The transposase-derived transcription factors FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED-IMPAIRED RESPONSE1 (FAR1) are two best-characterized members from the FAR1-RELATED SEQUENCE (FRS) family, which control phyA signaling in a partially redundant manner (Wang and Deng, 2002). As a type of transcription factors derived from the ancient Mutator-like transposase, FAR1/FHY3s regulate the expression of the downstream genes by binding the FHY3/FAR1-binding site (FBS) cis-acting elements within their promoters in the form of homodimer or heterodimer (Lin and Wang, 2004). FAR1/FHY3s are considered as the founding members of the FRS and FRS-RELATED FACTOR (FRF) gene families, which are conserved among land plants (Ma and Li, 2018).

Light is one of the pivotal environmental signals that regulate plant growth and development. It has been reported that the loss-of-function mutants of fhy1 together with fhy2 and fhy3 were shown in Arabidopsis thaliana (L.) to display an elongated hypocotyl in far-red (FR) light but not in white light (Whitelam et al., 1993), indicating their functions in light response in plants. Understanding the mechanisms by which the light controls the plant development has long been of great interest to plant biologists (Li et al., 2012). Plants can perceive and absorb the light of different quality, duration, and wavelength through different types of photoreceptors, including phytochromes (phys) (Chen et al., 2004; Li et al., 2010, 2011; Chen and Chory, 2011; Ma et al., 2016). In Arabidopsis, there are five phys designated phyA to phyE, encoding different phytochromes, which sense red (R) and FR light, and regulate various growth and development processes, including seed germination, low-quality growth, chlorophyll synthesis, stomatal membrane opening, and flower initiation (Shin et al., 2009; Franklin and Quail, 2010; Li et al., 2010; Wang and Wang, 2015). Phytochrome exists in two photo-interconvertible forms, i.e., a R-light-absorbing Pr form and a FR light-absorbing Pfr form. AtFHY3 and AtFAR1 are essential for the Pfr formation of phyB together with phyD and phyE to maintain a regular expression pattern of ELF4 after the sunset in short days (Siddiqui et al., 2016).

AtFHY3 and AtFAR1 form homodimers or heterodimers to directly bind promoters and activate the transcription of AtFHY1 and AtFHL, which encode critical regulators of phyA translocating into the nucleus under FR light (Ma and Li, 2018). AtFHY3 and AtFAR1 are also involved in ultraviolet-B (UV-B) light signal transduction. In the presence of UV-B, they directly bind the promoter of AtCOP1 and activate its transcription. A recent study in Arabidopsis reported that FHY3, rather than FAR1, functioned in early photomorphogenic UV-B response (Huang et al., 2012). Although UV-B (wavelength ranging from 280 to 315 nm) only represents a small part of sunlight, it exerts a strong influence on the growth and development of plants after reaching the surface of the Earth (Huang et al., 2012). Based on the studies on model plants, AtFHY3 and AtFAR1 are regarded as essential for phytochrome signaling. For example, the loss-of-function of FHY3 and FAR1 exhibited an exaggerated shade avoidance phenotype in Arabidopsis (Liu et al., 2019). It has also been proposed that these two proteins may have divergent roles likely through protein sub-functionalization (Lin et al., 2008). Loss-of-function of either AtFHY3 or AtFAR1 resulted in the loss-of-expression of the target genes (Hudson et al., 2003; Li et al., 2011).

AtFHY3 and AtFAR1 are playing crucial roles in responses to the dark–light transition in mature plants (Ma and Li, 2018). Loss-of-function of FHY3 and FAR1 resulted in an increased reactive oxygen species (ROS) accumulation and sensitivity to oxidative stress in various plant species (Letunic and Bork, 2018). Several previous studies have concluded that FHY3 and FAR1 are partially redundant in function, and FHY3 plays a more critical role than FAR1 in various plant species (Lin et al., 2007, 2008; Ouyang et al., 2011; Huang et al., 2012; Tang et al., 2012, 2013). The systematic biological analysis showed that the FHY3/FAR1-related gene family evolved into one or more dysfunctional MULE transposons in eukaryotic genomes (Feschotte and Pritham, 2007). Protein structure analysis revealed that FHY3 and FAR1 contain three main functional domains, including C2H2 zinc finger domain at N-terminal with DNA-binding ability, putative core transposase domain in middle-terminal regions, and SWIM zinc finger domain at C-terminal with transcriptional activation activity in Arabidopsis (Lin et al., 2007, 2008). FAR1 appears to act at the downstream of the regulation cascade as a link between the signal transduction pathway and the cell cycle in yeast (Chang and Herskowitz, 1990). Previous findings indicated that overexpression of FAR1 in Arabidopsis increased cell size and nuclear localization of FHY3/FAR1 proteins (Hudson et al., 1999; Wang and Deng, 2002; Alberghina et al., 2004), inspiring the idea that AtFHY3/FAR1 might function as Mutator-like transposase-derived transcription factors (Lin et al., 2007). A recent genome-wide analysis suggested that FHY3 has numerous putative targets in Arabidopsis, implying that FHY3 might have broader functions in plant growth and development, most of which are unknown (Tang et al., 2013).

Eucalyptus (Eucalyptus grandis Hill ex Maiden) is a large evergreen tree species grown initially in Australia, Philippines, Papua New Guinea, Indonesia, and Timor. Some E. grandis trees were spread to other areas and then widely planted in short-rotation woodlots worldwide. Eucalyptus oil extracted from its leaves, fruit, buds, and bark has a wide range of antibacterial, antiseptic, antioxidant, anti-inflammatory along with anti-cancer effects. Therefore, it is traditionally used to treat respiratory diseases, common cold, influenza, and sinus congestion (Sebei et al., 2015; Sharifi-Rad et al., 2017). However, abiotic stresses, including salt, cold, and heat stresses, can generate numerous ROS in E. grandis, which is a major threat to the timber accumulation for E. grandis. Among 25 CsFHY3/FAR1 genes in tea, there were 11 upregulated genes and 13 downregulated genes, 1 upregulated gene and 23 downregulated genes, and 9 upregulated genes and 10 downregulated genes under 200 mM NaCl, 4°C, and 40°C stresses, respectively (p < 0.05) (Liu et al., 2021). These suggested that FHY3/FAR1 genes were responsive to abiotic stress in some species. Therefore, further functions on EgFAR1/FHY3 proteins are greatly necessary to be carried out.

At present, our understanding of FAR1/FHY3 protein family is mainly from Arabidopsis, in which 14 FAR1/FHY3 family members have been identified. In the present study, genome-wide identification of FAR1/FHY3 genes from E. grandis was performed using bioinformatic methods. We showed that these genes also responded to salt and temperature stresses, confirming FAR1/FHY3 had broader functions in addition to responding to light. It is envisaged that the findings in this study would not only improve our current understanding on the FAR1/FHY3 proteins in E. grandis and broad woody plant species as a whole but also provide a set of gene tools for their genetic improvement.

Eucalyptus grandis is a fast-growing evergreen tree belonging to the Myrtaceae family with a wide-spreading and relatively thin crown. Usually E. grandis grows 40–55 m high but exceptionally to 75 m (Myers et al., 1996). Eucalyptus grandis is one of the most important commercial trees widely used as the sources of wood, paper materials, and forage. Due to its characteristics of fast-growing and various usages, E. grandis has become a global renewable resource of fiber and energy. Eucalyptus grandis also has strong adaptability, especially to salt and drought stresses, making it an ideal ecological plant species being planted in a wide range of areas (Young, 1983; Lahr et al., 2018; Chen et al., 2020b). Moreover, its metabolites, for example, terpenes acting as defensive substance, are now used as unique pharmaceutical oils. It has been demonstrated that E. grandis has the highest genes diversity for those specialized metabolites (Ma and Li, 2018). In 2014, the genome of E. grandis was sequenced and released (Myburg et al., 2014), which facilitated and accelerated the functional and comparative genomic studies.

Genetic studies have identified two pairs of homologous genes essential for phyA signaling: FAR1/HY3 and FHY1/HL, which mediate the light-dependent nuclear accumulation of phyA (Lin et al., 2007). In model plants Arabidopsis and rice, FHY3/FAR1 has been confirmed to be involved in phytochrome signaling by controlling the phytochrome accumulation through FHY1 (Genoud et al., 2008; Lao et al., 2011). However, the specific biochemical function of FHY3 and FAR1 is still ambiguous and needs to be elucidated (Lin et al., 2007). In this study, genome-wide identification of FAR1/FHY3 gene family was conducted in E. grandis using bioinformatic methods.

Comparing with the protein sizes and molecular weights in Arabidopsis, their large difference in E. grandis (Supplementary Table S2) might suggest new functions different from their homologies in Arabidopsis. Similar to R2R3-MYB (Soler et al., 2015), NAC (Hussey et al., 2015), lignin (Carocha et al., 2015), and FLA (MacMillan et al., 2015) gene families, it was interesting to observe more FAR1/FHY3 members (33) than Arabidopsis (Figure 1) and 21 pairs of duplicated repeats (Figure 5), which is common to find duplicated genes in E. grandis. Moreover, as a Mutator-like transposase-derived transcription factor, maybe it will accelerate this process partly (Hudson et al., 2003; Feschotte and Pritham, 2007; Chae et al., 2021).

It seems that the segmental duplication (Figure 5) has been extended to the members of FAR1/FHY3 genes in E. grandis, and mutations in gene structure (Figure 3) may affect the function of new members (Abdullah et al., 2021; Musavizadeh et al., 2021). Genome-wide identification of FHY3/FAR1 family in other plants has rarely been reported, and the function of FHY3/FAR1 genes in other biological processes other than phytochrome signaling remains to be elucidated (Lin et al., 2007). Recently, several studies indicated multifaceted roles of FHY3/FAR1 in diverse physiological and developmental processes, such as plant architecture and flowering, chloroplast biogenesis and chlorophyll biosynthesis, circadian clock entrainment, UV-B signaling, ABA signaling, ROS homeostasis, and programmed cell death (PCD) (Lin and Wang, 2004; Allen et al., 2006; Li et al., 2011; Ouyang et al., 2011; Huang et al., 2012; Stirnberg et al., 2012; Gao et al., 2013; Tang et al., 2013; Kiseleva et al., 2017).

Studies in Arabidopsis and rice suggested that FHY3/FAR1s control phytochrome accumulation through regulating the expression of FHY1 (Lao et al., 2011). G-box and Box-4 indicated that the FAR1/FHY3 genes in E. grandis might involve in the photoreceptive response (Figure 6). There was a link between photoresponse and response to salt/temperature stress, for example, the EgFAR33 promoter had 3 Box-4s, 3 LTRs, and 5 MYBs (Figure 6) and more importantly, it was upregulated to salt/temperature stress (Figure 8). However, this link was ambiguous and needs more research. Fixed growth of plants forces them to evolve many adapting mechanisms to environmental stimuli, among which abiotic stresses are challenging plants all the time. In the presence of stresses, plants adjust their growth and development for surviving through various pathways, such as cell cycle regulation and hormone regulation. Additionally, the regulation of photosynthesis is another strategy for plants to respond to stresses. Publicly available microarray data have shown a significant expression of FHY3/FAR1 in response to biotic/abiotic stresses, indicating some potential functions of FAR1/FHY3 genes in plants (Wang and Deng, 2002; Allen et al., 2006; Stirnberg et al., 2012). High soil salinity is considered as a major abiotic threat for agricultural productivity in semi-arid or coastal areas (Espinosa-Ruiz et al., 1999). Previous studies showed that more than half of C3H, WRKY, and FAR1 family genes were upregulated in response to salt stress, suggesting that these transcription factor families may participate in salt response and tolerance (Mansouri et al., 2019). In this study, we investigated the expression profiles of representative FAR1/FHY3 genes after salt treatments and showed that FAR1/FHY3s were a group of the early-responsive genes in E. grandis under salt treatment. Interestingly, under 200 mM of salt concentration, the relative expression level of the FAR1/FHY3 gene started to upregulate following the treatment for 12 h (Figure 8), indicating that FAR1/FHY3 genes show a lagged response when plants are treated with a relative stronger salt stress (Figure 9).

Temperature is another critical environmental factor regulating plant growth and development (Penfield, 2008). In the current study, the expression of the most FAR1/FHY3 genes did not change significantly in response to the cold treatment. However, under 40°C heat stress, the expressions of four FAR1/FHY3 genes were significantly increased (Figure 8). This suggested that the FAR1/FHY3 genes might have been involved in the responses to heat shock rather than the cold stress, despite of their lagged response under high-temperature stress. In conclusion, FAR1/FHY3 genes in E. grandis showed a diverse expression in response to abiotic stresses, indicating that they are involved in different cellular processes and cell signaling (Faraji et al., 2021; Heidari et al., 2021).

In E. grandis, abiotic stresses, especially salt and temperature stresses, are increasingly affecting wood productivity. Using bioinformatics methods and expression profile analysis, we identified FAR1/FHY3 genes and showed they were responsive to salt treatment. In addition, EgFAR33 showed great potential for understanding on the response to temperature stress. This study could provide potential to wood yield increase.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

YC and SC conceived this experimental study. JD performed most of the experiments and wrote the manuscript. JS was involved in all the bioinformatics and their visual analysis. WP, WL, and YZ analyzed some of the data. X-RZ made key comments on the revision of the manuscript. X-RZ, YQ, YC, and SC were involved in revising and improving the manuscript. All authors contributed to the article and approved the final version of the manuscript.

This work was supported by National Natural Science Foundation of China (32170380), Fujian Agriculture and Forestry University Forestry Peak Discipline Construction Project (71201800739), and the Science and Technology Program of Fujian Province (2019N5008).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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