Discovering the TUB Homologs
In Arabidopsis thaliana, there are 10 TUBBY (AtTLP1–7 and AtTLP9–11) genes. For each of the genes, we searched for Zea mays orthologs. Out of the 10 genes in Arabidopsis thaliana, we found five to have homologs in Zea mays. Those are the AtTLP2 and AtTLP3 that have two homologs each in Zea mays, namely ZmTLP6 and ZmTLP9, and ZmTLP10 and ZmTLP11, respectively. AtTLP5 has five homologs in Zea mays, namely, ZmTL2, ZmTLP5, ZmTLP7, ZmTLP8 and ZmTLP14. Also, AtTLP7 has three homologs ZmTLP3, ZmTLP4 and ZmTLP12 and finally AtTLP8 with one homolog, ZmTLP13. As far as ZmTLP10 concerns, in the process of manual curation, we did not find enough evidence supporting the gene module and therefore it was eliminated. In total, we came up with thirteen unique Zea mays TUBBY TFs genes homologs to Arabidopsis thaliana named as ZmTLP. Three ZmTLP genes are located in chromosome four, two genes in each of chromosomes three and five, and one gene in each of chromosomes six, eight, nine and ten (Table 1; Fig. 3). Moreover, we found one paralog gene -ZmTLP1- unique to maize that is located in chromosome 8.
Table 1
Comparative analysis of TUBBY Transcription Factor Gene Family between A.thaliana and Z. mays. The first column refers to the general TUBBY gene module. The second and third columns refer to the Zea mays gene, and the Arabidopsis thaliana gene module, respectively. In the next two columns the alternative-splicing events in Zea mays are presented: more specifically the Before manual annotation refers to the alternative-spliced modules found for each locus initially, and the After manual annotation column refers to the modules we kept after performing the manual annotations. In the last three columns under the Z. mays Genomic location part, the chromosome number, start and end coordinates of each gene locus in Zea mays is shown
Gene name | Z.mays genes | Homolog A.thaliana | Alternative-splicing events in Z.mays | Z.mays Genomic location |
| | | Before manual annotation | After manual annotation | Chr | Start | End |
TUBBY14 | ZmTLP1 | - | 4 | 2 | 10 | 8711082 | 8725025 |
TUBBY13 | ZmTLP10 | AtTLP3 | - | - | 9 | 115435278 | 115437104 |
TUBBY11 | ZmTLP11 | AtTLP3 | 4 | 1 | 1 | 60879438 | 60888390 |
TUBBY5 | ZmTLP12 | AtTLP7 | 5 | 2 | 6 | 162624149 | 162628635 |
TUBBY8 | ZmTLP13 | AtTLP8 | 2 | 1 | 5 | 93095266 | 93097731 |
TUBBY2 | ZmTLP14 | AtTLP5 | 6 | 2 | 3 | 178236350 | 178240117 |
TUBBY12 | ZmTLP15 | - | - | - | | | |
TUBBY3 | ZmTLP2 | AtTLP5 | 5 | 3 | 4 | 218421396 | 218425679 |
TUBBY15 | ZmTLP3 | AtTLP7 | 7 | 5 | 3 | 213249521 | 213254004 |
TUBBY1 | ZmTLP4 | AtTLP7 | 8 | 1 | 8 | 154851163 | 154856045 |
TUBBY4 | ZmTLP5 | AtTLP5 | 4 | 2 | 2 | 26117809 | 26122010 |
TUBBY9 | ZmTLP6 | AtTLP2 | 6 | 6 | 5 | 206380608 | 206384416 |
TUBBY10 | ZmTLP7 | AtTLP5 | 4 | 3 | 6 | 154166396 | 154172068 |
TUBBY7 | ZmTLP8 | AtTLP5 | 4 | 4 | 4 | 200038358 | 200042350 |
TUBBY6 | ZmTLP9 | AtTLP2 | 4 | 3 | 4 | 162975759 | 162979667 |
We also searched for alternative-splicing events. We found in total 63 alternative-splicing events that per ZmTLP gene ranged from two to eight as shown in Table 1. Specifically, the maximum of eight splicing events were found in ZmTLP4, and the least of two splicing events in ZmTLP13. After manual annotation, we ended up with 35 alternative-splicing events.
Annotating the TUBBY genes
Once we identified the Zea mays TUBBY transcription factors, we used a web version of Apollo to validate the annotations (Table 1; Fig. 1). In this process, we took into consideration expression data (both PacBio and Illumina) from different tissues. We validated 12 TUBBY genes (Table 1; Fig. 1) from which 35 alternative-spliced models were confirmed by expression data (Table 1). By going over with the manual annotation of the TUBBY genes, we deleted 44.4% of the automatically predicted alternative-spliced transcripts.
Phylogenetic analysis
We found that all ZmTLP genes have homologs in all vascular eukaryotic plants but not in non-vascular plants (Supplementary Fig. 1). ZmTLP11 has no homologs in Hordeum vulage, while ZmTLP13 has no homologs in any of the Corchorus capsularis, Trifolium pretense and Lupinus angustifolius vascular plants. In Selaginella moellendorfii and ancient vascular seedless plant species, most of the ZmTLPs genes have homologs with the exception of ZmTLP1, ZmTLP3 and ZmTLP4. Finally, ZmTLP1 is not present in most of the vascular plants, except of several Oryza species (Supplementary Fig. 1).
To find the evolutionary relationship of the TLP genes in maize, we constructed the phylogenetic tree of the Zea mays genes (Fig. 4). We observed four sub-groups to be formed. One group consisted ZmTLP11 and 13, another group with ZmTLP3, 4 and 12, a third group with ZmTLP6 and 9, and finally, the last and largest subgroup contained ZmTLP2, 5, 7, 8, 14 and ZmTLP1. These results are in agreement with the findings in Table 1.
Discovering motifs and domains
We were further interested in characterizing the annotated products/proteins (Fig. 5. The majority of the ZmTLP loci including the alternative-splicing products contained the TUB1 and TUB2 motifs. The ZmTLP8.1, 8.3 and 8.4 have a prokaryotic lipoprotein (ID PS51257) motif at the N-terminus of the sequences, while ZmTLP8.2 follows the pattern of the rest of the proteins having both TUB1 and TUB2 motifs. The ZmTLP7 locus has three final products as a result of alternative splicing, but ZmTLP7.3 has no predicted TUB1 and TUB2 motif. From locus ZmTLP6, all alternative spliced products have the TUB1 and TUB2 motifs with the exception of ZmTLP6.5 that has only TUB2 motif. The ZmTLP3 locus has five alternative spliced products and have only TUB1 motif. Interestingly, the maize-specific gene ZmTLP1 has X alternative-spliced products and consists of both TUB1 and TUB2 motifs. Finally, ZmTLP13 has no TUB1 or TUB2 predicted motif.
Further, we searched the maize TLP genes for cis-regulatory elements using the PlantPan 3.0 database and selecting all available plant genomes. In total, 55 elements in the 13 genes were identified (Supplementary table 1), out of which 22 cis-regulatory elements were found present in all genes. On our set of genes, we identified motifs for binding site of other transcription factors that are responsible for organ development. Those are motifs recognized by B3, SRS, AT-hook, Trihelix. We also found motifs that other regulatory transcription factors involved in both stress and development bind to, such as bHLH, bZIP, C2H2, HD-ZIP, ZF-HD transcription factors. Moreover, additional motifs for binding of transcription factors involved only in the stress responses such as WRKY, MADF, MADS and Homeodomain were identified. Finally, there are binding sites for transcription factors such as the C3H involved in embryo development, the MYB involved in organ identity, the NAC involved in leaf senescence, the SBP involved in flower development, and the VOZ involved in pollen maturation.
Digital Expression across developmental stages
The next step was to use available data to study gene expression of the different homologs and across different developmental stages. We clustered the results for both genes and developmental stages (Fig. 6). Two major groups are formed regarding the developmental stages, the dough stage and fruit formation called Group 1, where we do not observe high expression of the genes. Group 2 is sub-divided into two subgroups SG1 subgroup with relative higher expression values, and the SG2 subgroup with even highest expression than SG1. The developmental stages in SG1 subgroup are the germination, stem elongation and the seedling stage, while the SG2 subgroup is the inflorescence formation and anthesis. Specifically, the group composed of ZmTLP2, 4, 5, 6, 7, and 9 displayed higher expression than the second group composed of ZmTLP3, 8, 11, 12 and 14. Finally, the ZmTLP1 has the highest expression and the ZmTLP13 has the lowest expression of all (Fig. 6).
In the process of the gene annotation, we took into consideration the tissues that genes were expressed. We found all genes expressed in tassel, ear, and embryo tissues, while ZmTLP1 and ZmTLP13 are additionally expressed in root tissues. In endosperm tissue, the ZmTLP2, ZmTLP4, 5, and 6 as well the ZmTLP9, 11 and 14 are expressed. In pollen tissues, only ZmTLP1, 3 and 14 are expressed (example gene, TUBBY 11, is shown in Fig. 2b). To verify the expression of the genes we used cDNA from roots, leaves and stems. Here, we confirmed the expression of ZmTLP6.2, ZmTLP14.1, ZmTLP3.1, ZmTLP3.2 and ZmTLP11.1 in stem tissue (Fig. 7).