Identification of WOX genes in Triticeae plant species
Totally, 43 TaWOX transcripts were obtained using the recently released IWGSC wheat genome [3], and there were still 6 pseudo gene copies (Table 1). Specifically, 15 WOX transcripts in H. vulgare (Table 2), 13 WOX transcripts in A. tauschii (Table S1), 23 WOX transcripts in T. dicoccoides (Table S2), 28 WOX transcripts in T. turgidum (Table S3), and 8 WOX transcripts in T. urartu (Table S4) were identified from IWGSC genome database, respectively. Some homologous alleles of WOX genes were not annotated as transcripts in the database, but were also collected and listed in the tables. For example, TaWUSb and TaWUSd were located on chromosomes 2B and 2D in T. aestivum, respectively (Table 1). The WUS genes in other five Triticeae plant species were also located on their group 2 chromosomes (Table 2, Table S1-S4). TdWOX12a, TdWOX12b, TdWOX7b and TdWOX13b were located on chromosomes 1A, 1B, and 3B in T. dicoccoides, respectively (Table S2).
Table 1
Characteristics of TaWOX gene family members in T. aestivum.
Gene | Gene locus | Chromosome | Gene stretch region | mRNA length (bp) | Protein Sequence Length (aa) | UniProt ID |
TaWOX2a | TraesCS1A02G052000 | 1A | 33,397,501 − 33,398,955:-1 | 1314 | 263 | A0A1D5S1T3 |
TaWOX12a | TraesCS1A02G399400 | 1A | 563,818,671 − 563,823,103:1 | 1854 | 486 | A0A1D5RPD4 |
TaWOX2b | TraesCS1B02G069000 | 1B | 53,364,615 − 53,365,864:-1 | 1119 | 264 | A0A1B1XWM5 A0A1B1XWM7 W5ABB5 |
TaWOX12b | TraesCS1B02G427400 | 1B | 652,781,930 − 652,786,496:1 | 1983 | 485 | A0A1D5SDQ8 |
TaWOX2d | TraesCS1D02G054000 | 1D | 35,059,826 − 35,061,088:-1 | 1138 | 267 | W5ANF9 |
TaWOX12d | TraesCS1D02G406900 | 1D | 470,219,711 − 470,224,514:1 | 2028 | 486 | A0A1D5SWV6 |
TaWUS | TraesCS2A02G491900 | 2A | 724,513,458–724,514,647:1 | 927 | 308 | A0A1D5TC72 |
TaWOX4a | TraesCS2A02G514000 | 2A | 738,371,677–738,372,966:1 | 1061 | 234 | A0A1D5TF70 |
TaWOX11a | TraesCS2A02G100700 | 2A | 53,782,606 − 53,785,288:1 | 1380 | 265 | A0A1D5TJV0 |
TaWOX11b | TraesCS2B02G117900 | 2B | 81,755,546 − 81,758,516:1 | 1366 | 261 | A0A1D5U6K9 |
TaWOX4b | TraesCS2B02G542600 | 2B | 740,320,190–740,321,561:-1 | 1002 | 237 | W5BBK8 |
TaWOX11d | TraesCS2D02G100200 | 2D | 52,227,203 − 52,229,885:1 | 1379 | 264 | A0A1D5TJV1 A0A1D5V0E6 |
TaWOX4d | TraesCS2D02G515600 | 2D | 606,709,221–606,710,431:1 | 979 | 237 | A0A1D5UH04 |
| | 2D | 590146287–590147498:1 | | | |
TaWOX10a | TraesCS3A02G073500 | 3A | 45,776,166 − 45,777,448:1 | 992 | 260 | A0A1D5VKG7 |
TaWOX7a | TraesCS3A02G247200 | 3A | 465,225,214–465,228,773:1 | 1968 | 515 | A0A1D5V4S9 |
TaWOX8a | TraesCS3A02G341700 | 3A | 588,932,808 − 588,937,056:1 | 2230 | 265 | A0A077RTA5 A0A1D5VD81 A0A1D5VQY0 A0A1D5WIZ6 A0A1D6RQB3 A0A1D6RQB4 A0A1D6RQB5 W5CGX8 |
TaWOX14.1a | TraesCS3A02G358200 | 3A | 606,515,981 − 606,519,197:-1 | 1162 | 288 | A0A1D5VFV1 |
TaWOX13a | TraesCS3A02G358100 | 3A | 606,444,775 − 606,446,830:-1 | 1138 | 301 | A0A1D5VA42 |
TaWOX14.2a | TraesCS3A02G358400 | 3A | 606,573,438–606,576,220:-1 | 1133 | 290 | A0A1D6RQ92 |
TaWOX9a | TraesCS3A02G368100 | 3A | 617,060,395–617,061,453:-1 | 949 | 212 | T1WFN3 |
TaWOX10b | TraesCS3B02G087800 | 3B | 56,055,903 − 56,057,760:-1 | 1196 | 261 | A0A1D5VWS6 |
TaWOX7b | TraesCS3B02G272200 | 3B | 438,378,936 − 438,382,259:-1 | 1776 | 515 | A0A077RSZ6 |
TaWOX8b | TraesCS3B02G373800 | 3B | 586,694,870 − 586,698,391:1 | 1216 | 261 | A0A077S168 A0A1D5WT92 |
TaWOX13b | TraesCS3B02G391100 | 3B | 616,425,121–616,426,978:-1 | 900 | 299 | A0A1D5VST7 |
TaWOX14b | TraesCS3B02G391200 | 3B | 616,645,332–616,647,892:-1 | 1216 | 290 | A0A1D5WB93 |
TaWOX9b | TraesCS3B02G399800 | 3B | 631,036,656 − 631,037,718:-1 | 948 | 209 | D8L9N7 |
TaWOX10d | TraesCS3D02G073300 | 3D | 33,294,918 − 33,295,992:1 | 786 | 261 | A0A077RHG9 A0A1D5WSB5 A0A341T564 |
TaWOX7d | TraesCS3D02G244300 | 3D | 339,473,290–339,476,679:-1 | 1834 | 513 | A0A1D5WHW6 |
TaWOX8d | TraesCS3D02G335500 | 3D | 447,560,283–447,562,999:1 | 792 | 263 | A0A1D5VD82 A0A341TAX4 |
TaWOX13d | TraesCS3D02G352500 | 3D | 463,197,196–463,199,275:-1 | 1112 | 298 | A0A1D5WMN9 |
TaWOX14.1d | TraesCS3D02G352600 | 3D | 463,227,796 − 463,230,501:-1 | 895 | 285 | A0A1D5WPP9 |
TaWOX14.2d | TraesCS3D02G352700 | 3D | 463,378,560 − 463,381,808:-1 | 942 | 291 | A0A1D5WNX7 |
TaWOX9d | TraesCS3D02G361100 | 3D | 474,614,857 − 474,615,873:-1 | 901 | 210 | T1WGQ3 |
TaWOX6a | TraesCS4A02G130200 | 4A | 170,708,103–170,711,065:-1 | 1350 | 307 | A0A341TSN5 |
TaWOX6b | TraesCS4B02G174400 | 4B | 382,691,977 − 382,694,806:1 | 1254 | 309 | A0A1D5XNI6 A0A1D5Y4Y9 |
TaWOX6d | TraesCS4D02G176400 | 4D | 306,795,298–306,798,208:1 | 1262 | 306 | A0A341UK30 |
TaWOX5a | TraesCS5A02G085000 | 5A | 111,588,730 − 111,590,895:1 | 1220 | 318 | A0A341UT17 |
TaWOX3a | TraesCS5A02G157300 | 5A | 336,949,988 − 336,951,183:1 | 1060 | 241 | A0A1D5YD57 |
TaWOX5b | TraesCS5B02G091000 | 5B | 118,451,983 − 118,454,221:1 | 1302 | 321 | A0A1D5ZG91 A0A1D6A0K9 |
TaWOX3b | TraesCS5B02G156400 | 5B | 288,891,901 − 288,893,003:-1 | 968 | 241 | W5F9A2 |
TaWOX5d | TraesCS5D02G097400 | 5D | 108,103,399 − 108,105,722:1 | 1381 | 322 | W0Z680 |
TaWOX3d | TraesCS5D02G162600 | 5D | 254,023,305 − 254,024,410:1 | 1006 | 242 | W5FQU4 |
TaWOX8u | TraesCSU02G204800 | Un | 304,503,012–304,503,827:1 | 617 | 156 | A0A077RQB3 A0A096UQ47 A0A1D6RTL8 A0A1D6RTL9 |
TaWUSb | | 2B | 714,777,526–714,778,733:1 | 921 | 306 | |
TaWUSd | | 2D | 590,146,287–590,147,498:1 | 927 | 308 | |
| | 1D | 6,219,571-6,220,231:1 | | | |
| | 3A | 64,319,914 − 64,325,218:-1 | | | |
| | 3B | 83,465,544 − 83,470,232:-1 | | | |
| | 3B | 83,471,253 − 83,471,941:-1 | | | |
| | 3D | 52,801,752 − 52,812,298:-1 | | | |
| | 3D | 463,261,309–463,261,744:-1 | | | |
Table 2
Characteristics of HvWOX gene family members in H. vulgare
Gene | Gene locus | Chromosome | Gene stretch region | mRNA length (bp) | Protein Sequence Length (aa) | Uniprot ID |
HvWOX2 | HORVU1Hr1G010580 | 1H | 24,444,001–24,445,742:1 | 1742 | 279 | A0A287ELV0 |
HvWOX12 | HORVU1Hr1G087940/50 | 1H | 540,693,806 − 540,698,431:-1 | 1470 | 489 | A0A287GM87 A0A287GM65 |
HvWOX11 | HORVU2Hr1G017270 | 2H | 40,107,707 − 40,111,565:1 | 927 | 308 | A0A287H773 |
HvWOX4 | HORVU2Hr1G113820 | 2H | 729,806,496–729,808,073:1 | 1151 | 228 | A0A287JHP1 |
HvWOX10.1 | HORVU3Hr1G013290 | 3H | 28,673,837 − 28,674,948:-1 | 786 | 261 | M0Y8G7 |
HvWOX10.2 | HORVU3Hr1G013330 | 3H | 28,785,048 − 28,786,156:-1 | 815 | 261 | A0A287K575 |
HvWOX7 | HORVU3Hr1G060950 | 3H | 464,417,446–464,421,050:1 | 2027 | 516 | A0A287L9L2 |
HvWOX8.1 | HORVU3Hr1G080660 | 3H | 589,829,423–589,834,968:-1 | 3229 | 267 | M0 × 0 × 0 |
HvWOX8.2 | HORVU3Hr1G080690 | 3H | 590,115,430–590,116,290:1 | 584 | 130 | A0A287LWD8 |
HvWOX9 | HORVU3Hr1G085050 | 3H | 610,834,437–610,835,788:-1 | 1165 | 209 | F2E473 |
HvWOX14 | HORVU3Hr1G086430 | 3H | 616,993,938 − 616,996,482:-1 | 1216 | 283 | M0XTJ6 |
HvWOX13 | HORVU3Hr1G086450 | 3H | 617,085,484–617,087,698:1 | 824 | 274 | A0A287M365 |
HvWOX6 | HORVU4Hr1G051530 | 4H | 423,508,136–423,511,456:-1 | 1710 | 306 | M0Y4Z0 |
HvWOX5 | HORVU5Hr1G022120 | 5H | 111,001,136 − 111,003,388:1 | 1046 | 276 | A0A287QMF0 |
HvWOX3 | HORVU5Hr1G049190 | 5H | 381,765,625 − 381,766,908:1 | 1126 | 186 | A0A287R4V3 |
HvWUS | | 2H | 717,822,805 − 717,905,740:-1 | 942 | 313 | |
Identification of WUS homologous genes in Triticeae plant species
In the six Triticeae plant species, only one transcript of WUS gene was annotated as TaWUSa on chromosome 2A in wheat in the database (Table 1). We found the homologous fragments of TaWUSa on chromosomes 2B and 2D in wheat (Table 1), 2D in A. tauschii (Table S1), 2A and 2B in T. dicoccoides and T. turgidum (Tables S2 and S3), and 2H in barley (Table 2). According to the results of multiple sequence alignment, the full length of the open reading frame (ORF) of these homologous genes can be achieved, and their deduced amino acid sequences were highly consistent with TaWUS (Fig. 1A). To understand if these genes can normally transcribe and express, promoter analysis was performed. It was showed that the promoter region of the WUS genes in the six Triticeae plant species all contained core promoter elements including transcription start TATA-box and AT ~ TATA-box,indicating they possessed potential transcriptional activity (Fig. 1B). In the promoter region of TaWUSa, TdWUSa, TtWUSa, and TuWUS, a fragment of GGTCCAT was existed, which is a cis-acting regulatory element involved in auxin responsiveness. Nevertheless, this element was not detected in the promoter of AtaWUS, TaWUSb, TaWUSd, TdWUSb, and TtWUSb.
Chromosomal location of WOX genes in Triticeae plant species
In general, no WOX gene was found on homologous groups 6 and 7 for the genomes of the six Triticeae plant species, i.e., T. aestivum, T. turgidum, T. dicoccoides, H. vulgare, A. tauschii, and T. urartu, (Tables 1 and 2, and Tables S1-S4). In T. aestivum, except TaWUS, all the TaWOX genes had three copies of transcripts on its genomes A, B, and D. Three homologous alleles of TaWUS were located on chromosomes 2A, 2B, and 2D. The homologous genes of TaWOX2 or TaWOX12 were located on chromosomes 1A, 1B, and 1D. Three copies of TaWOX4 or TaWOX11 were located on chromosomes 2A, 2B, and 2D. The three homologous genes of TaWOX7 to TaWOX10, TaWOX13 and TaWOX14 were all located on chromosomes 3A, 3B, and 3D. The three alleles of TaWOX6 were located on chromosomes 4A, 4B, and 4D. The three alleles of TaWOX3 or TaWOX5 were located on chromosomes 5A, 5B, and 5D. Further investigation would be needed for the unknown chromosomal location of an incomplete transcript of TaWOX8. No WOX gene was found on homologous groups 6 and 7 (Table 1, Fig. 2A). The HvWOX genes in H. vulgare showed the similar chromosomal localization to the TaWOX genes in T. aestivum and AtaWOX genes in A. tauschii. HvWOX2 and HvWOX12 were located on chromosome 1H; HvWOX4 and HvWOX11 were located on chromosome 2H; HvWOX7 to HvWOX10, HvWOX13, and HvWOX14 were located on chromosome 3H; HvWOX6 was located on chromosome 4H, and HvWOX3 and HvWOX5 were located on chromosome 5H. (Table 2; Fig. 2B). There are additional copies of HvWOX8 and HvWOX10 on chromosome 3H. The HvWOX10.1 and HvWOX10.2 showed complete sequence consistency, but HvWOX8.2 was shortened compared with HvWOX8.1.
Similar situation was observed in A. tauschii. AtaWOX2 and AtaWOX12 were located on chromosome 1D. AtaWOX4 and AtaWOX11 were located on chromosome 2D. AtaWOX7 to AtaWOX10, AtaWOX13, and AtaWOX14 were all located on chromosome 3D. AtaWOX6 was located on chromosome 4D, AtaWOX3 and AtaWOX5 were located on chromosome 5D (Table S1, Fig. S1A). Similar results were also obtained in T. dicoccoides and T. turgidum. As expected, all the TdWOX and TtWOX genes were located on the corresponding chromosomes of their genomes A and B because the two species only have the two genomes (Table S2, Table S3, Fig. S1B, Fig. S1C). Additional copies of TdWOX8a and TtWOX14a were also existed on the corresponding chromosomes.
To verify the chromosomal locations of those WOX genes in the six Triticeae species, partial sequences of some of the WOX genes were amplified by their specific primers using a set of T. durum-T. aestivum genome D substitution lines (Fig. 3). The TaWUSa and its two homologs (named as TaWUSb and TaWUSd) were detected in T. aestivum L. cv CS (ABD genome), T. durum cv Langdon (AB genome), and other substitution lines except 2D(2A), indicating that the two copies TaWUSa and TdWUSa were located on chromosome 2A. TaWUSb was amplified in CS, Langdon, and other substitution lines except 2D(2B), indicating that TaWUSb was located on chromosome 2B. TaWUSd only appeared in CS, 2D(2A) and 2D(2B), indicating that it was located on chromosome 2D (Fig. 3). Similarly, WOX2a, WOX2b, WOX6a, and WOX6b were absent in 1D(1A), 1D(1B), 6D(6A), and 6D(6B), respectively. WOX2d and WOX6d were only detected in CS and the substitution lines which contain chromosome 1D or 4D (Fig. 3).
Evolution of WOX family proteins in Triticeae plant species
Phylogenetic trees of WOX family proteins in Triticeae species were constructed based on the deduced protein sequences. From the phylogenetic trees, it was suggested that WOX proteins in Triticeae plants were also divided into three clades, like those in many other plant species [44, 45]. However, the WOX protein classification in wheat was closer to that in rice in comparison with that in Arabidopsis. TaWUS, TaWOX2 to TaWOX5, TaWOX9, TaWOX13, and TaWOX14 were assigned to the same clade with the homologous proteins in rice, corresponding to Arabidopsis WUS clade (AtWUS and AtWOX1 to AtWOX7). TaWOX6, TaWOX7, and TaWOX10 to TaWOX12, and their homologous proteins from rice were classified into a clade, corresponding to an Arabidopsis intermediate clade (AtWOX8, 9, 11, and 12). TaWOX8 and OsWOX8 were clustered in separated branches, showing correspondence to an Arabidopsis ancient clade (AtWOX10, 13, and 14) (Fig. 4).
Barley WOX proteins were also divided into three clades: the first clade harbored HvWOX2, 3, 5, 9, 13 and 14; the second clade was for HvWOX8 only; and the third clade included HvWOX6, 7, and 10 to 12 (Fig. S2A). Similar to wheat, one branch in A. tauschii contained AtaWOX2 to AtaWOX5, 9, 13 and 14. AtaWOX6, 7, and 10 to 12 were clustered into the same branch, but AtaWOX8 was belonged to another branch alone (Fig. S2B). In T. turgidum, TtWOX proteins were also divided into three clades: TtWOX2 to TtWOX5, 9, 13 and 14 were in the first branch; TtWOX6, 7, and 10 to 12 were in the second branch; and the three copies of TtWOX8 were clustered into the same group with OsWOX8 (Fig. S2C). In T. dicoccoides, TdWOX2 to TdWOX5, 9, 13 and 14 were clustered in one branch, TdWOX8 was in other branch alone, and TdWOX6, 7, and 10 to 12 were in another branch (Fig. S2D). In T. urartu, only eight sequences coding WOX family proteins were retrieved because there was no complete genome information on T. urartu yet. The deduced protein sequences from gene sequences of TuWOX and OsWOX were used to construct a phylogenetic tree, in which TuWOX2, 5, and 9 were grouped together, and TuWOX10 and TuWOX6/11 were in the same branch, and the two homologous sequences of TuWOX8 were clustered together (Fig. S2E).
The phylogenetic tree of the WOX family proteins from the six Triticeae species was also constructed via maximum likelihood method (Fig. 5). Based on the tree, it was clearly seen that the WOX proteins with the same names from the six Triticeae species were clustered together (Fig. 5), indicating that the WOX proteins were conserved in these plant species.
Analysis for the conserved motifs of WOX proteins in Triticeae species
All the amino acid sequences of WOX proteins in the six Triticeae species were deduced from their transcripts mentioned above. Each member contained HOX homeodomain, which were the most noteworthy symbol and defining feature of this protein family (Fig. 6, Fig. S3). Sequences of HOX homeodomain of the three clades of WOX proteins were conserved in the six Triticeae species (Fig. 7A). The conserved WUS-box motif TLXLFPXX (TL-[DEQP]-LFP-[GITVL]-[GSKNTCV]) was found in TaWUS, WOX2 to WOX5, and WOX9 in these Triticeae species (Fig. 6A, Fig. 7B). While, there was one amino acid residue change in ELXLFPXX of TaWUS and LLXLFPXX of WOX13 and WOX14 in the Triticeae species (Fig. 7B). The carboxy-terminal ERF-associated amphiphilic repression (EAR) domain of L-[ED]-L-[RST]-L only exists in WUS and WOX9 (Fig. 6A), and EAR domain of WOX9 in these Triticeae species showed highly conserved (Fig. 7C).
Expression patterns of TaWOX genes in different organs of wheat
The WOX genes mainly expressed in the meristematic region, and played a regulatory role in the process of plant growth and tissue differentiation. We retrieved the data from expVIP website (http://wheat-expression.com), and sketched the contours of expression pattern of TaWOX genes. It is showed that TaWUS expressed in root during seedling stage, in spike during vegetative stage, and in spike and leave/shoot during productive stage. Its expression level was higher in spike than other organs (Fig. S4A). All the three homologous of TaWOX2 to 4, 7, 8, and 12 showed higher expression level in developing spike than other organs, and even higher at vegetative stage than reproductive stage (Fig. S4B-D, G, H, and L). The expression level of TaWOX5 was higher in grain than that in other organs at reproductive stage (Fig. S4E). TaWOX6, 9 to 11 showed a high transcriptional activity in root (Fig. S4F, I-K). The transcripts of TaWOX10 and TaWOX11 mainly accumulated in root at seedling stage while the expression level of TaWOX9 was high in root at vegetative stage (Fig. S4I-K). The transcript levels of TaWOX6b and TaWOX6d in root were increased at productive stage compared with vegetative stage (Fig. S4F).
Further, we used wheat root, stem, leave, spike at booting stage, and anther at heading stage as well as immature embryo, callus derived from the immature embryos at proliferative and differential stages as materials to perform expression profiling analysis of TaWOX genes by qPCR assay. The results indicated that expression patterns of TaWOX genes changed greatly in different organs at different stages (Fig. 8). The expression levels of TaWUS and TaWOX6 to 8 were relative high in spike (Fig. 8A, B), and the expression levels of TaWOX9 and TaWOX11 were high in root (Fig. 8B, C). Additionally, TaWOX2 showed high activity in embryo, and TaWOX3 and TaWOX4 showed high expression levels in embryogenic callus and differential callus, respectively (Fig. 8A).