**1. Introduction**

Poplar (Populus) has enormous economic and ecological benefits. It has a relatively small genome (485 ± 10 Mb; 2*n* = 38) and is the model forest species for genomic and genetic studies of woody plants owing to the relative ease of experimental manipulation and range of available genetic tools [1,2]. It is characterized by its height, thickness, and rapid growth, but biotic and abiotic stresses have limited its growth [3,4]. Among these stresses, drought is commonly pervasive with the characteristics of repressing stomata, photosynthesis, respiration, altering gene expression, and reducing biomass [5,6]. Drought is destructive and economically damaging for poplar, with important research and economic values [1,2,7,8]. Accordingly, investigating the stress resistance mechanisms of poplar has research and practical implications.

RNA editing primarily occurs in mitochondria and plastids of land plants and plays an important role in transcript maturation by insertions/deletions and conversion editing [9–11]. Conversion editing includes C-to-U, U-to-C, and A-to-I editing. U-to-C and A-to-I editing are likely less than C-to-U, which account for the vast majority of RNA editing events [12,13]. The cis-regulatory region, between nucleotide positions −40 and −5 of upstream of the edited site, interacts with trans-regulatory elements involved in identifying the site and catalyzing the conversion, from cytidine to uridine [14]. The organelle RNA recognition motif-containing protein (ORRM), organelle zinc finger (OZ), protoporphyrinogen IX oxidase (PPO), pentatricopeptide repeat (PPR), and RNA editing factor interacting protein (RIP)/multiple organellar RNA editing factor (MORF) families are key trans-acting elements for RNA editing [11,15,16]. The *ORRM* genes are required for RNA editing; the family contains six genes named *ORRM1* to *ORRM6*. Plastid and mitochondrial editing sites are impaired in almost all *orrm*-mutants [16–20]. OZ1 and PPO1 have also recently been implicated in RNA editing and are all located in plastids [21,22]. PPR proteins include many family members and have been studied extensively [23]. Several PPR functions are in a broad range of events, including developmental and physiological processes and response to various biotic and abiotic stresses [24]. For instance, a PPR96 deficiency in *A. thaliana* is associated with insensitivity to ABA and oxidative stress [23]. Mutations in SLO2 of the E+ subclass of the P-L-S subfamily of PPRs retard leaf emergence, restrict root growth, and result in late flowering, and these parameters are enhanced in the absence of sucrose. Additionally, four RNA editing defects and reduced editing at three sites in *slo2* mutants have been identified [25]. The conversion of nucleotides can not succeed without protein–protein interactions between certain PPRs and MORFs. *(PLS)3PPR* and *LPA66*—two PPR genes—are associated with increased RNA-binding activity based on the presence of *MORF9* binding in *A. thaliana* [26]. MORF proteins interact with specific PPR proteins based on pull down in vivo and yeast two-hybrid assays [15].

MORFs are a small protein family in land plants (nine members in *A. thaliana* and seven members in maize) [27,28]. The name of DAG-like (DAL) gene family in maize was previously adopted based on the first identified member (DAG) of the gene family in *Antirrhinum majus* [27,28]. In *A. thaliana*, MORF proteins were also named RNA editing factor interacting proteins (RIP). There are nine members, defined as MORF1–9, while there are ten RIPs. RIPs or MORFs have been used interchangeably, except for RIP1, which corresponds to MORF8, and RIP8, which has been referred to MORF1. Only one gene, *RIP10* (At1g53260), was specially not defined as belonging to the MORF family [9]. In *A. thaliana*, all MORF proteins have no annotated domains but shared a similarly conserved domain. MORF2 and MORF9 are targeted to plastids, and MORF8 is located in chloroplasts and mitochondria, and the others are located in mitochondria. Some *MORF* genes work together and influence each other during some RNA editing events, i.e., they exhibit homo- and heteromeric interactions. For example, both mitochondrial MORF1 and plastid MORF2 proteins can interact with the dual-targeted MORF8 protein. MORF2 and MORF9 are both required for editing at several sites. The presence or absence of MORF8 influences edited sites targeted by MORF2 and/or MORF9 [27,29].

Several members of the RNA editosome interacted with *MORF* family genes towards their important roles in plant growth, development, and RNA editing efficiency [30,31]. Plant development would be negatively affected without MORF proteins. In rice, owing to impaired chloroplast development, the *wsp1* mutant has the variegated phenotype and reduced chlorophyll content. Further, photosynthetic efficiency, CO<sup>2</sup> gas conductance, and transpiration rate of *wsp1* plants are lower than those of the wild type [32]. In *A. thaliana*, *morf2* and *morf9* mutants show a lack of chlorophyll in leaves, and the T-DNA insertional *rip1* (RIP1 also named as MORF8) mutant demonstrates dwarfism [27,33].

In poplar, functional studies of MORF proteins focused on biotic and abiotic stresses are sparse. In this study, we predicted nine putative *MORF* genes in the *P. trichocarpa* genome. A comprehensive

analyses of the poplar MORF family, including phylogenetic, gene structure, chromosomal distribution, and synteny analyses, were performed. The expression profiles of *PtrMORF* genes under drought were determined using public microarray and quantitative RT-PCR data. Our results provide insight into the fascinating properties and biological functions of *MORF* genes in response to drought stress in poplar. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 3 of 19 In poplar, functional studies of MORF proteins focused on biotic and abiotic stresses are sparse. In this study, we predicted nine putative *MORF* genes in the *P. trichocarpa* genome. A comprehensive analyses of the poplar MORF family, including phylogenetic, gene structure, chromosomal distribution, and synteny analyses, were performed. The expression profiles of *PtrMORF* genes under

> drought were determined using public microarray and quantitative RT-PCR data. Our results provide insight into the fascinating properties and biological functions of *MORF* genes in response
