**1. Introduction**

As a widely distributed ornamental genus around the world, it is difficult to deny that *Rhododendron* has more dimensions of ornamental diversity than other popular woody horticultural plants. In China, *Rhododendron simsii* is the most widely cultivated *Rhododendron* species in landscaping, and numerous cultivars have been obtained through hybridization [1]. *Rhododendron* flower buds generally undergo a period of dormancy between flower bud differentiation and anthesis [2] to cope with the environmental conditions of autumn and winter. Previous studies have shown that *Rhododendron* flower buds are more sensitive to cold than are leaf buds, leaves and stems, and that the cold-tolerance gap between flower tissues and vegetative tissues is more obvious in plants with strong cold tolerance [3]. The critical low temperature that flower buds can tolerate before damage occurs is regarded as an important trait indicator [4]. Therefore, cold tolerance is one of the most important prerequisites for maintaining ornamental characteristics. In addition to temperature, various abiotic stress factors such as drought and high soil salt concentrations, not only affect the overall health of plants but also cause damage to flower buds [5,6]. Gardeners formerly used cross-breeding to obtain varieties with high abiotic stress tolerance. However, this method was less efficient in terms of time and the resulting varieties were often difficult to combine ornamental traits together with abiotic stresses resistance [4]. It is necessary to understand the molecular mechanism related to flower stress resistance to meet the

**Citation:** Guo, Z.; He, L.; Sun, X.; Li, C.; Su, J.; Zhou, H.; Liu, X. Genome-Wide Analysis of the *Rhododendron* AP2/ERF Gene Family: Identification and Expression Profiles in Response to Cold, Salt and Drought Stress. *Plants* **2023**, *12*, 994. https://doi.org/10.3390/ plants12050994

Academic Editors: Aiping Song and Yu Chen

Received: 2 February 2023 Revised: 20 February 2023 Accepted: 20 February 2023 Published: 22 February 2023

**Copyright:** © 2023 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/).

demand. Recent research on the rose APERALA2/Ethylene Responsive Factor (AP2/ERF) gene family showed that the isolated member gene *RcDREB2B* is repressed under drought stress, while the overexpression of *RcDREB2B* was found to promote sensitivity to salt, Abscisic Acid (ABA) and poly (ethylene glycol) [7]. Enhancing abiotic stress tolerance through molecular breeding can enable the barriers of traditional breeding methods to be overcome and can influence the regional distribution of cultivation [4].

The AP2/ERF superfamily is one of the most conserved and important Transcription Factor (TF) families and is mainly found plants [8]. This TF family has various functions for regulating plant biological and physiological processes, including responses to abiotic stress and contributions to developmental processes and plant morphogenesis [9]. AP2/ERF proteins are recognized with AP2 binding domains, and the APE/ERF family can be divided into five subfamilies: AP2, Dehydration-Responsive Element-Binding (DREB) protein, ERF, ABI3/VP1 (RAV) and soloist, according to the features of the domains [8]. Different structural characteristics determine the differences in function among these subfamilies, while the AP2 subfamily members contribute to the regulation of anthesis and development of flower organs [10,11]. The ERF and DREB subfamily members mainly regulate the response toward abiotic stresses such as cold and salt [12,13], and the RAV subfamily members participate in the regulation of plant hormones such as ethylene and brassinolide [14]. The soloist subfamily function requires more research to clarify.

It has been reported in *Arabidopsis thaliana* that exogenously applied abscisic acid, cold, dehydration and high salinity will induce the expression of genes with cis-acting elements sharing a conserved 'A/GCCGAC' core sequence [15–18]. One of the cis-elements, TACC-GACAT, was named the Dehydration-Responsive Element (DRE) and can bind to the TFs named DRE-Binding Protein 1/C-repeat Binding Factor (DREB1/CBF) and DREB2 [19,20]. Overexpression of DREB1/CBF ultimately improves the tolerance to cold, drought and high salinity in *Arabidopsis*, while DREB2 can help counter the stress caused by drought, high salinity and heat shock through a series of expression changes in downstream genes [21–23]. AP2/ERF transcription factors have been widely detected in plants, and their functions in flower physiology and development have been studied, including in *Arabidopsis* [24], tomato [25] and maize [26], while their functions in abiotic stress response have also been studied, mainly in model and crop plants such as rice [27] and wheat [28]. However, in ornamental plants, where research on flower development is more important and prominent, there is no comprehensive understanding of the AP2/ERF transcription factor family as there is in food-crop plants. In flowers, an important reproductive organ, there is still a lack of research on the AP2/ERF gene family and the mechanism toward abiotic stress resistance, and the need for obtaining more bioinformatics data. The achievement of the *Rhododendron* whole-genome sequence provides opportunities to comprehensively study the *Rhododendron* AP2/ERF gene family.

In this study, we identified AP2/ERF genes from the *Rhododendron* genome using bioinformatics methods and obtained complete information such as sequence features, classification, and chromosomal and promoter locations. We generated more understanding of the RsAP2 genes' function through determining the expression patterns of the AP2/ERF genes in different *Rhododendron* flower developmental stages and the expression levels in closed buds under different abiotic stresses, including cold, salt and drought. This study provides a theoretical basis for the further validation of RsAP2 gene function and protein interaction.
