*3.6. qRT-PCR Analysis of Expression Patterns of HhMYBs under Temperature and Hormonal Treatments*

Seven selected *HhMYB* genes with relatively large fold changes under the PEG and NaCl treatments were tested for their response to salt and drought stress; high and cold temperatures; and SA, ABA, and MeJA treatments by qRT-PCR analysis. It can be seen from Figure 3 that the expression of these genes underwent significant changes under the different stress treatments. The expression trends of HhMYB75 were essentially opposite under low and high temperature conditions. All the selected *HhMYB*s were responsive to the various hormone treatments but showed differing expression trends, indicating that *HhMYB* genes are differentially deployed in response hormones.

**Figure 3.** Expression pattern of *HhMYB* genes in *H. hamabo* under 500 mM mannitol (Drought), 400 mM NaCl (Salt), 42 ◦C (Heat), 4 ◦C (Cold), 200 μM salicylic acid (SA), 50 μM abscisic acid (ABA), or 1 mM methyl jasmonate (MeJA) treatments. D0, D6, D24, CL0, CL6, and CL24 represent treatments for 0 h, 6 h, and 24 h under drought stress and 0 h, 6 h, and 24 h under salt stress, respectively.

#### **4. Discussion**

MYBs constitute one of the largest transcription factor families in plants, with a conserved MYB–DNA binding domain composed of about 52 amino acids at the Nterminus [23]. MYB transcription factors play a key role in plant development, secondary metabolism, hormone signal transduction, disease resistance, and abiotic stress tolerance [11,24,25]. With the increasing number of sequenced plant genomes, members of the MYB family have been systematically identified in various plant species [17,26]. For example, 197 candidate MYBs were identified in *Arabidopsis*, and a different number of candidate MYBs has also been identified in other species, such as 155 in rice and 245 in *Helianthus annuus* L. [27,28]. However, the MYB gene family in *H. hamabo* has not been reported, and its functions remain unclear. *H. hamabo*, with strong adaptability to saline alkali soil and seawater immersion, can adapt to the land–sea transition zone. Because *H. hamabo* is a good material for studying plant resistance, MYB is an important plant regulator. Genome-wide identification and analysis of MYB transcription factor family in *H. hamabo* were performed for the first time in this study. The results of this study showed that *H. hamabo* has a family of 204 *MYB*s, which may be due to large-scale fragment replication in the genome of *H. hamabo* [2].

Based on a phylogenetic analysis, the 204 candidate HhMYB proteins and AtMYBs were divided into 28 subfamilies. Almost all of these subfamilies contained both HhMYBs and AtMYBs, but in differing proportions. HhMYBs clustered in the same subfamily, in many cases, shared similar and highly conserved profiles of MYB motifs, suggesting that they may be functionally related. Only a few HhMYB proteins located in the same branch contained different motif profiles, indicating that these HhMYB members may have arisen as the result of functional differentiation during *H. hamabo* evolution. Additionally, HhMYB members have introns varying from 0 to 15, which are similar to those found in studies on, for example, *Hedychium coronarium* [29]. The results again indicated complex differentiation during evolution.

As a semi-mangrove plant, *H. hamabo* has a high resistance to saline and alkaline soils and drought and barren environments [8]. Some *Arabidopsis* MYB transcription factors have been reported to be involved in plant resistance to salt and drought stresses [30,31]. *AtMYB60* can influence the drought response by regulating the ABA signal transduction pathway [32]. The wheat *MYB33* was reported to enhance the salt and drought tolerance of transgenic *Arabidopsis* through its restoration of osmotic balance and increase in ROSscavenging capabilities [33]. Here, the analysis of transcriptomic data indicated that several *HhMYB*s were involved in the plant responses to both saline and drought stresses. The expression patterns of the genes were different under drought or saline stress, suggesting that these *HhMYB*s may have regulatory functions in the tolerance to saline and drought stress and deserve further investigation.

Other biotic stresses, such as high and low temperatures, also seriously affect the growth of *H. hamabo*. MYBs have been reported to regulate the response and resistance to temperature stress. Transgenic rice overexpressing *OsMYB3R-2* exhibited an enhanced cold tolerance as well as an increased cell mitotic index [34]. Therefore, the expression levels of *HhMYB*s under temperature stress were also analyzed in this study. The expression trends of *HhMYB75* were essentially opposite under low and high temperature conditions. Similarly, AtMYB104 has been reported to be down-regulated by cold stress but up-regulated under high temperature, while the expression trend of *AtMYB81* displayed the opposite [12].

The involvement of MYBs in plant responses to environmental factors may be mediated by hormones [35]. *AtMYB*s *2*, *13*, *15*, and *101* have been shown to respond to ABA, and the ABA regulation of *AtMYB2* can influence the response to saline stress [36]. Many potato *StMYB* genes were induced under ABA, GA, and IAA treatments [36]. The results in this study showed that a high proportion of *HhMYB*s are responsive to ABA, SA, or MeJa, but with differing levels of intensity and at different time points after hormonal stimulation. This study provides a reference for future research into the role of MYB transcription factors in the response of *H. hamabo* to abiotic stresses.
