PeCLH2 Gene Positively Regulate Salt Tolerance in Transgenic Populus alba × Populus glandulosa

Salt is an important environmental stress factor, which seriously affects the growth, development and distribution of plants. Chlorophyllase plays an important role in stress response. Nevertheless, little is known about the physiological and molecular mechanism of chlorophyll (Chlase, CLH) genes in plants. We cloned PeCLH2 from Populus euphratica and found that PeCLH2 was differentially expressed in different tissues, especially in the leaves of P. euphratica. To further study the role of PeCLH2 in salt tolerance, PeCLH2 overexpression and RNA interference transgenic lines were established in Populus alba × Populus glandulosa and used for salt stress treatment and physiologic indexes studies. Overexpressing lines significantly improved tolerance to salt treatment and reduced reactive oxygen species production. RNA interference lines showed the opposite. Transcriptome analysis was performed on leaves of control and transgenic lines under normal growth conditions and salt stress to predict genes regulated during salt stress. This provides a basis for elucidating the molecular regulation mechanism of PeCLH2 in response to salt stress and improving the tolerance of poplar under salt stress.


Introduction
Due to unreasonable irrigation, environmental pollution and other factors, soil salinization is becoming more and more serious, and salt stress has become one of the important reasons to limit plant growth and restrict the development of the agricultural industry [1][2][3]. At present, about 1/5 of the arable land in China is salinized, with a total area of up to 98 million hm 2 [4]. China is the world's most populous country with less than 0.1 hm 2 of arable land per capita, and it is a difficult issue to utilize the salinized land and promote the increase in food production [5]. Therefore, the study of salt tolerance and physiological mechanisms of plants and the screening and breeding of salt-tolerant crop varieties have become some of the hot spots in the field of agricultural research.
P. euphratica is a precious forest resource unique to desert areas. Growing in the desert all year round, it has a strong integrated resistance to salinity, barrenness, cold and drought [6][7][8][9]. Therefore, it is important to screen the excellent resistance genetic resources in P. euphratica and combine them with genetic engineering to breed resistant and high-quality tree species suitable for the needs of arid and semi-arid regions in China.
Chlorophyll (Chlase, CLH) is an important pigment for photosynthesis in plants, and its synthesis and degradation exist in a dynamic form in the plant. Chlorophyll degradation is closely related to plant physiological phenomena such as plant senescence, fruit ripening and tree defoliation [10][11][12]. Chlorophyllase has been of interest as the key enzyme that catalyzes this degradation reaction. Globally, about 10 9 kg of chlorophyll is reported to be degraded in plants annually [13][14][15]. Among them, CLH is the key rate-limiting enzyme in the chlorophyll degradation process, which can hydrolyze chlorophyll into dephytolized chlorophyll and phytol of carboxylic acid [16]. It has been shown in the literature that the expression of chlorophyllase genes and changes in CLH activity in plants may change significantly under multiple exogenous stimuli (such as heavy metals, low temperature, high temperature, salinity, drought, injury, pathogens, pest infestation, salicylic acid/SA) [17][18][19][20]. It has been reported that ERF72 can directly bind to the promoter region of the iron deficiency response gene CLH1 and reduce CLH gene expression [21]. Overexpression of ERF4 leads to degradation of chlorophyll content and up-regulation of the CLH1 gene in tobacco [22]. In addition, CLH genes have been isolated successively in Citrus reticulata [14], Chlamydomonas reinhardtii [23], Ginkgo biloba [24] and Chenopodium album [25] plants by previous authors, and preliminary studies and have been conducted to find that CLH genes of different species have different expression patterns and functions during plant growth and development [26]. Despite the fact that chlorophyll controls various important traits, so far, there have been few studies on the molecular mechanisms of chlorophyll genes for salt tolerance in forest trees.
In this study, a salt-tolerant chlorophyll gene, PeCLH2, was identified through transcriptome analysis of P. euphratica in the early stage, and heterologous expression of the PeCLH2 gene was performed in P. alba × P. glandulosa [27,28]. Transcriptome analyses were performed on overexpression, RNA interference expression and wild-type plants. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that differential genes were mainly enriched in isoquinoline alkaloid biosynthesis, tyrosine metabolism-α-linolenic acid metabolism and phytohormone signaling pathways after salt stress. The results indicated that the PeCLH2 gene positively regulates salt tolerance in transgenic P. alba × P. glandulosa. It provides a theoretical basis for the in-depth study of its regulatory mechanism and the functional study of related genes.

Plant Materials
The experimental material used in this study was P. alba × P. glandulosa and P. euphratica asexual line histoculture seedlings from natural P. euphratica seed propagation in Xinjiang, preserved in the State Key Laboratory of Tree Genetics and Breeding of the Chinese Academy of Forestry. It was grown in culture flasks containing 1/2 MS medium. The culture conditions were a 16/8 h light/dark cycle with an average temperature of 25 • C.

Genetic Phylogenetic Analysis
Protein sequences of CLH2 genes of nine different species were obtained from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/ (accessed on 22 January 2022)) and multiple sequence alignment was performed using clustal W [29]. Phylogenetic tree analysis was performed by MEGA 6 [30] using the neighbor-joining method.

Molecular Cloning of the Gene
The CLH2(POPULUS_EUPHRATICA_20786) gene coding sequence was found on the P. euphratica genome website to design primers, the CLH2 gene sequence was cloned from P. euphratica, and the coding region of PeCLH2 was cloned into the plant expression vector PMDC32 using the Gateway vector construction method to obtain a 35S::PeCLH2 overexpression vector; PeCLH2 RNA interference vector (RNAi-PeCLH2) was obtained on pH7GWIWG2-I vector [31]; and the PeCLH2 promoter sequence was cloned into vector PBI121 to obtain PPeCLH2::GUS vector and transform P. alba × P. glandulosa [32].

GUS Experiment
The GUS tissue staining method was carried out with reference to previous studies [33]. The 2kb promoter of PeCLH2 was cloned into PBI121 vector to obtain PPeCLH2::GUS transgenic plants (Table S1).The 1-month-old transgenic plants were fixed in 90% acetone at 4 • C for 2 h and transferred to GUS staining buffer (0.2M sodium dihydrogen phosphate, 0.2 M disodium hydrogen phosphate, 2.0 mM potassium ferricyanide, 2.0 mM potassium ferricyanide, ready-to-use) for washing; then, it was transferred to GUS staining solution (GUS staining buffer with 0.2% Triton X-100) for incubation at 37 • C for 12 h, decolorized with 70% ethanol, and stored at 4 • C (the solution was changed to ethanol when it turned green). The localization of GUS signals in different tissues was observed using an Olympus microscope. GUS staining experiments contained at least 6 transgenic strains, with at least 6 replicates of each transgenic strain. GUS staining experiments were repeated at least three times.

Breeding of Transgenic Plants with Overexpression and RNA Interference Expression
Transgenic lines were obtained by callus transformation [34]. One-month-old transgenic resistant buds were transferred to a rooting medium containing plants cultured on 200 mg L −1 cephalexin and 1.5 mg L −1 hygromycin B. Transgenic poplars were examined at the molecular-level DNA and by real-time PCR. RT-qPCR was performed on a Light-Cycler 480 thermal (Roche, Basel, Switzerland) cycler using the SYBR PreMix Ex Taq kit (Dalian Takara, China) according to the manufacturer's instructions (Table S1). The qPCR procedures were: 95.0 • C for 30 s, 95.0 • C for 5 s, 60.0 • C for 34 s, 95.0 • C for 15 s, 60.0 • C for 1 min, and 95.0 • C for 15 s. Data were processed using the 2 −∆∆CT method. Each sample had at least three biological replicates.

Morphometry
Two high-expression lines, OC1 and OC9, were overexpressed, and RC6 and RC10 with high silencing efficiency were selected for salt stress test in RNA interference transgenic lines. For the convenience of analysis and reading, we have simplified this. OC1 is marked as OC1, OC9 as OC2, RC6 as RC1, and RC10 as RC2.Transgenic lines and wild-type (WT) seedlings in consistent growth status were transferred to 1/2 MS medium containing 0, 50, 75 and 100 mM NaCl and grown.
The plant height, root length and fresh weight of 1-month-old plants were determined. There were at least 6 individual replicates for each line, and the experiment was repeated at least 3 times.

Transgenic Salt Stress Experiment, Physiological Index Determination and Histochemical Staining
Two transgenic lines each with the same growth status of PeCLH2 gene overexpression (OC1; OC2), repressed expression (RC1; RC2) and WT were transplanted into soil pots in the greenhouse for one month. They were divided into six groups on average, with three plants per strain in each group. The three groups were treated with 150 mM NaCl for seven days, and the other three groups were treated with water as control. Each treatment contained nine plants. Phenotypic changes were observed and recorded. The second and third leaves below the top were taken for DAB and NBT staining, and the fourth and sixth leaves were taken for POD, SOD, MDA and other physiological indicators. Physiological indicators and histochemical staining including peroxidase (POD), superoxide dismutase (SOD), malondialdehyde (MDA), relative conductivity, chlorophyll content, soluble sugar content, diaminobezidin3,3 (DAB), and tetranitroblue tetrazolium chloride (NBT) were determined in transgenic lines and WT according to the manufacturer's instructions (Solarbio, Beijing, China).

Expression Analysis of Stress Resistance Related Genes
Transgenic and WT tissue culture seedlings grown in culture flasks for 30 days with consistent growth were extracted from leaf RNA for Quantitative Real-time PCR (RT-qPCR) analysis, and information on the primers for the internal reference primers, POD and SOD-related genes are shown in Table S1.

RNA Sequencing
Transgenic and WT plants grown in soil pots for one month were treated with 150 mM NaCl for 24 h. Three overexpression transgenic lines (OC), three RNA interference transgenic lines (RC), and one wild-type control top leaf (WT) were collected, immediately frozen in liquid nitrogen, and saved in a −80 • C freezer for similar evaluation and sequencing (each strain had three biological replicates). Total RNA was extracted using the plant total RNA extraction kit (TIANGEN, Beijing, China). The purity and integrity of RNA were checked by nanodrops, agarose gel electrophoresis and the Agilent Biological Analyzer 2100 system (Agilent Technologies Co. Ltd., Beijing, China).Total RNA was extracted and sent to Gene Denovo (Guangzhou, China) for sequencing using the Illumina HiSeq2000 platform. The experimental procedure for transcriptome sequencing included library construction, library quality control and up-sequencing.

GO and KEGG Enrichment Analyses of DEGs
This study used the Populus trichocarpa genome as the reference genome. The R package DESeq2 was used to identify differentially expressed genes between transgenic lines and WT with a threshold of logFC ≥ 1.5 and q-value ≤ 0.05 [35]. Gene Ontology (GO) enrichment analysis and visualization of results were performed using the GOseq R package [36]. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using KEGG Orthology-Based Annotation Systerm2.0 (KOBAS) [37].
Differentially expressed genes were compared to the Plant Transcription Factor Database v3.0 (Plant TFDB v3.0) database [38] to screen for differentially expressed transcription factor genes.

RT-qPCR Examination of RNA Sequencing Results
To verify the reliability of transcriptome sequencing data, the expression levels of 16 stress-responsive genes were analyzed by RT-qPCR (Table S1). The relative expression levels of the genes in the transgenic strains were calculated based on their expression levels in the WT strains. Statistical analysis was performed using t-test in GraphPad Prism 8 software (GraphPad Company, San Diego, CA, USA) [39]. A p-value < 0.05 was considered statistically significant.

Phylogenetic Analysis and Gene Cloning
The cDNA fragment of PeCLH2 was cloned from P. euphratica. The coding sequence region had 1005 bp and encoded 335 amino acids, the molecular weight was 83.53631 KDa, the isoelectric point was 5.08, the molecular formula was C 3068 H 5133 N 1005 O 1286 S 214 , the total number of atoms was 10,706, the fat coefficient was 28.26, the half-life was >20 h, the instability coefficient was 49.39, and the overall average hydrophilicity (GRAVY) was 0.756, and the protein was predicted to be a hydrophobic protein. The multiple sequence alignment and evolution analysis of PeCLH2 amino acid sequence were carried out by BioEdit and MEGA5.0 software. The results showed that the PeCLH2 protein sequences of 10 plants all had a conserved chlorophyll domain. The amino acid sequence identity is 78-100%. Among them, the PeCLH2 protein has high homology with the CLH2 protein of P. trichocarpa,(XM_002315716.3) Hevea brasiliensis (XM_021836258.1), and Jatropha curcas (XM_012214305.2) ( Figure 1).

Expression Analysis of PeCLH2
To explore the expression pattern of PeCLH2 in different tissues, we used RT-qPCR to detect its expression levels in roots, stems, and leaves in P. euphratica, respectively. Results indicated that PeCLH2 had the highest expression level in leaves, followed by stems and roots. The significant differences are shown in Figure 2A.  In order to investigate the expression pattern of PeCLH2 in P. euphratica under salt stress, we used RT-qPCR to determine its expression levels in roots, stems and leaves treated with 150 mM NaCl or 300 mM NaCl concentrations for 0, 12, 24, and 48 h, respectively. As shown in Figure 2B-D, the salt response of PeCLH2 to leaves and stems segments showed an up-regulation trend from 0 h to 12 h, and then showed a gradual down-regulation trend. In addition, it is worth noting that the salt response of PeCLH2 to P. euphratica root was up-regulated from 0 h to 24 h and then down-regulated.

Gus Staining
In order to study the function of PeCLH2 gene promoter, the transgenic PPeCLH2::GUS promoter were taken out from the culture flask, the root medium was washed with running water, and GUS staining was performed. The top young parts of the transgenic plants were stained blue, indicating that the PPeCLH2::GUS promoter was active and could drive the expression of the GUS gene in the young tissues of the plants ( Figure S1).

Identification of Transgenic Plants
The expression of foreign genes was detected at the transcriptional level by RT-qPCR. The real-time monitoring of the entire PCR process by the accumulation of fluorescent signals showed that the expression of fluorescent signals was detected in each gene transgenic line ( Figure S2). The results showed that 35S::PeCLH2 (OC) and RNAi-PeCLH2 (RC) transgenic plants were expressed at the RNA level, and the relative expression levels were different.

Morphological Characteristics of Transgenic Plants
To investigate the salt tolerance of PeCLH2, seedlings of 35S::PeCLH2, RNAi-PeCLH2 and WT were treated with 0, 50, 75 and 100 mM NaCl, respectively. The results showed that the plant height, root length and fresh weight in the 35S::PeCLH2 transgenic lines are superior than that of the WT at control and stress conditions. RNAi-PeCLH2 shows the opposite situation ( Figure 3A-D).
Under normal conditions, the plant height, root length and fresh weight of 35S::PeCLH2 values were 1.2, 1.08 and 1.2 times than those of WT, respectively. RNAi-PeCLH2 values was 0.85, 0.94 and 0.88 times, respectively. Under the condition of 50 mM NaCl, 35S::PeCLH2 values were 1.58, 1.08 and 2 times higher than WT, respectively. RNAi-PeCLH2 was 0.9, 0.76 and 0.7 times, respectively. Under the condition of 75 mM NaCl, 35S::PeCLH2 values were 1.81, 1.06 and 2.3 times, respectively. RNAi-PeCLH2 was 0.85, 0.51 and 0.77 times higher than WT, respectively. Under the condition of 100 mM NaCl, 35S::PeCLH2 values were 1.28, 1.39 and 2.7 times, respectively. RNAi-PeCLH2 was 0.75, 0.08, and 0.9 times higher than WT, respectively. ted to Journal Not Specified  To investigate the role of PeCLH2 in salt stress, we treated transgenic and wild-type plants for 7 days, which were grown in soil for one month at a NaCl concentration of 150 mM. As shown in Figure 3E, under control conditions, 35S::PeCLH2 transgenic lines showed better growth compared to WT, while RNAi-PeCLH2 transgenic lines did not grow differently from WT. Under salt stress, 35S::PeCLH2 transgenic plants grew normally and showed greater salt tolerance. The WT and RNAi-PeCLH2 transgenic appeared to wilt and show intolerance, and RNAi-PeCLH2 transgenic line leaves wilted completely and even died. This evidence suggests that PeCLH2 plays a crucial role in salt tolerance.

Physiological Index Determination and Histochemical Staining Analysis
Under salt-stress conditions, the POD and SOD activities, soluble sugar content and chlorophyll content of 35S::PeCLH2 were significantly higher than the WT, while RNAi-PeCLH2 was significantly lower than WT. the results of MDA content and relative conductivity showed that 35S::PeCLH2 were significantly less damaged than the WT under salt stress, while RNAi-PeCLH2 was significantly higher than the WT (Figure 4A-E).  Transgenic plants with PeCLH2 under salt-stress conditions were stained for NBT and DAB histochemistry ( Figure 5A,B). The results showed that the staining levels of leaves of transgenic plants were similar to the WT under normal conditions, indicating that there was no significant difference in the accumulation of O 2− and H 2 O 2 . The staining level of 35S::PeCLH2 under salt-stress conditions was significantly lower than the WT, while RNAi-PeCLH2 was significantly higher than the WT.  The above results showed that the damage of 35S::PeCLH2 transgenic plants under salt stress was significantly higher than that of WT, and the damage of RNAi-PeCLH2 under salt stress was significantly lower than WT, indicating that PeCLH2 has certain salt tolerance.

Expression Analysis of Stress Resistance Related Genes
The quantitative results showed that the relative expression level of stress-resistancerelated genes in transgenic lines was significantly higher than WT. The relative expression levels of SOD-and POD-related genes in 35S::PeCLH2 were 1.16-3.42 times and 1.43-2.49 times higher than WT, respectively. The relative expression levels of SOD-and POD-related genes in RNAi-PeCLH2 were 0.16-0.74 times and 0.24-0.76 times higher than those in the WT, respectively ( Figure 6A,B).

Sequencing Data Quality Assessment
Transcriptome analysis of WT and transgenic lines was conducted using RNA-seq technology. After the sequencing data quality control was completed, clean data were evaluated. The Q20 value of all sample data reached more than 97.37%, the Q30 value (base misjudgment rate of 0.1%) reached more than 92.73%, the GC content was between 43.73% and 44.63%, and the sequencing depth was more than ten times (Table S2). It shows that transcriptome sequencing has obtained high-quality clean data, which can meet the requirements of subsequent analysis.

Gene Expression Level Analysis
Under normal condition, almost no significant differences were found between WT and transgenic line in terms of gene expression of the stress-related genes. However, when treated with salt, a lot of differentially expressed genes were observed in both WT and transgenic plants ( Figure 7A-C). Based on the difference analysis results, we screened genes with FDR < 0.05 and |log2FC| > 1 as significantly different genes. Compared with WT, 11 differentially expressed genes (including 6 up-regulated genes and 5 downregulated genes) were identified from 35S::PeCLH2 (OC) in the control group. A total of 27 differentially expressed genes (including 20 up-regulated genes and 7 down-regulated genes) were identified in RNAi-PeCLH2 (RC). In the treatment group, 2146 differentially expressed genes (including 745 up-regulated genes and 1401 down-regulated genes) were identified in OC. Six differentially expressed genes (including four up-regulated genes and two down-regulated genes) were identified in RC (Table S3). The results showed that OC and WT had the most differential genes after salt treatment, indicating that OC mainly functions after salt stress.

GO and KEGG Enrichment Analyses of DEGs
To determine the most important biochemical metabolic pathways and signaling pathways involved in differential gene expression in transgenic plants during salt stress, we performed GO and KEGG enrichment analysis. Global functional analysis of DEGs was performed on GO annotations into biological processes, molecular functions, and cellular components. GO enrichment analysis of all comparison groups revealed that the most common categories in the ontology of biological processes were "metabolic processes" and "single organism processes", followed by "cellular process". Within the category of molecular function ontology, "catalytic activity" and "binding" acted as two primary functional groups. Within the cellular component, "cell", "cell part" and "organelle" were predominant ( Figures S3 and S4).
In addition, prediction of the biochemical pathways associated with the DEGs was performed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers. In the untreated condition, WT and OC were enriched into 10 KEGG pathways. Sesquiterpenoid and triterpenoid biosynthesis, nitrogen metabolism, arginine biosynthesis, alanine, aspartate and glutamate metabolism, and porphyrin and chlorophyll metabolism were all significant enrichment pathways ( Figure 8A). WT and RC were enriched into 10 KEGG pathways, and sesquiterpenoid and triterpenoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis RNA polymerase, flavonoid biosynthesis, and pyrimidine metabolism were significant enrichment pathways ( Figure 8B). In conclusion, the biosynthesis of semi terpenoids and triterpenoids is the most abundant pathway in the untreated condition.
Under salt treatment, WT and OC were enriched in 116 KEGG pathways. Isoquinoline alkaloid biosynthesis, tyrosine metabolism, alpha-Linolenic acid metabolism, plant hormone signal transduction metabolic pathway, biosynthesis of secondary metabolites, and fatty acid degradation were significant enrichment pathways ( Figure 9A). Among them, Isoquinoline alkaloid biosynthesis was significantly enriched with 16 differential genes, followed by tyrosine metabolism with 21 differential genes and alpha-Linolenic acid metabolism with 17 differential genes. Under salt treatment, WT and RC were enriched into four KEGG pathways, and RNA polymerase, pyrimidine metabolism, purine metabolism and metabolic pathway were significant enrichment pathways ( Figure 9B). Taken together, these results suggest a fundamental change in the gene function/metabolic network in the expression of PeCLH2 under normal and NaCl stress conditions.

Transcription Factors Involved in Salt Stress Response
The response of PeCLH2 transgenic plants to salt stress is synergistically regulated by multiple genes, among which transcription factors play an extremely important role. Therefore, further analysis of transcription factor differentially expressed genes in this study revealed that a total of 90 differentially expressed transcription factors, including various types of WRKY, MYB, NAC, and bHLH, were annotated under salt-stress conditions (Table S4). It is hypothesized that the effect of salt-stress treatment on transgenes was mainly concentrated on WRKY, MYB, NAC, and bHLH transcription factor families. This shows that the expression of these transcription factors is regulated by salt stress.

Reliability of Transcriptome Sequencing Data
To verify the accuracy of the transcriptome sequencing results, we selected 16 genes related to stress and designed primers within their conserved structural domains for RT-PCR experiments, and later compared them with the transcriptome results, which are shown in the figure below, and the results shown in the figure are almost identical to the transcriptome sequencing results ( Figure 10). This indicates that the results of transcriptome data analysis are reliable.

Discussion
Chlorophyllase is one of the earliest discovered plant enzymes and one of the most important pigments in the biological world [40,41]. Chlorophyll degradation occurs when plants are subjected to biotic or abiotic stresses such as low temperature, strong light, pest and disease infestation and during plant senescence, resulting in a series of physiological responses, such as leaf fading, wilting, yellowing and premature end of life cycle [42][43][44]. CLH genes have been studied in plants, for example, CLH1 and CLH2 were identified as candidate genes by genome-wide association analysis (GWAS) of 107 Arabidopsis accessions, and loss of function or down-regulation of CLH1 and CLH2 expression promoted tolerance to the polyamine oxidase inhibitor guazatine [45,46]. Metabolomic and transcriptomic analyses of cabbage 1-methylcyclopropene treatment revealed significant inhibition of the expression of most Chl degradation-related genes (BcPPH1/2, BcSGR1/2, BcPAO, BcNYC1, and BcNOL) [47]. Chlorophyllase genes associated with Chl degradation were found to be up-regulated in yellow leaves in the transcriptome analysis of yellow leaf coloration in Populus deltoides Marsh [48]. Although CLH was discovered more than 100 years ago [49] and many studies have focused on its function and related mechanisms, little research has been reported on CLH in poplar for salt stress.
P. euphratica is a tall tree mainly distributed in extreme saline and semi-desert areas of Xinjiang, Gansu, Inner Mongolia in China. The strong salt tolerance of this species makes it a good model plant for studying salt tolerance, mining salt resistance genes and improving saline lands. However, the underlying molecular mechanisms controlling salt stress tolerance still have not been identified. In our previous study, we performed transcriptome analysis of salt-treated P. euphratica and screened a series of key salt-tolerance regulatory genes by combined WGCNA and GWAS analysis and selected the PeERF1 and PeCLH2 gene for functional validation. PeERF1 has been identified for salt tolerance. PeCLH2 is a chlorophyll family gene cloned from P. euphratica. Transgenes were obtained for 35S::PeCLH2 and RNAi-PeCLH2 gene. Morphological measurements, physiological characterization and histochemical analysis of transgenic P. alba × P. glandulosa in salt assays showed that 35S::PeCLH2 enhanced salt tolerance in transgenic plants and that RNAi-PeCLH2 was sensitive to salt treatment, demonstrating that PeCLH2 plays an important role in salt-stress response.
Under high salt concentrations, antioxidant systems such as SOD and POD can scavenge ROS, and hence avoid the build-up of oxidative damage in plants to improve salt tolerance [50]. As the duration of stress increased, membrane lipid peroxidation was enhanced and MDA content increased [51]. Salt accumulation in plants disrupts cell membrane permeability, leading to an increase in conductivity; meanwhile, the content of osmoregulatory substances such as soluble sugar increases, scavenging free radicals and reducing the salt damage to plants. In this study, the changes in POD and SOD activities, MDA content, soluble sugar content, chlorophyll content and relative conductivity of transgenic poplar showed that the PeCLH2 gene could significantly enhance salt tolerance. In addition, the up-regulation of SOD, POD, and other stress-related genes in transgenic plants 35s::PeCLH2 and the decrease in SOD, POD, and other stress-related genes in RNAi-PeCLH2 transgenic plants indicated that this gene enhanced salt tolerance of poplar by regulating the expression of stress-related genes.
RNA-seq data analysis showed that the overall gene expression profile of the overtransgenic plants was different from that of the control plants. Compared with the WT, 11 genes in the overexpressed transgenic lines showed significantly different expression levels before salt treatment, of which 6 were up-regulated and 5 were down-regulated. After 24 h of salt treatment, the number of DEGs in overexpressing transgenic plants increased to 2146, of which 745 were up-regulated and 1401 were down-regulated, suggesting that CLH2 may regulate many stress-related genes. Interestingly, there were 27 RNAi-PeCLH2 transgenic lines (20 up-regulated and 7 down-regulated DEGs) before salt stress and only 6 DEGs (4 up-regulated and 2 down-regulated DEGs) after stress, and the changes in the RNAi-PeCLH2 transgenic lines before and after salt stress were not significant. This may be due to the changes in PeCLH2 expression levels before and after salt stress, resulting in different patterns of genetic regulation in transgenic plants.
Isoquinoline alkaloids are major secondary metabolites in a variety of plants, and the biosynthetic pathways of various isoquinoline alkaloids have been clarified through previous studies [52][53][54][55]. In the present study, the "isoquinoline alkaloid synthesis" pathway ranked first among the top 20 KEGG pathways of DEGs identified in 35S::PeCLH2 and WT salt stress, indicating that the PeCLH2 gene could respond to salt stress by regulating the isoquinoline biosynthesis pathway in transgenic plants ( Figure 8A).
Among the downstream genes regulated by PeCLH2, there are many stress-resistancerelated genes that play important roles in plant response to abiotic stresses. Among them, PtrANS, PtrPER64, and PtrWRKY6 genes are important genes that are significantly upregulated in expression (Table S5). It has been demonstrated that PtrANS, PtrPER64 and PtrWRKY6 genes play an important role in protecting plants from abiotic stress damage. In plants, PtrANS, PtrPER64 and PtrWRKY6 genes can significantly improve their tolerance to abiotic stresses such as salt, osmotic and drought [56][57][58][59][60]. However, whether PeCLH2 can directly bind to the promoters of PtrANS, PtrPER64, and PtrWRKY6 needs further validation.
To date, most studies on CLH2 resistance have focused on leaf senescence, fruit ripening and chlorophyll degradation. In this study, the salt tolerance of PeCLH2 was verified by transgenic experiments. This will provide a basis for the direction of PeCLH2 gene resistance. In addition to the salt tolerance of PeCLH2 gene to poplar in this study, it may also have other resistances, and further studies to verify its resistance need to be continued in the future.

Conclusions
In this study, we cloned a key regulatory gene for salinity tolerance in P. euphratica and CLH2 and then transformed it in P. alba × P. glandulosa. The overexpression transgenic lines showed morphological, physiological and biochemical advantages compared to WT, while the repressed expression showed the opposite. In addition, POD and SOD related stress tolerance genes expression levels were significantly higher in the transgenic lines than in WT, indicating that CLH2 gene could improve salt tolerance of transgenic poplars by regulating the expression of stress related genes. Many genes related to salt tolerance were identified by salt-stress transcriptome analysis of the transgenic lines, and CLH2 functioned mainly after salt stress. A series of salt-stress-related resistance genes were screened to provide candidate genes and theoretical basis for genetic engineering breeding in forest trees.

Supplementary Materials:
The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/genes14030538/s1. Figure S1. Promoter transgenic P alba × P glandulosa analysis of PeCLH2. Image of GUS activity assay of PPeCLH2::GUS transformed P alba × P glandulosa. Scale bar is 5 mm. Figure S2. PeCLH2 expression analysis in transgenic P. alba × P. glandulosa. (A) and (B), Analysis of PeCLH2 expression levels in various transgenic strains. The expression level of PeCLH2 was estimated in relation to that of the wild-type plant. WT, wild type poplar; OC1-32, PeCLH2 overexpression lines; RC1-10, RNAi-PeCLH2 expression lines. The expression level of each line was calculated relative to its expression level at WT (t test, p < 0.05). Figure S3. Gene ontology (GO) term analysis of untreated transgenic transcriptome DEGs. A: GO enrichment of the significant gene expression difference between the transgenic with expression of the PeCLH2 overexpression and the WT under normal conditions. The enrichment analysis was performed for compartmentalization; B: GO enrichment of the significant gene expression difference between the transgenic with expression of the RNAi-PeCLH2 and the WT under normal conditions. Figure S4. Gene ontology (GO) term analysis of salt-treated transgenic transcriptome DEGs. A: GO enrichment of the significant gene expression difference between the transgenic with expression of the PeCLH2 overexpression and the WT under salt stress. The enrichment analysis was performed for compartmentalization; B: GO enrichment of the significant gene expression difference between the transgenic with expression of the RNAi-PeCLH2 and the WT under salt stress. Table S1. The primers used in this study. Table S2. Statistics of PeCLH2 transgenic transcriptome sequencing data comparison results. Table S3. DEGs identification of PeCLH2 transgenic for the salt stress transcriptome. Table S4. Differential expression of TFs after salt stress. Table S5. Differentially expressed genes related to stress.  Data Availability Statement: Additional data supporting the findings in this study are available in the Supplementary Information of this article. The raw datasets are available from the first author or corresponding author on reasonable request.