**1. Introduction**

Soil salinization is a serious and growing global problem [1], and salinized areas are growing at a rate of 10% per year. Soil salinity is a severe abiotic stress that affects plant seed germination, growth and development, and reproductive development by causing oxidative stress, ionic toxicity, osmotic stress, and metabolic disturbances in plants [2,3]. Several investigations on several plant species have highlighted the complicated and crucial role of transcription factors in abiotic stress reduction [4]. The dynamic coordination of salt stress-responsive transcription factors in the interaction pathway, as well as their unique integration into the stress adaption cellular network, will serve as a stepping stone for plant tolerance to environmental stresses [5].

Transcription factors (TFs), also called trans-acting factors, interact with cis-acting elements in a particular genetic promoter region to regulate gene transcription and ensure target gene expression at a specific time, place, and intensity [6,7]. Typical transcription factors contain functional regions such as the DNA binding domain, nuclear localization signal region, oligomerization site, and transcription activation domain [8,9]. Transcription factors widely regulate plant growth and development and deeply engage in biotic and abiotic stress responses.

Studies have shown that Dof (DNA binding with one finger) proteins appear to be unique to plants. The first Dof transcription factor identified was *ZmDof* from maize [10].

**Citation:** Cao, X.; Wan, W.; Mao, H.; Yin, D.; Deng, X.; Yan, H.; Ren, L. Genome-Wide Identification and Expression Analysis of Dof Transcription Factors in Lotus (*Nelumbo nucifera* Gaertn.). *Plants* **2022**, *11*, 2057. https://doi.org/ 10.3390/plants11152057

Academic Editors: Aiping Song and Yu Chen

Received: 14 June 2022 Accepted: 3 August 2022 Published: 6 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

With the development of bioinformatics technology, more Dof proteins have been found in the genomes of different plant species, such as *Solanum lycopersicum* [11], *Manihot esculenta* [12], *Prunus persica* [13], and *Capsicum annuum* L. [14]. Dof TFs usually consist of 200–400 amino acids with highly conserved DNA-binding domains at their N-termini, transcriptional regulatory domains at their C-termini, and nuclear localization signals [10,15]. The highly conserved domain consisting of 50–52 amino acids at the N-terminus, containing a C2-C2 zinc finger domain is composed of CX2CX21CX2C. Different transcriptional regulatory domains at the C-terminus indicate the diversity of Dof protein functions [16]. The *Dof* family was divided into seven sub-populations by Yanagisawa [17]. Several researchers collected Arabidopsis and rice *Dof* genes and classified them into four subfamilies: Aa, Bb, Cc, and Dd [18]. The 116 *Dof* genes from seven species were more comprehensively classified by Moreno-Risueno into seven subgroups, A–G [19]. *Dof* proteins play multiple roles in different biological processes, including growth and development [20,21], flowering regulation [22,23], carbon and nitrogen metabolism [24], hormone response [25], and abiotic stress [26,27] in various plant species. Overexpression of *OBP4* in Arabidopsis promotes cell proliferation in the differentiation zone and induces callus formation [28]. *ZmDof3* controls starch accumulation and aleurone development in maize endosperm by binding to the *Dof* core element promoters of *Du1* and *Su2* [29]. In addition, *ZmDof36* is important in regulating starch synthesis. Its overexpression can increase starch content and reduce soluble sugars and reduce sugars [30]. Cycling Dof factor 2 (*CDF2*) leads to photoperiod-insensitivity and delayed flowering in Arabidopsis by reducing CO mRNA levels [31].

RNA-Seq data showed that most *TaDof* genes respond to heat and PEG-induced drought stress in wheat [26]. Overexpression of *GhDof1* could notably enhance tolerance to salt and cold stresses by increasing proline content during the seedling stage [32]. Some *ClDof* genes showed significantly different expressions under salt stress, suggesting that they may contribute to salt stress adaptation in watermelon [33]. *OsDof15* coordinates the regulation of salt and ethylene, inhibiting primary root growth by affecting cell proliferation in the root apical meristem [34]. *SlDof22* can be combined with the promoter of the *SlSOS1* gene, and inhibiting *SlDof22* by significantly downregulating the *SlSOS1* gene leads to reduced tolerance to salt stress [35].

Sacred lotus (*Nelumbo nucifera* Gaertn.), which has been grown in the Far East for 5000–7000 years, is a large aquatic plant with significant ecological, scenic, and economic value [36,37]. Lotus cultivars are categorized depending on their usage and morphological characteristics: rhizome lotus, seed lotus, and ornamental lotus [38]. Lotus has high ornamental value throughout the growing period, with large flowers, various petal types, gorgeous color, green leaves, tall and straight habit, and remains attractive even during the dry leaf period. Because of its ornamental value, lotus is considered a theme plant in waterscape garden layouts. In addition, lotus has high economic value and medicinal value [39]. Lotus tea is traditionally used to clear away "summer heat", that is, relieve symptoms of heat injury, and lotus seed is rich in phospholipids, alkaloids, and flavonoids, which in Chinese traditional medicine are used to clear the heart, nourish the mind, and tonify the spleen and kidney [40].

Although salinity stress causes certain harm to the growth and development of lotus, there are few reports on the salt tolerance of ornamental lotus. Furthermore, Dof TFs have been found to be resistant to salt stress in many different plant species, but the *Dof* gene has not been identified in lotus. In this study, we identified and characterized 29 *Dof* family genes in lotus. They were unevenly distributed on the seven chromosomes and divided into six groups based on phylogenetic analysis. Its physicochemical properties, gene structure, conserved motifs, and cis-acting elements upstream of the gene were also analyzed. Tissuespecific expression of *NnDofs* and gene response to salt treatment were investigated using RNA-seq data and qRT-PCR. We predicted possible interacting proteins and regulatory networks of *NnDofs* related to these stress responses. The results provide a reference for

further functional research of lotus *Dof TFs*, and they can be used as genetic resources to make lotus and other crops more tolerant of salt through molecular genetic breeding.
