*3.1. Identification, Structural and Phylogenetic Analysis, and Evolutionary Characterization of NnDofs*

Assessing the structure and function of transcription factors facilitates the analysis of species-specific gene regulatory networks [41]. *Dof* genes are plant-specific transcription factors with important roles in plant growth and development [17]. Other functions of most *Dof* genes remain to be explored. *Dof* genes have been identified in Arabidopsis (36), rice (30), maize (46), tomato (34), and chrysanthemum (20) species. A total of 29 *Dof* genes were identified in the lotus genome, which is close to the number of *Dof* genes in rice and tomato. Systematic classification has important implications for the analysis of *NnDofs*. Systematic classification has important implications for the analysis of *NnDofs*. The Arabidopsis Dof proteins were incorporated with NnDof proteins to construct a phylogenetic tree, and *NnDofs* were classified into six of seven subfamilies. Interestingly, none of the *NnDofs* were classified into group I, while *AtDOF4.2* and its close homolog *AtDOF4.4*, which are classified in group I, are associated with regulation related to the regulation of branching meristem seed coat formation in Arabidopsis [42]. In addition, overexpression and RNAimediated silencing of *AtDOF4.2* have opposite effects on the expression levels of flavonoid biosynthesis-related genes and flavonoid accumulation [43].

Gene structure and motif distribution can be used as supporting evidence for evolutionary relationships between species or genes [44,45], while generally, members of the same subfamily have similar exon/intron structure and number distribution [19]. However, the distribution of the number of introns in the taxonomic subgroups of lotus did not follow this pattern exactly. Multiple sequence alignment was used to compare the amino acid sequences in the *Dof* structural domain of 29 different species of lotus. It was found that the *NnDofs* structural domain sequences were very similar, that they all had the typical CX2CX21CX2C pattern, and that they all had nuclear localization signals (NLS).

Gene duplication is an essential mechanism for generating new evolutionary templates in eukaryotes [46]. In the evolution of angiosperm genomes, genomic duplications have occurred extensively, including whole-genome duplications (WGDs) and segmental duplications [47,48]. Analysis of gene duplications can help us better understand how genes and organisms evolved over time [49]. Ten pairs of segmental duplications and two sets of tandem duplicated *NnDofs* were identified in the lotus genome. Most of the segmental duplicated *NnDofs* had high sequence similarity, while the Ka/Ks of the duplicated gene pairs were less than one. Indicating that all duplicated pairs of *NnDofs* were negatively selected during the evolutionary process, suggesting that to some extent, segmental duplications may be the main amplification mode of the *NnDof* gene family. To some extent, genome duplication may be the main amplification mode of the *NnDof* gene family. In a homozygous analysis of *NnDofs* in lotus and three representative plants, it was found that some *NnDof* genes, such as *NnDof1* and *NnDof10*, were linked to at least three homozygous gene pairs. It is thought that these *NnDofs* may be vital to the generation of the *NnDof* gene family.

## *3.2. Expression Profiling of NnDofs*

*NnDofs* exhibit tissue-specific expression patterns, confirming previous research on *Dof* genes in other species. For instance, *NnDof2*, *NnDof5*, *NnDof13*, and *NnDof20* were highly expressed in the seed coat and rhizome (mixed stage), root and mature receptacle, mature stamen, and petiole and leaf, respectively. In addition, except for *NnDof16*, *NnDof19*, and *NnDof28*, all other *NnDofs* showed lower expression levels in the cotyledon. *Dof5.6*/*HCA2* has been reported to positively regulate the formation of interfascicular cambium during vascular tissue development in Arabidopsis [50]. *AtDof2.4*/*AtDof5.8* may be essential in the primary but distinct processes of vasculature formation [51]. Arabidopsis root hair development is ABA-dependent inhibited, attributed to *OBP4*-mediated transcriptional regulation of *RSL2* [52]. Dof genes are definitely widely involved in plant tissue differentiation and development, which explains several *NnDofs*' differential expression in rhizome (mixed stage), rhizome internode, rhizome elongation zone, and other tissues. In addition, Dof has distinct expression patterns in different tissues and developmental processes in plants. In pepper, *CaDof18* was preferentially expressed in the early stage of flower [14]. Ethylene induces specific increased expression of several *MaDofs* in bananas during fruit ripening [53]. More than half of the A and B1 Dof group members in *Brassica napus* were more highly expressed in the stems and young roots, respectively [54]. *CsDof33* was highly expressed in the terminal buds of tea plants, whereas the expression in young leaves was reversed [55].

Cis-acting elements are essential in gene expression [56], and gene promoter investigation is crucial to understanding the general control of gene expression in plants [57]. In this study, a large number of elements related to light response and meristematic tissue expression were found in the promoter region of *NnDofs*, suggesting that these *NnDofs* may be involved in light signaling pathways or meristematic tissue development. Various hormone responsive elements (ABA, GA, and MeJA response elements) and abiotic stress responsive elements are predicted.

Various investigations into the response of lotus to abiotic stresses have been published. Isolation of bZIP TFs from salt-tolerant lotus root tips enhances the adaptation of transgenic tobacco plants under salt stress [58]. The *NnCIPK6* gene was highly expressed under NaCl treatment in lotus resistant cultivars and was successfully cloned [59]. *NuSTP5*, a monosaccharide transporter family gene, produces stress responses to NaCl, drought, and cold stress [60]. All *NnWRKYs* responded to at least one of SA, JA, and submergence treatments, suggesting that they are extensively involved in abiotic stress [61]. However, studies on the lotus Dof TFs have not been reported yet. According to qRT-PCR analysis, the expression pattern of *NnDofs* was dramatically changed after salt treatment. Under salt stress, the expression of most *NnDofs* was significantly upregulated within 1 h but then progressively decreased after 4 h. It is proposed that several specific *NnDofs* may be engaged in response to salt stress in lotus. Published studies have shown that *Dof* TFs are involved in salt stress resistance of plants through various physiological pathways. Overexpression of *GhDof1* in cotton resulted in a substantial improvement throughout salinity tolerance in wild-type plants, according to prior research [32]. Several *CaDof* genes in pepper were determined as being particularly sensitive to salt stress [14]. The expression of the *ZmDof* gene in maize seedlings was dramatically elevated in those that had been exposed to salt [62]. Salt stress inhibits the transcription of *OsDof15*, which regulates ethylene generation and limits primary root growth in rice by direct contact with the *OsACS1* promoter [34]. *SlDof22* inhibition significantly reduces the expression of the *SlSOS1* gene in tomato, resulting in lower tolerance to salt stress [35]. Under salt conditions, tomato plants that overexpressed the *CDF3* gene maintained growth and boosted yield [22].
