*2.5. Expression Profiles of Populus MORF Gene*

differences: \* *p* < 0.05; \*\* *p* < 0.01.

*2.5. Expression Profiles of Populus MORF Gene*  We then examined the tissue-specific expression of nine Populus *MORF* genes by qRT-PCR: we evaluated various tissue types, including buds, freshly expanded leaves, expanding young leaves, mature leaves, old leaves, cortex, xylem, and roots. The expression levels of *PtrMORFs* in these tissues were comparable to those in buds. Some *PtrMORF* genes exhibited clear tissue-specific expression (Figure 9). Five *PtrMORF* genes—*PtrMORF1.1*, *PtrMORF8.1*, *PtrMORF2.2*, *PtrMORF3*, and *PtrMORF9*—were higher or weakly expressed in over four tissues significantly. Among them, the latter three genes were highly expressed level in almost all leaves types, and *PtrMORF8.1* and *PtrMORF1.1* had higher expression levels in freshly expanded leaves and old leaves, respectively. All but one gene—*PtrMORF2.1*—had higher expression in xylem. *PtrMORF1.2*, *PtrMORF1.3*, and *PtrMORF8.2* had no significant difference in their expression in leaves and merely higher expressed in xylem compared to buds. Additionally, The *PtrMORF* genes with closest evolutionary relationship had different expression patterns. *PtrMORF1.1*, *PtrMORF1.2*, and *PtrMORF1.3* had the closest homology relationship with *MORF1* of *A. thaliana.* Among them, *PtrMORF1.1* had high expression levels in old leaves, cortex, xylem, and roots, while *PtrMORF1.2* and *PtrMORF1.3* only in xylem. With respect to *PtrMORF2.1* and *PtrMORF2.2*, which were orthologous to *MORF2* of *A. thaliana*, the latter had high expression level in four leaves types and the former exhibited high expression only in old We then examined the tissue-specific expression of nine Populus *MORF* genes by qRT-PCR: we evaluated various tissue types, including buds, freshly expanded leaves, expanding young leaves, mature leaves, old leaves, cortex, xylem, and roots. The expression levels of *PtrMORFs* in these tissues were comparable to those in buds. Some *PtrMORF* genes exhibited clear tissue-specificexpression (Figure 9). Five *PtrMORF* genes—*PtrMORF1.1*, *PtrMORF8.1*, *PtrMORF2.2*, *PtrMORF3*, and *PtrMORF9*—were higher or weakly expressed in over four tissues significantly. Among them, the latterthree genes were highly expressed level in almost all leaves types, and *PtrMORF8.1* and *PtrMORF1.1* had higher expression levels in freshly expanded leaves and old leaves, respectively. All but onegene—*PtrMORF2.1*—had higher expression in xylem. *PtrMORF1.2*, *PtrMORF1.3*, and *PtrMORF8.2* had no significant difference in their expression in leaves and merely higher expressed in xylemcompared to buds. Additionally, The *PtrMORF* genes with closest evolutionary relationship had different expression patterns. *PtrMORF1.1*, *PtrMORF1.2*, and *PtrMORF1.3* had the closest homologyrelationship with *MORF1* of *A. thaliana.* Among them, *PtrMORF1.1* had high expression levels in old leaves, cortex, xylem, and roots, while *PtrMORF1.2* and *PtrMORF1.3* only in xylem. With respectto *PtrMORF2.1* and *PtrMORF2.2*, which were orthologous to *MORF2* of *A. thaliana*, the latter had high expression level in four leaves types and the former exhibited high expression only in old leaves. A similar inconsistency in expression was observed between *PtrMORF8.1* and *PtrMORF8.2*.

leaves. A similar inconsistency in expression was observed between *PtrMORF8.1* and *PtrMORF8.2*.

**Figure 9.** Expression analysis of 9 *MORF* genes in eight representative samples by qRT-PCR. Eight various tissues including buds (B), freshly expanded leaves (FL), expanding young leaves (YL), mature leaves (ML), old leaves (OL), cortex (C), xylem (X), and roots (R). Data were normalized to UBQ gene. The buds were defined as 1 in the figure. The data were presented as the mean ± SE of three separate measurements. Asterisks denote significant differences: \* *p* < 0.05; \*\* *p* < 0.01. **Figure 9.** Expression analysis of 9 *MORF* genes in eight representative samples by qRT-PCR. Eight various tissues including buds (B), freshly expanded leaves (FL), expanding young leaves (YL), mature leaves (ML), old leaves (OL), cortex (C), xylem (X), and roots (R). Data were normalized to UBQ gene. The buds were defined as 1 in the figure. The data were presented as the mean ± SE of three separate measurements. Asterisks denote significant differences: \* *p* < 0.05; \*\* *p* < 0.01.

#### **3. Discussion 3. Discussion**

RNA editing plays an irreplaceable role in plant growth and development and C-to-U RNA editing events occur frequently in vascular plants. Trans-acting factors (RNA editosome) are required to recognize nucleotides to be edited, including OZ1, PPR, ORRM, and others. MORF proteins that interact with these factors have been found in *A. thaliana* and *O. sativa* [22,26,32]*. MORF* genes, which are important subunits of the RNA editosome, play a vital role in the regulation of RNA editing [11]. It is worth noting that the functions of most MORF proteins in response to stress, especially drought, remain unclear in woody plant. In this study, we identified the whole *MORF* gene family in poplar and examined the expression patterns of these genes in different plant tissues and in response to drought. The structure of the MORF family, evolutionary events, transcriptional changes responded to drought, and tissue-specific expression pattern are discussed below. Members of the MORF family have been identified and characterized in many taxa. For example, RNA editing plays an irreplaceable role in plant growth and development and C-to-U RNA editing events occur frequently in vascular plants. Trans-acting factors (RNA editosome) are required to recognize nucleotides to be edited, including OZ1, PPR, ORRM, and others. MORF proteins that interact with these factors have been found in *A. thaliana* and *O. sativa* [22,26,32]. *MORF* genes, which are important subunits of the RNA editosome, play a vital role in the regulation of RNA editing [11]. It is worth noting that the functions of most MORF proteins in response to stress, especially drought, remain unclear in woody plant. In this study, we identified the whole *MORF* gene family in poplar and examined the expression patterns of these genes in different plant tissues and in response to drought. The structure of the MORF family, evolutionary events, transcriptional changes responded to drought, and tissue-specific expression pattern are discussed below.

10 *MORF* genes have been identified in *A. thaliana*, but At1g53260 (RIP10) may exhibit a partial lack of partial functionality owing to incomplete *MORF* box. We focus on nine *MORF* genes in *A. thaliana* in this study [9,27]. In maize, seven putative the DAG-like (*DAL*) genes have also have been identified. It is worth noting that DAL, MORF, and RIP genes are the same [28]**.** We screened the *P. trichocarpa* genome for putative *MORF* genes using a tailor-made HMM file derived from multiple MORF domain alignments. We identified nine *PtrMORF* genes consistent with the findings of previous report [28]. Compared with *MORF* genes in *A. thaliana* and in maize (*Zea mays*), the family has not been extensively studied in poplar, indicating that *P. trichocarpa* MORF proteins might perform functions similar to those preformed by their homologs in herbaceous plants. In our study, to better understand the evolution of the *MORF* gene family in poplar, the Members of the MORF family have been identified and characterized in many taxa. For example, 10 *MORF* genes have been identified in *A. thaliana*, but At1g53260 (RIP10) may exhibit a partial lack of partial functionality owing to incomplete *MORF* box. We focus on nine *MORF* genes in *A. thaliana* in this study [9,27]. In maize, seven putative the DAG-like (*DAL*) genes have also have been identified. It is worth noting that DAL, MORF, and RIP genes are the same [28]. We screened the *P. trichocarpa* genome for putative *MORF* genes using a tailor-made HMM file derived from multiple MORF domain alignments. We identified nine *PtrMORF* genes consistent with the findings of previous report [28]. Compared with *MORF* genes in *A. thaliana* and in maize (*Zea mays*), the family has not been extensively studied in poplar, indicating that *P. trichocarpa* MORF proteins might perform functions similar to those preformed by their homologs in herbaceous plants.

structure, conserved motifs, phylogenetic relationships, and collinearity of *PtrMORF* genes were characterized. Four conserved motifs were located in the MORF domain, suggesting that the MORF domain is conserved among *A. thaliana* and *Populus* proteins. Most of the *PtrMORF* genes exhibited similar numbers of exons. A phylogenetic analysis revealed that *PtrMORF* genes and putative *MORF* genes from other species could be classified into six subgroups. The distribution of genes among the In our study, to better understand the evolution of the *MORF* gene family in poplar, the structure, conserved motifs, phylogenetic relationships, and collinearity of *PtrMORF* genes were characterized. Four conserved motifs were located in the MORF domain, suggesting that the MORF domain is conserved among *A. thaliana* and *Populus* proteins. Most of the *PtrMORF* genes exhibited similar numbers of exons. A phylogenetic analysis revealed that *PtrMORF* genes and putative *MORF* genes

from other species could be classified into six subgroups. The distribution of genes among the subclasses indicated that the expansion of the MORF family occurred before the divergence of the species. Most of *PtrMORF* genes were grouped with *MORF* genes from *A. thaliana* and *P. persica* genomes, indicating a close relationship among *MORF* genes from these species. Gene duplication is a major mechanism underlying the evolution of novel protein functions. We detected 2 (22.2%) *PtrMORF* genes that were tandemly duplicated and seven genes (77.8%) that were segmentally duplicated, implying low tandem and high segmental duplication rates in *PtrMORF* genes. Three homologous pairs of chromosomes included seven of the nine *PtrMORF* genes, with segmental duplication in the poplar genome. For example, homologous chromosomes 8 and 10 both contained one *MORF* gene each; similar findings were obtained for homologous chromosomes 1 and 11, as well as 3 and 4. Two *MORF* genes—Potri.010G007200 and Potri.011G032900—were detected on chromosomes 10 and 11, but their homologous chromosomes 8 and 1 lacked *PtrMORF* genes. Tandem duplication was detected (Table S3). Furthermore, the synteny block including *PtrMORF1.1*, *PtrMORF1.2*, and *PtrMORF1.3* was attributed to multiple copies of the chromosome. These results indicated that some *PtrMORF* genes were possibly generated by gene duplication and segmental duplication events likely served as driving force in *PtrMORF* evolution.

Additionally, synteny maps between two representative species and poplar were constructed to better understand the phylogenetic relationships. Four pairs were detected in *A. thaliana* and none was detected in rice indicating a weak homology relationship between poplar and rice. The ω values for duplicated gene pairs between *PtrMORF* genes and *MORFs* in *A. thaliana* were calculated to explore selective pressures (Tables 1–3). In general, the *MORF* genes in the two plants were under strong purifying selection using the branch model. The ω values for four groups were less than 1, except for group II, *MORF8* (At3G15000) and At1G53260 (RIP10), indicating that purifying selective pressure was strong. Positively selected sites might cause adaptive changes after gene duplication and during the evolution of *MORFs* in *A. thaliana*.

RNA editing could potentially contribute to plant resistance to abiotic tolerance on the basis of previous studies [38,41,42]. And some genes closely related to RNA editing, such as *PPR* genes in *A. thaliana* or rice, their mutants changed morphological characteristics due to environmental forces [32,43]. Additionally, *MORF* genes were interacted with *PPR* genes to establish complex editosomes in plant [26,27]. Therefore, we made an attempt to confirm whether the *PtrMORF* genes responded to stress. The eight *PtrMORF* genes distributed in all groups were upregulated or downregulated significantly under drought treatment. It was indicated that they may respond to drought stress. Additionally, the sensitivity of *PtrMORF* genes responding to drought stress was different. The expressions of *PtrMORF1.2*, *PtrMORF2.2*, and *PtrMORF8.2* changed significantly after nine or even 12 days of drought, while the other five genes had lower or higher expression only in three-day drought restriction. This might implied that the former three genes are less sensitive to drought than the latter five. However, not all the genes, which were responded to drought on day three, had consistent response as drought stress increases. This was similar to the response of rice some *PPR* genes to drought stress [44]. Additionally, five of the six genes from poplar chloroplasts and mitochondria showed obviously higher or lower expression compared with no drought treatment, and the edited site efficiency of these genes were affected when the *MORF* genes were mutated out in *A. thaliana* [27], suggesting that the *PtrMORF* gene family, as important component of RNA editing, might be involved in the response to drought stress. However, many questions were worth exploring: (1) How would RNA editing perform under stress? (2) What were the deeper mechanisms of *MORF* genes, as important editosome members of RNA editing?

The functional divergence of *MORF* genes was speculated depended on their tissue-specific expression. Firstly identified member in *A. majus*, the *DAG* gene is essential for chloroplast development in the leaves and etioplast formation of cotyledons [45]. In *A. thaliana*, the *DAG-like* gene is involved in early chloroplast differentiation [46]. Our study showed that six *PtrMORF* genes are expressed highly in the leaves of black poplar (Populus × *euramericana cv. 'Neva'*), suggesting that *PtrMORF*s might play an important role in chloroplast differentiation and development. Some genes that were highly expressed highly in both the leaves and other tissues, such as the cortex, xylem, and roots, indicating the expanded function of *PtrMORF*s. The expression of three genes, merely higher expressed in xylem compared to buds. There are homologous genes in *A. thaliana*; *MORF1* was located in both mitochondria and *MORF8* in both mitochondria and chloroplasts and possessed an extended C-terminus with unknown function. In previous reports, glycine-rich regions were observed in the C-termini of MORF1 and MORF8 in *A. thaliana* [16]. The genes exhibited glycine-rich regions may play key roles in the biotic and abiotic stresses [47]. In addition, the firstly discovered glycine-rich protein—*GRP-1*—was highly expressed in buds and vascular tissue in the petunia [48]. Taken together, these reports suggested this hypothesis that glycine-rich regions observed in *MORFs* might support them in responding to adversity and be highly expressed in xylem in poplar. Xylem was important for water transportation in plants and *PtrMORF* genes expressed highly in xylem might be closely related to water regulation, making the *MORF* genes likely respond to drought [49]. However, we had to point out that the exact roles within *PtrMORFs* need further experiments. Additionally, there were still living parenchyma cells in the xylem and the activity of parenchyma cells provided a critical metabolic and energetic role in woody stem [49]. They played a major role in editing implying that the three *MORF* genes in poplar may play unlikely similar roles in annual plants and were probably required in other critical functions in perennial woody plants.

Therefore, this research preliminarily provided insight into the roles of *PtrMORF*s in stress response and investigations of functions for these genes required further experimental validation in future studies.

#### **4. Materials and Methods**

#### *4.1. Plant Materials and Treatments*

Black poplar (Populus × *euramericana cv. 'Neva'*) were grown in a greenhouse at 25 ◦C under a 16/8 h light/dark cycle. The 3.5-month-old plants were subjected to drought stress. Twelve plants were encountered water-limited treatment, ranging from 3 to 12 days of drought in which the soil RWC was reduced from 70%; the other three plants were provided abundant water, with a the soil RWC of greater than 30% [50]. Three biological replicates were performed.

Total RNA was isolated from poplar mature leaves (sixth to twelfth) after drought stress. Different organs and tissues of 3.5-month-old plants, including buds, freshly expanded leaves (second to third), expanding young leaves (fourth to fifth), mature leaves (sixth to twelfth), old leaves (leaves below the mature leaves), cortex, xylem (the stem without cortex), and roots, were collected at the same time and immediately immersed in liquid nitrogen.

#### *4.2. Genome-Wide Identification and Sequence Analysis of MORF Genes in P. trichocarpa*

The potential *MORF* genes in *P. trichocarpa* were queried using a local BLASTP search with an E-value threshold of <10−<sup>10</sup> and a bit score of >100 in poplar genome annotation data (Ptrichocarpa\_210\_v3.0.protein.fa.gz downloaded from https://phytozome.jgi.doe.gov/pz/portal. html) based on preexisting MORF/RIP genes, including At1g11430, At1g32580, At1g72530, At2g33430, At2g35240, At3g06790, At3g15000, At4g20020, and At5g44780 from *A. thaliana*, which was then confirmed in previous studies [27,28]. Multiple sequence alignments of PtrMORF proteins and the known MORF proteins were generated using DNAMAN. The alignment results were used to build protein Hidden Markov Models (HMMs) to mine the conserved domain; the file was named morf.hmm and was generated using the hmmbuild program in the HMMER 3.0 package (version 3.1b2). No known motifs in PtrMORF proteins and MORF proteins of other plants were detected by screening the PFAM (http://pfam.sanger.ac.uk/) and INTERPRO (http://www.ebi.ac.uk/interpro/) databases [51,52]. The HMM file was used as a probe to search genome files of representative species downloaded from Phytozome V11.0, including *A. lyrata*, *B. distachyon*, *G. max*, *O. sativa Japonica*,

*P. persica*, and *V. vinifera* [53]. Protein hits with an E-value of <10−<sup>10</sup> and sequence score of "best 1 domain" >100 were collected.

MEME (http://meme.nbcr.net/meme/cgi-bin/meme.cgi) was used to investigate the putative conserved motifs among PtrMORF proteins with the following parameters: length between 15 and 50 aa, maximum number of motifs = 4, and one per sequence. To obtain the intact conserved MORF domain, different limits for the length of each motif were used between 100 and 120 aa [54]. In addition, TargetP, and Wolf PSORT were used to predict the putative organelle localization of PtrMORF proteins [55,56].

#### *4.3. Phylogenetic Analysis*

A multiple sequence alignment of 69 MORF proteins from *P. trichocarpa* and other species including *A. lyrata*, *A. thaliana*, *B. distachyon*, *G. max*, *O. sativa Japonica*, *P. persica*, and *V. vinifera* was generated using the MUSCLE method. A phylogenetic tree was constructed by using the NJ method implemented in MEGA V7.0 [57]. The parameters for tree construction were as follows. Phylogeny test and options: bootstrap (1000 replicates); gaps/missing data: pairwise deletion; model: Dayhoff model; pattern among lineages: same (homogeneous); and rates among sites: uniform rates. Finally, the phylogenetic tree was visualized using itol (http://itol.embl.de/) [58].

#### *4.4. Chromosome Location and Gene Structure Analysis*

Positional information and gene structures of *PtrMORF* genes on chromosomes of *P. trichocarpa* were obtained from the PopGenIE database [59]. The chromosomal locations were displayed with MapDraw program [60]. The numbers and organization of introns and exons and gene structures were drawn and displayed using the online PIECE2 server of GSDraw [61].

#### *4.5. Colinearity of PtrMORF Gene*

The chromosomal locations of *PtrMORF* genes were obtained from PopGenIE. Multiple Collinearity Scan Toolkit (MCScanX) was used to analyze gene duplication events with the default parameters and the colinearity information was showed by Circos [62,63]. Using Dual Systeny Plotter software (https://github.com/CJ-Chen/TBtools), the synteny relationships of orthologous *MORF* genes among poplar, *A. thaliana*, and rice were evaluated [64]. Nonsynonymous (*dN*) and synonymous (*dS*) substitutions, as well as *dN/d<sup>S</sup>* ratio (ω) for duplicated *MORF* genes, were calculated, using branch-specific, site-specific (one, neutral, selection, discrete, beta, and beta & ω > 1), and branch-site (Model A) of codon substitution models, as implemented in PAML version 4 [65–67]. Likelihood ratio tests (LRTs) were used to compare model fits. The sites under positive selection were identified using Bayesian methods [68].

#### *4.6. RT-PCR and qRT-PCR Analyses*

Total RNA was extracted using a Plant RNA EASYspin Plus Kit (Aidlab, Beijing, China) and first-strand cDNA synthesis was performed using approximately 2 µg of RNA using the FastQuant RT Kit (with gDNase) (TIANGEN, Beijing, China) following the manufacturer's protocols. qRT-PCR was conducted on the ABI StepOnePlus Real-Time PCR System (ABI, Foster City, CA, USA) based on the SYBR Green II method. Twenty microliters of cDNA was diluted 1:10 with nuclease-free water. Each reaction contained 10 µL of SYBR Green qPCR Mix (Aidlab), 0.2 µL of ROX Reference Dye (Aidlab), 1 µL of cDNA (corresponding to 10 ng of total RNA), 7.8 µL of nuclease-free water, and 0.25 µM each primer. The thermal profile for qRT-PCR: 3 min at 94 ◦C, 40 cycles of 10 s at 94 ◦C, 20 s at 60 ◦C, and 72 ◦C for 30 s. Primers were designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) [69]. A list of primers used was given (Table S4). Each experiment was performed in three biological replicates. The poplar housekeeping gene, UBQ, was used as an internal control. Relative expression was calculated by the 2−∆∆Ct method [70]. A Student's *t*-test was used to generate every *p*-value for

statistical analyses, and R project was used to identify significant variance (R version 3.5.1, one sample and two sample *t*-test: \* *p* < 0.05; \*\* *p* < 0.01).
