Introduction

Lipids are critical for maintaining cellular membrane composition and integrity and for storing energy for use in diverse cellular, developmental, and physiological events. In plants, the subcellular trafficking of lipids and their derivatives are pivotal for cellular signal perception and transduction, photosynthesis, and multiple metabolic processes (Ohlrogge and Browse 1995). Acyl-CoA-binding proteins (ACBPs) belong to an important housekeeping protein family involved in intracellular trafficking of acyl-CoA esters and in the formation and maintenance of a cytosolic acyl-CoA pool (Xiao and Chye 2011a).

On the basis of phylogenetic analysis, ACBPs have been proposed to be present in more than 66 plant species. They were experimentally identified in 27 species, ranging from algae, moss, ferns, and gymnosperms to angiosperms (Lung and Chye 2016; Wang et al. 2019). Plant ACBPs have been extensively investigated in two models, the eudicot Arabidopsis thaliana and the monocot Oryza sativa (hereafter, Arabidopsis and rice; Du et al. 2016; Lung and Chye 2016; Ye and Chye 2016). Based on the molecular mass, domain architecture, and phylogeny, plant ACBPs are differentiated into four classes including small ACBPs (class I), ankyrin-repeat ACBPs (class II), large ACBPs (class III), and kelch-ACBPs (class IV) (Lung and Chye 2016), which play nonredundant roles in different tissues and organs (Meng et al. 2011). The expression patterns of six A. thaliana ACBPs and six rice ACBPs and data from other molecular genetic analyses indicate that they have versatile functions at the cellular and subcellular levels, including transport of lipids (i.e., phospholipids, phosphatidylcholine, and acyl-CoA ester), saturated fatty acid metabolism, fatty acid β-oxidation, vesicular trafficking, and signal transduction during disparate developmental processes (e.g., pollen and seed development, seed gemination, and cuticle formation). They also participate in plant responses to diverse biotic and abiotic stresses such as ABA, indole-3-butyric acid, ethanol, heavy metal, hypoxia, cold, wounding, drought, salt, oxidative stress, and pathogen infection (Gao et al. 2009; Fan et al. 2010; Xiao and Chye 2011a; Liu et al. 2015; Du et al. 2016; Qin et al. 2016; Ye and Chye 2016; Narayanan et al. 2019, 2020; Wang et al. 2019; Zhu et al. 2021).

The diverse and specific functions of different ACBP members depend on their subcellular localization and spatiotemporal expression. AtACBPs and OsACBPs localize in a variety of subcellular components, such as the plasma membrane (PM) (Chye et al. 1999; Gao et al. 2009, 2010a, 2010b; Chen et al. 2010; Napier and Haslam 2010; Licausi et al. 2011; Du et al. 2013b), cytosol (Chen et al. 2008; Yurchenko et al. 2009, 2014; Hsiao et al. 2014; Meng et al. 2014), vesicle (Chye et al. 1999), endoplasmic reticulum (ER) reticulum/ space (Chye et al. 1999; Leung et al. 2006; Xiao et al. 2010; Xiao and Chye 2011b; Xia et al. 2012; Takato et al. 2013; Xue et al. 2014; Xie et al. 2015), endomembrane (Chye et al. 1999; Li and Chye 2003; Licausi et al. 2011; Du et al. 2013b), Golgi apparatus (Li and Chye 2003, 2004; Gao et al. 2009, 2010a; Licausi et al. 2011), chloroplast (Meng et al. 2014), peroxisome (Meng et al. 2014), nuclear periphery (Li et al. 2008), cytoskeleton (Takato et al. 2013), and apoplast (Leung et al. 2006; Xiao et al. 2010; Pastor et al. 2013; Lung and Chye 2016). Moreover, expression of plant ACBP members from Arabidopsis, rice, Cucumis sativus, Cucurbita maxima, Brassica napus, and Cocos nucifera has been detected in embryos, stem epidermis, guard cells, pollen, and phloem sap. Their expression has been linked to their specific functions in early embryogenesis (Chye et al. 1999; Chen et al. 2010; Du et al. 2013a, b; Guo et al. 2019), cuticle formation (Xue et al. 2014), drought response (Du et al. 2013a), pollen development (Hsiao et al. 2015), and systemic lipid transport (Walz et al. 2004; Suzui et al. 2006; Guelette et al. 2012). Results from model plant species have facilitated the understanding of the diverse representative functions of ACBPs.

However, key scientific issues underlying tree-specific traits, such as wood formation, long-term perennial growth, and seasonality, cannot be easily addressed using Arabidopsis or rice (Jansson and Douglas 2007). In many respects, trees are physiologically and genetically distinct from Arabidopsis due to their multiple origins throughout land plant evolution (Jansson and Douglas 2007).

Poplar (Populus spp.) is an important economic and ecological species (Bradshaw et al. 2000), a model system for tree and woody perennial plant biology, and relatively close phylogenetically to Arabidopsis in the angiosperm clade eurosid. The completion of a draft sequence of the black cottonwood (Populus trichocarpa) genome facilitated the rapid collection of genomic and molecular genetics data for Populus and the comparative analyses of Arabidopsis and Populus (Tuskan et al. 2006; Jansson and Douglas 2007). Comparative genomics and functional studies on Populus have provided important information on the mechanism of wood development, vascular cambium formation, seasonality, phenology, flowering, various stress responses, and interactions with other organisms (Jansson and Douglas 2007; Xu et al. 2021). However, the genome-wide ACBP gene family has not been comprehensively evaluated in P. trichocarpa.

In the present genome-wide analysis of ACBP genes in P. trichocarpa, we identified eight PtACBP genes and analyzed the phylogeny, gene structure, conserved motifs, chromosome locations, and expression patterns in different tissues and organs and in response to multiple abiotic stresses. These results will provide a theoretical basis for further investigation into the potential roles of PtACBP genes in growth, development, and stress response.

Materials and methods

Identification of ACBP family in P. trichocarpa

To identify ACBPs in P. trichocarpa, the protein database of Populus was downloaded from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html) (Goodstein et al. 2012). The updated Hidden Markov Model (HMM) profile file (ACB.hmm) for the acyl-CoA-binding domain (ACB) (PF00887) was downloaded from the Pfam database (http://pfam.xfam.org/), and then used in a searched of the poplar genome database. The amino acid sequences for six Arabidopsis acyl-CoA-binding proteins, AtACBP1 (NP_200159.1), AtACBP2 (OAP00506.1), AtACBP3 (Q9STX1.1), AtACBP4 (OAP02573.1), AtACBP5 (AED93708.1), and AtACBP6 (AEE31396.1) were used as the query sequences in a BLASTP search against the online genome database of P. trichocarpa. The ACB domain at the N-terminus, the ankyrin (ANK) domain, and the Kelch domain at the C-terminus were used as search criteria (Meng et al. 2011). The characteristics of amino acid sequences, such as molecular weight, isoelectric point (pI), number of amino acids, aliphatic index, chromosomal location, and grand average of hydropathicity GRAVY score, were determined using the online ExPASy program (http://www.expasy.org/). Cellular localization was determined using the online package Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/), WoLF PSORT (http://www.genscript.com/wolf-psort.html) and MultiLoc2 (http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc2).

Chromosomal locations of ACBP genes

The chromosomal location of the PtACBP genes was obtained from the Phytozome database and mapped using MapGene2Chrom web v2 (http://mg2c.iask.in/ mg2c_ v2.0/).

Structural analysis of ACBP proteins

The conserved domains of ACBPs in Arabidopsis, P. trichocarpa, and Picea sitchensis were referenced in the NCBI database (https://www.ncbi.nlm.nih.gov/protein/). IBS software (Liu et al. 2005) was used to map the protein primary structure.

Exon/intron structure and conserved motifs analysis

The Gene Structure Display Server (GSDS2.0, http://gsds.cbi.pku.edu.cn) was used to predict the distribution patterns of exons and introns in the PtACBP genes. Conserved motifs were identified in PtACBP genes by using the online MEME tool (http://meme-suite.org/tools/meme) with the options maximum distinct motifs to identify = 10, minimum width = 7, and maximum width = 50.

Analysis of cis-acting elements

Promoter sequences of the PtACBP family genes were downloaded from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html). The PlantCARE online database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) was used to predict regulatory elements. The GSDS2.0 website was used to visualize cis-acting elements.

Phylogenetic analysis and sequence alignments of plant ACBPs

In the NCBI protein database, the plant was searched by keyword " ACBP " to obtain ACBP protein in plants (https://www.ncbi.nlm.nih.gov/protein/), the ACBP protein sequences in woody plants were manually screened, and then these sequences had been multi-sequence alignment, After removing the same sequence in the same species (identify (A,B) > 95% & identify (B, A) > 95%), and after removed the protein sequence of the amino acid sequence length < 60, 125 sequences from 23 species were finally obtained, including eight ACBP proteins of P. trichocarpa. The sequences were phylogenetically analyzed using ClustalW (PMID: 17,846,036) with default parameters (gap opening penalty = 10, gap extension penalty = 0.2) and a tree constructed in MEGA 7 (https://www.megasoftware.net) (Larkin et al. 2007) using the maximum likelihood method (Kumar et al. 2016) and 1000 bootstrap replications. In total, 11 lant protein sequences consisting of an ACB domain were obtained using a detailed BLAST search of nonredundant protein sequences from the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments of PtACBP and amino acid sequences from other plant species were performed using Bioedit software (Alzohairy 2011).

Plant materials, RNA extraction, cDNA synthesis, and RT-qPCR

P. trichocarpa sterile plantlets were grown in the State Key Laboratory of Tree Genetics and Breeding of Northeast.

Forestry University, Harbin, China. Sterile tissue of P. trichocarpa seedlings was cultured for 3 weeks with 16 h light/8 h dark at 25 °C. Samples from the roots, stems, and leaves were collected, immediately frozen in liquid nitrogen, and then stored at − 80 °C for further analysis. For abiotic stress assays, 3-week-old seedlings were transferred to 1/2 MS medium containing 200 mM NaCl, 100 mM NaHCO3, or 75 mM Na2CO3. The roots and leaves were harvested at 6, 12, 24, and 48 h after the start of the stress treatments, and then stored at − 80 °C for PCR analysis.

Total RNA was extracted using TRIzol reagent (Takara Bio, RNAiso Plus 9109, JPN) following the manufacturer’s protocol. RNA quality was detected using a UV spectrophotometer (BioMate 3S, Thermo Fisher Bio, USA). The PrimeScript RT Reagent Kit (Takara Bio, Perfect Real Time, RR047A, JPN) was used for cDNA synthesis. Three biological replicates of the cDNA samples were analyzed by qRT-PCR with primers for PtACBP genes designed using Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) (Supplemental Table S1). The qRT-PCRs were run in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using SYBR Premix Ex Taq II (Takara Bio, RR820A, JPN). The total volume of the reaction is 20 μl, and the reaction conditions are as follows: 95 °C, 10 min, then 95 °C, 15 s, 57 °C, 20 s, 72 °C, 35 s for 40 cycles. Three biological replicates of each treatment were conducted.

Expression of PtACBP genes in tissues and organs of P. trichocarpa

Tissue- and organ-specific expression data of PtACBP genes in mature leaves, young leaves, roots, nodes, and internodes were derived from PopGenIE (http://popgenie.org) and used to generate visualizations (Sundell et al. 2015).

Results

Identification of ACBP proteins and chromosome localization of ACBP genes in P. trichocarpa

The HMMER searches using the ACB domain profile PF00887 as a query against the P. trichocarpa protein database (http://pfam.xfam.org/) generated 22 candidate proteins, and BLASP search using the six Arabidopsis ACBP proteins as queries resulted in 30 candidates. Then, the 22 overlapping candidates were screened for Ankyrin and Kelch domains, which generated eight ACBP proteins in P. trichocarpa. Eight PtACBPs were designated as PtACBP1 to PtACBP8, with locus numbers in the P. trichocarpa gene database of Phytozome v3.0 of Potri.003G103700.1, Potri.001G130200.1, Potri.012G017700.1, Potri.015G010200.1, Potri.002G120200.1, Potri.014G018700.1, Potri.005G026900.1, and Potri.013G018800.1, respectively (Table 1). The protein molecular weight, pI, number of amino acids, aliphatic index, chromosomal location, and grand average of hydropathicity GRAVY score are given in Table 1.

Table 1 Information on members of the PtACBP family in the Populus trichocarpa genome
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Interestingly, on the basis of Populus genome database, eight PtACBP genes were physically mapped on chromosome 1, 2, 3, 5, 12, 13, 14, and 15, respectively, but not on the other seven chromosomes of P. trichocarpa (Fig. 1). This distribution pattern implied a distinct function for PtACBP genes, which needs to be further investigated.

Fig. 1
figure 1

Physical map of eight ACBP genes in Populus trichocarpa chromosome. The diagram was drawn using the MapGene2Chrom web v2 software, and the eight PtACBP genes were located on different chromosomes

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The PtACBP characterization and subcellular localization prediction

The eight members of PtACBPs differed considerably in the number of amino acids, ranging from 116 to 686 amino acids (aa; Table 1). Among the eight, PtACBP7 and PtACBP8 were relatively larger (677 and 686 aa, respectively), and PtACBP1 (116 aa) and PtACBP2 (117 aa) were the smallest (Table 1). Their molecular weights varied from 13.24 kDa for PtACBP1 to 75.13 kDa for PtACBP8, and their theoretical pIs ranged from 4.31 (PtACBP6) to 9.10 (PtACBP2).

The subcellular localization analysis of the PtACBPs predicted PtACBP1, PtACBP2, PtACBP3, PtACBP4, PtACBP6, PtACBP7, and PtACBP8 to be in the nucleus; PtACBP1, PtACBP3, PtACBP5, PtACBP7 and PtACBP8 in the cytoplasm; PtACBP5 and PtACBP6 in the chloroplasts; and PtACBP3, PtACBP5 and PtACBP6 in the mitochondria (Supplemental Table S2).

Architectural comparisons of ACBP domains from P. trichocarpa, Picea sitchensis, and A. thaliana

Based on domain compositions and amino acid sequence lengths, the eight PtACBPs were divided into four subfamilies (Fig. 2). All the PtACBPs had an ACB domain, and some had either an Ankyrin domain or a Kelch domain. The 10-kDa PtACBP1 (116 amino acids) and PtACBP2 (117 amino acids) had only an ACB domain and were categorized as Small-ACBPs in Class I. PtACBP3 (246 amino acids) and PtACBP4 (353 amino acids) had an ankyrin-repeat domain and were classified as ankyrin-repeat ACBPs (ANK-ACBP) in Class II. Two of the proteins with molecular weights greater than 40 kDa, PtACBP5 (364 amino acids) and PtACBP6 (427 amino acids), were categorized as Large-ACBP in Class III, and had an ACB domain in the middle. In addition, PtACBP7 (677 amino acids) and PtACBP8 (686 amino acids) had N-terminal ACB domains and at least one kelch domain and were classified as Kelch-ACBPs in Class IV (Fig. 2).

Fig. 2
figure 2

Schematic domain structures of ACBPs from Arabidopsis thaliana, Populus trichocarpa, and Picea sitchensis. Green indicates the acyl-CoA-binding domain (ACB), yellow indicates the ankyrin repeats domain (ANK), and pink indicates the kelch domain (Kelch). The “ + ” indicates that the actual number of ACBPs may be greater because sequencing of the genome is still incomplete

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When comparing the PtACBPs with those from A. thaliana and P. sitchensis, we found that the amino acid sequences of PtACBPs are more similar to those of A. thaliana, and the amino acid identities in each class between P. trichocarpa and A. thaliana were as follows: classes I (73%–74%), II (66%–73%), III (40%) and IV (72%–76%). Interestingly, in P. sitchensis, PsACBP1 has only the ACB domain and is in Class 0, and there were no PsACBPs in Class III. However, A. thaliana or P. trichocarpa had no ACBPs in Class 0 (Fig. 2). ACBPs in Class 0 have an ACB domain similar to those in Class III in higher plants, but they have fewer amino acids than in Class III members. Moreover, both A. thaliana and P. trichocarpa have two ACBPs in Class II and Class IV, respectively, but P. sitchensis only has one in each (Fig. 2). In addition, A. thaliana ACBP families have only one member in class I (AtACBP6) and class III (AtACBP3), but the PtACBP family has two members in class I (PtACBP1, PtACBP2) and class III (PtACBP5 and PtACBP6. These results imply that ACBP families arose during the evolution of lower plants to higher plants (Meng et al. 2011).

Analysis of gene structure and distribution of conserved motifs

In the analysis of the structure of the eight PtACBP genes using GSDS2.0 and comparison of the mRNA and genomic DNA nucleotide sequences, exon length was found to be mostly conserved, and the number of exons was similar among each subfamily except Class I (Fig. 3). In Class I, two exons were found in PtACBP1 (122 and 220 bp) and four exons in PtACBP2 (95, 117, 62, and 79 bp). In Class II, PtACBP3 and PtACBP4 contained six exons, five of which (the 2nd to the 6th) were similar in length (105, 100–103, 91, 97, 170 bp, respectively). Similarly, in Class IV, both PtACBP7 and PtACBP8 have 18 exons. In Class III, PtACBP5 and PtACBP6 have five and six exons, respectively, and four exons were mostly conserved (778–817, 105, 100, 74 bp, respectivel). Importantly, the length and position of introns varied considerably (Fig. 3). These results indicate that there is a close relationship between the phylogeny and gene structure of PtACBP genes.

Fig. 3
figure 3

Phylogenetic tree and structure of eight ACBP genes found in Populus trichocarpa. The tree was constructed using the neighbor-joining method using 1000 replicates; bootstrap percentages are shown at the branches

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Ten conserved motifs of PtACBP family members were identified using online MEME software and named motif 1 to 10 (Fig. 4A, B). Subfamily members had similar motif types and arrangements (Fig. 4A). Class I contained only one motif; this single-motif model was exclusive to this PtACBP subfamily. Class II had three motifs, Class III had two, and Class IV had 10. Importantly, motifs 4, 5, 7, 8, 9, and 10 only appeared in Class IV; thus, these motifs can serve to identify PtACBP members in Class IV (Fig. 4A).

Fig. 4
figure 4

Predicted conserved motifs and amino acid sequences of the eight ACBPs found in Populus trichocarpa. A Classes of eight PtACBPs based on conversed motifs and corresponding p-values. Motifs are in different colors, which are defined in B. B Amino acid sequence for each motif. Motifs were predicted by MEME motif searching, and 10 were selected as the maximum number parameter

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Analysis of PtACBP promoters for cis-acting element

The 2000-bp sequence upstream of the initial transcription position was analyzed to identify various cis-acting elements using the Plant CARE website. The distribution of 30 cis-acting elements in eight PtACBPs and their distinct functions are shown in Fig. 5.

Fig. 5
figure 5

Predicted cis-elements in the promoter region (2000 bp upstream from the transcription initiation site) of ACBP genes from Populus trichocarpa. The scale bar at the bottom indicates the length of the promoter sequence. Different colors indicate the functions of the cis-acting elements

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Among the light-signaling elements found in the PtACBP genes were G-box, Box 4, Gap-box, GT1-motif, AE-box, TCT-motif, TCCC-motif, MRE, AT1-motif, I-box, GATA-motif, AAAC-motif, Box II, L-box, AE-box, Sp1, chs-CMA1a, and ACE (Table S3). Seven cis-elements were classified as involved in hormone responses: ABRE, P-box, GARE-motif, TGA-element, CGTCA-motif, TGACG-motif, and TCA-element (Table S3). For instance, all PtACBP genes except for PtACBP7 contained the ABA-responsive element ABRE (Fig. 5). Four PtACBP genes (i.e., PtACBP2, PtACBP3, PtACBP6, and PtACBP7) contained gibberellin-responsive P-boxes and GARE-motifs, and four genes (i.e., PtACBP2, PtACBP4, PtACBP5, and PtACBP8) contained auxin-responsive element TGAs (Fig. 5). In addition, five genes (i.e., PtACBP2, PtACBP3, PtACBP5, PtACBP7, and PtACBP8) contained MeJA responsive elements CGTCA-motifs and TGACG-motifs, and six genes (i.e., PtACBP2, PtACBP3, PtACBP5, PtACBP6, PtACBP7, and PtACBP8) contained salicylic-acid-responsive elements TCAs (Fig. 5). Also found in different PtACBP members were cis-elements such as MBS, ARE, LTR, GC-motif, and TC-rich repeats that are related to various stress responses. For example, PtACBP2, PtACBP3, PtACBP5, and PtACBP6 had drought-inducible elements (MBS), seven genes (i.e., PtACBP1 to PtACBP4, PtACBP6 − 8) had the anaerobic induction element ARE, and four genes (PtACBP1, PtACBP2, PtACBP4, PtACBP8) had a low temperature responsive (LTR) element (Fig. 5). PtACBP5 contained anoxia-specific inducibility element GC-motifs (Fig. 5). These results imply that PtACBPs are critical in P. trichocarpa for diverse stress responses.

Phylogenetic analyses of ACBPs from 23 plant species

In the phylogenetic tree based on 125 ACBP sequences from 23 plant species using the maximum likelihood (ML) method, ll ACBPs clustered into five groups: Class 0, Class I, Class II, Class III, and Class IV, which contained 2, 36, 26, 32, and 29 members, respectively (Fig. 6). High-bootstrap support was obtained for classes 0, I, III, and IV, and ACBP Class I was basal to the other classes (Fig. 6). Class II was the least-supported clade with a bootstrap value of 47% (Fig. 6). PtACBP1 (XP_002303469.1) and PtACBP2 (XP_006368280.2) grouped into Class I, PtACBP3 (PNT08967.1) and PtACBP4 (XP_002321391.1) into Class II, and PtACBP5 (XP_024449804.1) and PtACBP6 (XP_006374836.1) grouped into Class III (Fig. 6). Additionally, PtACBP7 (XP_024458146.1) and PtACBP8 (XP_006375749.1) were grouped into Class IV (Fig. 6). Notably, seven of eight PtACBPs (i.e., PtACBP1, PtACBP3, PtACBP4, PtACBP5, PtACBP6, PtACBP7 and PtACBP8) except PtACBP2 were clustered closely with PeACBPs from Populus euphratica, indicating a close evolutionary relationship between PtACBPs and PeACBPs.

Fig. 6
figure 6

Phylogenetic tree based on complete amino acid sequences of 125 plant acyl-CoA-binding proteins (ACBPs) using the maximum likelihood in MEGA-X. The ACBPs grouped into four classes. Accession number for each gene is shown after the species code in the tree. Bootstrap replicates = 1000

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Sequence alignment of the ACB domain of ACBPs from different plant species

Amino acid sequence variations could influence binding affinities of ACBPs to acyl-CoA esters (Emanuelsson et al. 2000; Xie et al. 2015). Two conserved motifs of YKQA and KWDAW in ACBPs were previously considered as essential motifs for binding acyl-CoA esters (Kragelund et al. 1993; Xiao and Chye 2011a). In this study, multiple sequence alignment analysis indicated that amino acid sequences of eight PtACBPs had similarities with those from other plant species (Fig. 7). We found two conserved motifs in PtACBPs when compared with other plant species (Fig. 7). Importantly, the alignment analysis also revealed four conserved alpha-helices (H1-H4) containing five highly conserved potential binding sites for acyl-CoA esters in PtACBPs (Fig. 7). In addition, whether H4, a highly conserved site of alanine (Fig. 7), is critical for binding acyl-CoA esters still needs to be investigated.

Fig. 7
figure 7

Sequence alignments of ACB domains from the PtACBP family and other species. Blue circles show residues identical in all ACBPs. Arrowheads indicate potential binding sites for acyl-CoA esters; H1–H4 indicate the positions of four putative alpha-helices. motifs YKQA and KWDAW are underlined in red. Pt, Populus trichocarpa; At, Arabidopsis thaliana; Cr, Chlamydomonas reinhardtii; Ol, Ostreococcus lucimarinus; Pp, Physcomitrella patens; Sm, Selaginella moellendorffii; Ps, Picea stichensis; Os, Oryza sativa; Hb, Hevea brasiliensis; Pa, Populus alba; Pe, Populus euphratica

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Tissue- and organ-specific expression of PtACBPs

The qRT-PCR analyses of the PtACBP genes in roots, stems, and young leaves from P. trichocarpa seedlings in sterile tissue culture showed that expression of all the PtACBP genes was highest in the leaves, lowest in the stems, and intermediate in the roots (Fig. 8A). Interestingly, expression of PtACBP5 and PtACBP2 was relatively higher than the other PtACBP genes in the leaves (Fig. 8A).

Fig. 8
figure 8

Organ-specific expression profiles of PtACBP genes in Populus trichocarpa. A Expression in roots, stems, and leaves of 49-day-old seedlings. Data showed the mean values ± SE (p < 0.05). B Diagrams to show expression levels in mature leaves, young leaves, roots, nodes, and internodes. Profiles for organ-specific expression data were derived from http://PlantGenIE.org

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In addition, the tissue-specific expression patterns of PtACBP genes in mature leaves, young leaves, roots, nodes, and internodes were also predicted and visualized based on the data for PtACBP genes from the Plant Genome Integrative Explorer (http://PlantGenIE.org) (Sundell et al. 2015) (Fig. 8B). The expression data showed that the expression of PtACBP1 and PtACBP2 was highest expression in mature leaves and medium or low in roots, similar to our qRT-PCR results (Fig. 8A). Expression of PtACBP4 and PtACBP6 was high in the nodes and internodes (Fig. 8B). The expression of PtACBP5, PtACBP7, and PtACBP8 was higher in the nodes, internodes, and roots than in other organs (Fig. 8B).

Effect of salt-alkali stress on growth of P. trichocarpa

Three-week-old seedlings of P. trichocarpa were treated with 75 mM Na2CO3, 100 mM NaHCO3, or 200 mM NaCl for 0, 6, 12, 24 and 48 h (Fig. 9). Alkaline salt stresses (75 mM Na2CO3 and 100 mM NaHCO3) affected the growth of seedlings. Under 75 mM Na2CO3 for 6 h, the leaves appeared slightly curled, and old leaves turned dark brown. Under 100 mM NaHCO3 for 12 h, the old leaves were curled and/or chlorotic. After 24 h and 48 h treatments with Na2CO3 or NaHCO3, the old leaves start to turn yellow and wilt. However, seedlings treated with NaCl had no obvious differences in phenotype from the control (Fig. 9).

Fig. 9
figure 9

Phenotype of wild-type P. trichocarpa seedlings after different durations of salt-alkali stress. Three-week-old seedlings were transferred to 1/2 MS medium containing 75 mM Na2CO3, 100 mM NaHCO3 or 200 mM NaCl. Roots and leaves were harvested at 6, 12, 24, and 48 h after stress treatments

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Expression of PtACBP genes in response to salt-alkali stresses

The qRT-PCR analysis showed that PtACBP genes differed in response to various stress treatments, including 200 mM NaCl, 75 mM Na2CO3, and 100 mM NaHCO3 treatments for 6, 12, 24, and 48 h (Fig. 10; Supplemental Fig. S1).

Fig. 10
figure 10

Salinity-alkali responsive expression profiles of PtACBP genes in roots and leaves. Color scale shows 2 − △△Ct values, which were normalized to untreated controls and log2-transformed counts. Blue and red colors indicate downregulated and upregulated, respectively, in response to 75 mM Na2CO3, 100 mM NaHCO3, or 200 mM NaCl

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In roots, under 200 mM NaCl treatments, transcript levels of seven PtACBP genes (PtACBP1, PtACBP2, PtACBP3, PtACBP4, PtACBP6, PtACBP7, and PtACBP8) gradually increased from 0 to 48 h and were significantly induced by short-time stress (e.g., 1.5-fold increase after 6 h of NaCl treatments). However, PtACBP5 was downregulated threefold at 24 h (Fig. 10). In leaves under NaCl stress, the expression of PtACBP1, PtACBP3, and PtACBP4 increased similar to their expression in roots, and their differences from the control were the greatest after 24 h of NaCl stress: 4.1, 6.5, and 7.8 times higher, respectively, than the control (Fig. 10). However, the expression levels of PtACBP5 and PtACBP6 in leaves were significantly different from those in roots. PtACBP5 in leaves was downregulated more than twofold at 6 h and fourfold at 12 h, and PtACBP6 was downregulated at all sampling times (Fig. 10).

Under 75 mM Na2CO3 treatments, six genes (PtACBP1, PtACBP3, PtACBP4, PtACBP5, PtACBP7, and PtACBP8) were upregulated, then downregulated (Fig. 10). PtACBP1, PtACBP4, and PtACBP5 were upregulated after the short-time stress (6 h), with highest expression at 12 h (2.4, 6 and 2.9 times higher, respectively, than the control), then downregulated (Fig. 10). Expression of PtACBP3 increased eightfold in the roots at 24 h, whereas PtACBP2 and PtACBP6 were downregulated at 6 h, then up-regulated by 12 h (Fig. 10).

In addition, during the 100 mM NaHCO3 treatments, four genes (PtACBP1, PtACBP3, PtACBP4, and PtACBP8) were upregulated and four (PtACBP2, PtACBP5, PtACBP6, and PtACBP7) were downregulated by 6 h (Fig. 10). The expression pattern of PtACBP3 during the 100 mM NaHCO3 was similar to that under Na2CO3 treatments, which was eightfold higher than in the control. PtACBP5 and PtACBP6 in roots were repressed at all sampling times, and PtACBP5 was downregulated tenfold at 12 h.

Under 75 mM Na2CO3 treatments, the expression levels of PtACBP1, PtACBP3, PtACBP4, PtACBP5, PtACBP7, and PtACBP8 in leaves were similar to those in roots, showing a trend of upregulation, then downregulation (Fig. 10). Significantly, PtACBP3 was upregulated eightfold in leaves compared with the control after 48 h.

Under 100 mM NaHCO3 treatments, five genes (PtACBP2, PtACBP3, PtACBP4, PtACBP7, and PtACBP8) were upregulated in expression, and three (PtACBP1, PtACBP5, and PtACBP6) were downregulated at 6 h (Fig. 10). The expression of PtACBP1 and PtACBP2 in roots was opposite of that in leaves at 6 h. PtACBP5 was downregulated in roots and leaves, whereas PtACBP6 was downregulated in roots and upregulated in leaves at 24 h (Fig. 10). All these results imply that PtACBP genes act in different organs in response to various salinity-alkali conditions.

Discussion

Phylogenetic analysis reveals conservation of the PtACBP family

The search to find ACBP genes in the P. trichocarpa genome identified eight; thus, the PtACBP gene family is small, similar to the case in other plant, including six ACBP genes identified in Hevea brasiliensis (Nie et al. 2018), nine in Zea mays (Zhu et al. 2021) and five in Sorghum bicolor (Paterson et al. 2009). In the phylogenetic analysis, the PtACBP genes grouped into four subfamilies, consistent with the ACBP genes in A. thaliana and rice (Meng et al. 2011). We also compared the subfamily members from A. thaliana, rice and 21 other woody plant species (Fig. 6). As we noted earlier, Populus is relatively closely related to Arabidopsis (Janson and Douglas 2007). We found that all the PtACBP genes except PtACBP2 were evolutionarily close to the PeACBP genes form P. euphratica. This result implies that the ACBP genes in Populus are relatively conserved and have similar functions.

Cis-elements in the promoter and PtACBP expression patterns suggest crucial roles in stress responses

Cis-acting elements are essential in many biological processes and stress responses (Ibraheem et al. 2010). For example, the cis-elements G-Box, I-Box, ARE, ABRE, and CGTCA-motif respond to light, hormonal, and anaerobic induction (Guidotti et al. 1983; Busk and Pages 1998; Mattick and Gagen 2001; Hattori et al. 2002; Turner et al. 2002; Gómez-Porras et al. 2007; Staswick 2008; Yang et al. 2017; Yu et al. 2020). In the present study, several common elements that are involved in the responses to light, ABA, salicylic acid and MeJA were identified in most PtACBP promoter regions. However, only class III PtACBP5 contained an anoxia-specific inducibility element (GC-motif), implying that PtACBP5 is involved in plant abiotic stress defense and response to hypoxia, similar to class III AtACBP3 (Choi et al. 1994; Xie et al. 2015). In addition, stress-related cis-elements, including TC-rich repeats, evident in the 5’-flanking regions of PtACBP8 further indicate that the large PtACBPs might function in stress tolerance, similar to findings for rice (OsACBP4, OsACBP5 and OsACBP6) (Meng et al. 2011).

ACBPs participate in plant responses to diverse biotic and abiotic stresses, such as heavy metal, oxidative stresses, drought, hypoxia, cold, salt and pathogen infection (Gao et al. 2009; Fan et al. 2010; Liu et al. 2015; Du et al. 2016; Qin et al. 2016; Narayanan et al. 2019, 2020; Zhu et al. 2021). In A. thaliana, overexpression of AtACBP6 and AtACBP2 enhances tolerance to cold and drought stress, respectively (Du et al. 2013a, b; Liao et al. 2014). AtACBP3, AtACBP4 and AtACBP6 contribute to drought tolerance by playing a role in cuticle formation (Xia et al. 2012). Similarly, overexpression of OsACBP4 and OsACBP5 increased rice tolerance to drought and salt stresses (Meng et al. 2011). Virus-induced gene silencing reduced expression of GhACBP3 and GhACBP6 subclass genes in cotton, significantly decreasing tolerance to drought and salt stresses (Qin et al. 2016). Membrane-associated AtACBP1 and AtACBP2 bind the heavy metal Pb(II) and are also involved in response to oxidative stress (H2O2) (Gao et al. 2009, 2010b; Du et al. 2016). Interestingly, AtACBP2 interacts with a group VII ethylene response factor (ERF), RAP2.3, to regulate the hypoxia response (Li and Chye 2004). AtACBP3 also participates in the response to hypoxia by interacting with VLC acyl-CoA esters and modulating fatty acid/lipid metabolism (Xie et al. 2015). In addition, AtACBP3 and OsACBP5 participate in defense against pathogen infections. Overexpression of AtACBP3 and OsACBP5 enhance the resistance of A. thaliana and rice to the bacterial pathogen Pseudomonas syringae pv tomAto DC3000 (Narayanan et al. 2019, 2020) and to necrotrophic (Rhizoctonia solani and Cercospora oryzae) and hemibiotrophic (Magnaporthe oryzae and Fusarium graminearum) fungi and biotrophic (Xanthomonoas oryzae pv. oryzae) (Narayanan et al. 2020).

In the present study, we found that the expression patterns of eight PtACBP genes were altered in roots and leaves in response to salinity-alkali stress (Fig. 10). Two PtACBP genes (i.e., PtACBP1 and PtACBP2) were significantly induced by 75 mM NaHCO3 and three (i.e., PtACBP3, PtACBP4, and PtACBP8) by 100 mM NaHCO3 in leaves and roots. Interestingly, two class III genes (PtACBP5 and PtACBP6) were significantly induced in roots but not in leaves. However, class I PtACBP1 and PtACBP2 and class II PtACBP3 and PtACBP4 were upregulated in leaves and roots. Thus, members of the different subfamilies apparently have different roles in response to salinity and alkali stresses.

Subcellular localization of PtACBPs determines function

The subcellular localization of different ACBP members determines their function such as intracellular trafficking, protection, and pool formation of acyl-CoA esters (Lung and Chye 2016). In the eudicot A. thaliana and the monocot rice, the subcellular localization of six AtACBPs and six OsACBPs have been determined using various in silico analyses and experimental verifications, such as observations with immunoelectron microscopy and/or confocal laser-scanning microscopy after transient expression in onion/ tobacco epidermal cells and/or stable expression in transgenic plants, western blots using subcellular fractions, and subcellular proteomic analyses (Ito et al. 2011; Ye et al. 2016). Plasma-membrane-localized class II AtACBP1 and AtACBP2 bind various ligands such as heavy metals, acyl-CoA esters and phospholipids to sense early signals and convey them for downstream responses, which are critical for embryogenesis, seed germination, and diverse ABA and stress responses (Xiao et al 2008; Gao et al. 2009, 2010a; Chen et al. 2010; Du et al. 2013a). Moreover, AtACBP1 and AtACBP2 are localized to the ER or vesicles, as are class II OsACBP4 and class III OsACBP5, facilitating acyl exchange between acyl-CoA and phospholipid pools (Napier and Haslam 2010) and contributing to vesicular lipid trafficking (Chye et al. 1999), which were important for lysoPC turnover and repair of membrane structure. In addition, the apoplast-localized class III AtACBP3 binds to pathogen-secreted lipids after pathogen infection, then is rapidly degraded by apoplastic proteolysis and re-localized intracellularly to trigger the expression of defense genes (Xiao and Chye 2011b). Class IV AtACBP4 is in the cytosol and nuclear periphery and interacts with transcription factor AtEBP in response to ethylene and jasmonate (Li et al. 2008). Cytosolic class IV AtACBP4 cooperates with AtACBP5 and class I AtACBP6 during seed germination (Hsiao et al. 2014). In addition, the peroxisome-localized OsACBP6 is involved in conversion of fatty acids to carbohydrates (Meng et al. 2014). Loss of OsACBP6 not only results in the disruption of acyl-CoA homeostasis but also of peroxidase-dependent reactive oxygen species (ROS) homeostasis (Meng et al. 2020).

The subcellular location of ACBPs in woody plants is still unclear. In our silico analyses, four PtACBPs (i.e., PtACBP1, PtACBP3, PtACBP7 and PtACBP8) were localized in the cytosol and thus should be involved in intracellular acyl-CoA transport (Knudsen et al. 1999; Chen et al. 2008; Meng et al. 2014). Three ACBPs (i.e., PtACBP4, PtACBP5, and PtACBP6) were predicted to be associated with the ER and thus function in the transfer of oleoyl-CoA esters to ER for the biosynthesis of membrane phospholipids and triacylglycerol (Ohlrogge and Jaworski 1997; Xiao and Chye 2009). Besides, the predicted nuclear localization of seven PtACBPs except PtACBP6 implied that they were probably involved in regulating transcription (Li et al. 2008). In addition, the location of PtACBP3, PtACBP5 and PtACBP6 in the mitochondria and PtACBP5 in the chloroplast suggests that they are associated with membrane stability and the trafficking of lipids between these organelles and the cytosol (Li and Chye 2003; Meng et al. 2014; Block and Jouhet 2015).

Spatial expression patterns of ACBPs indicate function specialized for tissues/organs

ACBPs were expressed in a variety of A. thaliana tissues and organs, such as seeds, leaves, and roots, which function in various developmental processes (Gao et al. 2009; Du et al. 2016). During embryogenesis, AtACBP1 facilitates acyl-CoA metabolism and membrane phospholipid biogenesis and shares an overlapping role with AtACBP2 in lipid metabolism (Chen et al. 2010). Cytosolic AtACBP4, AtACBP5 and AtACBP6 facilitate acyl transfer from plastids to ER for glycerolipid synthesis during seed development (Xiao and Chye 2009; Hsiao et al. 2014). During seed germination and seedling development, AtACBP1 promots PLDα1 activity via protein–protein interaction at the plasma membrane, leading to phosphatidic acid accumulation, then triggering of ABA-mediated responses (Du et al. 2013a, b). Also, extracellular targeting of AtACBP3 mediates the modulation of VLC acyl-CoA esters, uniquely affecting membrane lipid metabolism during leaf senescence (Xiao et al. 2010; Xiao and Chye 2010). AtACBP1, AtACBP3, AtACBP4 and AtACBP6 function in cuticle development by maintaining appropriate FA/lipid levels (Xue et al. 2014). In addition, AtACBP5 and AtACBP6 have overlapping roles in pollen development (e.g., microspores, tapetal cells, and pollen grains) by transporting acyl-lipid precursors for sporopollenin biosynthesis (Hsiao et al. 2015).

In this study, we found that all eight PtACBP genes had relatively high expression in leaves, moderate expression in roots, and low expression in stems, similar to their homologs in rice (Meng et al. 2011), suggesting that the genes have similar organ specificities among different plant species.

Conclusions

ACBPs are abundant and widely distributed in various plants and are involved in important processes such as lipid metabolism, enzyme and gene expression regulation, and coping with biotic and abiotic stresses. Here, we provided genome-wide information about the ACBP family of P. trichocarpa. We identified eight P. trichocarpa ACBPs, classified into four subfamilies, which displayed considerable evolutionary divergence in P. trichocarpa and conserved domains. The organ-specific expression profiles of ACBPs indicated potentially divergent functions in P. trichocarpa growth and development. The analyses of cis-acting elements and stress-responsive gene expression profiles suggest that ACBPs are involved in responses to diverse abiotic stresses and hormonal stimuli. Our results establish a foundation for further functional characterization of P. trichocarpa ACBPs.