PYL Family Genes from Liriodendron chinense Positively Respond to Multiple Stresses

The phytohormone abscisic acid (ABA) plays important roles in response to abiotic and biotic stresses in plants. Pyrabactin resistance 1-like (PYR/PYL) proteins are well-known as ABA receptors, which are responsible for ABA signal transduction. Nevertheless, the characteristics of PYL genes from Liriodendron chinense, an endangered timber tree, remain unclear in coping with various stresses. In this study, five PYLs were identified from the genome of Liriodendron chinense by sequence alignment and conserved motif analysis, which revealed that these LcPYLs contain a conserved gate and latch motif for ABA binding. The LcPYL promoters possess a series of cis-acting elements involved in response to various hormone and abiotic stresses. Moreover, the transcriptome data of Liriodendron hybrid leaves reveal that LcPYL genes specifically transcript under different abiotic stresses; Lchi11622 transcription was induced by drought and cold treatment, and Lchi01385 and Lchi16997 transcription was upregulated under cold and hot stress, respectively. Meanwhile, the LcPYLs with high expression levels shown in the transcriptomes were also found to be upregulated in whole plants treated with the same stresses tested by qPCR. Moreover, under biotic stress caused by scale insect and whitefly, Liriodendron hybrid leaves exhibited a distinct phenotype including disease spots that are dark green in the middle and yellow on the margin; the qPCR results showed that the relative expression levels of Lchi13641 and Lchi11622 in infected leaves were upregulated by 1.76 and 3.75 folds relative to normal leaves, respectively. The subcellular localizations of these stress-responsive LcPYLs were also identified in protoplasts of Liriodendron hybrid. These results provide a foundation to elucidate the function of PYLs from this elite tree species and assist in understanding the molecular mechanism of Liriodendron hybrid in dealing with abiotic and biotic stresses. In future research, the detailed biological function of LcPYLs and the genetic redundancy between LcPYLs can be explored by gene overexpression and knockout based on this study.


Introduction
Higher plants have evolved a high plasticity for adaptation to environmental challenges such as drought, cold, hot and insect attack stresses, which severely influence normal plant development and productivity [1][2][3][4]. Phytohormone abscisic acid (ABA) plays a critical role in plant growth and development, including cell division and elongation, embryo maturation, seed dormancy and germination, leaf senescence, root growth, fruit ripening and stomatal aperture [5,6]. ABA also serves as an endogenous messenger to transduce the signals of various stresses and plays a major role in plant adaptation to the environment [7][8][9]. For instance, in response to drought stress, plants synthesize the

Genome-Wide Analysis and Chromosome Distribution of LcPYL Family Genes
To explore the molecular mechanism of the ABA signaling pathway in L. hybrid under abiotic and biotic stresses, we identified the ABA receptors from the genome of L. chinense. As a result, 13 LcPYL genes were obtained by searching with the conserved domains, including Polyketide_cyc2 domain (PF10604) and Polyketide_cyc domain (PF03364) [16]. To further verify the LcPYL members, we used the NCBI-CDD database to identify whether the candidates have a PYR/PYL (RCAR)-like domain (cd07821). As a consequence, six PYLs were identified from the L. chinense genome. Combined with ABA binding conserved motif analysis, we finally obtained five PYL genes from the L. chinense genome.
The ORF sizes of these PYLs vary from 534 bp to 648 bp ( Figure 1A). Analysis of the physical and chemical properties of LcPYL proteins showed that Lchi16997 has the smallest protein sequence (197 aa), followed by Lchi11622 (178 aa), and Lchi01385 has the largest number of amino acids (204 aa) ( Table 1). The molecular weights of identified The ORF sizes of these PYLs vary from 534 bp to 648 bp ( Figure 1A). Analysis of the physical and chemical properties of LcPYL proteins showed that Lchi16997 has the smallest protein sequence (197 aa), followed by Lchi11622 (178 aa), and Lchi01385 has the largest number of amino acids (204 aa) ( Table 1). The molecular weights of identified proteins ranged from 19.75 KDa to 23.75 KDa. The range of isoelectric points (pI) is from 5 to 7.65. Additionally, the predicted subcellular locations of LcPYL proteins showed that Lchi16997 and Lchi01385 are located in the cytoplasm, whereas Lchi13641, Lchi00864 and Lchi11622 are located in chloroplast ( Table 1). The chromosomal distributions of LcPYLs showed that five PYLs were located in five chromosomes of L. chinense (19 chromosomes in total), and Lchi00864, Lchi11622, Lchi13641, Lchi01385 and Lchi16997 were distributed on chromosomes 1, 3, 5, 11 and 15 of L. chinense, respectively ( Figure 1B).

Conserved Domains and Phylogenetic Study of LcPYL Proteins
To confirm the conserved sequences, we compared the protein sequences of all LcPYLs. The result revealed that although a high difference exists in the LcPYL protein sequences, the conserved motifs that are necessary for ABA receptors are included in all LcPYL proteins. A gate motif containing the SGPLA sequence and a latch motif containing the HRL sequence were found to be the common characteristics in all LcPYL proteins (16) (Figure 2A). The gate-latch-lock structure for ABA signaling transduction was also observed in the predicted 3D structures for each LcPYL protein ( Figure 2B-F). The prediction of secondary structures showed that alpha helix and random coil are the most common secondary structures in all LcPYL proteins, followed by extended strand and beta turn ( Table 2). The amino acids with grey shade are similar sequences; ʹ*ʹ indicates that the amino acid residues are completely consistent; ʹ:ʹ represents amino acid residues with particularly similar properties; ʹ.ʹ represents amino acid residues with weakly similar properties. The amino acids with purple shade are hydrophobic. The red box indicates the conserved gate, and the black box indicates the latch residues. The key amino acids of gate and latch are noted with blue arrows. (B-F) Three-dimensional structures of Lchi01385 (B), Lchi00864 (C), Lchi11622 (D), Lchi13641 (E) and Lchi16997 (F) with conserved gate and latch motif for ABA binding were predicted using SWISS MODEL software. Orange and blue arrows represent the conserved gate and latch of ABA signal receptors, respectively. The pink dots indicate the skeleton of key amino acids of gates (SGLPA) and latches (HRL). '*' indicates that the amino acid residues are completely consistent; ':' represents amino acid residues with particularly similar properties; '.' represents amino acid residues with weakly similar properties. The amino acids with purple shade are hydrophobic. The red box indicates the conserved gate, and the black box indicates the latch residues. The key amino acids of gate and latch are noted with blue arrows. (B-F) Three-dimensional structures of Lchi01385 (B), Lchi00864 (C), Lchi11622 (D), Lchi13641 (E) and Lchi16997 (F) with conserved gate and latch motif for ABA binding were predicted using SWISS MODEL software. Orange and blue arrows represent the conserved gate and latch of ABA signal receptors, respectively. The pink dots indicate the skeleton of key amino acids of gates (SGLPA) and latches (HRL). PYLs from Arabidopsis thaliana (A. thaliana, AtPYLs) have been well-studied both in terms of function and structure. Therefore, to further verify the candidate genes from L. chinense as PYL family members, we analyzed the phylogenetic relationship between LcPYLs and AtPYLs and explored the conserved motifs in two groups of PYLs. The phylogenetic study showed that 5 LcPYLs and 14 AtPYLs were classified as three clades in general ( Figure 3A). Clade I includes Lchi01385, Lchi00864, AtPYR1 and AtPYL1-3; Lchi01385 is a sister clade with AtPYR1 and AtPYL1; and Lchi00864 is classified to the subgroup containing AtPYL2 and AtPYL3. Clade II contains Lchi13641, Lchi11622, AtPYL4-6 and AtPYL11-13; Lchi13641 is a sister branch with AtPYL4; and Lchi11622 is a sister branch of AtPYL11-13. Clade III contains Lchi16997 and AtPYL7-10; however, Lchi16997 is closer to AtPYL8 and AtPYL10 ( Figure 3A). Although the result of conserved motif exploration by MEME software with 10 conserved motifs as the cutoff revealed the specific motifs in each group of PYLs, the numbers of all PYLs are no more than five. In addition, all PYLs harbor motifs 1-3, and motif 1 includes the gate and latch sequences for ABA binding ( Figure 3B,C).
To better understand the phylogenetic relationship between LcPYLs and their homologous proteins, the amino acid sequences of PYLs from A. thaliana, Oryza sativa (O. sativa L.) and Vitis vinifera (V. vinifera L.) were used to construct a phylogenetic tree using TBtools (v1.108) and the maximum-likelihood method with 1000 bootstrap replications ( Figure 4). Five LcPYLs, fourteen AtPYLs, twelve OsPYLs and nine VvPYLs were classified into three clades (clade I, clade II and clade III) based on the classification of AtPYLs [12] (Figure 4). Overall, clade I contains 11 PYL members, clade II contains 15 PYLs, and clade III contains 14 PYLs (Figure 4). These results further confirm that the candidates from L. chinense are PYL family genes.

Cis-Acting Element Exploration of LcPYL Promoters
The result of the phylogenetic study and conserved motif analysis revealed the common and unique motifs in different groups of PYLs that may indicate the specific functions of different PYLs. Therefore, to explore the potential function of LcPYLs, we analyzed the cis-acting elements of LcPYL promoters to understand the transcription regulation. To this end, 3000 bp upstream sequences of LcPYL ORFs (including 5 UTR) were extracted from the L. chinense genome to predict cis-acting elements using the PlantCARE database. The results show that the regulatory elements of the LcPYLs were abundant in number and variety. There were six hormone-related cis-acting elements from the prediction, including ABA, MeJA, gibberellin, salicylic acid (SA), ethylene and auxin. All five LcPYL promoters harbor at least two hormone response elements. 'ABRE' and 'AAGAA' identified as ABA response elements were found in all LcPYL promoters as expected ( Figure 5A). Additionally, cis-acting elements involved in stress response were also found in LcPYL promoters. Lchi11622, Lchi00864 and Lchi01385 promoters harbor drought-inducible element 'MBS', whereas Lchi00864 and Lchi13641 promoters contain the cis-acting element involved in defense and stress-responsiveness 'TC-rich repeats' ( Figure 5A). The statistics showed that all LcPYL promoters contain at least six kinds of cis-acting elements related to hormone and multiple stresses ( Figure 5B). The diversities of cis-acting elements in the LcPYL promoters suggest that the transcription of these genes might be regulated by the corresponding hormone and stress factors. To better understand the phylogenetic relationship between LcPYLs and their homologous proteins, the amino acid sequences of PYLs from A. thaliana, Oryza sativa (O. sativa L.) and Vitis vinifera (V. vinifera L.) were used to construct a phylogenetic tree using TBtools (v1.108) and the maximum-likelihood method with 1000 bootstrap replications ( Figure 4). Five LcPYLs, fourteen AtPYLs, twelve OsPYLs and nine VvPYLs were classified into three clades (clade I, clade II and clade III) based on the classification of AtPYLs [12] (Figure 4). Overall, clade I contains 11 PYL members, clade II contains 15 PYLs, and clade III contains 14 PYLs (Figure 4). These results further confirm that the candidates from L. chinens PYL family genes.

Cis-Acting Element Exploration of LcPYL Promoters
The result of the phylogenetic study and conserved motif analysis revealed the mon and unique motifs in different groups of PYLs that may indicate the specific func of different PYLs. Therefore, to explore the potential function of LcPYLs, we analyze cis-acting elements of LcPYL promoters to understand the transcription regulation. To end, 3000 bp upstream sequences of LcPYL ORFs (including 5′UTR) were extracted the L. chinense genome to predict cis-acting elements using the PlantCARE database results show that the regulatory elements of the LcPYLs were abundant in number variety. There were six hormone-related cis-acting elements from the prediction, in ing ABA, MeJA, gibberellin, salicylic acid (SA), ethylene and auxin. All five LcPYL moters harbor at least two hormone response elements. 'ABRE' and 'AAGAA' ident as ABA response elements were found in all LcPYL promoters as expected (Figure Additionally, cis-acting elements involved in stress response were also found in L promoters. Lchi11622, Lchi00864 and Lchi01385 promoters harbor drought-inducible ment 'MBS', whereas Lchi00864 and Lchi13641 promoters contain the cis-acting ele involved in defense and stress-responsiveness 'TC-rich repeats' ( Figure 5A). The stat showed that all LcPYL promoters contain at least six kinds of cis-acting elements re to hormone and multiple stresses ( Figure 5B). The diversities of cis-acting elements i LcPYL promoters suggest that the transcription of these genes might be regulated b corresponding hormone and stress factors.

Expression Pattern of LcPYLs in Different Tissues of L. hybrid
To analyze the expression pattern of PYL genes in different tissues of L. hybrid, previously published RNA-seq data of phloem, stigma, xylem, bud, stamen, leaf, bark and sepal were used for elucidation ( Figure 6). The transcript abundance of Lchi00864 in all harvested tissues was hardly detected. On the contrary, Lchi16997 has high transcription in most of tissues, except phloem and xylem tissues. Lchi13641 expresses in the sepal, bark, leaf, stamen and stigma but not in the phloem, xylem or bud. Lchi11622 specifically transcripts in bark, bud and xylem, whereas Lchi01385 specifically expresses in bark, leaf and stamen but with a relatively lower level than Lchi13641 and Lchi16997 ( Figure 6). These results indicate the potential function of the target proteins on different tissues.

LcPYL Genes Specifically Respond to Abiotic Stresses in L. hybrid
As one of the most important stress-induced phytohormones, ABA has been reported to control adaptive responses toward environmental stresses, such as drought and extreme temperature [29,30]. The PYR/PYL family genes act as the downstream factors of ABA signaling, mediating ABA perception and signal transduction [31]. To study the function of LcPYL genes, we analyzed the gene transcription pattern in L. hybrid under abiotic stresses. According to the reported transcriptome of L. hybrid leaves, the LcPYL genes were observed to differently respond to drought stress simulated with 15% PEG6000. The FPKM values showed that Lchi11622 was highly upregulated under drought stress, Lchi13641 was relatively upregulated, and Lchi01385 and Lchi00864 were not changed obviously; however, Lchi16997 was downregulated ( Figure 7A). To verify the expression pattern of drought-responsive genes, we tested the expression level of Lchi11622 and Lchi13641 in root, stem and leaf of L. hybrid treated with 20% PEG6000 by qPCR ( Figure 7B,C). The results showed that Lchi11622 was only induced by drought in leaves (not in root or stem), and its expression level in leaves under drought stress for 1 h and 3 days, was 6.98 and 12.04 folds of the gene expression in roots under normal condition, respectively ( Figure 7B). The transcription of Lchi13641 was found to be induced in leaves and stems under drought stress, but its expression level in roots was downregulated compared with the gene expression in stems under normal conditions ( Figure 7C). The expression trends of genes in leaves tested by qPCR are in accordance with the transcriptome under drought treatment.

Expression Pattern of LcPYLs in Different Tissues of L. hybrid
To analyze the expression pattern of PYL genes in different tissues of L. hybrid, previously published RNA-seq data of phloem, stigma, xylem, bud, stamen, leaf, bark and sepal were used for elucidation ( Figure 6). The transcript abundance of Lchi00864 in all harvested tissues was hardly detected. On the contrary, Lchi16997 has high transcription in most of tissues, except phloem and xylem tissues. Lchi13641 expresses in the sepal, bark, leaf, stamen and stigma but not in the phloem, xylem or bud. Lchi11622 specifically transcripts in bark, bud and xylem, whereas Lchi01385 specifically expresses in bark, leaf and stamen but with a relatively lower level than Lchi13641 and Lchi16997 ( Figure 6). These results indicate the potential function of the target proteins on different tissues.

LcPYL Genes Specifically Respond to Abiotic Stresses in L. hybrid
As one of the most important stress-induced phytohormones, ABA has been reported to control adaptive responses toward environmental stresses, such as drought and extreme temperature [29,30]. The PYR/PYL family genes act as the downstream factors of ABA signaling, mediating ABA perception and signal transduction [31]. To study the function of LcPYL genes, we analyzed the gene transcription pattern in L. hybrid under abiotic stresses. According to the reported transcriptome of L. hybrid leaves, the LcPYL genes were observed to differently respond to drought stress simulated with 15% PEG6000. The FPKM values showed that Lchi11622 was highly upregulated under drought stress, Lchi13641 was relatively upregulated, and Lchi01385 and Lchi00864 were not changed obviously; however, Lchi16997 was downregulated ( Figure 7A). To verify the expression pattern of drought-responsive genes, we tested the expression level of Lchi11622 and Lchi13641 in root, stem and leaf of L. hybrid treated with 20% PEG6000 by qPCR ( Figure 7B, C). The results showed that Lchi11622 was only induced by drought in leaves (not in root or stem), and its expression level in leaves under drought stress for 1 h and 3 days, was 6.98 and 12.04 folds of the gene expression in roots under normal condition, respectively ( Figure 7B). The transcription of Lchi13641 was found to be induced in leaves and stems under drought stress, but its expression level in roots was downregulated compared with the gene expression in stems under normal conditions ( Figure 7C). The expression trends of genes in leaves tested by qPCR are in accordance with the transcriptome under drought treatment. The transcriptome data of L. hybrid suffering from temperature stresses revealed that the LcPYLs reacted a different level compared with those under drought stress. The transcription of Lchi11622 and Lchi01385 was not obviously changed, and Lchi00864 and Lchi13641 were lightly downregulated; Lchi16997 was the most responsive member and was upregulated under 37 °C for 1 h, 3 h and 3 days ( Figure 8A). Accordingly, the qPCR data showed a similar expression pattern for Lchi16997 ( Figure 8B). However, low-temperature treatment at 4 °C revealed another expression pattern for LcPYL members ( Figure  9A). The expression level of Lchi11622, Lchi01385 and Lchi16997 was upregulated after cold The transcriptome data of L. hybrid suffering from temperature stresses revealed that the LcPYLs reacted a different level compared with those under drought stress. The transcription of Lchi11622 and Lchi01385 was not obviously changed, and Lchi00864 and Lchi13641 were lightly downregulated; Lchi16997 was the most responsive member and was upregulated under 37 • C for 1 h, 3 h and 3 days ( Figure 8A). Accordingly, the qPCR data showed a similar expression pattern for Lchi16997 ( Figure 8B). However, low-temperature treatment at 4 • C revealed another expression pattern for LcPYL members ( Figure 9A). The expression level of Lchi11622, Lchi01385 and Lchi16997 was upregulated after cold treatment, but they responded to cold stress at various treatment stages ( Figure 9A). Lchi16997 responded to cold when treated for 1 h and 3 h; however, Lchi11622 and Lchi01385 exhibited high transcription under cold treatment for 1 day and 3 days ( Figure 9A). Lchi00864 responded to cold 1 h treatment ( Figure 9A). The qPCR data for Lchi11622 and Lchi01385 indicate that these genes positively responded to cold stress, with gene expression further improved by ABA treatment (Figure 9B-E). Compared with normal conditions, the expression levels of Lchi11622 and Lchi01385 in L. hybrid under low temperature for 3 days increased by 14 and 24 times, respectively, and slightly increased under low temperature and ABA treatment for three days compared with only low-temperature treatment. According to the results, we found that Lchi11622 positively responded to cold stress but not hot stress, whereas Lchi01385 positively responded to cold stress but negatively responded to hot treatment. Interestingly, Lchi16997 was found to positively respond to both cold and hot treatment. These results suggest that LcPYLs positively responded to drought, hot and cold stresses but with specific expression patterns in response to various abiotic stresses.

LcPYL Genes Specifically Respond to Biotic Stresses in L. hybrid
To explore the response of LcPYLs to biotic stresses, the L. hybrid leaves infected by scale insect and whitefly were harvested for qPCR tests. The leaves covered by the insects show disease spots that were dark green in the middle and yellow at the margin ( Figure 10A-E). However, the LcPYLs were differentially transcribed in these leaves, as revealed by the results of the qPCR test. The relative expression levels of Lchi13641 and Lchi11622 in infected leaves increased by 1.76-and 3.75-fold relative to normal leaves, respectively. The relative expression levels of Lchi00864 and Lchi01385 in infected leaves were downregulated, while the transcription of Lchi16997 was not changed obviously ( Figure 10F). These results indicate that the LcPYLs from L. hybrid variously responded to biotic stresses caused by scale insect and whitefly.

LcPYL Genes Specifically Respond to Biotic Stresses in L. hybrid
To explore the response of LcPYLs to biotic stresses, the L. hybrid leaves infected by scale insect and whitefly were harvested for qPCR tests. The leaves covered by the insects show disease spots that were dark green in the middle and yellow at the margin ( Figure  10A-E). However, the LcPYLs were differentially transcribed in these leaves, as revealed by the results of the qPCR test. The relative expression levels of Lchi13641 and Lchi11622 in infected leaves increased by 1.76-and 3.75-fold relative to normal leaves, respectively. The relative expression levels of Lchi00864 and Lchi01385 in infected leaves were downregulated, while the transcription of Lchi16997 was not changed obviously ( Figure 10F). These results indicate that the LcPYLs from L. hybrid variously responded to biotic stresses caused by scale insect and whitefly.

Subcellular Localization of LcPYL Genes
To verify the subcellular localization of stress-responsive ABA receptors, Lchi11622, Lchi16997 and Lchi01385 were inserted into the pJIT166-GFP vector to overexpress LcPYL-GFP fusion proteins. As shown in Figure 11, the four fields from left to right are bright field, GFP field, H2B-mChrry field and merged field, and the empty GFP vector is used as the positive control. The results show that Lchi11622, Lchi16997 and Lchi01385 proteins were located in the cytoplasm and nucleus, which is consistent with the reported results of the localization of AtPYLs and OsPYLs family members in the cytoplasm and nucleus. This experiment shows that PYLs proteins are located in similar subcellular structures in different plant species, supporting their roles as ABA receptors.

Subcellular Localization of LcPYL Genes
To verify the subcellular localization of stress-responsive ABA receptors, Lchi11622, Lchi16997 and Lchi01385 were inserted into the pJIT166-GFP vector to overexpress LcPYL-GFP fusion proteins. As shown in Figure 11, the four fields from left to right are bright field, GFP field, H2B-mChrry field and merged field, and the empty GFP vector is used as the positive control. The results show that Lchi11622, Lchi16997 and Lchi01385 proteins were located in the cytoplasm and nucleus, which is consistent with the reported results of the localization of AtPYLs and OsPYLs family members in the cytoplasm and nucleus. This experiment shows that PYLs proteins are located in similar subcellular structures in different plant species, supporting their roles as ABA receptors.

A Limit Number of LcPYL Proteins
PYL family proteins are critical factors for plant adaptation to abiotic and biotic

A Limit Number of LcPYL Proteins
PYL family proteins are critical factors for plant adaptation to abiotic and biotic stresses in the ABA signaling pathway, which have been identified from various plant species [32,33]. In Arabidopsis, 14 AtPYLs have been confirmed as ABA receptors, which were classified into three clades in the phylogenetic analysis [19]. In our study, only five PYL proteins were identified as ABA receptor members from the genome sequence of L. chinense, comprising 42% OsPYLs, 36% AtPYLs and 17% GhPYLs. Nevertheless, the phylogenetic study of LcPYLs and AtPYLs showed that Lchi01385, Lchi00864, AtPYR1 and AtPYL1-3 were classified into clade I; Lchi13641, Lchi11622, AtPYL4-6 and AtPYL11-13 were classified into clade II; and Lchi16997 and AtPYL7-10 were classified into clade III. These results suggest that although the LcPYL members are limited, the types of LcPYLs are complete compared with the classification of AtPYLs [11].
A previous phylogenetic study revealed the interesting correlations between the phylogeny of the MADS-box gene family and functional evolution of plants, which indicates that the genes falling into the same clade trend to function similarly. Therefore, the classification of LcPYLs and AtPYLs might suggest a limited number of LcPYL members but with complete function belonging to this gene family. Our results are consistent with the VvPYLs identification from V. vinifera, which revealed that only nine proteins were identified as PYL family members in the V. vinifera genome [20]; compared with other plant species, VvPYLs comprise 75% OsPYLs [21], 64% AtPYLs [19] and 33% GhPYLs [26].
L. chinense, as a perennial arbor, has shorter iteration times, resulting a lower probability of gene mutants affecting gene duplication than plant species with a short lifespan. For instance, Arabidopsis and rice complete their life in a few months, which leads to the generation of more reproductive processes than L. chinense [34,35]. New generations may evolve for a better adaptability to the environment, resulting in gene mutation and duplication. In a similar manner, V. vinifera is one kind of fruiter but also a perennial plant that has a much longer lifespan but fewer PYL members than Arabidopsis and rice. Therefore, we suppose that a limited number of LcPYL genes might be caused by the fewer iterations during evolutionary history.

LcPYLs Specifically Respond to Various Abiotic Stresses
In our study, five LcPYLs were identified from the genome of L.chinense that were found to specifically respond to different abiotic stresses (Figures 7-9). Lchi11622 and Lchi13641 positively and significantly responded to drought stress simulated by 15% PEG6000; however, the transcription of other LcPYLs was not changed obviously under drought treatment ( Figure 7A). Only the expression level of Lchi16997 was found to be upregulated under 37 • C treatment ( Figure 8A). The expression levels of Lchi11622 and Lchi01385 were upregulated under cold stress, but the expression levels of the other three LcPYLs were not significantly changed ( Figure 9A). These results demonstrate the specific expression of LcPYLs in response to various abiotic stresses. According to the analysis of cisacting elements, we found that the promoters of LcPYLs contain various cis-acting elements in response to hormones, such as auxin, MeJA, salicylic acid and ABA, or abiotic stresses, such as low temperature and drought. These LcPYL promoters share some common cisacting elements but also include their specific elements ( Figure 5B). In our study, we found that all LcPYL promoter sequences contain ABA response element but at different levels. For instance, Lchi00864 has three ABRE elements and is identified as a cis-acting element involved in ABA responsiveness, Lchi01385 contains three ABA-responsive elements (one ABRE element and two AAGAA-motifs) and Lchi16997 has two AAGAA motifs. Lchi13641 and Lchi11622 contain one ABRE element and one AAGAA-motif but different numbers of other hormone or stress cis-elements. All these identities might be the reason for different spatiotemporal responses of LcPYL genes to various environmental stresses [36,37].

Plant Materials and Stress Treatment
In this study, L. hybrid seedlings were generated from callus-based somatic embryogenesis of L. hybrid. The L. hybrid seedlings were planted in pots with soil in a greenhouse at 22 • C under16 h light/8 h dark and 75% relative humidity for four weeks, then irrigated with 15% PEG6000 to simulate drought stress for three days. For temperature stress treatment, the prepared plants were moved into a chamber at 4 • C or 37 • C for three days. In order to study the response of LcPYL genes to ABA, the L. hybrid plants in a cold environment were sprayed with 50 mg/L ABA. The plants cultured under the normal condition were set as the control group. During the stress treatment, the L. hybrid plants were harvested at 1 h, 3 h and 3 d using liquid nitrogen and stored at −80 • C for RNA extraction. To check the transcription of LcPYLs in response to biotic stress, the first and second L. hybrid leaves infected by female whitefly and scale insect were harvested for qPCR testing. The plants grown under normal conditions were taken as the control. The samples were frozen in liquid nitrogen and stored at −80 • C for RNA extraction. Three biological replicates were conducted for each treatment.

Genome-Wide Identification of LcPYL Family Genes
The protein sequences of 14 AtPYLs were downloaded from the Arabidopsis information resource (TAIR 10, https://www.arabidopsis.org/, accessed on 7 August 2022) and employed as queries to search the L. chinense genome data using BLASTp [28]. The obtained proteins were then searched in the Pfam database (https://www.ebi.ac.uk/interpro/entry/ pfam/#table, accessed on 7 August 2022) [38]. The Polyketide_cyc2 domain (PF10604) and Polyketide_cyc domain (PF03364) were found in the 13 harvested LcPYLs. The NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 7 August 2022) database was further used to verify whether the candidate sequences have a PYR/PYL (RCAR)-like domain (cd07821) [39,40]. As a consequence, 6 PYLs were identified from the L. chinense genome. Based on the ABA receptor characteristics, 5 LcPYLs were finally confirmed by the conserved gate and latch motifs for ABA binding [16].

Cis-Acting Elemental Analysis of LcPYL Gene Promoters
In order to explore the cis-acting elements existing in the promoter sequences of LcPYL genes, we used TBtools (v1.108) to extract 3000 bp upstream sequences of LcPYL open reading frames (ORFs) (including the 5 untranslated region) from the L. chinense genome. Then, the obtained promoter sequences of 5 LcPYL genes were submitted to PlantCARE online software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 25 August 2022.) for cis-acting element prediction [49].

Phylogenetic Analysis
The sequence alignment and evolutionary study of 5 LcPYLs and 14 AtPYLs were performed using the maximum likelihood method in Mega 11 in with 1000 bootstrap replications and the JTT model. The AtPYL accession numbers and gene names are shown in Figure 3A. The phylogenetic tree of PYLs from L. hybrid, A. thaliana, O. sativa and V. vinifera was built using TBtools (v1.108) with maximum-likelihood estimation and 1000 bootstrap replications. Phylogenetic tree beautification was performed using iTOL online software (https://itol.embl.de/, accessed on 25 August 2022.) [50].

Quantitative Real-Time PCR Analyses
Quantitative real-time PCR (qPCR) analysis was performed to confirm the response of LcPYLs to drought, cold and hot treatment. The total RNA was extracted from root, stem and leaf tissue of L. hybrid plants treated with 15% PEG6000 at 4 • C and 37 • C, respectively. Total RNA extraction was conducted with a Eastep ® Super Total RNA Purification Kit (Promega, Shanghai, China), followed by genomic DNA digestion using DNase I prepared in the kit. The quality of total RNA was evaluated by ultraviolet spectrophotometry and gel electrophoresis. The cDNA was synthesized using a HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech, Nanjing, China) following the manufacturer's instructions.
qPCR was performed using TB Green ® Premix Ex Taq™ (Takara, Dalian, China) on a LightCycler ® 480 qPCR detection system (Roche, Basel, Switzerland) following the manufacturer's instructions. The expression level of LcPYL genes was normalized according to the expression level of 18S RNA in L. hybrid [51]. Three biological repeats and three experimental replicates were performed for the qPCR test. Specific primers of LcPYL genes and 18S RNA for qPCR are listed in Supplementary Table S1.

Gene Clone and Subcellular Localization Analysis
To verify the subcellular localization of stress-responsive LcPYLs predicted by the WoLF PSORT online tool, specific primers (listed in Supplementary Table S2) of Lchi01385, Lchi11622 and Lchi16997 were designed for PCR amplification using L. hybrid cDNA as templates to obtain their ORFs. The sizes of electrophoresis bands of PCR products were consistent with the expected sizes of the target genes. The target fragments obtained from gel extraction purification were inserted into the pJIT166-GFP vector to transiently express LcPYL-GFP fusion proteins under the control of the 2 × CaMV 35S promoter (35S). Then, each p2 × 35S:LcPYL-GFP vector and p35S:H2B-mCherry vector were simultaneously transformed into L. hybrid protoplasts, which were prepared based on the method proposed by Huo (2017) [52]. After three days, the images of green GFP and red H2B-mCherry fluorescence from the transformed protoplasts were observed and captured by a Zeiss LSM 800 laser scanning confocal microscope.

Conclusions
In this study, we identified five stress hormone ABA receptors from the L. chinense genome through sequence blast and characteristic confirmation that were further verified by sequence alignment and conserved motif analysis. The results revealed that these five LcPYLs contain a conserved gate and latch motif for ABA binding. The study of cis-acting elements existing in LcPYLs promoters indicates the potential response of these LcPYLs to various hormones and stresses. The transcriptome data of L. hybrid leaves and the results of qPCR test revealed the differential expression of LcPYL genes under different stresses, suggesting that these LcPYLs specifically respond to various stresses. The subcellular localizations of these stress-responsive LcPYLs were also analyzed in L. hybrid protoplasts. These results provide a foundation for functional exploration of PYLs from this elite tree species and support the understanding of the molecular mechanism of L. hybrid in coping with stresses. However, there are still some issues that need further verification and resolution. For example, the function of LcPYLs in response to various stresses could be explored by gene overexpression and knockout in L. hybrid. Additionally, the genetic redundancy between LcPYLs also needs to be clarified in this tree species.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12142609/s1, Table S1: Specific primers of genes for quantitative real-time PCR (qPCR) analysis; Table S2: Specific primers designed for the gene clone. Table S3: Protein names and sequences of PYL.