Price et al. (35). Nodes with more than 70% support from 1000 bootstrap replications were considered significant and indicated by a black circle. Different colors represent CSL proteins from different species. The scale bar indicates a radial distance equal to 0.5 amino acid substitutions per site. To keep the gene family nomenclature uniform, maize gene models from Gramene were renamed as follows: Zm, first four digits of the locus number, Csl, and the class identifier as described in Schwerdt et al. (9)

Fig. 2

Distribution of the TaCsl genes and their splice variants in seven subfamilies and their corresponding pfam domains used to identify TaCsl gene family members

Splice variants of Csl genes

Twenty two of the 108 genes appeared to encode two or more proteins because of the presence of alternative splicing sites, as predicted by Ensembl database, which would result in 137 probable Csl protein products (Table 2). Splice variants were predicted in all the subfamilies of the TaCsl genes except TaCslD (Table 2). In the subfamily TaCslA, 6 genes alternatively spliced to form 13 putative proteins whereas in the subfamily TaCslC, 5 genes were alternatively spliced resulting in 14 putative proteins. Similarly, for the subfamilies TaCslE and TaCslF, alternative splicing resulted in 7 and 10 splice variants, respectively. Alternative splicing of 1 and 2 genes respectively generated 3 and 4 putative proteins in the CslH and CslJ subfamilies (Fig. 2). More than half (51%) of the splice variants stemmed from exon skipping, ~24% from alternative 5′ and 3′ splice sites, and the rest, ~24%, from intron retention (Table 2).

Table 2

Splice variants of the bread wheat Csl genes

Ensembl gene ID	Gene name	Predicted amino acids	Spliced exon/introns	Status
TRIAE_CS42_6BS_TGACv1_513375_AA1639370.1	TaCslA1_6BS	581	–	Wild type
TRIAE_CS42_6BS_TGACv1_513375_AA1639370.2		390	Exon 1 and 2	Exon skipping
TRIAE_CS42_6BS_TGACv1_513376_AA1639390.2	TaCslA4_6BS	528	–	Wild type
TRIAE_CS42_6BS_TGACv1_513376_AA1639390.1		393	Exon 1 and 2	Exon skipping
TRIAE_CS42_7AS_TGACv1_569190_AA1809650.1	TaCslA3_7AS	551	–	Wild type
TRIAE_CS42_7AS_TGACv1_569190_AA1809650.2		380	Exon 7, 8 and 9	Exon skipping
TRIAE_CS42_7AS_TGACv1_569190_AA1809650.3		503	Exon 9	Exon skipping
TRIAE_CS42_7DL_TGACv1_602617_AA1962870.2	TaCslA10_7DL	515	–	Wild type
TRIAE_CS42_7DL_TGACv1_602617_AA1962870.1		555	Intron 8	Intron retention
TRIAE_CS42_3DL_TGACv1_249033_AA0835410.2	TaCslA6_3DL	524	–	Wild type
TRIAE_CS42_3DL_TGACv1_249033_AA0835410.1		572	Intron 1	Intron retention
TRIAE_CS42_3B_TGACv1_221079_AA0729630.1	TaCslA6_3B	571	–	Wild type
TRIAE_CS42_3B_TGACv1_221079_AA0729630.2		538	Exon 2	Exon skipping
TRIAE_CS42_5BL_TGACv1_404820_AA1311790.1	TaCslC10_5BL	712	–	Wild type
TRIAE_CS42_5BL_TGACv1_404820_AA1311790.2		468	Exon 5	Alternative 5′ site
TRIAE_CS42_5BL_TGACv1_404820_AA1311790.3		504	Exon 1	Exon skipping
TRIAE_CS42_5DL_TGACv1_435778_AA1454840.1	TaCslC10_5DL	708	–	Wild type
TRIAE_CS42_5DL_TGACv1_435778_AA1454840.2		502	Exon1	Exon skipping
TRIAE_CS42_5AL_TGACv1_374268_AA1195590.3	TaCslC10_5AL	703	–	Wild type
TRIAE_CS42_5AL_TGACv1_374268_AA1195590.2		496	Exon 5	Alternative 5′ site
TRIAE_CS42_5AL_TGACv1_374268_AA1195590.1		501	Exon 5	Exon skipping
TRIAE_CS42_3DL_TGACv1_251593_AA0882850.1	TaCslC1_3DL	704	–	Wild type
TRIAE_CS42_3DL_TGACv1_251593_AA0882850.2		493	Exon 5	Exon skipping
TRIAE_CS42_3DL_TGACv1_251593_AA0882850.3		679	Exon 1	Alternative 3′ site
TRIAE_CS42_3AL_TGACv1_197197_AA0665370.1	TaCslC1_3AL	704	–	Wild type
TRIAE_CS42_3AL_TGACv1_197197_AA0665370.2		560	Exon 5	Alternative 3′ site
TRIAE_CS42_3AL_TGACv1_197197_AA0665370.3		679	Exon 5	Alternative 5′ site
TRIAE_CS42_6AL_TGACv1_471004_AA1500600.1	TaCslE2_6AL	667	–	Wild type
TRIAE_CS42_6AL_TGACv1_471004_AA1500600.2		737	Intron 8	Intron retention
TRIAE_CS42_6AL_TGACv1_471004_AA1500600.3		635	Exon 4	Alternative 5′ site
TRIAE_CS42_5DL_TGACv1_433536_AA1415830.1	TaCslE1_5DL	728	–	Wild type
TRIAE_CS42_5DL_TGACv1_433536_AA1415830.2		684	Exon 4	Exon skipping
TRIAE_CS42_5BL_TGACv1_406235_AA1342600.1	TaCslE1_5BL	734	–	Wild type
TRIAE_CS42_5BL_TGACv1_406235_AA1342600.2		728	Exon 1	Exon skipping
TRIAE_CS42_2DS_TGACv1_177641_AA0581710.1	TaCslF3_2DS	847	–	Wild type
TRIAE_CS42_2DS_TGACv1_177641_AA0581710.2		735	Exon 2	Alternative 3′ site
TRIAE_CS42_2DS_TGACv1_179076_AA0604160.1	TaCslF4_2DS	783	–	Wild type
TRIAE_CS42_2DS_TGACv1_179076_AA0604160.2		700	Exon 1	Exon skipping
TRIAE_CS42_2BS_TGACv1_147667_AA0486240.1	TaCslF9_2BS	877	–	Wild type
TRIAE_CS42_2BS_TGACv1_147667_AA0486240.2		796	Exon 1	Exon skipping
TRIAE_CS42_5BL_TGACv1_409916_AA1366600.1	TaCslF7_5BL	745	–	Wild type
TRIAE_CS42_5BL_TGACv1_409916_AA1366600.2		815	Intron 2	Intron retention
TRIAE_CS42_5AL_TGACv1_374191_AA1193100.1	TaCslF7_5AL	792	–	Wild type
TRIAE_CS42_5AL_TGACv1_374191_AA1193100.2		807	Intron 1	Intron retention
TRIAE_CS42_2AL_TGACv1_094351_AA0296300.1	TaCslH1_2AL	737	–	Wild type
TRIAE_CS42_2AL_TGACv1_094351_AA0296300.2		660	Exon 9	Exon skipping
TRIAE_CS42_2AL_TGACv1_094351_AA0296300.3		480	Exon 6, 7, 8 and 9	Exon skipping
TRIAE_CS42_3AS_TGACv1_210908_AA0681280.1	TaCslJ1_3AS	738	–	Wild type
TRIAE_CS42_3AS_TGACv1_210908_AA0681280.2		766	Intron 4	Intron retention
TRIAE_CS42_3DS_TGACv1_272756_AA0924850.2	TaCslJ2_3DS	609	–	Wild type
TRIAE_CS42_3DS_TGACv1_272756_AA0924850.1		734	Intron 1	Intron retention

Conserved motifs and domains

All predicted TaCSL proteins contain either the pfam glycosyltransferase family 2_3 (GT) domain (PF13641) or the cellulose_synt domain (PF03552), considered to be the signature domains of the GT2 superfamily [12, 26]. Subfamilies TaCslA and TaCslC contained GT 2_3, and CslD, CslE, CslF, CslH,and CslJ contained the cellulose_synt domain (Fig. 2). All the TaCsl translanted products contained the motifs D, DXD, D and QXXRW except eight truncated genes that lacked some of these motifs apparently because of the missing sequence (TaCslA7_2DS, TaCslD4_1BS, TaCslD4_5BS, TaCslF2_7BL, TaCslF6_7AL, TaCslF6_7DL, TaCslH3_3AS, TaCslH2_3B). Rice CesA10, 11 and CslH3 also contained only the DXD but lacked the D and QXXRW motifs [38]. The variable amino acids in the conserved motifs DXD and QXXRW were diverse in different subfamilies of Csl genes, for example, for TaCslA (DMD, QQH/FRW); TaCslC (DMD, QQHRW); TaCslD (DCD, QVLRW); TaCslE (DCD, QHKRW); TaCslF (DC/GD, QI/VL/VRW); TaCslH (DCD QF/YKRW); TaCslJ (DCD, QNKRW). These motifs are highlighted in alignment files in the text file S_2a-f.

Phylogenetic analysis of the CslD subfamily

The evolutionary history of the CslD subfamily from Arabidopsis, Brachypodium, rice, maize and wheat was inferred using the Neighbor-Joining method, in MEGA6 [36], after grouping the orthologs from various species into different clades (Fig. 3). Rice Csl genes were used as reference because their complete nomenclature is well documented. All the genes grouped into three clades. The first clade contained CslD2 and CslD1 genes from rice and their orthologs from the remaining species. The three homeologous genes of wheat branched together with OsCslD1; wheat genes under this clade were named TaCslD1_1AL, TaCslD1_1BL, and TaCslD1_1DL. The second clade contained two subgroups with the orthologs of rice genes CslD3 and CslD5 from different species. The genes in the first subgroup were named TaCslD3_2AS, TaCslD3_2BS, and TaCslD3_2DS, and those of the second subgroup TaCslD5_7AL, TaCslD5_7BL, and TaCslD5_7DL. The last clade was composed of the orthologs of the rice CslD4 and wheat genes TaCslD4_5BS, TaCslD4_1BS and TaCslD4_5DS. Here we found only two homeologs of TaCslD4, but a gene from the 1BS genome (TaCslD4_1BS) of wheat grouped together with TaCslD4 genes (bootstrap = 1000), pointing to a translocation from its original A genome (Table 1). This gene shared sequence identity of 85% with TaCslD4_5BS at the amino acid level. OsCslD genes shared 73–86% sequence identity with the corresponding wheat orthologs.

Fig. 3

An unrooted phylogenetic tree representing the CslD subfamily from Arabidopsis, Brachypodium, maize, rice and wheat using Neighbour Joining (NJ) method with 1000 replicates to generate bootstrap values that are shown beside the each node forming the Csl clusters. Different colors and shapes represent orthologous Csl genes from different species. Arabidopsis-blue circles, Brachypodium- sky blue triangels, maize-brown rectangles-, rice-no marker, and wheat-black circles

Gene structure and intron evolution of TaCslD subfamily

The 12 TaCslD genes identified from bread wheat ranged in size from 1519 to 5864 bp. The TaCslD4_1BS gene was the shortest and TaCslD1_1AL was the longest. Homeologous copies of all the genes shared sequence identity ranging from 87 to 94% at the nucleotide level. The variation in size among different genes was primarily because of the number and length of introns but also because of a lack of the complete sequences in the database (Fig. 4). The number of introns in all the genes varied from 2 to 4. Two homeologs: TaCslD1_1AL and TaCslD1_1BL each contained three introns whereas, a third homeolog (TaCslD1_1DL) had four. The genes TaCslD3, TaCslD4 and their homeologs contained three introns each, except TaCslD4_1BS with only two introns. TaCslD5 and its homeologs also had two introns each. For the phases of introns, the genes from the TaCslD subfamily exhibited variable patterns of distribution. Introns 1, 2 and 3 of TaCslD1_1AL, TaCslD1_1BL and TaCslD1_1DL were in 2, 0, and 0 phase whereas the 4th intron of TaCslD1_1DL was in 0 phase. Introns 1 and 2 of TaCslD3_2AS, TaCslD3_2BS and TaCslD3_2DS both were in 0 phase. The third intron of these genes was in phase 2, 1 and 2 respectively. The genes TaCslD4_5BS, TaCslD4_5DS, TaCslD5_7AL, TaCslD5_7BL and TaCslD5_7DL had intron 1 and 2 in phases 2 and 0, respectively, and the third intron of TaCslD4_5BS and TaCslD4_5DS was in phase 0 and 2, respectively. TaCslD4_1BS had introns 1 and 2 in phases 1 and 0. The largest proportion of introns (60%) of all the genes was in phase 0, followed by phase 2 (34%) with a few in phase 1 (6%).

Fig. 4

Structural features and phases of intron evolution of the CslD subfamily genes. Drawn to scale, exons are represented by red boxes and introns by back lines. Corresponding phases of intron evolution (0, 1, and 2) for the CslD genes are shown on the top of the black lines

Expression analysis of TaCsl genes from bread wheat

Publicly available RNA-Seq datasets were used to analyse the expression of TaCsl genes over three developmental stages and different tissues of wheat including root, stem, leaf, spike, and grain. Expression data were available for 32 of the TaCslA genes. Two genes (TaCslA1_6AS and TaCslA1_6BS) were expressed in all the tissues except reproductive stem and leaves. Four genes (TaCslA5_2BS, TaCslA5_2DS, TaCslA6_3B, and TaCslA6_3AL) were expressed moderately. TaCslA9 gene was highly expessed in the leaf tissue from the reproductive stage while the transcript abundance of the remaining genes was low (Fig. 5). TaCslC subfamily genes, wtht the exception of TaCslC3, TaCslC9 and two homeologs of TaCslC10, were expressed highly in root and spike tissues. Two genes, TaCslC1 and TaCslC7 and their homeologs displayed moderate to high expression in all the tissues at seeding and vegetative stage. One gene (TaCslC10_5DL) exhibited moderate to high expression in all the tissues studied except reproductive stem and grain (Fig. 6). Expression of most of the genes of the TaCslD subfamily ranged from moderate to a high in the spike and root tissues but was very low in all the other tissues (Fig. 7). Three of the 10 TaCslE subfamily genes (TaCslE2_6AL, TaCslE2_6BL and TaCslE3) were expressed from moderate to high levels in all the tissues.The remaining genes were expressed at a very low level in all the tissues (Fig. 8). A mixed pattern of expression was observed in the large TaCslF subfamily. Three genes (TaCslF6_7AL, TaCslF6_7BL and TaCslF6_7DL) were highly expressed in all the tissues except the leaves at the reproductive stage. Two genes (TaCslF4_2BS and TaCslF4_2DS) were highly expressed in the stem tissue, but only at a low or moderate level in all other tissues. All other genes expressed at low or moderate levels in one or more tissues (Fig. 9). In the TaCslH subfamily, one of the eight genes, TaCslH1_2BL, was expressed from moderate to high levels in the leaf, stem and spike tissues. The remaining genes were expressed from low to moderate levels in all the tissues (Fig. 10). Three out of four members of the subfamily TaCslJ were expressed from low to moderate levels in the leaf and root tissues while one gene (TaCslJ1_3DS) was poorly expressed in all the tissues studied (Fig. 10).

Fig. 5

Heat map showing the expression profiling of wheat TaCslA genes at seedling, vegetative and reproductive stages. RNA-seq data were obtained from root, leaf, stem, spike and grain of the Chinese spring cultivar. The respective transcripts per 10 million values were used to construct heat map with the scale bar showing expression of the genes

Fig. 6

Heat map of the expression profiling of wheat TaCslC genes at seedling, vegetative and reproductive stages. RNA-seq data were obtained from root, leaf, stem, spike and grain of Chinese spring cultivar. The respective transcripts per 10 million values were used to construct heat map with scale bar showing expression of the genes

Fig. 7

Heat map of the expression profiling of wheat TaCslD genes at seedling, vegetative and reproductive stages. RNA-seq data were obtained from root, leaf, stem, spike and grain of Chinese spring cultivar. The respective transcripts per 10 million values were used to construct heat map with scale bar showing expression of the genes

Fig. 8

Heat map of the expression profiling of wheat TaCslE genes at seedling, vegetative and reproductive stages. RNA-seq data were obtained from root, leaf, stem, spike and grain of Chinese spring cultivar. The respective transcripts per 10 million values were used to construct heat map with scale bar showing expression of the genes

Fig. 9

Heat map of the expression profiling of wheat TaCslF genes at seedling, vegetative and reproductive stages. RNA-seq data were obtained from root, leaf, stem, spike and grain of Chinese spring cultivar. The respective transcripts per 10 million values were used to construct heat map with scale bar showing expression of the genes

Fig. 10

Heat map of the expression profiling of wheat TaCslH and TaCslJ genes at seedling, vegetative and reproductive stages. RNA-seq data were obtained from root, leaf, stem, spike and grain of Chinese spring cultivar. The respective transcripts per 10 million values were used to construct heat map with scale bar showing expression of the genes

Discussion

Grass cell walls contain 20–40% non-cellulosic polysaccharides. The proportion and composition of these polysaccharides varies in different plant species [39]. After the first report demonstrating the β-glucan synthase activity in a Csl-encoded protein was published [15], several members of the Csl gene family have been reported to be involved in the formation of the backbone of the hemicellulosic polysaccharides [16, 18, 19, 26, 38, 40, 41]. As information on the identify of the Csl genes in wheat was lacking, we undertook this study to fill this gap.

We retrieved 108 TaCsl genes from wheat using two conserved domains, PF00535, and PF03552, which were previously shown to be present in the derived proteins of all the Csl genes [12]. These genes include homeologs from A, B and D genome of bread wheat. Similar patterns of homeologous genes were found for FLOWERING LOCUS T (FT), Pairing homeologous 1 (Ph1) and ADP-glucose pyrophosphorylase (AGPase) gene families of hexaploid wheat. Approximately, a quarter of the identified Csl genes were predicted to be alternatively spliced, possibly contributing to the diversity of encoded enzymes. A recent study suggested that alternative splicing was common in plants and accounted for about 20% of the loci transcribed in the leaf and spike tissues of Aegilops tauschii. In the case of germinating barley embryos, 14–20% of intron-containing genes were alternatively spliced [42]. This phenomenon, apparently meant to increase the fitness of an organism, has not thus far been reported for the Csl genes from other species [43].

The TaCsl genes were distributed across all the wheat chromosomes except one, chromosome 4 (Fig. 11). A similar trend of Csl gene distribution was observed in barley [9, 44, 45]. More than half the TaCsl genes were located on only two chromosomes: 2 (32%) and 3 (22%). This suggests hyper-multiplication of the Csl genes on these chromosomes although the reasons for this phenomenon are unknown. It appears, though, that cis duplication of the Csl genes was favored over trans duplication in wheat. Five of the nine CslF genes in barley were located on chromosome 2H [40]. In fact, the barley CslF gene was assigned its role in mixed-linked glucan (MLG) formation via syntenic orthology with rice long before the barely genome sequence became available [40] A detailed analysis of the rice syntenic region corresponding to a known QTL for MLG from barley, which had been published previously, initially led to the breakthrough of the role of CslF in the formation of this polysaccharide [40]). A similar cluster of CslF genes was also detected in the conserved syntenic regions of Brachypodium and sorghum on chromosomes 1and 2, respectively [9].

Fig. 11

Pie chart showing the percentage of TaCsl genes on wheat chromosomes

The observation that only half of genes from the subfamily CslA were expressed at varying levels in the studied tissues suggests that the apparently silent genes may provide a backup under stressful conditions. Alternatively, they may express only transiently in specialized cells or cell parts at levels too low to be detected by the method used to study expression. The first biochemical evidence for the relationship of CslA genes with mannan synthase activity came from the expression of a guar CSLA cDNA in soybean somatic embryos [15]. Subsequent studies in insect cells demonstrated the role of CslA family members in the glucomannan synthases [16, 46]. Reverse genetic and biochemical approaches in Arabidopsis and Dendrobium officinale have also allowed association of certain CslA genes with glucomannan biosynthesis [41, 47]. A recent study in wheat suggested the involvement of a gene from the CslA subfamily in the development of tillers, cell wall composition and stem strength. This study further suggested the probable role of CslA gene transcript levels in carbon partitioning throughout the plant [48].

For the subfamilies TaCslC and TaCslD, most of the genes were relatively highly expressed in the root and spike tissues during the vegetative as well as reproductive phases. Heterologous expression in Pichia revealed that the CslC-encoded enzymes made β-1,4-glucan, the backbone of xyloglucan [19]. The CslD subfamily is conserved in all land plants and is most closely related to the CesA gene family with 40–50% sequence similarity at the amino acid level [49]. Similar to CesAs, the CslD subfamily is ubiquitous in all plant genomes examined to date, unlike other, taxa-specific Csl subfamilies [50]. Previous reports also showed the involvement of certain members of the CslD subfamily in tip growth, for example development of root hairs and pollen tube elongation [51, 52], normal plant growth [50, 53], and meristem morphology [53, 54]. More recently, their role in resistance against biotic stresses has been described [55]. Adding to this discussion, our in silico expression analysis suggests the involvement of certain TaCslD genes in spike development. This suggestion is supported by the observation that a mutant, slender leaf 1 (sle1), which encodes the CSLD4 protein in rice, reduces the number and width of spikelets in the panicle [56].

Two groups of Csl genes, CslF and CslH, have evolved independently in grasses [57]. A third group CslJ, originally believed to be specific to grasses, was recently identified in some dicots [11, 13]. Although TaCslF6 gene showed higher expression in all the studied tissues except the leaf tissue from reproductive stage, it was the only member of the TaCslF subfamily which expressed highly in the grain tissue. Several studies have demonstrated the functional role of CslF6 and CslH in the synthesis of MLG [18, 44, 58, 59]. Only one member of all the genes in these families, CslF6, was expressed in the grain, suggesting that it was responsible for MLG formation. MLG is a desirable polysaccharide as a dietary fiber but undesirable for the brewery industry because it causes haze in beer. It should be possible to select natural variants for the expression of the CslF6 gene to select for an increased or reduced MLG content depending upon the target market for the grain.

Differential expression patterns were observed among homeologous copies from three different genomes of bread wheat, which agree with the previous studies reporting unequal contributions of the three genomes toward gene expression. Interestingly, the homeologous copies of TaCslD genes also differed from each other in terms of intron phase evolution, indicating structural and functional divergence of homeologous gene copies (Fig. 4). Most introns were present in phase 0, which is in accordance with previous findings showing an intron bias in favour of phase 0 [7, 60, 61]. The three homeologs of each gene were not observed for all the genes reported in this study. This could be because of the incomplete sequencing information or because of the elimination of the genes during the allopolyploidization of wheat.

Conclusions

We have identified 108 TaCsl genes in bread wheat and classified them into seven subfamilies (CslA, CslC, CslD, CslE, CslF, CslH, and CslJ). Two or three homeoalleles were identified for most of the Csl genes. Although located on all the wheat chromosomes except chromosome 4, the Csl genes were especially concentrated on chromosomes 2 and 3, suggesting selective, localized duplication in cis phase. Only one of the 29 CslF genes, CslF6, was expressed in the grain, suggesting its role in mixed-linked glucan formation. Neither CslJ nor CslH was expressed in the grain. Information in this report will be helpful in designing experiments to alter wall composition in wheat for improving grain quality, culm strength, or culm composition for biofuels.

FASTA sequences of CSL proteins used for the phylogenetic analysis. (PDF 453 kb)

List of Csl subfamily genes, their protein sizes (number of amino acids), and multiple protein sequence alignments for different subfamilies. The conserved motifs (D, D, DXD, QXXRW) diagnostic of CSL proteins are highlighted with red boxes for each of the subfamilies. (PDF 465 kb)