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Article

PYL Family Genes from Liriodendron chinense Positively Respond to Multiple Stresses

by
Xinru Wu
1,2,3,
Junjie Zhu
1,2,3,
Xinying Chen
1,2,3,
Jiaji Zhang
1,2,3,
Lu Lu
1,2,3,
Zhaodong Hao
1,2,3,
Jisen Shi
1,2,3 and
Jinhui Chen
1,2,3,*
1
State Key Laboratory of Tree Genetics and Breeding, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2
Key Laboratory of Forest Genetics and Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
3
College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Plants 2023, 12(14), 2609; https://doi.org/10.3390/plants12142609
Submission received: 29 May 2023 / Revised: 30 June 2023 / Accepted: 7 July 2023 / Published: 11 July 2023
(This article belongs to the Special Issue Molecular Mechanisms of Biotic and Abiotic Stress Tolerance in Plants)
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Abstract

:
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.

1. 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 hormone ABA to reduce water loss by triggering stomata closure [10]. However, although rapid production of ABA in response to environmental stresses is necessary to define ABA as a stress hormone, decoding ABA signaling is also essential in that role. Therefore, the perception and integration of environmental stresses induced by ABA are considered as important research interests for improving plant performance [11].
ABA receptor proteins consist of the pyrabactin resistance1 (PYR1) and PYR1-like (PYL) proteins [12]. Following a series of crystallographic studies, a well-accepted mechanism of ABA recognition by PYLs was proposed. ABA signaling is perceived and transduced by a gate–latch–lock mechanism; the binding of ABA induces conformational changes in the PYL receptor ligand binding pocket. This creates an interaction surface for downstream clade A type 2C protein phosphatases (PP2Cs), leading to the release of class III sucrose non-fermenting 1-related protein kinase 2s (SnRK2s) [13,14,15,16]. Free and activated SnRK2s phosphorylate the downstream targets such as ABA-responsive elements (ABREs)/ABA binding factors (ABFs) [17,18]. This unique ABA perception mechanism reveals the importance of PYLs in the ABA signaling transduction module. Therefore, numerous studies have identified PYR/PYLs from various plant species. A total of 14 AtPYR/AtPYLs were verified from model plant Arabidopsis [19]. A total of 9 PYLs from grape [20], 12 PYLs from rice [21], 14 PYLs from tomato [22], 14 PYLs from rubber tree [23], 14 PYLs from Camellia sinensis [24], 21 PYL homologs from soybean [25], and 27 PYLs from cotton [26] have already been identified by researchers. However, the function of most PYLs is still a conundrum, which might be caused by genetic redundancy in ABA signaling [19].
The Liriodendron genus belongs to the magnolia family, containing one pair of sister species: Liriodendron tulipifera (L. tulipifera) and Liriodendron chinense (L. chinense) [27,28]. Liriodendron hybrid (L. hybrid) was obtained via the cross between L. tulipifera and L. chinense, which is a type of ornamental tree used for landscaping because of its straight trunk, distinctive leaf shape and tulip-shaped flowers. L. hybrid plants are also widely cultivated as industrial timber trees due to their versatile wood with excellent working properties. Furthermore, L. hybrid can survive in areas with harsh environments, such as drought mountains with cold temperature. To investigate the mechanisms of L. hybrid in coping with environmental stresses, in this study, we identified PYLs from the L. chinense genome (LcPYLs) and studied their response to drought, extreme temperature and insect infection. The results of sequence alignments and conserved domain analysis further verified the five isolated candidates as PYL members. The evolutionary study of LcPYLs and their homologous proteins from other plants showed a consistent classification with phylogenetic analysis of AtPYLs. Moreover, the identified LcPYLs were found to specifically respond to drought, hot, cold, and insect attack stresses. Our results provide evidence for the response of L. hybrid ABA receptors under various stresses, providing a foundation for functional exploration of LcPYL genes and better understanding the mechanism of stress tolerance in this relict tree species.

2. Results

2.1. 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 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).

2.2. 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).
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.

2.3. 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.

2.4. 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.

2.5. 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 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.
Plants 12 02609 g008 550
Figure 8. LcPYLs respond to heat stress. (A) The expression levels of LcPYLs are displayed as colored boxes with FPKMs from transcriptome data of L. chinense treated with 37 °C for the indicated times. Red represents a high expression level, and blue represents a low expression level. We marked the differentially expressed genes in the Figure 8A and Figure 9A with green FPKM values for downregulated genes and dark FPKM values for upregulated genes. (B) The relative expression levels of Lchi16997 in plants treated with 37 °C for the indicated times were quantified by qPCR. h: hour; d: day.
Figure 8. LcPYLs respond to heat stress. (A) The expression levels of LcPYLs are displayed as colored boxes with FPKMs from transcriptome data of L. chinense treated with 37 °C for the indicated times. Red represents a high expression level, and blue represents a low expression level. We marked the differentially expressed genes in the Figure 8A and Figure 9A with green FPKM values for downregulated genes and dark FPKM values for upregulated genes. (B) The relative expression levels of Lchi16997 in plants treated with 37 °C for the indicated times were quantified by qPCR. h: hour; d: day.
Plants 12 02609 g008
Plants 12 02609 g009 550
Figure 9. LcPYLs respond to cold stress. (A) The expression levels of LcPYLs are displayed as colored boxes with FPKMs from transcriptome data of L. chinense treated with 4 °C for the indicated times. Red represents a high expression level, and blue represents a low expression level. We marked the differentially expressed genes in the Figure 8A and Figure 9A with green FPKM values for downregulated genes and dark FPKM values for upregulated genes. (B) Phenotype of plants treated with 4 °C or 4 °C combined with 50 mg/L ABA for 3 d. (C) Proportion of plants with wilting leaves under the above treatments. (D,E) The relative expression levels of Lchi11622 (D) and Lchi01385 (E) in plants from (B) were quantified by qPCR. h: hour; d: day.
Figure 9. LcPYLs respond to cold stress. (A) The expression levels of LcPYLs are displayed as colored boxes with FPKMs from transcriptome data of L. chinense treated with 4 °C for the indicated times. Red represents a high expression level, and blue represents a low expression level. We marked the differentially expressed genes in the Figure 8A and Figure 9A with green FPKM values for downregulated genes and dark FPKM values for upregulated genes. (B) Phenotype of plants treated with 4 °C or 4 °C combined with 50 mg/L ABA for 3 d. (C) Proportion of plants with wilting leaves under the above treatments. (D,E) The relative expression levels of Lchi11622 (D) and Lchi01385 (E) in plants from (B) were quantified by qPCR. h: hour; d: day.
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2.6. 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.

2.7. 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.

3. Discussion

3.1. 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.

3.2. 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 (Figure 7, Figure 8 and Figure 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 cis-acting 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 cis-acting 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].

4. Materials and Methods

4.1. 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.

4.2. 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].

4.3. Analysis of Gene Exon–Intron Structures and Protein Conserved Motifs

L. chinense genome data and the CDS of 5 LcPYL genes were uploaded to TBtools (v1.108) for gene structure analysis [41]; the alignment of LcPYLs was conducted using Clustal Omega (v1.2.4) online software [42]. Prediction of 3D structures for LcPYL proteins was conducted using SWISS-MODEL online software (https://swissmodel.expasy.org/, accessed on 25 August 2022) and PyMOL (https://pymol.org/2/, accessed on 25 August 2022) [43,44]. The conserved motifs of 5 LcPYLs and 14 AtPYLs were analyzed by MEME online software (https://meme-suite.org/meme/doc/meme.html, accessed on 7 August 2022) with 10 as the maximum motif number setting and keeping default values for the remaining parameters [45,46].

4.4. Basic Information of LcPYL Proteins

The number of amino acids, molecular weight (MW), isoelectric point (pI), instability index, aliphatic index and grand average of hydropathicity of LcPYLs were studied with ExPASy online software (https://web.expasy.org/protparam/, accessed on 25 August 2022) [47]. The subcellular localization of LcPYL proteins was predicted by Wolf PSORT online software (https://wolfpsort.hgc.jp/, accessed on 25 August 2022) [48]. In addition, the secondary structures of LcPYL proteins were predicted by SOPMA using NPS online software (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html, accessed on 25 August 2022) [40].

4.5. 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].

4.6. 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].

4.7. 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.

4.8. 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.

5. 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.

Author Contributions

Conceptualization, X.W.; methodology, L.L.; software, L.L.; validation, J.Z. (Junjie Zhu); resources, X.C.; formal analysis, J.Z. (Jiaji Zhang); writing—original draft preparation, X.W.; writing—review and editing, X.W., L.L. and Z.H.; supervision, Z.H.; project administration, J.C. and J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Key R&D Program of China during the 14th Five-year Plan Period (2021YFD2200103), the Youth Foundation of the Natural Science Foundation of Jiangsu Province (Grant No. BK20210614) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability Statement

The abiotic stress transcriptome data of L. hybrid are annotated with accession number PRJNA679101 and can be downloaded through NCBI (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA679101/, accessed on 7 August 2022). The complete genome, transcript/protein sequences and genome feature files of Lchi were downloaded from https://www.ncbi.nlm.nih.gov/assembly/GCA_003013855.2, accessed on 7 August 2022.

Acknowledgments

The authors thank Yuhao Weng, Bojun Liao, Yao Tang and all other laboratory colleagues for the constructive discussion and technical support.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The gene architectures and distributions of LcPYLs on L. chinense chromosomes. (A) Architectures of LcPYLs including coding sequences (CDS) and the untranslated region (UTR). (B) The locations of LcPYLs on L. chinense chromosomes from top (start) to bottom (end) according to genome annotation. Chr: chromosome; numbers following ‘Chr’ are the chromosomal numbers.
Figure 1. The gene architectures and distributions of LcPYLs on L. chinense chromosomes. (A) Architectures of LcPYLs including coding sequences (CDS) and the untranslated region (UTR). (B) The locations of LcPYLs on L. chinense chromosomes from top (start) to bottom (end) according to genome annotation. Chr: chromosome; numbers following ‘Chr’ are the chromosomal numbers.
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Figure 2. The sequence alignment and overall structures of LcPYL proteins. (A) Alignment of LcPYL protein sequences by Uniprot online software. The amino acids with grey shade are similar sequences; ‘*’ 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).
Figure 2. The sequence alignment and overall structures of LcPYL proteins. (A) Alignment of LcPYL protein sequences by Uniprot online software. The amino acids with grey shade are similar sequences; ‘*’ 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).
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Figure 3. Phylogenetic study and conserved motif analysis for LcPYL proteins. (A) Phylogenetic relationship between 5 LcPYLs and 14 Arabidopsis PYLs. The phylogenetic tree was generated using the maximum likelihood method in Mega 5 with 1000 bootstraps. (B) Conserved motif distributions of PYL proteins. The 10 boxes with different colors on the right side indicate 10 conserved motifs. (C) The conservative sequences of 10 conserved motifs from (B). The black and blue boxes represent the conserved gate and latch of ABA signal receptors, respectively.
Figure 3. Phylogenetic study and conserved motif analysis for LcPYL proteins. (A) Phylogenetic relationship between 5 LcPYLs and 14 Arabidopsis PYLs. The phylogenetic tree was generated using the maximum likelihood method in Mega 5 with 1000 bootstraps. (B) Conserved motif distributions of PYL proteins. The 10 boxes with different colors on the right side indicate 10 conserved motifs. (C) The conservative sequences of 10 conserved motifs from (B). The black and blue boxes represent the conserved gate and latch of ABA signal receptors, respectively.
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Figure 4. Phylogenetic study of PYLs from L. chinense and other plant species. The phylogenetic tree of LcPYL family genes and their homologous genes from A. thaliana, O. sativa and V. viniferaother was constructed using Mega 11 via the maximum likelihood method with 1000 bootstrap replications and the JTT model. Red gene IDs represent the genes from L. chinense.
Figure 4. Phylogenetic study of PYLs from L. chinense and other plant species. The phylogenetic tree of LcPYL family genes and their homologous genes from A. thaliana, O. sativa and V. viniferaother was constructed using Mega 11 via the maximum likelihood method with 1000 bootstrap replications and the JTT model. Red gene IDs represent the genes from L. chinense.
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Figure 5. Cis-acting elements existing in the promoters of LcPYLs. (A) The distributions of cis-acting elements involved in various hormones and stresses are displayed with different color boxes. 3 kb upstream sequences of LcPYL ORFs including 5′UTR were extracted from the L. chinense genome for cis-acting elemental analysis. (B) Numbers of cis-acting elements from (A).
Figure 5. Cis-acting elements existing in the promoters of LcPYLs. (A) The distributions of cis-acting elements involved in various hormones and stresses are displayed with different color boxes. 3 kb upstream sequences of LcPYL ORFs including 5′UTR were extracted from the L. chinense genome for cis-acting elemental analysis. (B) Numbers of cis-acting elements from (A).
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Figure 6. Expression level of LcPYL genes in different tissues of L. chinense. The transcriptome data of various L. chinense tissues including phloem, stigma, xylem, bud, stamen, leaf, bark and sepal reveal the expression levels of LcPYL genes. FPKM, fragments per kilobase million reads.
Figure 6. Expression level of LcPYL genes in different tissues of L. chinense. The transcriptome data of various L. chinense tissues including phloem, stigma, xylem, bud, stamen, leaf, bark and sepal reveal the expression levels of LcPYL genes. FPKM, fragments per kilobase million reads.
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Figure 7. LcPYLs respond to drought stress. (A) The expression levels of LcPYLs are displayed as colored boxes with FPKMs from transcriptome data of L. chinense treated with drought stress simulated with 15% PEG6000. Red represents a high expression level, and blue represents a low expression level. We marked the differentially expressed genes in the Figure 8A and Figure 9A with green FPKM values for downregulated genes and dark FPKM values for upregulated genes. (B,C) The relative expression levels of Lchi11622 (B) and Lchi13641 (C) in root, stem and leaf treated with 20% PEG6000 for 0 h, 1 h and 3 d, respectively, were quantified with qPCR. h: hour; d: day.
Figure 7. LcPYLs respond to drought stress. (A) The expression levels of LcPYLs are displayed as colored boxes with FPKMs from transcriptome data of L. chinense treated with drought stress simulated with 15% PEG6000. Red represents a high expression level, and blue represents a low expression level. We marked the differentially expressed genes in the Figure 8A and Figure 9A with green FPKM values for downregulated genes and dark FPKM values for upregulated genes. (B,C) The relative expression levels of Lchi11622 (B) and Lchi13641 (C) in root, stem and leaf treated with 20% PEG6000 for 0 h, 1 h and 3 d, respectively, were quantified with qPCR. h: hour; d: day.
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Figure 10. LcPYLs variously respond to biotic stresses. (A–D) Phenotype of L. hybrid leaves under normal conditions or infected with scale insect and whitefly. (E) Two types of insects (scale insect and whitefly) causing the biotic stresses in this study. (F) The relative expression levels of PYLs in L. hybrid leaves from (A–D) were tested by qPCR.
Figure 10. LcPYLs variously respond to biotic stresses. (A–D) Phenotype of L. hybrid leaves under normal conditions or infected with scale insect and whitefly. (E) Two types of insects (scale insect and whitefly) causing the biotic stresses in this study. (F) The relative expression levels of PYLs in L. hybrid leaves from (A–D) were tested by qPCR.
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Figure 11. Subcellular localization of stress-responsive LcPYLs including Lchi11622, Lchi16997 and Lchi01385 in L. chinense protoplast. The cells overexpressing LcPYL-GFP fusion protein and H2B-mCherry (nuclear marker protein) are shown with fields as indicated. The vector only with GFP overexpression was taken as the positive control. Scale bar: 5 µm.
Figure 11. Subcellular localization of stress-responsive LcPYLs including Lchi11622, Lchi16997 and Lchi01385 in L. chinense protoplast. The cells overexpressing LcPYL-GFP fusion protein and H2B-mCherry (nuclear marker protein) are shown with fields as indicated. The vector only with GFP overexpression was taken as the positive control. Scale bar: 5 µm.
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Table
Table 1. Characteristics of LcPYL proteins.
Table 1. Characteristics of LcPYL proteins.
Gene IDLchi01385Lchi00864Lchi11622Lchi13641Lchi16997
aa 1204215178209197
MW (kd) 222.6623.7519.7522.5522.53
pI 355.915.747.655.75
Instability index38.3133.2860.2148.3247.14
Aliphatic index78.288685.990.4390.41
GRAVY 4−0.415−0.226−0.157−0.056−0.427
Subcellular locationCytoplasmChloroplastChloroplastChloroplastCytoplasm
1 Amino acid; 2 molecular weight; 3 isoelectric point; 4 grand average of hydropathicity.
Table
Table 2. Secondary structure prediction of LcPYL proteins.
Table 2. Secondary structure prediction of LcPYL proteins.
Lchi01385Lchi00864Lchi11622Lchi13641Lchi16997
Alpha helix %35.78
(73 aa 1)		37.67
(81 aa)		42.7
(76 aa)		32.06
(67 aa)		40.61
(80 aa)
Extended strand %18.63
(38 aa)		19.07
(41 aa)		20.22
(36 aa)		19.14
(40 aa)		17.26
(34 aa)
Beta turn %4.41
(9 aa)		6.05
(13 aa)		4.49
(8 aa)		4.78
(10 aa)		4.06
(8 aa)
Random coil %41.18
(84 aa)		37.21
(80 aa)		32.58
(58 aa)		44.02
(92 aa)		38.07
(75 aa)
1 Amino acid.
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MDPI and ACS Style

Wu, X.; Zhu, J.; Chen, X.; Zhang, J.; Lu, L.; Hao, Z.; Shi, J.; Chen, J. PYL Family Genes from Liriodendron chinense Positively Respond to Multiple Stresses. Plants 2023, 12, 2609. https://doi.org/10.3390/plants12142609

AMA Style

Wu X, Zhu J, Chen X, Zhang J, Lu L, Hao Z, Shi J, Chen J. PYL Family Genes from Liriodendron chinense Positively Respond to Multiple Stresses. Plants. 2023; 12(14):2609. https://doi.org/10.3390/plants12142609

Chicago/Turabian Style

Wu, Xinru, Junjie Zhu, Xinying Chen, Jiaji Zhang, Lu Lu, Zhaodong Hao, Jisen Shi, and Jinhui Chen. 2023. "PYL Family Genes from Liriodendron chinense Positively Respond to Multiple Stresses" Plants 12, no. 14: 2609. https://doi.org/10.3390/plants12142609

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