Allele specific expression of Dof genes responding to hormones and abiotic stresses in sugarcane

Dof transcription factors plant-specific and associates with growth and development in plants. We conducted comprehensive and systematic analyses of Dof transcription factors in sugarcane, and identified 29 SsDof transcription factors in sugarcane genome. Those SsDof genes were divided into five groups, with similar gene structures and conserved motifs within the same groups. Segmental duplications are predominant in the evolution of Dof in sugarcane. Cis-element analysis suggested that the functions of SsDofs were involved in growth and development, hormones and abiotic stresses responses in sugarcane. Expression patterns indicated that SsDof7, SsDof23 and SsDof24 had a comparatively high expression in all detected tissues, indicating these genes are crucial in sugarcane growth and development. Moreover, we examined the transcription levels of SsDofs under four plant hormone treatments, SsDof7-3 and SsDof7-4 were down-regulated after ABA treatment, while SsDof7-1 and SsDof7-2 were induced after the same treatment, indicating different alleles may play different roles in response to plant hormones. We also analyzed SsDofs’ expression profiling under four abiotic stresses, SsDof5 and SsDof28 significantly responded to these four stresses, indicating they are associate with abiotic stresses responses. Collectively, our results yielded allele specific expression of Dof genes responding to hormones and abiotic stresses in sugarcane, and their cis-elements could be crucial for sugarcane improvement.

with a conversed target DNA sequence [1]. The C-terminal of Dof transcription factors play important roles in transcription regulation, including interaction with diverse regulatory proteins [2]. The functions of Dof TFs have been identified in many plants. AtDAG1, a Dof transcription factor, was identified to be involved in light-quality response in Arabidopsis. In maize, Dof1 and Dof2 were identified to promote regulation of carbohydrate metabolism [3]. In potato, researches have been confirmed that Dof transcription factors StCDF1 was involved in the regulation of tuber development through restraining the expression of CO1/2 in potato [4]. In rice, over-expression of OsDof12 promoted early flowering [5]. In tomato, over-expression of Dof transcription factors SICDF3 promoted late flowering in transgenic Arabidopsis plants [6]. In Jatropha curcas, JcDof3 was regulated by circadian clock and identified to regulate flowering time [7]. Dof transcription factors were also identified to play important roles in plant hormonal signaling. In barley, HvDof19 was reported to repress the hydrolase gene when the barley aleurone was germinating [8]. OsDof3 was associate with gibberellin-related expression during germination in rice [9,10]. In Arabidopsis, Dof transcription factor OBP1 could regulate gene expression when responding to plant hormones, such as salicylic acid and auxin [11]. In addition, previous studies showed that Dof TFs are involved in abiotic stresses responses. In Arabidopsis, OBP1 was identified to play important roles in regulating the gene expressions responding to the signals of oxidative stresses [11]. In tomato, over-expression of Dof transcription factors SlCDF1 and SlCDF3 could influence the salt and drought responses of transgenic plants in Arabidopsis [6].
Dof transcription factors in different plant species have been studied in past years, such as Arabidopsis [12], rice [10], cucumber [13] and soybean [14]. However, information about Dof genes is lacking in sugarcane (Saccharum spp., Poaceae). Sugarcane is a major crop in producing biofuel and sugar, accounting for about 40% of ethanol production and 80% of sugar production all over the world [15]. The sugarcane (Saccharum spontaneum) genome was sequenced and genomic resources are available for detailed analysis of target genes [16]. We performed a comprehensive and systematic analysis to investigate the Dof genes in sugarcane genome and 29 SsDof genes were identified in sugarcane. These transcription factors were thoroughly analyzed on sequence phylogeny, exon and intron structure, motif patterns, chromosome location, duplication events and cis-element analysis. We examined the expression profiling of SsDofs in various developmental stages and tissues in sugarcane. We also analyzed the transcription levels of SsDofs under different treatments of abiotic stresses and plant hormones.

Plant material and treatments
We used SES208 (Saccharum spontaneum, 2n = 8x = 64) as plant materials in our study. And these sugarcane plants grew in the green house at Fujian Agriculture and Forestry University.
For analyzing transcription levels of SsDof genes in different tissues and stages: root samples were obtained from root in seedling stage (45 days old), including the top of root (below the root hair, root-t), the middle of root (root-m) and the base of the root (root-b). Stem and leaf samples were from 9 months old premature internode (pre-m-stem3, pre-m-stem6 and prem-stem9), 12 months old mature internode (m-stem3, m-stem6 and m-stem9) and leaf (leafb, leaf-m and leaf-u) as previously described [17][18][19].
For analyzing transcription levels of sugarcane Dof genes under four plant hormones: the whole sugarcane seedlings (45 days old) were subjected to four plant hormones (ABA, GA, Auxin and Ethylene, purchased from Solarbio company), the leaves were collected at 24 h after treatments.
For analyzing transcription levels of sugarcane Dof genes in seedling stage and under four treatments by RT-qPCR: tissue samples were obtained from root, stem and leaf in seedling stage (45 days old). As for the cold and heat applications, the sugarcane seedlings were grown at 4˚C and 38˚C (artificial climate chamber from Yiheng company) for 4, 8, 12 and 24 h, respectively. In addition, the whole seedlings were performed with 15% PEG6000 (purchased from Takara company) and 100 mM NaCl (purchased from Takara company) for 4, 8, 12 and 24 h respectively.

Identification of Dof genes in Saccharum spontaneum
We obtained the sequences of Dof genes in Arabidopsis thaliana and Oryza sativa from Arabidopsis genome (http://www.arabidopsis.org/) and rice genome (http://rice.plantbiology.msu. edu/). Then we performed BLASTN to identify all Dof homolog hits in Saccharum spontaneum genome. We collected all non-redundant hits whose values were less than 1E -5 . And we used the PFAM program (http://pfam.sanger.ac.uk/) and SMART program (http://smart.emblheidelberg.de/) to further confirm the existence of Dof domain (PF002701).Then we used the GENSCAN program (http://genes.mit.edu/GENSCAN.html) to verify the sequences identified [20]. We used the ExPASy program (https://web.expasy.org/protparam/) to check the molecular weights (MW) and isoelectric points (PI) of all sequences.

Phylogenetic analysis of SsDof genes in sugarcane
Based on multiple sequence alignments of SsDof and AtDof proteins and all sugarcane Dof genes could be divided into various groups. We performed phylogenetic analysis with MEGA5.0. Sequence of Dof proteins from Arabidopsis and sorghum were obtained from literature [23]. The phylogenetic tree image was enhanced by the Evolview online program (http:// www.evolgenius.info/evolview).

Chromosomal distribution and gene duplication
The genomic and CDS sequences of SsDof genes were obtained from Saccharum spontaneum genome. We checked the gene duplications of SsDof genes by BLAST search in the genome. The chromosomal distribution of SsDof genes was generated by Circos software (http://circos. ca/).

Expression profiling of sugarcane Dof genes by RNA-seq
RNA-seq was carried out using Illumina NovaSeq. We use the S. spontaneum AP85-441 genome as the reference genome to align the reads of SES208. Using Trinity software (https:// github.com/trinityrnaseq/trinityrnaseq/wiki), we counted mappable reads from SES208 and normalized the FPKM values of each sample. Then, we used the TBtools software to generate the heatmap. RPKM value of SsDofs used in this study were shown in S5 Table.

Identification of SsDof genes in sugarcane
The Open Reading Frame length of SsDofs ranged from 504 bp (SsDof15-2) to 2337 bp (SsDof13-4) ( Tables 2 and 3 To explore the distribution of the homologous sequences at each position, we performed the multiple alignment analysis with SsDofs' amino acid sequences. It was indicated that all SsDofs possess a representative DNA binding domain of 52 amino acids that included a single C2/C2 zinc finger structure. (Fig 1).

Motif composition and gene structure of sugarcane Dof gene family
We performed the MEME program to investigate the motif patterns of SsDof proteins. And 25 motifs were checked in SsDofs protein sequences (Fig 3A and 3B). Similar to the results in Arabidopsis [35], soybean [14], cucumber [13] and tomato [36], our results suggested that SsDof genes were highly conserved in sugarcane. The motif1 was the conserved Dof domain and distributed in each SsDof proteins. In addition, the motif patterns of SsDof proteins have similar compositions within the same group. For instance, in group Ⅰ, 10 motifs (1,4,7,8,10,11,15,16,21,25) were the conserved motifs. There were 12 conserved motifs (1,7,8,11,12,13,14,15,16,18,21,24) in group Ⅱ. And group Ⅲ contained the most numbers of motifs, including 17 conserved motifs, while group Ⅳ had only one conserved motif (Dof domain). These results indicated that there would be some similar functions of SsDof genes within the same group.
To investigate the evolution of SsDof genes in sugarcane, we examined the gene structure of SsDof genes. As depicted in Fig 3A and 3C, the number of introns of SsDofs was no more than 5. Thirty-nine (43.8%) alleles and paralogs were intronless, whereas thirty-seven (41.6%) alleles and paralogs contained one intron. In addition, some SsDofs groups showed similar gene structure compositions. For instance, SsDofs in group Ⅲ had the most numbers of introns, in which SsDof13-4 had five introns. SsDofs in group Ⅳ were intronless except SsDof23 including one intron. In groupⅠ, the number of introns of SsDofs various from 0 to 3.

Chromosomal location and duplication of sugarcane Dof genes
SsDofs were unevenly distributed in 27 of the 32 chromosomes of S. spontaneum AP85-441 except chromosome 6C, 6D, 8A, 8C and 8D (Fig 4). Chromosome 1A and 1D contained eight SsDofs followed by seven SsDofs in chromosomes 1B and 1C. There was only one SsDofs in chromosomes 4A, 4D, 5A, 5B, 5C, 7D and 8B. There was no correlation between the number of SsDof genes and the length of sugarcane chromosomes.
The Ks, Ka and Ka/Ks ratio were calculated to investigate the divergence time of the duplication blocks. The duplications of SsDofs in S. spontaneum AP85-441 occurred approximately 0.21 Mya (million years ago) to 15.60 Mya with an average of 1.97 Mya (Table 4). SsDof1, SsDof2, SsDof3, SsDof11, SsDof14, SsDof20, SsDof23, SsDof27 and SsDof28 had undergone purifying selection because their Ka/Ks ratio were lower than 1, whereas SsDof8 had undergone positive selection as its Ka/Ks ratio was higher than 1. These results indicate that different SsDofs were under different selective constraints relating to their functions.

Cis-elements analysis of SsDof genes in sugarcane
We checked the cis-elements of SsDof genes and collected the cis-elements for growth and development, plant hormones and abiotic stresses responses in plants (Fig 5). For plant growth and development, the most frequent cis-elements identified were G-box and Sp1elements, which are related to light responses. The ABRE elements and TGACG motifs and CGTCA motifs were the most frequent elements for plant hormones-related cis-elements. For abiotic stress responses, ARE element included the most numbers of elements (S4 Table).  Allele specific expression of Dof genes responding to hormones and abiotic stresses in sugarcane Additionally, the promoter of SsDof13 contained most cis-elements of MYB binding site motifs. The promoter of SsDof20 contained most ABA responsive cis-elements, whereas SsDof3 contained most MeJA responsive elements and SsDof13 contained most anaerobic induction elements (Fig 5).
The cis-elements of SsDofs' alleles distributed differently (Fig 5). The number of cis-elements for plant hormone responses distributed differently in SsDofs' alleles. For example, the numbers of MeJA-responsive elements were different between alleles of SsDof3, SsDof4, SsDof5, SsDof6, SsDof9, SsDof13 and SsDof25. The numbers of gibberellin response elements were detected differently in alleles of SsDof3, SsDof4, SsDof5 and SsDof26. And the numbers of auxin responsive elements were also detected differently between the alleles of SsDof2, SsDof3, SsDof15 and SsDof28. In addition, the number of cis-elements for plant growth and development distributed differently in some SsDofs' alleles, such as the numbers of light responses elements between alleles of SsDof1, SsDof3, SsDof5, SsDof13, SsDof18, SsDof20 and SsDof27, the numbers of the MYB binding site elements between alleles of SsDof3, SsDof4, SsDof5, SsDof13 and SsDof22.

Expression profiling of SsDof genes
To investigate the expression profiling of SsDofs, we examined the transcription levels of SsDofs in different tissues and stages, including root in seedling stage, stem in premature and mature stage, and leaf in mature stage (Fig 6). Among these SsDofs, SsDof1-2, SsDof26-2 and SsDof13-5 was not expressed in all samples, which may have special temporal expression patterns not examined in our libraries. And forty SsDofs (44.9%) were expressed in all samples. SsDof7, SsDof23 and SsDof24 had a high expression in all detected tissues. The expression of SsDof1-1, SsDof3-2, SsDof3-3, SsDof4-2, SsDof4-3, SsDof11-1, SsDof26-1, SsDof26-3 and SsDof26-4 were only detected in leaves, indicating that they may be involved in leaf development. Additionally, SsDof4-1, SsDof11-2, SsDof11-3, SsDof22-2 and SsDof28-3 only expressed in roots and leaves, indicating that these SsDofs may be associated with leaf and root development.
In order to verify the transcriptome data, we carried out the quantitative real-time PCR experiments. SsDof10, SsDof20 and SsDof23 showed comparatively higher levels of expression in root, while SsDof3, SsDof4, SsDof5, SsDof13, SsDof18, SsDof22, SsDof24 and SsDof28 showed relatively higher levels of expression in leaf (Fig 7). All the 12 SsDofs showed a very low level of expression in stem except SsDof17 and SsDof23. Our results were identical to the expression profiling of SsDof genes detected by RNA-Seq.

Expression profiling of SsDof genes responding to plant hormones
To investigate the expression profiling of SsDof genes responding to plant hormones, we examined their transcription levels under four plant hormones treatments (ABA, GA, Auxin and Ethylene). As shown in Fig 8, SsDof7 and SsDof10 were up-regulated under ABA treatment, but SsDof18 were down-regulated. After GA treatment, SsDof13 and SsDof24 were up-regulated, but SsDof18 were down-regulated. The expression of SsDof10, SsDof13 and SsDof24 increased after IAA treatment, but the transcription levels of SsDof8 and SsDof18 reduced. Under ET treatment, SsDof7 were up-regulated whereas SsDof9 and SsDof18 were down-regulated. Interestingly, after four plant hormones treatment, SsDof10 and SsDof13 were up-regulated whereas SsDof18 were down-regulated.

Expression profiling of SsDof genes responding to abiotic stresses
12 SsDof members were selected from 29 sugarcane SsDof genes to investigate the expression profiling under various abiotic stresses. Then we conducted qRT-PCR experiments to observe their expression patterns after four treatments (4˚C, 38˚C, NaCl, PEG). As shown in Figs 9 and 10, SsDof5 and SsDof28 were obviously responding to all four treatments. The expression of SsDof5, SsDof10, SsDof18 and SsDof28 increased after these four treatments. All 12 SsDof genes were induced after cold treatment whereas SsDof3, SsDof4, SsDof5, SsDof17 and SsDof28 were induced after heat treatment. The transcription levels of SsDof4 and SsDof17 decreased after salt treatment. After different treatments, some SsDof genes showed opposite expression patterns. For example, SsDof17 was obviously up-regulated after cold and heat treatment whereas was down-regulated by salt treatment.

Discussion
Gene expression profiles provide valuable clues for gene function. In Arabidopsis, AtDof5.8 was involved in processes of vascular development [40,41]. SsDof23 is orthologous to AtDof5.8 and had a high expression in roots and stem, indicating that SsDof23 may contain some similar functions in the development of sugarcane vascular tissues. Moreover, SsDof1, SsDof8, SsDof25 and SsDof27 are orthologous to AtDof5.7, which had been confirmed to control the differentiation of guard cells by controlling the transcription levels of genes [42]. Interestingly, the expression profiling of most SsDofs' alleles displayed differently, such as the alleles of SsDof1, SsDof1-4 had a high expression in roots, whereas SsDof1-1 and SsDof1-5 had a low expression in roots. And the promoter regions of SsDof1-4, SsDof1-1 and SsDof1-5 contained different numbers of cis-elements for plant growth and development. These results indicated that allele specific expression of SsDof genes may be associated with cis-elements for plant growth and development.
There are previous studies about Dof genes in response to plant hormones. In potato, StDof genes showed either ABA-independent or ABA-dependent expression profiling [43]. In tobacco, NtBBF1was reported to facilitate the auxin-inducible gene expression [44]. In our study, SsDof10, SsDof13-1, SsDof13-2, SsDof13-3, SsDof13-6 and SsDof24-1 were up-regulated under four plant hormone treatments, whereas SsDof18-2 and SsDof18-3 were down-regulated, indicating these SsDofs may play important roles in response to phytohormones. Interestingly, SsDof7-1 and SsDof7-2 were up-regulated after ABA treatment, while SsDof7-3 and SsDof7-4 were repressed. Meanwhile, SsDof7-3 and SsDof7-4 had more abscisic acid responsive elements than SsDof7-1 or SsDof7-2 in their promoter regions. Our results suggested that allele specific expression of SsDof genes responding to hormones may be associated with cis-elements for plant hormones.
Cis-elements play critical roles in regulating phytohormones and abiotic stresses responses in plants [45,46]. The most cis-elements we have identified are those associated with light responsive, indicating light signals may play critical roles in transcriptional regulation of SsDofs in S. spontaneum AP85-441. Moreover, we also identified numbers of cis-elements about plant hormones and abiotic stresses in promoter regions of SsDofs. Meanwhile, most SsDofs were responsive to phytohormones and abiotic stresses detected by our data. These results suggested that SsDof genes may be involved in responding to phytohormones and abiotic stresses.
Dof genes have been reported to be associate with abiotic stresses responses. In Arabidopsis, the transcription levels of AtDof1.1 was up-regulated for three times under MeJA treatment, damaging the plant tissues [47]. In Chinese cabbage, many BraDof genes were induced obviously after cold, heat, salt and drought stresses. In tomato, SlCDF1-5 was obviously up-regulated after osmotic, cold, heat and salt treatments. Similar to previous researches, many SsDof genes were induced or repressed under cold, heat, salt and drought stresses, indicating that SsDof genes may be involved in responding to abiotic stresses. Interestingly, those SsDof genes induced were always detected about 4 hours after abiotic stresses treatments, indicating SsDofs' expression increased immediately under cold and heat stresses. Under diverse treatments, some SsDof genes presented reverse expression patterns. For instance, SsDof17 was significantly induced by cold and heat treatment, whereas was repressed by salt treatment. Our study demonstrated that SsDof genes may play important roles in responding to various abiotic stresses in sugarcane.
Gene, genome, and segmental duplications are reported to be associate with genetic novelty [48][49][50]. The sugarcane genome was identified to undergo two WGD events after divergence from its closest relative and detailed analysis of the genome showed duplications in other gene families [51][52][53][54][55][56]. The duplications of SsDofs in sugarcane originated from approximately 0.21 Mya to 15.60 Mya, which indicated the duplications of SsDofs in sugarcane took place prior and after the divergence of sugarcane and sorghum. Moreover, we identified forty-nine pairs of duplicated SsDof gene pairs, including forty-six pairs of segmentally duplicated genes and three pairs of tandemly duplicated genes. This result suggested that segmental duplications are predominant in the evolution of SsDof in sugarcane.

Conclusions
We performed a comprehensive and systematic analysis to investigate the Dof genes in sugarcane genome and 29 SsDof genes were identified. Those SsDof genes were divided into five groups, with similar gene structures and motif patterns in the same group. Forty-nine pairs of duplicated SsDof genes were identified in sugarcane chromosomes. The duplications of SsDof genes originated from approximately 0.21 Mya to 15.60 Mya. Cis-element analysis suggested that the functions of SsDofs were involved in growth and development, hormone and abiotic stress responses in sugarcane. Expression patterns indicated that SsDof genes are crucial in sugarcane growth and development. The transcription levels of SsDofs under plant hormone treatments indicated that different alleles may play different roles in response to plant hormones. SsDofs' expression profiling under four abiotic stresses indicated that they are involved in abiotic stress responses in sugarcane. This work provides a foundation for further functional analysis of SsDof genes in sugarcane.
Supporting information S1