Molecular characterization, expression and functional analysis of acyl-CoA-binding protein gene family in maize (Zea mays)

doi:10.21203/rs.2.15850/v1

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Molecular characterization, expression and functional analysis of acyl-CoA-binding protein gene family in maize (Zea mays)

https://doi.org/10.21203/rs.2.15850/v1

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published 15 Feb, 2021

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Background: Acyl-CoA-binding proteins (ACBPs) have a conserved acyl-CoA-binding (ACB) domain which facilitates binding to the acyl-CoA ester and trafficking in eukaryotic cells. Although the various functions of ACBP have been characterized in several plant species, its structural features, molecular evolution, expression profiling and function in Zea mays L . are still not clear.

Results: Genome-wide analysis identified nine ZmACBP genes in Z. mays , which were divided into four groups (class I, class II, class III, and class IV) by a phylogenetic tree constructed with 48 ACBP genes from six plant species. The subcellular localizations of the ZmACBPs were displayed subcellular multi-localization via transient expression of green fluorescence protein (GFP) fusions in tobacco ( Nicotiana tabacum ) epidermal cells. The expression profiles of ZmACBPs are dramatically different in different organs and normal growth, as well as abiotic or biotic stress by Quantitative RT-PCR (qRT-PCR). Eight of nine ZmACBP genes located in metaQTL regions associated with agronomic traits in maize. The heterologous constitutive expression of ZmACBP1 and ZmACBP3 in Arabidopsis enhanced the resistance to salinity and osmotic stress.

Conclusion: The ACBP genes family in Z. mays were highly conserved. ZmACBP genes had clear tissue and organ expression preference and were involved in abiotic or biotic stress response, suggesting their roles in plant growth and stress resistance.

Plant Molecular Biology and Genetics

Zea mays

acyl-CoA binding protein (ACBP)

evolution

subcellular localization

expression profiles

stress

Lipids are major and critical components that not only provide the structural basis for cell membranes and energy for metabolic processes, but also serve as signals responses to environmental stress in plant [1]. To enable lipids and their derivatives to function properly, they need to be translocated by various proteins, such as the lipid-transfer proteins (LTPs) and the acyl-CoA-binding proteins (ACBPs) [2; 3]. In fatty acid synthesis (FAS) of eukaryotes, the majority of long-chain fatty acids (LCFA, e.g. C16 and C18) is first converted into acyl-CoAs by long-chain acyl-CoA synthetase (LACS), and then exported to the ER for further acyl editing and biosynthesis of lipids [4]. As a lipid-binding protein in eukaryote, ACBPs with an acyl-CoA-binding (ACB) domain that binding to long-chain acyl-CoA esters contribute to the transport of acyl-CoAs/fatty acids from the chloroplasts to the ER [5].

Previous studies have reported many characterizations of ACBPs in eukaryotes including plants and mammals, which indicating their important biological function [6; 7; 8]. In yeast, ACBP plays important role in the assembly of plasma membrane, and ACBP mutation could elevate the expression of genes involved in many pathways, such as the biosynthesis of fatty acid, the metabolism of glycerol, and stress responses [9; 10; 11]. Mammals ACBPs are involved in the protection and regulation of enzymes, for example, acyl-CoA:lysophospholipid acyltransferase [12], acyl-CoA:cholesterol acyltransferase [13], carnitine palmitoyltransferase I [14] and glycerol–3-phosphate acyltransferase [15]. As a highly specialized acyl-CoA pool former and transporter, ACBPs are crucial molecules in biosynthesis of membrane, in regulation of enzyme activities and gene expression in lipid metabolism of plants [11]. Plant ACBPs could be divided into four categories based on their domain architecture: class I (small ACBPs) consists of peptides of 90 amino acid residues; class II (ankyrin-ACBPs) with ankyrin repeats in C-terminal; class III (Large-ACBPs) are large proteins with a C terminal ACB domain; and class IV (kelch-ACBPs) are multi-domain proteins containing C-terminal kelch motifs [16; 17; 18].

In Arabidopsis, there are six genes encoding ACBP proteins (designated AtACBP1-AtACBP6), which differ in structure, expression, subcellular location, acyl-coA ester affinities and functions [19]. AtACBP1 (338 amino acids, 37.5kDa) and AtACBP2 (354 amino acids, 38.5 kDa), belonging to class II ACBPs, are subcellularly localized to the endoplasmic reticulum (ER) and plasma membrane [20], and both of them could bind C18:2-coA and C18:3-coA esters [21]. AtACBP1 is highly expressed in seeds and siliques and AtACBP2 is expressed in roots, stems and flowers [21]. AtACBP1 and AtACBP2 could maintain a membrane-associated acyl pool, and involved in many plant stress response, for example, AtACBP1 was involved in the modulation of membrane lipids in response to freezing stress; the up-regulation of AtACBP2 contributed to improve the tolerance to drought, Cd(II) and oxidative stress [20; 21; 22]. AtACBP3 (362 amino acids, 39.3 kDa), being class III ACBPs, has an extracellular location and high affinity for binding arachidonyl-coA (C20:4), and is highly expressed in siliques and young shoots [5; 19]. AtACBP3 played an important role in maintaining normal lipid balance, and is involved in many biological functions, such as regulating leaf senescence, responding to pathogen infection, etc [23]. Class IV ACBPs contains AtACBP4 (668 amino acids, 73.3 kDa) and AtACBP5 (648 amino acids, 71 kDa), which both conserve five kelch motifs to bind C18:1-coA and PC and were found in the cytosol [24]. The expression of AtACBP4 and AtACBP5 were detected in all tissues, but AtACBP4 was higher expressed in roots and a lower expression in siliques, while AtACBP5 was mainly expressed in young shoots and mature leaves [24]. AtACBP4 and AtACBP5 were actively involved in pollen development and defense reaction by affecting lipid metabolism [25; 26]. Class I are the smallest ACBPs, usually named AtACBP6 (92 amino acids, 10.4 kDa), which was found to be localized in the cytosol and expressed in all plants organs [19]. AtACBP6 with binding 16:0-CoA and 18:2-CoA was involved in intracellular binding and trafficking of phosphatidylcholine in plant phospholipid metabolism, and enhanced freezing tolerance to the host [19; 27].

Like AtACBPs, ACBPs in Oryza sativa could also be subdivided into four classes: OsACBP1, OsACBP2, and OsACBP3 were small ACBP, OsACBP4 was ankyrin repeats ACBP, OsACBP5 was large ACBP, and OsACBP6 was kelch motif ACBP [16]. Although there are some similarities between AtACBPs and OsACBPs, however, the OsACBPs displayed difference in its affinity for substrates, tissue expression, and response to stress. For example, OsACBP1 and OsACBP4 could bind C18:1-coA and C18:2-coA, respectively, whereas others OsACBPs could bind C18:3-coA [16]. Additionally, OsACBPs are expressed in seed, leaf, stem, and root of rice, but there are significant differences in the expression level in different developmental stages among OsACBPs [16; 27]. The expression of OsACBP1, OsACBP2, and OsACBP3 were not affected under drought and high salinity treatments, while OsACBP4 and OsACBP5 were induced. Except for OsACBP6, the expression of others OsACBPs were suppressed under cold treatment [16].

Now, ACBPs have been investigated in some plant species, such as Arabidopsis, rice, Brassica napus, while no reports have analyzed the sequences of maize ACBP genes family. In this study, we reported the molecular characterization, phylogenetic evolution and expression profiles of ACBP genes in different tissues and stress treatments in maize. These findings could provide valuable information for further investigations into the biological functions of ZmACBPs during development and stress responses in maize.

Distribution of ACBP family genes in plant kingdom

To identify the distribution of ACBP proteins in plant kingdom, the ACBPs sequences of Arabidopsis were used as probes to search the Phytozome database, and the numbers of genes encoding ACBP proteins in 22 representative species were obtained (Fig. 1). Although ACBP proteins are found in all plant kingdom with the evolution of species, the number and classification of ACBPs have likely undergone extensive changes (Fig. 1). In Chlorophyta before the evolution of land plants, Ostreococcus lucimarinus and Chlamydomonas reinhardtii have four and three ACBP genes, respectively (Fig. 1). There is four ACBP genes in S. moellendorffii, which belongs to the lycophyte and is the origin of seed plants. However, the number of ACBP genes are explosively increased to eight in the moss Physcomitrella patens, which evolved into the vascular plants (Fig. 1). Although monocots are higher in evolution than moss, the gene numbers of the ACBP sharply decrease to five in Brachypodium distachyon, six in rice, six in Sorghum bicolorFig. 1). However, there are nine ACBP genes in maize and 11 genes in Triticum aestivumFig. 1). Interestingly, it seems that there is no significant ploidy relationship between the number of ACBP and the size of the species genome, for example, the maize genome is eight-fold larger than that of B. distachyon whereas the number of ACBP genes only increases by four (Fig. 1). Compared with monocot plants, the numberof ACBP genes in eudicot plants varies greatly. For example, there are only three ACBP genes in the Medicago truncatula genome, however, as many as 11 ACBP genes exist in Glycine max (Fi. 1). Similarly, there are only six ACBP genes in Arabidopsis, but up to 12 in Brassica napus (Fig. 1).

Identification and characterization of ACBP genes in maize

A total of nine putative ZmACBP sequences were identified by searching against the Maize Genome Database (https://www.maizegdb.org),, and designated as ZmACBP1 to ZmACBP9, respectively. Table 1 lists the general characteristics of the ZmACBP gene family including ID, gene name, open reading frame size and exon number of each gene, and length, molecular weight and isoelectric point of each gene coding protein. The number of amino acids (aa) in ZmACBPs varies from 89 aa (ZmACBP1) to 783 aa (ZmACBP9). Similar to A. thaliana, the conserved ACB domain is located at the N-terminal segment in of most ZmACBP proteins, while ZmACBP5 and ZmACBP6 contain the C-terminal ACB domain (Additional file 1: Figure S1a). Nine ZmACBPs were divided into four classes based on the domain structures of the ACBPs (Additional file 1: Figure S1a). Class I containing only one ACB domain had two members, ZmACBP1 and ZmACBP2 (Additional file 1: Figure S1a). ZmACBP3 and ZmACBP4 possessed an ACB domain and ankyrin repeats, and belonged to Class II. ZmACBP5 and ZmACBP6 were in Class III, which is a large ACBP class (Additional file 1: Figure S1a). ZmACBP7, ZmACBP8, and ZmACBP9 possessed an ACB domain and a kelch motif, respectively, and belonged to class IV (Additional file 1: Figure S1a). Nine ZmACBP genes were unevenly distributed on five different chromosomes based on their data of the gene locus (Table 1, Additional file 1: Figure S1b). For example, three ZmACBP genes are localized on chromosome 1, two ZmACBP genes are distributed on chromosomes 5 or 10, whereas only one ZmACBP gene is located on chromosomes 2 and 9, respectively (Additional file 1: Figure S1b).

Phylogenetic relationship, gene structure and conserved motifs of Zm ACBP genes in maize

To analysis of the evolutionary relationships among different species, the sequences of nine ZmACBPs, six AtACBPs, six OsACBPs, 11 TaACBPs, five BdACBPs, and 11 GlACBPs were downloaded for alignment and used to construct an unrooted phylogenetic tree using the Neighbor-Joining method. A total of 48 ACBP protein sequences, including nine ZmACBPs, could be classified into four ACBP classes (Fig. 2). The result of phylogenetic tree clearly shown that every ZmACBP was clustered with those of other closely related species, such as rice，B. distachyon, and T. aestivum, but were further from those of A. thaliana, a distantly related species (Fig. 2).

Analysis of the exon/intron structures of the ZmACBP genes revealed that the number of exons differed among members of the ZmACBP genes family, mostly ranging from three to six, whereas ZmACBP7/–8/–9 had the greatest number of exons (up to 18 exons, Fig. 2). It is interesting to find a similar exon/intron pattern in each group, for instance, most of the ACBPs in Clade I containing four exons; the number of ACBPs with seven exons accounted the majority in Clade II or Clade III; and the vast majority of ACBPs in the Clade IV have 18 exons (Fig. 2). In addition, a total of 10 conserved motifs were detected from 48 ACBPs, and all of the ACBP proteins have two different conserved motifs, motif 1 and 2, which were ACB domain (Fig. 2). The ACBPs in class II have two conserved motifs (10 and 8) in C-terminal, that are ankyrin repeats, and the class IV ACBPs are multi-motifs proteins containing C-terminal kelch domain (Fig. 2). Interestingly, the ACBP genes clustered in the same clade have different gene structures and domain architectures, which providing some clues for the functional investigation of maize ZmACBP genes.

Cis-regulatory elements in the promoters of ZmACBP genes

To further explore the function and the regulation patternsof ZmACBP genes, the cis-acting regulatory elements at the regions of 1,500 bp upstream from the initiation codons of nine ZmACBP genomic sequences were searched in the Plant CARE database. The results indicated that many conserved cis-regulatory elements resided within the ZmACBP promoters, and the number and distribution of the cis-regulatory elements were very different among different ZmACBPs (Fig. 3, Additional file 2: Table S1). The cis-acting regulatory elements could be divided into three important physiological processes: development-related, hormonal responses, and environment responses. The CAAT-box motif, involved in meristem expression element, was found in the promoters region of all nine ZmACBP, suggesting that ZmACBP genes play important roles in differentiation. What’s more, cis-elements involved in hormone responsiveness, including ABRE, AuxRR-core, CGTCA-motif, GARE motif, and TGA-element, resided within most ZmACBP gene promoters (Fig. 3), indicating that ZmACBP genes are involved in growth and development. In addition, some cis-elements involving in environment responses, such as G-Box, LTR, and TC-rich repeats, were only found in a few of ZmACBP gene promoters. For example, the LTR-motif, a cis-element involving in low-temperature responsiveness, could only be detected in ZmACBP4 and ZmACBP5 gene promoters (Fig. 3), while the TC-rich repeats involving defense and stress responsiveness were found in the promoters of ZmACBP1 and ZmACBP9 gene (Fig. 3).

Subcellular localization of ZmACBP proteins

To determine the location of ZmACBPs, eight ZmACBPs were selected for subcellular localization analysis by fusing to the N-terminus of GFP and transforming into N. benthamiana transiently. Notably, the fluorescence of the vector control was observed in the localization of nuclear and cytoplasmic (Fig. 4a). The GFP fluorescent signals of ZmACBP1::GFP and ZmACBP2::GFP were found in the cytoplasmic (Fig. 4b and c), indicating that they are located to cytosol. In N. benthamiana leaf epidermal cells, the GFP fluorescent signals of ZmACBP3::GFP, ZmACBP4::GFP, and ZmACBP5::GFP were distributed at the ER surrounding the nuclei (Fig. 4d, e, and f). To confirm above results, ZmACBP3::GFP, ZmACBP4::GFP, and ZmACBP5::GFP were co-transfected into N. benthamiana leaf epidermal cells with ER marker ZmBiP-RFP, and the signals of GFP (Fig. 4d, e, and f) and RFP (Fig. 4v1, w1, and x1) were overlapped (Fig. 4v, w, and x), indicating that ZmACBP3::GFP, ZmACBP4::GFP, and ZmACBP5::GFP were ER-associated proteins. The distribution of ZmACBP6::GFP, ZmACBP7::GFP, and ZmACBP8::GFP were similar to the GFP-vector control (Fig. 4g, h, and i), while disappearing in nuclear localization, indicating that they are targeted to the cytosol and plasma membrane.

Expression profiles of ZmACBP in different organs and developmental stages

To analyze the expression profiles of nine ZmACBP genes during maize development, their transcriptional patterns were detected using published microarray data (Maize eFP Browser, http://bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi) in 60 different tissues or developmental stages in maize. Based on the expression patterns of nine ZmACBPs in this database were represented as heatmaps (Fig. 5a). Some of these genes are preferentially expressed in particular organs. For example, four genes (ZmACBP2, 6, 5 and 8) show the highest expression levels in leaf compared with those in other tissues, three genes (ZmACBP3, 4 and 7) with the highest mRNA levels in endosperm, and ZmACBP1 and ZmACBP9 with the highest expression levels in embryo and husk, respectively (Fig. 5a).

The expression levels of the nine ZmACBP mRNAs were detected in different tissues by using qPCR, including root, stem, leaf, silk, immature cob, anther, kernel (10 DAP) (Fig. 5b). The expressions of nine ZmACBP transcripts were all detected in all different tissues, but the patterns were dramatically different. ZmACBP1, ZmACBP2, and ZmACBP5 were more highly expressed in the leaf than in other organs or tissues (Fig. 5b). ZmACBP3 was more highly expressed in kernel, and had almost the same expression level in others organs (Fig. 5b). ZmACBP4 was relatively highly expressed in root, moderately expressed in 10 DAP of kernel and showed low expression in leaf (Fig. 5b). ZmACBP6 had the highest expression level in cob (Fig. 5b). ZmACBP7 and ZmACBP8 had similarly expression patterns with relatively high expressions in kernel (Fig. 5b). ZmACBP9 was relatively highly expressed in silk and 10 DAP of kernel, followed by stem, and showed low expression in leaf (Fig. 5b). The qRT-PCR results were consistent with the microarray data, suggesting that different ZmACBP might play different roles in the development of different organs.

Rigional association mapping for ZmACBP genes

To explore the potential functions of the ZmACBP genes for agronomic traits in maize, an association mapping panel from different maize inbred lines was applied to explore correlation between ZmACBP genes and maize agronomic traits. A total of eight ZmACBP genes were found to be significantly correlated with more than one agronomic traits at the P ≤ 0.05 level, such as plant height (PH), ear height (EH), ear diameter (ED), 100 grain weight (HGW), silking time (ST), heading date (HD), kernel width (KW), and so on (Additional file 3: Table S2). What’s more, some ZmACBP genes were found to associate with some important agronomic traits at the P ≤ 0.01 level, such as grain yield, flowering time, and kernel-related traits. For example, ZmACBP3 was significantly correlated with EH (Fig. 6a), ZmACBP4 significantly affected on KW (Fig. 6b), and ZmACBP7 significantly effected on HD (Fig. 6c). These results suggested that these ZmACBP genes in different maize inbred lines could be regarded as important candidate genes related to maize development and kernel-related traits for subsequent functional validation in maize.

Expression profiling of ZmACBP genes in response to various abiotic and biotic stress

To gain further insight into the role of ZmACBPs in response to abiotic or biotic stress, the expression patterns of ZmACBPs were analyzed using qRT-PCR under different treatments, including 200 mM NaCl, 20% PEG6000, wounding, 4°C，20 μmol/L Cu²⁺ and fungus infection (Fig. 7). The results shown that ZmACBPs genes were induced by high salinity or osmotic treatments, peaking at 6 or 12 h after two treatments and then remained at relatively high levels (Fig. 7a and b). ZmACBP1, ZmACBP2, ZmACBP5 and ZmACBP6 were rapidly induced and peaked at 0.5 h after wounding treatment, following by a decrease in expression to levels lower than untreated, however, the expression of ZmACBP3, ZmACBP4, ZmACBP7, ZmACBP8, and ZmACBP9 were suppressed (Fig. 7c). ZmACBP1, ZmACBP4, ZmACBP7, ZmACBP8 and ZmACBP9 mRNAs were reduced after cold treatment and then recovered to minimum level in 12 or 24 h (Fig. 7d). While, ZmACBP2, ZmACBP3, ZmACBP5 and ZmACBP6 mRNAs were induced with cold treatment, and peaked within 12 h, then remained at relatively high levels at 24 h (Fig. 7d). In copper ion treatments, ZmACBP1 and ZmACBP8 mRNAs were suppressed within 48 h, and ZmACBP3 mRNAs was almost unaffected (Fig. 7e). ZmACBP4, ZmACBP5, ZmACBP6, ZmACBP7 and ZmACBP9 were rapidly induced by copper ion treatments, and peaked at 3 or 6 h after salt treatment, then remained at relatively high levels at 48 h (Fig. 7e). Fungus infection (Ustilago maydis) induced the expression of ZmACBP1, ZmACBP2, ZmACBP3, ZmACBP5, ZmACBP6 and ZmACBP8, while the expression of ZmACBP4, ZmACBP7 and ZmACBP8 were suppressed (Fig. 7f). These results suggest ZmACBPs play important roles in stress defense responses.

The constitutive expression in A. thaliana of two ZmACBPs had a positive effect on stress tolerance

To confirm ZmACBPs response to stress, ZmACBP1 and ZmACBP3 were selected for further overexpression (OE) in A. thaliana. Given the inducibility of ZmACBP1 and ZmACBP3 by salinity and osmotic (Fig. 7a and b), the OEs were exposed to 100 mM NaCl or 200 mM mannitol. There was no observable phenotypic effect between OE and VC plants with respect to either leaf or root growth on a medium containing no abiotic stress agent (Fig. 8a and e), however, in the presence of 100 mM NaCl, the OE lines heterogeneously expressing ZmACBP1 or ZmACBP3 grew more vigorously than VC (Fig. 8b and f), forming larger leaves and longer roots (Fig. 8d and h). In addition, the OE and VC lines performed similarly when challenged with 200 mM mannitol stress (Fig. 8c and d).

ACBP proteins have been annotated functionally in other higher plants, such as Arabidopsis, rice, and B. napus [27]. In this study, ACBP genes were found to exist in all plants, including the green microalga, moss Physcomitrella, and other land plants (Fig. 1), indicating that ACBP genes firstly originated in the green microalga and gradually appeared in other plants as species evolving. However, the different number of ACBP genes in different plant species maybe suggested that natural selection pressure of the ACBP genes are variant for different plant species. This study identified nine ZmACBPs and clustered them into four different subfamily groups based on the phylogenetic analysis (Additional file 1: Figure S1; Fig. 2). Alignment of the ZmACBPs protein sequences with ACBPs homologues from A. thaliana revealed a relatively conservation among function domains (Additional file 1: Figure S1a). Through gene structure analysis of ACBP family members, genes within the same branch generally showed the similar exon-intron structures (Fig. 2). The similarity of ACBPs in exon-intron structures in different species might be due to the loss or increase of individual introns during the evolution of the gene structure. In addition, the motif analysis of ACBP proteins in different species found that the ACBP proteins clustered in the same subfamily shared a similar motif composition, whereas the motif compositions differed markedly among the different subfamilies (Fig. 2). The subcellular localizations of maize ACBPs were mapped to various subcellular compartments, similar to A. thaliana and rice ACBPs [16; 19; 20] (Fig. 4). These results suggested that ACBP might have some conservation in evolution and the similarity of function from different species.

The results of tissue expression analysis shown that ZmACBPs were widespread in all organs of maize, however, the expression level of each ZmACBP was found to be significantly different (Fig. 5a), which indicating that ZmACBPs might have multiple biological function in different organs of maize. This may be associated with cis-regulatory elements in the promoters of ZmACBP genes. For example, the “MYB motif” recognized by the MYB transcription factor was distributed in the 1,500 bp promoters region of partial ZmACBPs (Fig. 3), and involved in regulate the biosynthesis of phenylpropanoid and lignin in plants [28]. The lignin was one of the components of the cell wall and cellulose skeleton and contributed to improve the mechanical strength of the plant, which facilitating the transport of water, and affecting the growth and development of organs [29]. In addition, there were some Gibberellins (GAs)-responsive elements in the promoter of the genes ZmACBP8 and ZmACBP9 (Fig. 3). GA played important role in many aspects of plant development, such as seed germination, the morphogenesis of leaf, and the growth of shoot and root, and so on [30]. These findings are consistent with studies in Arabidopsis and rice ACBP family [19], which indicating that the members of the same subfamily in different species seems to have similar functions.

It is well-known that plants are effected by multifarious environmental stresses, such as salt, drought, cold, and heavy metal, which could influence plant growth, development and crop productivity [31]. In previous research, Arabidopsis ACBPs could respond to stress including biotic and abiotic by different pathways. Mutation of AtACBP1 reduced the tolerance to Pb(II) due to its unfavorable binding to linoleoyl-CoA, which was the precursors of phospholipid repair after heavy metal stress [21; 32]. Additionally, AtACBP3, AtACBP4 and AtACBP6 played an important role in cuticle formation, which contributing to enhance the tolerance of drought [21; 33]. Compared with wild-type Arabidopsis, AtACBP6-overexpressing markedly improved the tolerance of flowers under freezing [34], whereasthe overproduction of AtACBP3 could enhance the resistance to the bacterial pathogen Pseudomonas syringae pv tomato DC3000 [33]. These findings are consistent with the results of maize ACBP family in this study, for example, the expression of ZmACBP genes were both induced by salt and osmotic treatment (Fig. 7a and b), and the overexpression of ZmACBP1 and ZmACBP3 in A. thaliana were effective in increasing the plant’s level of salinity and drought tolerance (Fig. 8). Additionally, the expression of Class I ZmACBP2 was induced by cold stress (Fig. 7d), whereas the expression of AtACBP6 belonging to Class I could also be induced by cold stress [35]. Furthermore, the transcripts of ZmACBP1/–2/–5/–6 were induced by wounding, and the ZmACBP4/–5/–6/–7/–9 transcripts were induced by heavy metal (Fig. 7c and e). Except for ZmACBP4/–7/–9, all other ZmACBP genes could be induced by fungal infections (Fig. 7f). The difference in expression pattern between the same subfamily members of maize and Arabidopsis under stress treatment indicate that ZmACBPs may have different biological functions, and are not mere duplicates of their Arabidopsis homologues.

Different ZmACBP genes showed great changes under stress, which may be related to the presence of cis-elements in their promoter regions. The cis-element of ABRE could be bound by ABA-responsive transcription factors (AREB⁄ABF) and is responsive to the ABA signaling pathway, while DRE⁄CRT is recognized by the C-repeat binding factor (CBF) family and plays an important role in responding to drought or salinity stress [36]. In this study, most of ZmACBPs containing one or more ABRE or C-repeat/DRE (Fig. 3; Additional file 2: Table S1), were induced under drought or salinity stress (Fig. 7a and b), indicating that they function in stress tolerance. LTR motifs responding to low temperature stress were found to distribute in the promoters of the ZmACBP4 and ZmACBP5 genes (Fig. 3), and the results of qRT-PCR also confirmed that these two genes were induced by low temperature (Fig. 7d). Salicylic acid and MeJA were regulated by the TCA and TGACG element, and involved in the plant responses to many stresses, including defense against insects, wounding, and bacterial pathogens [37]. The results of qRT-PCR shown that ZmACBP genes containing TCA and TGACG element in the promoters regions (Fig. 3; Additional file 2: Table S1) were induced by wounding and fungal infections (Fig. 7c and f). These findings indicated the expression profiling against abiotic or biotic stresses provided novel insights into the functions of ZmACBP genes.

In plants, ACBPs are involved in many stages of plant growth and development, including flowering, embryo development, seed germination and seedling development [27]. Arabidopsis overexpressing grapevine VvACBP1 shown slowed-down floral transition by suppressing the photoperiodic pathway [38]. During embryogenesis of Arabidopsis, AtACBP1 and AtACBP2 were involved in the synthesis of phospholipid and galactolipid in ER [39]. Additionally, the overexpression of B. napus ACBP1 affected the composition of fatty acid in Arabidopsis seeds, while acbp4/acbp5/acbp6 mutants of Arabidopsiscaused to accumulate 18:1-coA in the embryos and decrease the seed weight [40]. Therefore, normal expression of related ACBP genes is important to maintain normal morphology and kernel development in plants. In this study, a majority of ZmACBP genes were found to continuously express at different tissues in maize (Fig. 5). Through the analysis of correlation, several ZmACBP genes were identified to associate with agronomic traits (Fig. 6; Additional file 3: Table S2). For example, ZmACBP3 and ZmACBP4 might be regarded as important candidate genes associated with maize kernel-related traits with functions that are worth investigating through a reverse genetics approach. These analyses will contribute to the studies for unraveling the molecular mechanisms of signal transduction, stress response, and developmental processes.

In this study, nine members of the Z. mays ACBP family were identified. The basic informatics analyses of ZmACBPs, including evolution, gene structure, conserved motif analysis, and subcellular localization, shown that ZmACBPs were relatively conservative, and also had its own unique characteristics. qRT-PCR analysis revealed that ZmACBPs had a tissue and organ expression preference, as well as abiotic or biotic stresses. In addition, association analysis between ZmACBP genes and agronomic traits showed that some ZmACBP genes are significantly correlated with development and kernel-related traits. This study is a prelude for ACBP investigation in Z. mays, and multiple interesting functions are expected for crop improvement purpose.

Plant materials and growth conditions

Maize inbred line W22 (Zea mays L, 2n = 20) provided by Dr. Jianbing Yan, Huazhong Agricultural University, China. Maize seeds were first surface sterilized in 2% (w/v) sodium hypochlorite (NaClO) and then incubated at 28°C for germination. The seedlings were grown in a greenhouse with a regime of 16 h light and 8 h dark and at 28 °C until raising to the three-leaf stage, and then were selected for stress treatments. For the drought treatment, the seedlings were treated with 20% PEG 6000 and collected at various time intervals (0, 3, 6, 12, 24, and 48 h). For the salinity treatment, the seedlings were subjected to 200 mM NaCl and harvested at 0, 3, 6, 12, 24, and 48 h. For the cold treatments, seedlings at the three leaf stage were incubated at 4°C for 0, 3, 6, 12, and 24 h [41]. For the hormone treatments, the seedlings were subjected to 20 μmol/L Cu²⁺ copper solutions and harvested at 0, 3, 6, 12, 24, and 48 h. For wounding treatment, leaves of seedlings were scratched using a needle. Wounded leaves were subsequently maintained on water-saturated filter paper and sampled at 0, 0.5, 2 and 24 h after treatment [42]. In fungus infection (Ustilago maydis),, pathogenicity assays were performed as described [43], and maize leaves were sampled at 6 day post infection (dpi). Each treatment consisted of three replicates. Adult plants were grown in the field, and then all organs and tissues were harvested. All materials were immediately frozen in liquid nitrogen after harvesting and maintained at −80°C prior to RNA isolation.

Identification and phylogenetic analysis of the ACBP genes in maize

Genes encoding protein ZmACBP sequences that possess one ACB domain were identified using the conserved A. thaliana or rice ACB domains as a query to search the CDS sequences from the Maize Genome Database (https://www.maizegdb.org),, Phytozomev12.0 (http://www.phytozome.net) and NCBI (http://www.ncbi.nlm.nih.gov/). The matched sequences with one ACB domain were further analyzed for conserved domains using the motif database Pfam (http://pfam.sanger.ac.uk/) or the Pfam27.0 web server (http://pfam.xfam.org/). Based on the searched ACBP chromosome locus, we downloaded the genome sequences from Maize Genome Database. Therefore, nine independent ZmACBP genes were identified in maize. To study the phylogenetic relationships among ACBP genes from maize, Arabidopsis, rice, Triticum aestivum, Brachypodium distachyon and Glycine max, a phylogenetic tree based on the alignment of ACBP conserved domains was generated using MEGA7.1 by the Neighbor Joining (NJ) method with 1000 bootstrap replicates in p-distance model. Since the ACBP family in Arabidopsis has been identified and studied well, we named ZmACBPs based on the phylogenetic relationship with AtACBPs and Blast analysis against the Arabidopsis genome in TAIR.

Chromosomal locations, gene structure and protein characteristics of ACBP genes in maize

The putative ACBP protein sequences were further confirmed and analyzed using PFAMHMMs (http://pfam.sbc.su.se/) and CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The gene locations were based on phytozome v9.1 database and mapped by MapInspect software. The exon/intron organization was analyzed by Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/). The 1,500 bp upstream sequences before the translation initiation codon ATG of each maize ACBP genes were selected as gene promoters. Cis-regulatory elements of each promoter sequences were predicted though searching the Plant CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) with a FDR<0.1% [44]. The protein pI/Mw was calculated using Compute pI/Mw tool (http://web.expasy.org/compute_pi/). And the conserved motifs were identified and located with MEME (http://meme.sdsc.edu/meme4_3_0/intro.html)..

Subcellular localization of ZmACBPs

For subcellular localization, PCR was performed to amplify the coding sequences of ZmACBPs genes using gene-specific primers which are listed in Additional file 4: Table S3. Then, the PCR products were purified and cloned into pEarleyGate 103 [45] using the Gateway LR reaction (Invitrogen). The Agrobacterium tumefaciens EHA105 strain harboring relevant plasmids was transformed into tobacco epidermal cells. The ZmACBP-GFP fusion proteins were examined using a confocal laser scanning microscope LSM 800 (Zeiss).

Expression analysis and quantitative real-time PCR

Total RNAs were extracted from various maize tissues with Trizol reagent according to the user manual (Takara, Dalian, China). Reverse transcription reactions were performed using 5 μg RNA with a PrimeScript^TMRT reagent Kit with gDNA Eraser (Tiangen, Beijing, China) according to supplier instructions. The primers for real-time qRT-PCR were designed using Primer Premier 5.0 (Additional file 4: Table S4), and TUB-ribosylation factor was selected as the internal reference gene. The qRT-PCR was performed on Bio-Rad CFX Connect^TM using SYBR-Green to detect gene expression abundances. For all qRT-PCR analysis, triplicate biological samples were collected. Data were analyzed using Bio-Rad CFX Manager software.

Regional association mapping for ZmACBP genes

Regional association tests were performed in an association mapping panel composed of 508 inbred lines [46]. The genotype of this panel was detected by two genotyping platforms, resulting in 550,000 high quality SNPs [47; 48]. The 17 agronomic traits included plant height (PH), ear height (EH), tassel branch number (TBN), ear diameter (ED), 100 grain weight (HGW), silking time (ST), heading date (HD), leaf number above ear (LNAE), tassel main axis length (TMAL), ear length (EL), kernel width (KW), cob weight (CW), pollen shed (PS), kernel weight (KWe), kerner number perrow (KNP), ear leaf width (ELW), ear leaf length (ELL).

Only SNPs within the range of 100 kb upstream and downstream of candidate genes were used. And then an association mapping analysis was conducted using a mixed liner model that took into account population structure and relative kinship to test for statistical association between agronomic traits phenotypes and genotypes of each ZmACBP gene. Association mapping analysis and linkage disequilibrium (LD) among SNPs were conducted in TASSEL (v 5.0) [49]. The physical location of the SNPs was identified based on the maize genomic sequence version 5b.60 [50].

Transformation of A. thaliana and stress treatment of transgenic plants

The coding region of ZmACBP1 and ZmACBP3 were inserted into pSTART under the control of the CaMV 35S promoter. The resulting construct (or the empty pSTART vector) was transformed into A. thaliana ecotype Col–0 using the floral-dip method [51]. Seeds harvested from both homozygous transgenic Arabidopsis carrying ZmACBP1 and ZmACBP3 (OE lines) and the pSTART empty vector (VC line) were surface-sterilized and plated on the surface of half-strength Murashige and Skoog (1/2MS) agar medium. The plates were initially held at 4 °C in the dark for two days to break seed dormancy, and then removed to a chamber set to deliver a 16 h photoperiod and a constant temperature of 22 °C. After two days, the seedlings were transferred to a fresh plate containing the same medium supplemented with either 100 mM NaCl or 200 mM mannitol (for 10 days). All these experiments were replicated three times.

ACBP Acyl-CoA-binding proteins;

GFP Green fluorescent protein;

MW Molecular weight;

PEG Polyethylene glycol;

qRT-PCR: Real-time quantitative reverse transcriptional-polymerase chain reaction.

Acknowledgements

We thank Dr. Jianbing Yan (Huazhong Agricultural University, China) for maize inbred line W22.

Authors’ Contribution

JZ and HL wrote the paper. JZ, WL, and YZ performed the experiments. LP, JL, XX，and RC contributed to data analysis. All authors read and approved the manuscript.

Founding

This research was supported by grants from the Natural Science Foundation of China (31771797) and Joint Fund for Excellent Young Talents in Universities of Shandong Province (ZR201807061228) for financial support.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

Authors’ Information

¹School of Biological Science and Technology, University of Jinan, Jinan, 250022, China.

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Table 1 General information of ACBP genes in maize

Gene ID	Name	Chr.	ORF (bp)	Exon no.	Protein (aa)		PI	MW (KD)
GRMZM2G079908 (Zm00001d024518)	ZmACBP1	10	270	4	89	4.99			10.13
GRMZM2G344634 (Zm00001d045539)	ZmACBP2	9	276	3	91	5.83			9.97
GRMZM2G049495 (Zm00001d001798)	ZmACBP3	2	987	6	328	4.60			34.79
GRMZM2G173636 (Zm00001d026639)	ZmACBP4	10	987	6	328	4.59			35.12
GRMZM2G108138 (Zm00001d028397)	ZmACBP5	1	1635	5	544	4.15			57.93
GRMZM2G060781 (Zm00001d015957)	ZmACBP6	5	924	1	307	4.57			35.23
GRMZM2G053803 (Zm00001d034758)	ZmACBP7	1	1980	18	659	5.11			72.06
GRMZM2G351160 (Zm00008a005731)	ZmACBP8	1	1929	18	642	5.18			70.33
GRMZM2G326195 (Zm00001d012892)	ZmACBP9	5	2352	18	783	5.37			85.24

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Molecular characterization, expression and functional analysis of acyl-CoA-binding protein gene family in maize (Zea mays)

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Journal Publication

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Abstract

Figures

Background

Results

Distribution of ACBP family genes in plant kingdom

Identification and characterization of ACBP genes in maize

Phylogenetic relationship, gene structure and conserved motifs of Zm ACBP genes in maize

Cis-regulatory elements in the promoters of ZmACBP genes

Subcellular localization of ZmACBP proteins

Expression profiles of ZmACBP in different organs and developmental stages

Rigional association mapping for ZmACBP genes

Expression profiling of ZmACBP genes in response to various abiotic and biotic stress

The constitutive expression in A. thaliana of two ZmACBPs had a positive effect on stress tolerance

Discussion

Conclusion

Methods

Plant materials and growth conditions

Identification and phylogenetic analysis of the ACBP genes in maize

Chromosomal locations, gene structure and protein characteristics of ACBP genes in maize

Subcellular localization of ZmACBPs

Expression analysis and quantitative real-time PCR

Regional association mapping for ZmACBP genes

Transformation of A. thaliana and stress treatment of transgenic plants

Abbreviations

Declarations

Acknowledgements

Authors’ Contribution

Founding

Availability of data and materials

Ethics approval and consent to participate

Consent for publication

Competing interests

Authors’ Information

References

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