**3. Discussion**

*B. halophila* is a plant with high salt tolerance, so it is important to discover the salt tolerant genes of *B. halophila* for breeding salinity resistant varieties of trees. However, to our best knowledge, there is no report about genes associated with salt tolerance in *B. halophila*. In this study, we analyzed the transcriptomic data from the leaves of wild type *B. halophila* plants and plants with salt treatment. As a result, approximately 460 M raw reads were generated and were further assembled into 117,091 unigenes, among these unigenes, 64551 unigenes (55%) were annotated with gene descriptions, while the other 45% were unknown. This is the first report of transcriptome data from *B. halophila*. This transcriptome data provides an important genus resource for insight into the molecular mechanism of salt tolerance and facilitates discovery of novel genes responsive to salt stress in *B. halophila.*

In plants, salt stress responsive mechanisms are very complicated, which involve a complex interaction of physiological processes, metabolic pathways, and regulation at the molecular and cellular levels. Although plant response to salt stress has been extensively studied at different levels, the mechanisms underlying salinity tolerance are far from being completely understood. In addition, salt stress responsive mechanisms in different plants are also different. At present, the main mechanisms for which plants respond to salt stress include ion homeostasis and compartmentalization, ion transport and uptake, biosynthesis of osmoprotectants and compatible solutes, activation of antioxidant enzyme, and synthesis of antioxidant compounds [4–7,11]. In this study, 168 up-regulated genes and 351 down-regulated genes were identified in *B. halophila* under salt stress, respectively. These DEGs include dehydrin proteins, sodium/hydrogen exchanger, potassium transporter, sarcoplasmic/endoplasmic reticulum calcium ATPase, Ca2+ antiporter/cation exchanger, Nodulin MtN21/EamA-like transporter, heat shock protein, phosphoenolpyruvatecarboxykinase, NADH dehydrogenase, highly ABA-induced PP2C gene, homeobox-leucine zipper protein, phosphoglycerate kinase, WRKY transcription factor, AP2/ERF and B3 domain-containing transcription factor, flavonoid 30 ,50 -hydroxylase, which is consistent with the other plants that are reported to be responsive to salt stress [20–24].

The analysis of GO enrichments suggested that the 519 DEGs response to salt stress was mainly involved in plant-type cell wall organization biological process, plant-type cell wall organization or biogenesis biological process, cell wall cellular component and structural constituent of cell wall molecular function. KEGG pathway enrichment results showed that the top-four enriched pathways for DEGs was 'Fatty acid elongation', 'Ribosome', 'Sphingolipid metabolism', and 'Flavonoid biosynthesis'. The expression patterns of sixteen of these DEGs were analyzed by qRT-PCR to verify the RNA-seq results. It revealed that the qRT-PCR results were consistent with RNA-seq data.

Based on the functional annotations of the 519 DEGs and the physiological evidence of *B. halophila* in response to salt stress [25], the possible mechanism of salt tolerance in the leaves of *B. halophila* was summarized in Figure 7. The possible salt tolerance mechanism is coordinately linked with ion homeostasis, osmotic protection, antioxidant regulation, ABA signal pathway, transcription factors and chaperons. When the plant is treatment with 200mM NaCl, multiple signal pathways are activated to cope with salt stress such as the SOS pathway, antioxidant pathway and ABA signal pathway and so on. Meanwhile, the osmoprotectants such as proline and polyols were accumulated to protect the cell. In addition, the transcription factors (WRKY, ERF, ZIP and AHL) and (LEAs, HSPs and AQPs) were activated to regulate the genes involved in the above pathways [2–6]. Overall, the salt tolerance mechanism in *B. halophila* is a complex network that involved the interactions at multiple levels. This information will be useful in elucidating the salt tolerance mechanisms in *B. halophila.*

*Int. J. Mol. Sci.* **2018**, *19*, x 9 of 13

**Figure 7.** The possible mechanism of salt tolerance in the leaves of *B. halophila.* **Figure 7.** The possible mechanism of salt tolerance in the leaves of *B. halophila.*

In the present study, we observed that one dehydrin (DHNs) which is the most down-regulated gene among these DEGs and two other dehydrins showed a distinct salt responsive expression, suggesting that these dehydrin proteins may play different roles in response to salt stress in *B. halophila*. Dehydrins, also known as group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response to abiotic stresses [26–28]. Their expression is often induced under salinity, dehydration, cold and frost stress. Dehydrins are divided into five structural subgroups: Kn, SKn, KnS,YxKn and YxSKn [28]. The three dehydrins protein features of *B. halophila* were all SK3 subclass. It has been shown that SK3 dehydrins play an important protective role in plant stress tolerance, including drought, cold, and salinity [27]. For example, the expression of the durum wheat DHN-5 in A. thaliana led to an increase in salt and osmotic stress tolerance [28]. Rab16A in salttolerant Indica rice variety Pokkali can enhance tolerance to drought and salt stress in tobacco plants [29]. Similarly, overexpression of the wheat dehydrin PMA80 (as well as the LEAI protein PMA1959) enhances rice tolerance to drought and salt stress [30]. Although experimental evidence suggests that dehydrins have diverse roles (membrane protection, cryoprotection of enzymes, and protection from reactive oxygen species) in response to stresses [27–31], further efforts are still needed to precisely confirm the roles of these dehydrins and explore the regulatory mechanism underlying these functions in plant adaptive response to abiotic stresses. In the present study, we observed that one dehydrin (DHNs) which is the most down-regulated gene among these DEGs and two other dehydrins showed a distinct salt responsive expression, suggesting that these dehydrin proteins may play different roles in response to salt stress in *B. halophila*. Dehydrins, also known as group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response to abiotic stresses [26–28]. Their expression is often induced under salinity, dehydration, cold and frost stress. Dehydrins are divided into five structural subgroups: Kn, SKn, KnS, YxKn and YxSKn [28]. The three dehydrins protein features of *B. halophila* were all SK3 subclass. It has been shown that SK3 dehydrins play an important protective role in plant stress tolerance, including drought, cold, and salinity [27]. For example, the expression of the durum wheat DHN-5 in A. thaliana led to an increase in salt and osmotic stress tolerance [28]. Rab16A in salt-tolerant Indica rice variety Pokkali can enhance tolerance to drought and salt stress in tobacco plants [29]. Similarly, overexpression of the wheat dehydrin PMA80 (as well as the LEAI protein PMA1959) enhances rice tolerance to drought and salt stress [30]. Although experimental evidence suggests that dehydrins have diverse roles (membrane protection, cryoprotection of enzymes, and protection from reactive oxygen species) in response to stresses [27–31], further efforts are still needed to precisely confirm the roles of these dehydrins and explore the regulatory mechanism underlying these functions in plant adaptive response to abiotic stresses.

In addition, our results indicated that the transcription factor AT-Hook Motif Nuclear Localized gene (AHL) was the most up-regulated gene in leaves after salt stress, implying that it might play an important role in response to salt stress in *B. halophila*. Previous studies showed that the AHL genes regulate diverse aspects of growth and development in plants. Such as the homeostasis of phytohormones [32], and defense responses [33–40]. However, there is no report about the function of AHL genes associated with salt stress. Further studies are still needed to understand the function of AHL genes in salt stress. In addition, our results indicated that the transcription factor AT-Hook Motif Nuclear Localized gene (AHL) was the most up-regulated gene in leaves after salt stress, implying that it might play an important role in response to salt stress in *B. halophila*. Previous studies showed that the AHL genes regulate diverse aspects of growth and development in plants. Such as the homeostasis of phytohormones [32], and defense responses [33–40]. However, there is no report about the function of AHL genes associated with salt stress. Further studies are still needed to understand the function of AHL genes in salt stress.

Therefore, our results provide a list of candidate genes for further investigation to determine whether they have a role in allowing *B. halophila* to tolerate high salt levels, and may be helpful in the

understanding of the molecular mechanisms of salt stress response in *B. halophila*.

**4. Materials and Methods**

Therefore, our results provide a list of candidate genes for further investigation to determine whether they have a role in allowing *B. halophila* to tolerate high salt levels, and may be helpful in the understanding of the molecular mechanisms of salt stress response in *B. halophila*.
