PyuARF16/33 Are Involved in the Regulation of Lignin Synthesis and Rapid Growth in Populus yunnanensis

(1) Background: Lignin is a unique component of the secondary cell wall, which provides structural support for perennial woody plants. ARFs are the core factors of the auxin-signaling pathway, which plays an important role in promoting plant growth, but the specific relationship between auxin response factors (ARFs) and lignin has not been fully elucidated with regard to rapid plant growth in forest trees. (2) Objectives: This study aimed to investigate the relationship between ARFs and lignin with regard to rapid plant growth in forest trees. (3) Methods: We used bioinformatics analysis to investigate the PyuARF family, find genes homologous to ARF6 and ARF8 in Populus yunnanensis, and explore the changes in gene expression and lignin content under light treatment. (4) Results: We identified and characterized 35 PyuARFs based on chromosome-level genome data from P. yunnanensis. In total, we identified 92 ARF genes in P. yunnanensis, Arabidopsis thaliana, and Populus trichocarpa, which were subsequently divided into three subgroups based on phylogenetic analysis and classified the conserved exon–intron structures and motif compositions of the ARFs into the same subgroups. Collinearity analysis suggested that segmental duplication and whole-genome duplication events were majorly responsible for the expansion of the PyuARF family, and the analysis of Ka/Ks indicated that the majority of the duplicated PyuARFs underwent purifying selection. The analysis of cis-acting elements showed that PyuARFs were sensitive to light, plant hormones, and stress. We analyzed the tissue-specific transcription profiles of PyuARFs with transcriptional activation function and the transcription profiles of PyuARFs with high expression under light in the stem. We also measured the lignin content under light treatment. The data showed that the lignin content was lower, and the gene transcription profiles were more limited under red light than under white light on days 1, 7, and 14 of the light treatments. The results suggest that PyuARF16/33 may be involved in the regulation of lignin synthesis, thereby promoting the rapid growth of P. yunnanensis. (5) Conclusions: Collectively, this study firstly reports that PyuARF16/33 may be involved in the regulation of lignin synthesis and in promoting the rapid growth in P. yunnanensis.


Introduction
Rapid growth is a complex quantitative trait controlled by external signals and endogenous signals, such as light [1]. Tree height and stem diameter are the most important traits associated with plants' rapid growth [2]. The tree diameter is mainly determined by the secondary cell wall containing lignin, cellulose and hemicellulose and composed of vessels and fibers [3]. Lignin is the main structural component of plants' secondary cell wall [4]. Although the lignin biosynthesis pathway was studied previously in woody

Plant Materials and Treatment Conditions
The plant material was grown for one year in a greenhouse (Southwest Forestry University, Kunming, China). We obtained cuttings of about 15 cm and preserved the plants in the soil. The cuttings with 5-6 leaves were used in experiments performed in March 2022. The cuttings with uniform growth were placed in a constant-temperature incubator (temperature: 25 • C, humidity: 70%, light intensity: 90 µmol·m −2 ·s −1 , photoperiod: 12 h light/12 h dark) for cultivation under red and white light (Telipu, Beijing Zhongyi Boteng Technology Company, power: 8 W, radiation intensity: 68 µW/cm 2 , wavelength: 620-760 nm, 380-760 nm) from lamps placed 35 ± 1 cm above the cuttings. For the quantification of gene expression by quantitative reverse transcriptase-PCR (RT-qPCR) and the measurement of lignin content, we collected the cuttings of P. yunnanensis after 0, 1, 7, and 14 days under red and white irradiation. We obtained samples from point A (1 cm from stem tip) to point B (1 cm from the base of the stem) of the stem and tissue samples without treatment of the roots, RAM (root apical meristem), stem, SAM (shoot apical meristem), young leaves (1st to 3rd leaves) and old leaves (7th to 9th leaves) [16], preparing three biological replicates for each sample. All the plant samples were frozen in liquid nitrogen and stored at −80 • C.

Identification and Sequence Analysis of ARFs in P. yunnanensis
The genome of P. yunnanensis was obtained from our laboratory group (Southwest Forestry University, Kunming, China). The hidden Markov model (HMM) file of the ARF family was extracted from the Pfam database (http://pfam.wustl.edu, accessed on 5 December 2021) [16], and SPDE was used to compare data from the protein database of P. yunnanensis (accessed on 5 December 2021) [17]. Then, the protein sequences of the ARF gene family members from A. thaliana and P. trichocarpa were obtained from the PlantTFDB database (http://planttfdb.gao-lab.org/, accessed on 5 December 2021) [18]. Based on the genome and GFF files of P. yunnanensis, we used TBtools to extract the proteome files of P. yunnanensis [19]. With Blastp, we compared the protein sequences of the ARF gene family members from A. thaliana and P. trichocarpa with those in the protein database of P. yunnanensis obtained by SPDE (accessed on 6 December 2021). Using the NCBI database (https://blast.ncbi.nlm.nih.gov/, accessed on 6 December 2021), we compared the data with those from the Uniprot protein database, to eliminate redundancy and predict the sequences of the ARF gene family [20]. We used SMART (http://smart. embl-heidelberg.de, accessed on 7 December 2021) and NCBI (CD-Search) (https://www. ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 7 December 2021) to identify protein domains in the ARF family members. Finally, the existence of the ARF gene family in P. yunnanensis genome was determined. Using ProtParam (http://web.expasy.org/ protparam/, accessed on 7 December 2021) [21], the analysis the ARF genes provided other information, including the number of amino acids, the molecular weight, and the isoelectric point of the ARF proteins [22].

Comparative Phylogenetic Analysis of ARFs
Multiple sequence alignments of the ARF protein sequences from P. trichocarpa, A. thaliana, and P. yunnanensis were performed using molecular evolutionary genetics analysis (MEGA) version.7 0 [23]. We used the Maximum Likelihood (ML) method to construct the evolutionary tree. Bootstrap was set to 1000 [24]. Subsequently, using the Interactive Tree of Life (iTOL) (https://pubmlst.org/, accessed on 8 December 2021), we visualized the ML phylogenetic tree [25]. Lastly, using the Tbtools software, we constructed a combination of phylogenetic tree, conserved domains, gene structures, and conserved motifs of PyuARFs.

Localization and Gene Duplication of ARFs
To map the physical positions of the identified ARF genes to the 16 chromosomes of the P. yunnanensis genom, we used the Tbtools software. The orthologous ARF genes were included in a local database using DIAMOND [26]. The P. trichocarpa and A. thaliana genomes (version = 3.0) were derived from the NCBI database. Using the Multiple Collinearity Scan toolkit (MCScanX) [27], we analyzed the gene duplication events with default parameters. To estimate the Ks (synonymous substitution rate) and Ka (nonsynonymous substitution rate) of collinearity pairs of the ARF gene family members in P. yunnanensis, we used the Tbtools software.

Prediction of Cis-regulatory Elements in the Promoters of PyuARFs
The promoters' upstream region (~2000 bp) sequences of PyuARFs were extracted from the genomic DNA sequence of P. yunnanensis and, subsequently, were submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 9 December 2021) [28] for cis-acting element prediction. The results were analyzed by TBtools.

RNA Isolation and cDNA Synthesis
According to the relative manual, total RNA was isolated using the E.Z.N.A @ Plant RNA kit (Omega Bio-tek Inc., Norcross, GA, USA). We detected the quality and purity of RNA using K5800C (KAIAO, Beijing, China). According to the manufacturer's protocols, 0.5 ug RNA of each sample was used for 1st strand cDNA synthesis using Hifair ® III Reverse Transcriptase (YEASEN, Shanghai, China). Subsequently, the cDNA was diluted 7-fold for RT-qPCR analysis.

Quantitative Real-Time PCR for the Evaluation of Gene Transcription Profiles
We determined the tissue-specific transcription profiles of PyuARFs and the transcription profiles of PyuARFs in the stems under light. We designed the primers for real-time PCR based on the CDS sequences using NCBI primer BLAST (Table S1). The internal reference gene was HIS (histone) [29]. QRT-PCR was executed with the Hieff UNICON ® Universal Blue qPCR SYBR Green Master Mix (YEASEN, Shanghai, China) using a LightCycler ® 96 Real-Time PCR Detection System (Roche, Hercules, Switzerland). A total volume of 20 µL contained 10 µL of Blue qPCR SYBR Green Master Mix, 1 µL of cDNA samples, 0.4 µL of each primer (1 µM), and 8.2 µL of ddH 2 O. The PCR thermal cycle conditions were: 95 • C for 2 min, 45 cycles at 95 • C for 10 s and at 56 • C for 30 s. The melting curve conditions were: 1 cycle at 95 • C for 10 s, then at 65 • C for 10 s and 97 • C for 1 s. The relative gene expression was calculated using the 2 −∆∆Ct method [30]. The experiments were performed in triplicate technological repeats.

Measurement of Growth Height
The stem length from point A to point B was measured using a straight ruler for 14 days [17].

Measurement of Lignin Content
Cuttings spanning from point A to point B were dried, crushed it into powder, and passed through a 30-50-mesh sieve before component analysis. According to the manufacturer's protocols, a 5 mg sample was weighed, placed in a test tube, and treated with 0.5 mL of reagent and 20 µL of perchloric acid, to obtain a blank sample. It was then sealed with a sealing film and mixed thoroughly. The samples were placed in a water bath at 80 • C for 40 min, shaken once every 10 min, and then cooled naturally. Next, we added 0.5 mL of reagent II in the tubes, fully mixed, and centrifuged the tubes at room temperature at 8000× g for 10 min. We then placed 10 µL of supernatant in tubes and added 0.99 mL of glacial acetic acid. After fully mixing, the absorbance was measured at 280 nm (Sangon Biotech, Shanghai, China). We named the samples A determination tube and A blank tube; therefore, ∆A = A determination tube -A blank tube was calculated, as reported [31].
Here, V 1 is the detection reaction volume, 1 mL, V 2 is the liquid supernatant volume, 0.02 mL, V 3 is the acetylation reaction volume, 1.02 mL, ε is the extinction coefficient of lignin, 23.35 mL/mg/cm. W is the sample quantity, g; 1000: conversion factor, 1 g = 1000 mg.

Data Analysis
Image processing was performed using Adobe Photoshop CS6, chart layout using Adobe Illustrator 2020, and data processing using Graphpad Prism 8.0.2 software.

Genome-Wide Identification and Sequence Analysis of PyuARFs
Thirty-five ARF family genes were identified and named PyuARF1-PyuARF35 according to their position on chromosomes (Table 1). They had different sequence lengths, resulting in different isoelectric points and molecular weights. The lengths in amino acids ranged from 391 aa (PyuARF2) to 1130 aa (PyuARF35), and the open reading frames ranged from 1176 bp (PyuARF2) to 3393 bp (PyuARF35). The molecular weight ranged from 43.46 kDa (PyuARF2) to 125.45 kDa (PyuARF35), and the isoelectric point ranged from 5.28 (PyuARF17) to 8.99 (PyuARF24). The number of exons was from 2 (PyuARF6) to 16 (PyuARF11).

Comparative Phylogenetic Analysis of the ARFs
In order to understand the evolutionary relationship of the ARFs from A. thaliana, P. trichocarpa, and P. yunnanensis and the differences in the ARF genes among different species, we constructed a rootless phylogenetic tree using the protein sequences of the ARF family members, i.e., those of 22 AtARFs from A. thaliana, 35 PtrARFs from P. trichocarpa, and 35 PyuARFs from P. yunnanensis ( Figure 1).

Conserved Motif Domain and Structural Analysis of PyuARFs
We analyzed the exons and introns of the PyuARFs to determine the structural characteristics of the genes ( Figure 2). The genes in the Class I family have fewer exons and introns, while others have more exons and introns. In addition, the length of the exons of PyuARFs increased from the 5 -terminus to the 3 -terminus. We analyzed the protein domains of PyuARFs to investigate the genes' functions. All PyuARFs contain B3 domains and MR domains ( Figure S1). In addition to the Class I and Class II features, the ARF (PyuARF2, PyuARF29, PyuARF15, PyuARF31, PyuARF1, PyuARF21 and PyuARF27) genes contain AUX/IAA binding domains. The MR domain of the ARF (PyuARF35, PyuARF34, PyuARF33, PyuARF27, PyuARF21, PyuARF18, PyuARF17, PyuARF16, PyuARF13, PyuARF5, PyuARF4 and PyuARF3) genes contains a high proportion of Q (glutamine), S (serine), and L (leucine) (Table S2). We focused on the motifs of the ARF genes to analyze the functional composition of the gene domains. There are three major types of ARF genes in P. yunnanensis, in which each type of motif has high similarity and is conserved. All genes contain at least 10 motifs ( Figure S2).

Conserved Motif Domain and Structural Analysis of PyuARFs
We analyzed the exons and introns of the PyuARFs to determine the structural char acteristics of the genes (Figure 2). The genes in the Class I family have fewer exons and introns, while others have more exons and introns. In addition, the length of the exons o We constructed a rootless phylogenetic tree using the protein sequences of the ARF family members, i.e., 22 AtARFs from A. thaliana, 35 PtriARFs from P. trichocarpa, and 35 PyuARFs from P. yunnanensis. The red circles represent the genes of P. yunnanensis, the green triangles represent the genes of A. thaliana, and the black five-pointed stars represent the genes of P. trichocarpa.

Localization and Duplication of PyuARFs
The high sequence similarity between repetitive gene pairs indicated that they were likely involved in regulating similar biological processes. We used MCScanX to analyze the collinear blocks and gene duplication type ( Figure 3). Twenty-seven duplicated PyuARF pairs were observed in the P. yunnanensis genome. The duplication events, including segmental and whole-genome duplications during the evolutionary process of the PyuARFs family, were analyzed. The twenty-seven duplicated gene pairs appeared located on different chromosomes; except for chromosomes 7, 13, 19, all other chromosomes appeared to contain PyuARFs family members, and chromosomes 1, 2, 3, 4, and 6 appeared to contain the majority of PyuARFs (Figure 4). To gain a detailed understanding of the evolutionary constraints on PyuARFs family (Figure 5), the Ka/Ks ratios of the PyuARF pairs were analyzed ( Figure 6).

. Localization and Duplication of PyuARFs
The high sequence similarity between repetitive gene pairs indicated that they were likely involved in regulating similar biological processes. We used MCScanX to analyze the collinear blocks and gene duplication type ( Figure 3). Twenty-seven duplicated PyuARF pairs were observed in the P. yunnanensis genome. The duplication events, including segmental and whole-genome duplications during the evolutionary process of the PyuARFs family, were analyzed. The twenty-seven duplicated gene pairs appeared located on different chromosomes; except for chromosomes 7, 13, 19, all other chromosomes appeared to contain PyuARFs family members, and chromosomes 1, 2, 3, 4, and 6 appeared to contain the majority of PyuARFs (Figure 4). To gain a detailed understanding of the evolutionary constraints on PyuARFs family (Figure 5), the Ka/Ks ratios of the PyuARF pairs were analyzed ( Figure 6).

Prediction of Cis-Regulatory Elements in the Promoters of PyuARFs
We then studied the potential regulation of cis-acting elements in PyuARFs expression. We analyzed 2 kb promoter sequences of PyuARFs. We found that MeJA (CGTCA motif), light-responsive (Box4), ABA (ABRE) anaerobic (ARE), gibberellin (GARE motif) motifs and response elements were abundant in the promoters of PyuARFs; salicylic acid (TCA motif), MYB drought (MBS), auxin (AuxRR core), low temperature (LTR), endosperm and meristem (GCN4), and circadian response elements were also found in the promoters (Figure 7).

Prediction of Cis-Regulatory Elements in the Promoters of PyuARFs
We then studied the potential regulation of cis-acting elements in PyuARFs expres sion. We analyzed 2 kb promoter sequences of PyuARFs. We found that MeJA (CGTCA motif), light-responsive (Box4), ABA (ABRE) anaerobic (ARE), gibberellin (GARE motif motifs and response elements were abundant in the promoters of PyuARFs; salicylic acid (TCA motif), MYB drought (MBS), auxin (AuxRR core), low temperature (LTR), endo

PyuARFs Transcription Profiles under Light Irradiation
The transcription profiles of the five selected PyuARFs were examined under red light irradiation. On day 1, the transcription profiles of PyuARF16/33 under red light were less rich than under white light. Similarly, on day 7 and on day 14, the transcription profiles of PyuARF16/33 under red light were more limited than under white light (Figure 9).

Measurement of the Lignin Content
Lignin plays an important role in the rapid growth of plants. We measured the content of lignin in the cuttings of P. yunnanensis under light and observed the changes of lignin content on days 1, 7, and 14. On day 1, the lignin content under red light was lower than under white light. Similarly, on day 7 and on day 14, the lignin content under red light was lower than under white light ( Figure 10).

PyuARFs Transcription Profiles under Light Irradiation
The transcription profiles of the five selected PyuARFs were examined under red light irradiation. On day 1, the transcription profiles of PyuARF16/33 under red light were less rich than under white light. Similarly, on day 7 and on day 14, the transcription profiles of PyuARF16/33 under red light were more limited than under white light ( Figure 9).

Measurement of the Lignin Content
Lignin plays an important role in the rapid growth of plants. We measured the content of lignin in the cuttings of P. yunnanensis under light and observed the changes of lignin content on days 1, 7, and 14. On day 1, the lignin content under red light was lower than under white light. Similarly, on day 7 and on day 14, the lignin content under red light was lower than under white light ( Figure 10). Lignin plays an important role in the rapid growth of plants. We measured the content of lignin in the cuttings of P. yunnanensis under light and observed the changes of lignin content on days 1, 7, and 14. On day 1, the lignin content under red light was lower than under white light. Similarly, on day 7 and on day 14, the lignin content under red light was lower than under white light ( Figure 10).

Measurement of Stem Length
We explored the effect of the different types of light on the growth of the stem in P. yunnanensis. We measured the length of the stems at the beginning of the irradiation and after 14 days of irradiation ( Figure 11).

Measurement of Stem Length
We explored the effect of the different types of light on the growth of the stem in P. yunnanensis. We measured the length of the stems at the beginning of the irradiation and after 14 days of irradiation ( Figure 11).

Identification and Analysis of PyuARFs Gene Family Members in P. yunnanensis
In this study, 35 ARF gene family members were identified from the genome of P. yunnanensis. We found that the PyuARF proteins vary greatly in both sequence length and protein characteristics, indicating that they have different characteristics. It is noteworthy that the isoelectric point of most ARF proteins from P. yunnanensis is less than 7, indicating that most PyuARFs may encode weakly acidic proteins and play biological functions in the acidic subcellular environment [32].
The 92 ARF family genes were divided into three categories (Class I, Class II and Class III). Class III included two subcategories, IIIa and IIIb. The MR domain of the ARF gene family of Class IIIb mainly contains Q (glutamine) and presumedly has a transcrip-

Identification and Analysis of PyuARFs Gene Family Members in P. yunnanensis
In this study, 35 ARF gene family members were identified from the genome of P. yunnanensis. We found that the PyuARF proteins vary greatly in both sequence length and protein characteristics, indicating that they have different characteristics. It is noteworthy that the isoelectric point of most ARF proteins from P. yunnanensis is less than 7, indicating that most PyuARFs may encode weakly acidic proteins and play biological functions in the acidic subcellular environment [32].
The 92 ARF family genes were divided into three categories (Class I, Class II and Class III). Class III included two subcategories, IIIa and IIIb. The MR domain of the ARF gene family of Class IIIb mainly contains Q (glutamine) and presumedly has a transcriptional activation function [33]. The MR domain of the ARF genes of class IIIb mainly contains SPL (serine, proline, and leucine) (Table S2), and presumedly has transcriptional inhibition activity. Class Ia and Class II members have uncertain functions.
We found Ka/Ks < 1 for 108 duplicated ARF gene pairs within A. thaliana, P. trichocarpa, and P. yunnanensis and Ka/Ks < 1 for 25 duplicated ARF gene pairs in P. yunnanensis, We speculate that PyuARFs undergwent selective pressure during evolution. Thus, we hypothesized that these gene duplication events led to a higher functional diversity in the PyuARF family members.
The promoter regions of 35 PyuARF genes also contain various cis-acting elements, such as MeJA (CGTCA motif) Light (Box4), ABA(ABRE) anaerobic (ARE), gibberellin (GARE motif), salicylic acid (TCA motif), MYB drought (MBS), low temperature (LTR), endosperm and meristem (GCN4), and circadian response elements. These results indicate that ARFs are involved in a variety of growth and development processes and in signal regulation in plants. Their functions in P. yunnanensis deserve further study.

PyuARF16/33 Are Involved in Lignin Biosynthesis
Red light plays an important role in photosynthesis and in the deposition of secondary cell walls [39,40]. The stem of P. yunnanensis showed a significant difference in height under red and white light. We found that different types of light had different effects on the growth and development of the stem in P. yunnanensis. The results showed that red light promotes stem elongation more than white light.
Previous studies reported that several transcription factors and hormones can regulate lignin biosynthesis and inhibit plant height by increasing lignin content [41,42]. Lignin can enhance cell wall strength and stem lodging resistance in soybean [43,44]. However, whether ARFs affect lignin synthesis to regulate the growth and development of the plant stem is still unclear. ARF6 and ARF8 show functional redundancy [45], because they regulate the development of secondary xylem in wood and hypocotyl elongation in A. thaliana [1,13]. According to homology alignment, ARF6 and ARF8 of A. thaliana match to Class IIb ARFs in P. yunnanensis. So, 12 PyuARFs were selected to analyze their transcription profiles and investigate their physiological functions according to their transcriptional activation in various tissues. Five PyuARFs (PyuARF5, PyuARF16, PyuARF18, PyuARF3¡,3 and PyuARF34) appeared highly expressed in the stem, indicating that these ARFs are important for stem development, such as xylem development. In this study, the lignin content of cuttings and seedlings was measured under light treatment. The lignin content was lower, and the gene transcription profiles were more limited in red light than in white light on days 1, 7, and 14, suggesting that PyuARF16/33 might be a good candidate gene regulating lignin synthesis and the rapid growth of poplar cuttings and seedlings under red light.
Previous studies have shown that hierarchical transcriptional regulatory networks composed of various transcription factors such as NACs and MYBs are responsible for regulating lignin biosynthesis in A. thaliana [46][47][48]. For example, PbrMYB169 positively regulates the lignification of stone cells in pear fruit [49], and OSNAC5 regulates drought tolerance and lignin accumulation in roots [50]. A recent study showed that ARF8-MYB26-NST1 modulates premature endothecium lignification in A. thaliana by regulating lignin synthesis [51]. This study predicts that PyuARF16/33 might also regulate lignin synthesis through the downstream genes MYBs. However, whether PyuARF16/33 affects lignin accumulation by regulating MYB26-NST1 needs further experimental verification.

Conclusions
We identified 35 ARF family genes in P. yunnanensis genome and verified the tissue specificity of 12 PyuARFs with transcriptional activation function. We then selected five PyuARFs for transcription profiles analysis under light treatment. Finally, we measured lignin content under light treatment. The results showed that the lignin content was lower, and the gene transcription profiles were more limited in red light than in white light on days 1, 7, and 14, suggesting that PyuARF16/33 might be a good candidate gene regulating lignin synthesis and the rapid growth of cuttings and seedlings under red light in P. yunnanensis.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/genes14020278/s1, Table S1: Gene-specific primers for RT-qPCR used in this study. Table  S2: Proportion of Gln (Q), Ser (S), Leu (L), Pro (P), and Thr (T) in the MR domain of PyuARFs. Figure S1: Sequence alignment and corresponding domains of PyuARFs. Figure   Data Availability Statement: Genomic data of P. yunnanensis can be obtained by contacting the corresponding author. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare that they have no competing interests.