Next Article in Journal
Molecular Alterations of the Endocannabinoid System in Psychiatric Disorders
Previous Article in Journal
Hemin-Induced Endothelial Dysfunction and Endothelial to Mesenchymal Transition in the Pathogenesis of Pulmonary Hypertension Due to Chronic Hemolysis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 9
10.3390/ijms23094765
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification, Phylogenetic and Expression Analyses of the AAAP Gene Family in Liriodendron chinense Reveal Their Putative Functions in Response to Organ and Multiple Abiotic Stresses

by
Lingfeng Hu
1,
Ruifang Fan
1,
Pengkai Wang
1,2,
Zhaodong Hao
1,
Dingjie Yang
1,
Ye Lu
1,
Jisen Shi
1 and
Jinhui Chen
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics and Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
2
College of Horticulture Technology, Suzhou Polytechnic Institute of Agriculture, Suzhou 215000, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(9), 4765; https://doi.org/10.3390/ijms23094765
Submission received: 14 March 2022 / Revised: 21 April 2022 / Accepted: 23 April 2022 / Published: 26 April 2022
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Review Reports Versions Notes

Abstract

:
In this study, 52 AAAP genes were identified in the L. chinense genome and divided into eight subgroups based on phylogenetic relationships, gene structure, and conserved motif. A total of 48 LcAAAP genes were located on the 14 chromosomes, and the remaining four genes were mapped in the contigs. Multispecies phylogenetic tree and codon usage bias analysis show that the LcAAAP gene family is closer to the AAAP of Amborella trichopoda, indicating that the LcAAAP gene family is relatively primitive in angiosperms. Gene duplication events revealed six pairs of segmental duplications and one pair of tandem duplications, in which many paralogous genes diverged in function before monocotyledonous and dicotyledonous plants differentiation and were strongly purification selected. Gene expression pattern analysis showed that the LcAAAP gene plays a certain role in the development of Liriodendron nectary and somatic embryogenesis. Low temperature, drought, and heat stresses may activate some WRKY/MYB transcription factors to positively regulate the expression of LcAAAP genes to achieve long-distance transport of amino acids in plants to resist the unfavorable external environment. In addition, the GAT and PorT subgroups could involve gamma-aminobutyric acid (GABA) transport under aluminum poisoning. These findings could lay a solid foundation for further study of the biological role of LcAAAP and improvement of the stress resistance of Liriodendron.

1. Introduction

As a result of natural selection, there are currently only two species of Liriodendron in nature, namely, L. chinense and L. tulipifera, which are distributed in Asia and North America, respectively. Both are widely grown in China, mainly for landscaping and timber supply [1]. However, in the process of planting Liriodendron, adverse external environmental conditions such as temperature extremes and the presence of heavy metals ions will still affect the growth of Liriodendron, which hinders its popularization. Therefore, understanding the growth, development, and resistance mechanism of Liriodendron is conducive to its promotion and application.
Amino acids, as the main circulation form of organic nitrogen in the process of plant growth and development, are involved in various life processes, including protein synthesis, hormone regulation, and energy storage, as well as nucleotides, chlorophyll, some plant hormones, and most secondary metabolites [2,3]. It is a necessary source of nutrition and an important regulatory mechanism. The absorption of amino acids in the substrate by plants is achieved mainly by acid transporter proteins (AATs), thereby completing the transmembrane transport of amino acids through the phloem and xylem of plants, thereby completing the transmembrane transport of amino acids through the phloem and xylem of plants in plants [4,5]. The AAT superfamily was divided into two families, amino acid/auxin permease (AAAP) and amino acid polyamine choline (APC) gene families [6]. Among them, the AAAP gene family includes eight subgroups: amino acid permeases (AAPs), lysine histidine transporters (LHTs), proline transporters (ProTs), γ-aminobutyric acid transporters (GATs), putative auxin transporters (AUXs), similar to ANT1-like aromatic and neutral amino acid transporters (ANTs), and amino acid transporter-like (ATLa and ATLb) [6]. The protein structures of different subfamilies of AAAPs are very different. It is generally believed that the diversification of the sequences and structures of AAAPs is due to the specificity of the amino acids corresponding to amino acid transporters [7]. As a class of transmembrane transporters, the conserved domain of AAAP contains multiple transmembrane domains, the number and position of which are relatively conserved in different proteins and species; and the changes of transmembrane domains in transporters also indicate that genes are involved in transport functional differentiation [8].
AAAP protein is mainly involved in regulating the long-distance transport of amino acids in the body in plants, mediating amino acid transport across membrane structures, and participating in a variety of other life processes [9]. So far, AAAP genes in several species have been identified, such as Arabidopsis [10], rice [11], moso bamboo [12], Fragaria vesca [13], and Brassica rapa [7] et al. The function of many AAAP genes verified in model species. In Arabidopsis thaliana and Oryza sativa, AtAAP1 mediates the amino acid transport in embryo and root cells [14,15]; AtAAP2 could impact the metabolism of seed yield and oil content through the xylem-phloem transfer [16]; AtAAP3 is exclusively expressed in the root involved in amino acid uptake from soil [8] and OsAAP3 could control the yield by influencing the amino acid transfer [17]; AtAAP4 also plays a role in phloem loading [9]; AtAAP5 may perform amino acid import in companion cells in different organs [18,19] and OsAAAP5. The amino acid regulates tiller number in rice [20]; AtAAP6 could uptake amino acid function in the xylem parenchyma [21,22] and overexpression of the OsAAP6 cold enhance root absorption and improve the nutritional quality in rice [23]. Inhibition of StAAP1 expression results in altered amino acid levels in the phloem of Solanum tuberosum [16]. Overexpression of GmAAP6a enhanced soybean nitrogen tolerance and source-sink transport capacity and improved soybean seed quality [24]. AtAAP8 plays a crucial role in the early seed development in Arabidopsis [25]. PtAAP11 mediates proline transport with high affinity, providing proline to cell wall proteins during xylem formation in Populus trichocarpa [26]. Other subgroups have also been researched comprehensively. Members of the LHT subfamily are specific for lysine and histidine. AtLHT1, 2, 4, 5, and 6 are thought to play an important role in plant sexual reproduction and are expressed in male and female floral organs [9,27]. In addition, AtLHT1, 4, and 6 were detected in the root and bud, respectively [11,27,28]. OsLHT6 is specifically expressed in new shoot meristems [27]. PgLHT is involved in the growth and development of Panax ginseng roots [29]. The AUX gene family mainly promotes the development of roots or shoots by maintaining the homeostasis of auxin inside and outside cells together with other auxin-related proteins [30,31,32]. In the Gossypium hirsutum, GhAux1, GhAux2, GhAux3, GhAux6, and GhAux7 are mainly expressed in the vegetative organs of cotton and participate in the vegetative growth of cotton. However, GhAux4 and GhAux5 are preferentially expressed in the ovule of cotton on the day of flowering, and are mainly involved in the initiation of cotton fibers [33]. As a specific transporter of GABA, the expression of AtGAT1 is increased under flower and GABA treatment [34]. The ProTs subfamily is responsible for the transport of proline, glycine betaine (GB) and GABA in the vegetative organs of roots, leaves, and phloem and phloem in barley [35,36]. Overexpression of GmProT1 and 2 can affect the synthesis and response of proline and alleviate the damage of salt and drought stress to plants [37]. GsGAT gene expression levels increase the ability of Glu to convert to GABA and transport GABA to maintain high nitrogen availability in Camellia sinensis [38].
Although many functional AAAP genes have been revealed, proving their importance in the process of plant growth and development and in resistance to abiotic stress [36,37], the AAAP gene in L. chinense has not been characterized. In order to explore the composition of AAAP gene family members in L. chinense and the possible biological processes involved, we identified and analyzed them based on the whole genome data of L. chinense and systematically analyzed the AAAP gene family from the aspects of phylogeny, gene function differentiation, expression pattern, and stress response, which has provided the foundation for further gene function research and genetic improvement.

2. Results

2.1. Identification of LcAAAP Gene Family

Through the local BLASTP and HMMER to search the AAAP protein in the L. chinense genomic database, 73 putative protein sequences were obtained. Next, the Pfam and Conserved Domain Databases were applied to verify the conserved domains. Finally, the 52 LcAAAP protein sequences were retained; these genes were designated based on the AtAAAP gene family (Table 1, Figure 1 and Figure S1). Gene characteristics, including chromosomal location, open reading frame length, amino acid length, molecular weight, isoelectric point, and subcellular location were analyzed (Table 1). Through analysis of the chromosome distribution based on the L. chinense genome, 48 LcAAAP genes were located on 14 chromosomes. Chromosome 4 had the most LcAAAP genes (14), followed by chromosome 2 (seven genes), chromosomes 1 and 17 (five genes), chromosome 14 (four genes), chromosomes 3 and 18 (three genes), chromosomes 11, 13, and 16 (two genes), and chromosome 8, 10 and 12 (one gene). The genes LcAPP6c, LcATL7d, LcATL7b, and LcATL8a were located on the contigs. The LcAAAP proteins ranged from 288 (LcAPP17) to 1109 (LcAPP7a) amino acids, with molecular weights between 31.31 (LcAPP17) and 123.62 (LcAPP7a) kDa. The predicted pI values ranged from 5.56 (LcATL8b) to 9.9 (LcPorT1). The subcellular location predicted result showed the 48 genes can be located in the cell membrane (Table 1). LcATL1b only was located on the chloroplast, and LcAPP6a, LcATL8a, and LcAPP6b only were located on the Golgi apparatus. Additionally, the 17 genes could be located in other parts of the cell without the cell membrane, such as the chloroplast, cytoplasm, mitochondrion, and nucleus. There was a certain relationship between multiple positioning sites and gene function.

2.2. Evolutionary Analysis of LcAAAP Genes

To explore the LcAAAP gene family’s evolutionary relationship with other species, a phylogenetic tree was constructed based on the AAAP protein sequences of L. chinense (Lc), Oryza sativa (Os), Arabidopsis thaliana (At), Amborella trichopoda (Atr), Sorghum bicolor (Sb), Populus trichocarpa (Pt), Zea mays (Zm), and Vitis vinifera (Vv) (Figure S1). The basic information on this protein is listed in Table S1. The AAAP gene family could be divided into eight subgroups: Lysine and Histidine Transporters (LHT), Amino acid permease (AAP), proline transporter (ProT), gamma-aminobutyric acid transporter (GAT), auxin transporter (AUX), Amino acid-like transporter (ATLa and ATLb), and Aromatic and neutral amino acid transporters (ANT). The LcAAAP gene family also was divided into eight subgroups, including 6 LHT members, 15 AAP members, 2 ProT members, 5 GAT members, 5 AUX members, and 2 ANT members. A total of 10 members were classed into ATLa, and seven members belong to the ATLb (Figure 2). The phylogenetic tree result showed that many members of LcAAAP were clustered into a clade with VvAAAP and AtrAAAP; it was indicated that the AAAP gene family of L. chinense, A. trichopoda and V. vinifera have a closer kinship (Figure 1 and Figure S1).
Understanding gene structure can provide information about evolution and gene function. Therefore, the gene structures, protein conserved motif., and transmembrane topology were analyzed. The LcAAAP contains different exon numbers from 2 to 15 (Figure 3). Different subfamilies show different patterns of gene structure. Most AAAP gene family members within each subgroup share the same or similar gene structure and gene length. However, there were also certain differences in the structure of genes between members of the same subfamily: for example, LHT4 only has two exons, but 15 exons were detected in LHT2. Gene structure analysis showed that the gene structure of the LcAAAP gene family was relatively diverse, which may indicate the evolutionary trend of diversity and diverse gene functions. At the same time, if the positions of exons and introns in the same subfamily are relatively conserved, this may indicate that members of the same subfamily share a close evolutionary relationship. In addition, we found that multiple LcAAAP gene family members possess ultra-long introns (>10 kb); this result may negatively affect gene transcription. Motif analysis indicated that each subfamily has its own unique motif distribution pattern (Figure 4 and Table S2). Most of the genes that were revealed to be closely related by the phylogenetic analysis had a conserve motif type and distribution. Most of the genes that were revealed to be closely related by the phylogenetic analysis had a conserved motif type and distribution. Moreover, some conserved motifs were widely distributed among AAAP members, such as motif1, 4, and 6. In contrast, most conserved motifs are specific, appearing only in specific subfamilies. The motifs 14,18,19, and 20 belong only to the AUX subgroup, and motif 5 only exists in the APP subgroup; this result may herald their association with functions unique to subfamilies. In addition, some motifs such as 9,11,12,13, and 17 were mainly distributed in one subgroup, but they were sporadically distributed among individual members of other subfamilies; this may affect changes in the function of certain genes.
Most AAAP gene family members are responsible for the transport of amino acids and mediate their transport across the cell’s membrane structure. This indicates that the transmembrane structure of the AAAP protein has a specific connection with the function of the gene. We predicted the LcAAAP gene family transmembrane domain (Figure 5 and Figure S3 and Table S3). The result showed that the LcAAAP gene family typically has 8–12 transmembrane domains, and the location and number of transmembrane domains were relatively conserved, indicating that members of the same subfamily may have similar functions. However, there were also some members whose transmembrane domains were quite different, such as LcATL1a (14), LcLHT2 (17), LcAPP4b (17), LcATL11 (4), LcAAP17 (5), LcATL9 (6), and LcPorT1 (6). Reduced or expanded numbers of transmembrane domains indicate that their functions may change.

2.3. Synteny Analysis of LcAAAP Genes in L. chinense, Grape, Arabidopsis, and Rice

The expansion and contraction of gene families are affected mainly by whole genomic duplication and by tandem and segmental duplication. L. chinense has a single lineage-specific WGD event that occurred approximately 116 million years ago. This may cause the expansion of the LcAAAP gene family members. In addition, the whole-genome collinearity analysis result showed that one pair of tandemly duplicated even members (LcGAT1a-LcGAT1c) and six pairs of segmentally duplicated members (LcAPP4a-LcAPP4b, LcAUX1a-LcAUX1b, LcLHT1-LcLHT2, LcLAX5a-LcLAX5b, LcATL5a-LcATL5c, LcLAX2-LcLAX5b) were detected in the LcAAAP gene family (Figure 6, Table S4). The GAT, APP, AUX, ATL, and LHT subgroups have member expansion through tandem and segmental duplication, especially the AUX subgroups. Moreover, the number of collinear genes shared by different species may reflect the gene family phylogenetic relationships. We performed the synteny analysis of the AAAP gene family between L. chinense, Arabidopsis thaliana, Oryza sativa, and Vitis vinifera (Figure 7). A total of 38 orthologous pairs were investigated between L. chinense and the other three species’ AAAP genes. Among them, 10, 4, and 24 orthologous pairs were presented with genes in A. thaliana, rice, and grape, respectively. The LcAAAP and VvAAAP gene families have a large number collinear gene pairs, which shows that LcAAAP and VvAAAP are closer in evolutionary position.
To verify whether the duplicated homologous genes of AAAP were selected during the evolution to adapt to external changes, we measured the Ka/Ks nucleotide substitution ratios of collinear genes to study the exerted selective pressure (Table S4). In the LcAAAP, we found that their Ka/Ks << 1, indicating that the duplicated LcAAAP genes have undergone a purifying selection during their evolutionary history. Compared with the Arabidopsis thaliana, Oryza sativa, and Vitis vinifera, most of the AAAP gene pairs Ka/Ks << 1, it was mean that the orthologous LcAAAP genes of the three species were subjected to purifying selection during evolution. However, for two gene pairs between LcAAAP and VvAAAP gene families Ka/Ks>1, it was indicated that the two pairs have positive selection during their evolution.

2.4. Codon Usage Bias Analysis

The codon usage pattern reflects the evolution and mutation of species or genes. The relative synonymous codon usage and the relative frequency of synonymous codons of LcAAAP genes were calculated with CodonW (Table S5). Here, 30, 33, and 1 codon had a positive bias, negative bias (RSCU < 1), and no bias (RSCU =1), respectively. In the more frequently used codons, some amino acids (RSCU > 1) such as Leucine (Leu), Isoleucine (Ile), serine (Ser), and threonine (Thr) have two or more codons with positive bias. Moreover, if an RFSC value exceeds 60% or is 0.5 times greater than the average frequency of synonymous codons, the codons are considered high frequency. The RFSC analysis result showed that UUC, AAG, and AGC showed high frequency. Compared with the AtAAAP, OsAAAP, and AtrAAAP codon usage frequency ratios, 1, 29, and 1 codon had ratios greater than 2.00 or lower than 0.50, respectively. Among them, 29 codons had significant differences with the AAAP genes of O. sativa. This result showed that the LcAAAP codons usage frequency is like that of AtAAAP and AtrAAAP and that Arabidopsis can be used as one of the choices for LcAAAP gene receptors.

2.5. Analysis of Cis-Acting Elements in LcAAAP Promoters

To further investigate which process could be regulated through the LcAAAP, the cis-acting element promoter was analyzed by the online tool PlantCARE. We detected 111 type cis-acting elements and multiple elements that could be involved in different processes. Among them, 43 type elements were related to light responsiveness within all the LcAAAP genes (Table S6). In addition, we mainly analyzed and classified cis-acting elements related to hormones and stress. (Figure 8). Here, 11 and 3 type elements related to hormones and stress were identified, respectively. Most obviously, the ABRE (cis-acting element involved in the abscisic acid responsiveness) elements were abundantly enriched in certain genes, such as LcATL7d/8b/9 and LcAVT6 et al. Furthermore, the elements related to drought induction (MBS), low temperature responsiveness (LTR), auxin response (AAuxRR-core and TGA-element), and MeJA response (CGTCA-motif and TGACG-motif) were also recognized in many LcAAAP promoters. It was suggested that these genes may be involved in hormone signaling and stress response. In general, different gene promoters have different regulatory elements, suggesting the complexity of gene regulatory networks. In Figure 6, we found that 51 LcAAAP genes have one or more stress response elements. Among them, 32 and 37 genes could respond to low temperature signals and drought signals by MYB gene binding sites, and 18 genes could be involved in defense and stress responsiveness. Auxin, abscisic acid, salicylic acid, gibberellin, and methyl-jasmone response elements were distributed on all LcAAAP gene promoters. The most widely distributed was the ABA element (50), followed by the MeJA element (44 genes), GA response element (40), auxin response element (33), and SA response element (1).

2.6. Expression Patterns of LcAAAP Genes in Various Organs, Somatic Embryogenesis, and Response to Different Stress

We combined the published transcriptome data to determine the expression patterns of individual LcAAAP genes in seven organs (bract, leaf, shoot apex, stamen, petal, pistil, and sepal) (Figure 9). A total of 52 LcAAAP genes were divided into four groups based on their expression profiles in a hierarchical clustering heat map. In group 1, the 13 LcAAAP genes (LcAUX1a/1b, LcLHT1/4/8/11, LcATL5a/7b/9, LcAPP2/7b, LcLAX5a, and LcAVT6) had a high transcripts per million (TPM) value in various organs, except for the LcLHT8 expression level in the leaf. It was suggested this group gene could have an important role in the flower. In group 2, the genes expression level of the six LcAAAP genes (LcATL1b/5c, LcAPP7a/7c, LcANT2, and LcPorT2) maintain a steady state in different organs. The remaining 7 genes (LcAPP1a/4a and LcATL1a/7b/8b/12a/12b) were only specifically expressed in certain organs. In groups three and four, 12 genes (LcAPP1b/4b/4c/6b/6c/6d/17, LcGAT1b/1c/4, LcLHT3, and LcPorT1) lacked expression or had low expression levels. The LcATL15 and LcLHT2 were specifically highly expressed in the stamen; it was suggested that they could be involved in stamen development. The LcLAX5b only was found in the shoot apex. In addition, some genes only were detected at a relatively high expression level in the leaf, shoot apex, stamen, and pistil, such as LcLAX5b and LcLHT6, which had higher expression levels only in the shoot, apex, and pistil.
We compared the expression levels of the LcAAAP gene family in mid-petal development (1–4) of L. chinense and L. tulipifera (Figure 9 and Table S7). The expressions of some members of the second and fourth groups caught our attention. In L. tulipifera, the expressions of LcAPP1a/1b/4a and LcANT8a were upregulated to a very high level (58-fold, 67-fold, 79-fold, and 23-fold, respectively) in the second or third stage of petal development, whereas in L. chinense petals, the expression levels of these genes were low or significantly reduced. In contrast, the expression level of LcLHT8, LcATL9, and LcAUX1a in the petals of L. tulipifera was higher than that of L. chinense through the development of petals. In addition, we also explored the expression pattern of LcAAAP during somatic embryogenesis of hybrid Liriodendron (Figure 9 and Table S7). Based on the laboratory-established hybrid Liriodendron somatic embryogenesis system, we divided the process into 11 stages (PEM: embryogenic callus; ES1: 10 days after liquid culture; ES2: 2 days after screening; ES3: ABA 1 day of treatment; ES4: ABA treatment for 3 days; ES5: globular embryo; ES6: heart-shaped embryo; ES7: torpedo embryo; ES8: immature cotyledon embryo; ES9: mature cotyledon embryo; PL: plantlet). The expression levels of LcLHT1 and LcATL5a were continually upregulated 23.9-fold and 22.5-fold in the embryogenic callus stage (ES1–ES4), respectively. In LcLHT1, LcATL5a, and LcAUX1a/1b, there were certain genes (LcLHT1/8, LcATL1a/5a/8, LcAUX1a/1b, LcAPP4/9, and LcLAX2/5a) that were consistently highly expressed in the somatic embryogenesis stage, and the LcAUX1a/1b were stably expressed in all 11 stages of hybrid Liriodendron somatic embryogenesis. These results indicate that these genes may be involved in some important regulatory pathways in the process of somatic embryogenesis of hybrid Liriodendron.
We determined the expression pattern of the LcAAAP gene family under drought, low temperature (4 °C), and heat stress in the leaf (Figure 10). The 52 members were clustered into multiple groups following the different stresses. In response to the heat (40 °C) treatment, four groups were found by gene expression level. The group 3 gene (LcAPP4a, LcLHT1, and LcATL12b) expression levels were downregulated quickly in a short period of time (12 h) and then upregulated. In group 2, the LcATL5a and LcATL9 gene expression levels showed a trend of downregulation and upregulation within 12 h after heat stress treatment, respectively, and then recovered. Downregulated expression levels were detected after 1h of treatment for six genes (AUX1a/1b, LcAPP1a/2/7b, LcLAX5b). In the other genes of groups 1 and 4, the expression levels of most genes in leaves were very low or not expressed. Under drought stress, we did not observe significant up- or downregulation of LcAAAP gene expression except for LcATL5a, which was downregulated as processing time grew. In addition, the expression levels of some members only changed at a certain point, and eventually all returned to the original pattern, such as the LcAPP4a and the LcAAP7b, which upregulated only at 24 h and 12 h, respectively. Regarding the effect of low temperature on gene expression, LcATL5a, LcAPP4a, and LcLHT1 upregulated after 12 h exposure to 4 °C. The LcAPP7b and LcAUX1a/1b showed a trend of downregulated expression. In addition, we found that the LcATL12b expression level increased rapidly in 12 h. In this result, the expression abundances of most genes changed in a short period of time during different stress treatments, but the changes were small. It was worth noting that LcATL5a, LcAPP4a, and LcLHT1 showed different expression patterns under multiple stress treatments. These results suggest that diverse mechanisms control LcAAAP gene responses to various stresses.

2.7. Prediction and Correlation Analysis of LcAAAP Interacting Proteins

The construction of protein interaction networks is of great significance for the study of gene interactions and regulatory relationships. We demonstrate the protein interaction between the LcAAAP gene family and the protein interaction between LcAAAP protein and LcMYB/WRKY transcription factor (Figure 11 and Table S8). The protein interaction network within the LcAAAP gene family was divided into three groups. The first group, LcANT2, can interact with LcAPP4c/6c/9 and LcGAT2/4, and LcPorT1 can interact with LcATL1b/8a as the second group. In addition, a complex network of interactions involving some of the other LcAAAP members was constructed in the third group. LcLAX2, LcAPP2, and LcATL9 can interact with each other. LcATL7d can interact with the LcLAX2, LcAPP2, LcATL12b, and LcANT1. Some transcriptional regulators that may be involved in the regulation of LcAAAP protein were also identified (Figure 11). The result showed that LcMYB118 and LcWRKY22 could interact with LcAPP2, and LcMYB40/108 could interact with LcLAX2, respectively. LcAUX1b can be independently regulated by multiple transcription factors (LcMYB77/89/108/186), and LcWRKY18, LcMYB91, and LcMYB25 can be regulated with LcAPP7b, LcAVT6, and LcLHT4, respectively.
To further explore whether LcAAAP was involved in heat, drought, and low temperature stresses (Figure 12 and Table S9), we analyzed the correlation between LcAAAPs and the LcMYB/WRKY transcription factors involved in abiotic stress. The result showed a positive correlation (r > 0.7) between the expression levels of LcAPP4a and LcWRKY28, LcMYB83, LcMYB141, LcWRKY5, LcMYB71, and LcMYB38. LcLHT1 showed a positive correlation with LcWRKY14, LcWRKY15, LcWRKY16, LcWRKY17, LcWRKY27, LcMYB9, LcMYB37, LcMYB38, LcMYB83, and LcMYB105. In the same way, there was a significant positive correlation among the expression levels of LcATL12b, LcMYB28, LcMYB38, LcMYB56, and LcWRKY17. These results further showed that LcAPP4, LcLHT1, and LcATL12b could be involved in the abiotic stress response process. In addition, some genes may be specifically regulated only in certain abiotic stresses: for instance, under heat stress, the expression level of LcAPP2 has a positive correlation with LcMYB32, LcMYB43, LcMYB84, LcMYB89, LcMYB110, LcMYB113, LcMYB138, and LcMYB144. A negative correlation (r < −0.7) was identified in LcMYB12 and LcMYB84. In summary, the correlation analysis shows that a few LcAPPPs participate in the stress response.

2.8. Response of GAT and PorT Subgroups to Al Stress

Wang et al. showed that gamma-aminobutyric acid (GABA) signaling could enhance tolerance of hybrid Liriodendron to Al stress by promoting organic acid transport and maintaining cellular redox and osmotic balance. The current study shows that GAT is specific for GABA transport, whereas PorT mainly transports proline, but PorT of some species can also respond to GABA. Therefore, we explored the responses of GAT and PorT family members under the three treatment methods: Al stress, GABA treatment (Figure 13, Figure 14 and Figure 15), and Al stress followed by GABA treatment. In this process, the six genes of the GAT and PorT subgroups have varying degrees of expression except for LcGAT1b, which were not detected.
In the Al stress, the LcGAT1a, LcGAT1c, LcGAT2, and LcGAT4 increase the expression abundance in root and stem after 48 h under Al stress. LcGAT2 was upregulated in leaves after 48 h treatment, while LcGAT4 expression in leaves and shoots began to increase after 24 h treatment. LcPorT1 was consistently upregulated in different organs. LcPorT2 showed two expression patterns in different organs, downregulated first and then upregulated in stem and bud, in contrast to expression in leaves.
Under GABA stress, the expression level of LcGAT1a in roots and shoots was downregulated after 24 h of treatment, and the expression of LcGAT1c in leaves and shoots was downregulated after 12 h of treatment. Compared with other LcGAT genes, the expression level of LcGAT2 in the stem continued to increase after 12 h of treatment. In the PorT subgroup, LcPorT1 could be upregulated in four organs after 12 h of stress. Interestingly, the expression trend of LcPorT2 in stems, leaves, and shoots was the inverse of that of LcPorT1, suggesting that their responses to GABA may have mutually antagonistic effects.
LcGAT1a and LcPorT1 were upregulated in stem and leaf at 12 h after the GABA was applied after the Al stress treatment. A similar situation exists in other GAT genes. For example, expression levels of LcGAT1a, LcGAT1c, and LcGAT2 were upregulated in leaves at 12 h or 48 h; LcGAT2 also was upregulated in stem and bud at 12 h and 24 h, respectively. In addition, the downregulated abundance genes compared with the Al stress in different organs also were detected. LcGAT1a, LcGAT1c, LcGAT2, and LcPorT1 were downregulated in root, stem, and bud after 48 h of treatment. LcPorT2 was downregulated in stem and bud at 48 h.
The above analysis results show that the hybrid Liriodendron may respond to changes in the external environment by changing the content of specific substrates in cells of different organs, thereby affecting the expression of the LcGAT and LcPorT subgroups’ genes.

3. Discussion

As one of the largest gene superfamilies of AAT, the AAAP gene family can be divided into eight subfamilies according to existing research, which plays an important role in plant physiology and stress resistance research and has great potential value. Relevant research work has been carried out in a variety of model plants. such as Arabidopsis, rice, potato, and polar et al. [10,11,39,40]. Although the functions and roles of AAAP genes have been explored in multiple species, systematic analysis of the AAAP gene family has not been completed after the publication of the genome of L. chinense. In this research, 52 LcAAAP genes were identified based on the genome of L. chinense, and eight subgroups were divided by the AAAP gene family phylogenetic tree of multiple species. Although the cluster clade was like other AAAP gene families, the numbers of members in different subgroups differs significantly, suggesting that different degrees of expansion occurred in different species.
In addition to LcAPP6c, LcATL7d, LcATL7b, and LcATL8a, the remaining 48 genes were unevenly mapped to 14 chromosomes, and most genes were distributed in chromosomes 4, 2, and 17. Subcellular localization results showed that 48 LcAAAP genes were located on the cell membrane; it was suggested that these genes were mainly used for membrane localization to experiment with the transport of substances inside and outside the cell. Among the LcAAAP genes, many genes have multiple locations (17) or are located only in the chloroplast or Golgi (4). It was indicated that these genes could also function in different organelles. To better understand LcAAAP gene function and evolutionary history, we explored different perspectives. Exon–intron structure analysis indicated that most LcAAAP genes in the same subgroup had the same or similar numbers. However, there are still structural inconsistencies in some genes, such as differences in the number of exons and the existence of ultra-long introns. Most AAAP gene family members are responsible for the transport of amino acids with different specificities and characteristics and mediate their transport across the cell’s membrane structure. It was suggested that the existence of the transmembrane (TM) domain plays an extremely important role in the AAAP genes. 7–11 TM were identified in most of the LcAAAP genes of the same subgroups. However, there are still some genes (LcLHT2/4, LcATL11, LcPorT1, and LcAPP4/17) whose numbers of TMs have expanded or shrunk. Compared with the results of the motif analysis, the conserved motif types of different subfamilies were found to be consistent with the transmembrane domains. This result indicates that the composition of the transmembrane domain is closely related to gene function and further suggests a direct connection between gene motifs and gene function.
WGD and gene duplication events were thought to be the main causes of gene family expansion and functional diversity [41,42]. In the Magnolia plants, two and one whole-genome duplication events occurred in C. kanehirae and L. chinense [1,43], which could lead to the members of the gene family expanding. Currently, 25% (13/52) of AAAP genes are duplicated genes in L. chinense, which exist as one pair of tandemly duplicated and six pairs of segmental duplicated events, especially in the LcAUX subgroup, including three pairs of gene duplication events. These results showed that the segmentally duplicated event was the main cause of expression of the LcAAAP gene family. In addition, we analyzed the collinear relationship of AAAP genes between L. chinense, Arabidopsis thaliana, Oryza sativa, and Vitis vinifera. The 24 orthologous gene pairs were identified between LcAAAPs and VvAAAPs, greater than the 10 and 4 orthologous gene pairs found in LcAAAP-AtAAAP and LcAAAP-OsAAAP. It was indicated that there may be a closer kinship between LcAAAPs and VvAAAPs. At the same time, there were collinear relationships between the LcAAAP genes and VvAAAP/AtAAAP/OsAAAP, which also proves that the LcAAAP genes may have been formed before monocotyledonous and dicotyledonous plants. These opinions can be verified from a genome-wide perspective: magnoliids arose before the divergence of eudicots and monocots, represented by Liriodendron [1]. The Ka/Ks ratio of orthologous genes’ duplicated events was used to evaluate the AAAP genes contribute to organism fitness: Ka/Ks < 1 for most paralog gene pairs, both within and between species, which indicated that these paralog gene pairs encountered various degree purifying selective pressures, demonstrating that complex selective pressures drove the evolution of the AAAP gene family. Gene duplication events provide raw materials for regulating physiological and morphological changes in plants and provide a basis for species to adapt to changes in the external environment [44]. LcAUX1a and LcAUX1b have similar expression patterns in various organs and stresses, which indicated that they may have redundant functions. However, the other paralog gene pairs all showed different expression patterns, which, because of the duplicated genes, always tend to be sub-functionalized, neo-functionalized, or both [45]. This result suggested that the strong purifying selection may have inflected the gene functional divergence to adapt to diversity in the environment.
There was a very important relationship between the biological function of genes and their expression level, and a comprehensive analysis of the expression level of the genes can analyze the putative gene function in the process of plant growth and development [46]. In this study, the expression profiles of the LcAAAP gene family were analyzed in different organs and stages of Liriodendron. Approximately 28 and 34 LcAAAP genes were expressed at relatively high levels (TPM > 1) in the leaves and shoot apex of L. chinense, respectively. Here, 40 genes showed relatively high expression levels in the flowers of L. chinense. We found that LcLHT1, LcLHT2, and LcATL15 have high expression in leaves, bract, and stamens, respectively. This result is like that of paralogous genes in Arabidopsis [19,28,47]. Therefore, LcLHT2 could play an important role in amino acid transport in pollen development and maturation, and LcALT15 also might be involved in the long-distance transport of amino acids in stamens. In addition, the expression levels of LcLHT8 were significantly higher in floral organs than in leaves; it was indicated that LHT8 could also have an important role in flowering. Nectar generally contains substances such as carbohydrates, amino acids, inorganic ions, proteins, and lipids [48] and the central area of the petals was thought to be the location of the Liriodendron nectary [49,50]. The LcAAP1a gradually upregulated during the development of nectar in L. chinense; and the expression levels of LcAAP1a, LcAAP1b, and LcAAP4 in the nectar of L. tulipifera were significantly upregulated in the second stage relative to L. chinense, which indicated that the increased levels of amino acid transport in the nectar implies that the amount of nectar secreted by L. tulipifera would be greater than that of L. chinense, which is consistent with reality. This result showed that a more complex amino acid transport network exists in the nectar secretion of L. tulipifera. As protein synthesis substrates, amino acids play an indispensable role in the process of plant somatic embryogenesis [51,52]. As the embryogenic cells develop toward the globular embryo stage in hybrid Liriodendron (PEM-ES4), the LcLHT1 and LcATL5a were specifically upregulated in this progress. During subsequent embryonic development (ES5-ES8), the LcLHT1/8, LcATL1a/5a, and LcAAP4a maintained stable levels, and the expression levels of LcAUX1a, and LcLAX2 were significantly upregulated relative to the previous stage. In addition, the LcATL5a and LcAAP9 were upregulated, and the LcLAX2 and LcLAX5a were downregulated during plant morphogenesis (ES9-PL). These results indicate that protein and auxin transport plays an important role during somatic embryogenesis. In the first stage, a large amount of protein accumulates as the basis for subsequent embryonic development. In the second stage, the content of intracellular auxin decreases, resulting in assists in embryonic development, similar to the expression pattern of LcPIN genes [53]. In the third stage, the specific regulation of auxins and proteins in cells provides the basis for plant morphogenesis.
According to previous studies, AAAPs were involved in the low temperature, heat temperature, drought stress treatments in many plants [35,54,55]. In this study, we analyze the LcAAAP gene expression pattern based on the stress transcriptome data of hybrid Liriodendron [56,57]. A total of 13 genes in hybrid Liriodendron leaves were able to respond to the three abiotic stress treatments. Most genes’ expression levels were downregulated in a short period of time. Interestingly, the significant changes occurred in the expression patterns of LcATL5a, LcAPP4a, and LcLHT1 in these data, and they showed completely opposite trends in heat and low temperature stress treatments. Cis-acting element analysis results showed that the promoters of LcAAAP genes’ response to the abiotic have at least one of the LTR (response to the low temperature), MBS (MYB binding site involved in drought-inducibility), and TC-rich repeat elements (cis-acting element involved in defense and stress responsiveness), which showed that promoters could control structural and morphological changes in plant–environment interactions to adapt to unfavorable external environments [58]. How plants adapt to external abiotic stresses involves a very complex gene regulatory network, and a variety of transcription factors are usually involved in this process [58,59,60]. In previous studies, we found that WRKY and MYB genes were involved in regulating the response of hybrid Liriodendron to stress [56,57]. Therefore, we constructed the co-expression network based on the LcAAAP, LcMYB, and LcWRKY genes to explore the interactions between them. Multiple MYB and WRKY genes showed a significantly positive correlation, and LcWRKY5/27 and LcMYB9/28/83 could rise to a relatively high level in a short time under abiotic stress [56,57]. These results indicated that the LcLHT1, LcAPP4a, and LcATL5a could transport amino acids under abiotic stress, provide support for the internal homeostasis of plants, and then adapt to changes in the external environment, and the specific expression pattern of these genes was directly or indirectly regulated by upstream transcription factors.
Gamma-aminobutyric acid (GABA) enhances aluminum tolerance in hybrid Liriodendron [61]. Therefore, we explored the gene expression pattern of LcGAT and LcPorT, where GAT can specifically transport GABA and the PorT can barely transport GABA in addition to its ability to transport proline [34,36]. The LcGAT1a/1c/2/4 were significantly upregulated in different organs after Al stress treatment; and the treatment of exogenous GABA causes GATs to respond at different rates in different organs. For example, the expression level of LcGAT1c/2 in roots was gradually increased from 12 to 48 h, whereas it was gradually downregulated in buds. When exogenous GABA treatment was applied to Al stressed plants, the expression levels of LcGAT1/2 in stems and leaves were rapidly upregulated within 12 h, indicating that this treatment may accelerate the transport of GABA in plants, thereby revealing that the treatment might accelerate the transport of GABA in the plant, more quickly alleviating the effect of Al poisoning on the growth and development of hybrid Liriodendron. For example, the accumulation of GABA in leaves facilitates the closure of plant stomata, thereby increasing its tolerance [62]. In addition, proline accumulated in hybrid Liriodendron leaves under the effect of Al toxicity based on previous studies; therefore, the expression level of LcPorTs showed different degrees of upregulation under aluminum stress treatment. LcPorT1/2 showed opposite expression patterns in stems, leaves, and buds under GABA treatment; this indicates that there is also a relationship between the transport of LcPorTs and GABA, but the specific mode and pattern need further research.

4. Materials and Methods

4.1. Collection of Query Sequences and Identification of the AAAP Gene Family Based on L. chinense Genomic Data

AtAAAP gene family protein sequences were acquired from the UniProT database (https://www.uniprot.org/ (accessed on 12 February 2022)) [63], and all the AAAP gene family sequences of other species were obtained from the JGI (https://phytozome.jgi.doe.gov/pz/portal.html (accessed on 13 February 2022)) [64]. The index sequences consisted of 46 AtAAAP protein sequences based on the BLASTP. The 69 putative sequences were found from the L. chinense protein database through a local BLASTP search with an E-value threshold of e−10. The Hidden Markov mode (HMM) profile for the AAAP domain (PF01490) downloaded from the Pfam database (http://pfam.xfam.org (accessed on 15 February 2022)) [65], was used to identify LcAAAP genes from the L. chinense genome with HMMER 3.3.2 (http://hmmer.janelia.org/ (accessed on 15 February 2022)), with an E-value of e−5. A total of 73 putative sequences were identified from the genomic database. After all putative LcAAAP protein sequences were merged, the candidate sequences were further analyzed with the online tools Conserved Domain Database (http://www.ncbi.nlm.nih.gov/cdd/ (accessed on 16 February 2022)) [66] and pfam [67]. Finally, the 52 target sequences were identified and used for further analysis. Molecular weight and isoelectric point of each protein were determined using the online tool ExPASy (https://web.expasy.org/protparam/ (accessed on 17 January 2022)) [68]. The subcellular localization of all LcAAAP proteins was predicted based on the online tool Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 17 January 2022)) [69].

4.2. Phylogenetic Tree, Gene Structure, and Conserved Motif and Transmembrane Region Analyses of LcAAAP Genes

Multiple sequence alignments analyses of AAAP amino acid sequences of Arabidopsis thaliana, Oryza sativa, Vitis vinifera, Sorghum bicolor, Populus trichocarpa, Zea mays, Amborella trichopoda, and L. chinense were performed with Muscle 3.8.31 [70]. We built the phylogenetic tree using the Maximum-Likelihood estimation with IQtree2.13 [71] and 1000 bootstrap replications; the optimal model was JTT+F+R7. The method for constructing the phylogenetic tree from the LcAAP protein sequences was consistent with the above; the optimal model was VT+F+G4. Evolutionary tree beautification was carried out using online tools iTOL (https://itol.embl.de/tree/2182103287321642042781 (accessed on 16 April 2022)) [72] and Adobe Illustrator CS3 software (version 13.0.0).
The exon/intron organization of LcAAAP genes was conducted by scanning the genomic annotation data. Conserved motifs were predicted with MEME (http://meme-suite.org/tools/meme (accessed on 20 February 2022)) [73] and a maximum of 20 motifs. The gene structure and motifs were visualized with TBtools [74]. Protein transmembrane topology was predicted using the ΔG prediction server v1.0 (https://dgpred.cbr.su.se/index.php?p=home (accessed on 16 April 2022)) [75]. Images were produced using the Tbtools software [74].

4.3. Chromosomal Location, Syntenic Analyses and Calculation of the Ka/Ks Value

The positions of the LcAAAP genes were acquired from the annotation file. Among them, 48 LcAAAP genes were mapped on 14 chromosomes, and the remaining four genes were located on the three contigs, with results listed in Table 1. We used MCScanX [76] with the default settings to identify LcAAAP gene pairs of segmental/tandem duplications in the L. chinense genome and analyzed the LcAAAP genes’ syntenic relation with the AtAAAP, OsAAAP, and VvAAAP gene pairs. The KaKs_Calculator2.0 [77] software was used to calculate the Ka/Ks values of homologous genes.

4.4. Analysis of the Codon Usage Pattern and The Cis-Acting Element

We obtained the coding sequences of L. chinense, Arabidopsis thaliana, Oryza sativa, and Amborella trichopoda based on the above AAAP genes. The CodonW1.4.4 software [78] was used to calculate the relative synonymous codon usage (RSCU) and the relative frequency of synonymous codons (RFSC). As promoter sequences, 2500 bp upstream from the translation start sites for LcAAAP genes were considered, and the cis-acting elements were predicted and analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 16 April 2022)) [79].

4.5. RNA-Seq Analysis of LcAAAP Gene Expression Levels in Different Organs and Multiple Stresses

To explore the expression patterns of the LcAAAP gene family, transcript data of different organs and hybrid Liriodendron at high temperature (0 h, 1 h, 3 h, 6 h, 12 h) and drought (0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 72 h) stress data were downloaded from NCBI with the following accession numbers: SRR8101040, SRR8101041, SRR8101042, SRR8101043, SRR9945429, SRR9945430, SRR9945433, SRR9948913, SRR9948914, SRR9948915, SRR9948916, SRR9948917, SRR9948918, SRR9948919, SRR9949005, SRR9949006, SRR9949007, SRR9949008, SRR9949009, SRR9949010, PRJNA679089, and PRJNA679101. The low temperature (4 °C (0 h, 12 h, 24 h, and 48 h)), Liriodendron petal development (Lc1-4 and Lt1-4 represent the developmental process of the middle part of the petals of L. chinense and L. tulipifera, respectively) and Hybrid Liriodendron somatic embryogenesis (PEM: embryogenic callus; ES1: 10 days after liquid culture; ES2: 2 days after screening; ES3: ABA 1 day of treatment; ES4: ABA treatment for 3 days; ES5: globular embryo; ES6: heart-shaped embryo; ES7: torpedo embryo; ES8: immature cotyledon embryo; ES9: mature cotyledon embryo; PL: plantlet) transcript data own the undisclosed data; The expression levels of related genes were listed in Table S4. All mRNA abundance values were measured by transcripts per million (TPM) based on the L. chinense genomic database.

4.6. Plant Materials and Stress Treatment

According to the previous research, the GAT and ProT could transport the gamma-aminobutyric acid (GABA), and GABA signaling could enhance the tolerance of hybrid Liriodendron to Al stress by promoting organic acid transport and maintaining cellular redox and osmotic balance [61]. To compare the GAT and ProT subgroup expression levels in organs of different stages in the Al stress, we chose the one-month hybrid Liriodendron with basically the same growth for stress treatment (Nanjing, China). The cultivation environment is the greenhouse of the Key Laboratory of Forest Genetics and Biotechnology of the Ministry of Education, Nanjing Forestry University, under white light (light for 16 h, darkness for 8 h). The concentrations of AlCl3 and GABA were 30 uM and 10 mM, respectively; the culture medium was 3/4 MS. Hybrid Liriodendron seedlings were soaked in Al solution for 3h and then inoculated on the corresponding medium; we obtained materials from three time periods, 12 h, 24 h, and 48 h, respectively. We quickly froze the material in liquid nitrogen and stored it at −80 °C.

4.7. RNA Extraction and Quantitative Real-Time PCR Analysis

Total RNAs were extracted using the KK Fast Plant Total RNA Kit (ZOMANBIO, Beijing, China). The Evo M-MLV RT Kit with gDNA Clean for qPCRII AG11711 (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) was applied to synthesize the first-strand cDNA from 1.0 mg RNA. Equalbit 1× dsDNA HS Assay Kit (EQ121-01, vazyme) completed quantification of all reversed cDNAs. PCR amplifications were carried out with SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) in 20 μL volumes using Roche LightCycler® 480 Real-Time PCR System. Three replicates were performed for each selected gene. The 18S was taken as a reference gene. The relative expression levels were calculated by the ∆∆ CT method [80]. All qRT-PCR primers were designed by Primer5.0 [81] and are listed in Table S5.

5. Conclusions

In this study, we identified 52 AAAP genes from the L. chinense genome. Multiple analyses, including the construct phylogenetic tree, gene exon–intron structure, motif, and transmembrane domain prediction, indicated that the LcAAAP gene family was divided into eight subgroups. Most of the identified LcAAAP proteins have a close evolutionary relationship with A. trichopoda proteins, implying that they were relatively primitive among angiosperms. WGD and gene duplication events suggested that the expansion of some subgroups during the evolutionary history led to divergent gene functions. The expression patterns in different organs showed that the LcAAAPs were specifically expressed in the nectary development and somatic embryogenesis of Liriodendron, indicating that the LcAAAP gene family has coordinated to regulate growth and development. In addition, the LcAAAP gene family can also respond to low temperature, heat, and drought stresses through the regulation of WRKY/MYB genes; and the members of the GAT and PorT subgroups members can participate in the process of hybrid Liriodendron to resist Al poisoning.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/article/10.3390/ijms23094765/s1.

Author Contributions

L.H., P.W., Z.H., J.S., J.C.: contributed to the management and manuscript review. L.H., R.F., P.W., Z.H., D.Y., Y.L.: designed experiments as well as provided the methodology of data collection and analysis; L.H.: performed the experiment; All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China during the 14th Five-year Plan Period (2021YFD2200103), the Natural Science Foundation of Jiangsu Province (BK20210614), the Nature Science Foundation of China (32071784, 32101546), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX20_0899).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets supporting the conclusions and description of a complete protocol can be found within the manuscript and its additional files. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

We thank AJE (http://www.aje.limited/ (accessed on 10 March 2022)) for its linguistic assistance during the preparation of this manuscript.

Conflicts of Interest

The authors declare that they have no competing interests.

References

  1. Chen:, J.; Hao, Z.; Guang, X.; Zhao, C.; Wang, P.; Xue, L.; Zhu, Q.; Yang, L.; Sheng, Y.; Zhou, Y.; et al. Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation. Nat. Plants 2019, 5, 18–25. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Tegeder, M.; Offler, C.E.; Frommer, W.B.; Patrick, J.W. Amino acid transporters are localized to transfer cells of developing pea seeds. Plant Physiol. 2000, 122, 319–326. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Fischer, W.N.; Kwart, M.; Hummel, S.; Frommer, W.B. Substrate specificity and expression profile of amino acid transporters (AAPs) in Arabidopsis. J. Biol. Chem. 1995, 270, 16315–16320. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Williams, L.E.; Miller, A.J. Transporters responsible for the uptake and partitioning of nitrogenous solutes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 659–688. [Google Scholar] [CrossRef]
  5. Tegeder, M. Transporters for amino acids in plant cells: Some functions and many unknowns. Curr. Opin. Plant Biol. 2012, 15, 315–321. [Google Scholar] [CrossRef]
  6. Saier, M.H., Jr.; Reddy, V.S.; Tsu, B.V.; Ahmed, M.S.; Li, C.; Moreno-Hagelsieb, G. The Transporter Classification Database (TCDB): Recent advances. Nucleic Acids Res. 2016, 44, D372–D379. [Google Scholar] [CrossRef] [Green Version]
  7. Hu, L.-Z.; Yin, W.-B.; Chen, Y.-H.; Hu, Z.-M. Functional Divergence and Evolutionary Dynamics of the Putative AAAP Gene Family in Brassica rapa. Plant Mol. Biol. Rep. 2014, 32, 517–530. [Google Scholar] [CrossRef]
  8. Okumoto, S.; Koch, W.; Tegeder, M.; Fischer, W.N.; Biehl, A.; Leister, D.; Stierhof, Y.D.; Frommer, W.B. Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3. J. Exp. Bot. 2004, 55, 2155–2168. [Google Scholar] [CrossRef] [Green Version]
  9. Tegeder, M.; Ward, J.M. Molecular evolution of plant AAP and LHT amino acid transporters. Front. Plant Sci. 2012, 3, 21. [Google Scholar] [CrossRef] [Green Version]
  10. Rentsch, D.; Schmidt, S.; Tegeder, M. Transporters for uptake and allocation of organic nitrogen compounds in plants. Febs Lett. 2007, 581, 2281–2289. [Google Scholar] [CrossRef] [Green Version]
  11. Zhao, H.; Ma, H.; Yu, L.; Wang, X.; Zhao, J. Genome-Wide Survey and Expression Analysis of Amino Acid Transporter Gene Family in Rice (Oryza sativa L.). PLoS ONE 2012, 7, e49210. [Google Scholar] [CrossRef] [PubMed]
  12. Liu, H.; Wu, M.; Zhu, D.; Pan, F.; Wang, Y.; Wang, Y.; Xiang, Y. Genome-Wide analysis of the AAAP gene family in moso bamboo (Phyllostachys edulis). Bmc Plant Biol. 2017, 17, 29. [Google Scholar] [CrossRef] [Green Version]
  13. Liang, W.; Ling, L.; Wang, M.; Du, B.; Duan, Y.; Song, P.; Zhang, L.; Li, P.; Ma, J.; Wu, L.; et al. Genome-wide identification and expression analysis of the AAAP family inFragaria vesca. Biotechnol. Biotechnol. Equip. 2020, 34, 790–799. [Google Scholar] [CrossRef]
  14. Lee, Y.-H.; Foster, J.; Chen, J.; Voll, L.M.; Weber, A.P.M.; Tegeder, M. AAP1 transports uncharged amino acids into roots of Arabidopsis. Plant J. 2007, 50, 305–319. [Google Scholar] [CrossRef] [PubMed]
  15. Sanders, A.; Collier, R.; Trethewy, A.; Gould, G.; Sieker, R.; Tegeder, M. AAP1 regulates import of amino acids into developing Arabidopsis embryos. Plant J. 2009, 59, 540–552. [Google Scholar] [CrossRef] [PubMed]
  16. Zhang, L.; Tan, Q.; Lee, R.; Trethewy, A.; Lee, Y.-H.; Tegeder, M. Altered Xylem-Phloem Transfer of Amino Acids Affects Metabolism and Leads to Increased Seed Yield and Oil Content in Arabidopsis. Plant Cell 2010, 22, 3603–3620. [Google Scholar] [CrossRef] [Green Version]
  17. Lu, K.; Wu, B.; Wang, J.; Zhu, W.; Nie, H.; Qian, J.; Huang, W.; Fang, Z. Blocking amino acid transporter OsAAP3 improves grain yield by promoting outgrowth buds and increasing tiller number in rice. Plant Biotechnol. J. 2018, 16, 1710–1722. [Google Scholar] [CrossRef] [Green Version]
  18. Brady, S.M.; Orlando, D.A.; Lee, J.-Y.; Wang, J.Y.; Koch, J.; Dinneny, J.R.; Mace, D.; Ohler, U.; Benfey, P.N. A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 2007, 318, 801–806. [Google Scholar] [CrossRef]
  19. Tegeder, M.; Rentsch, D. Uptake and Partitioning of Amino Acids and Peptides. Mol. Plant 2010, 3, 997–1011. [Google Scholar] [CrossRef]
  20. Wang, J.; Wu, B.; Lu, K.; Wei, Q.; Qian, J.; Chen, Y.; Fang, Z. The Amino Acid Permease 5 (OsAAP5) Regulates Tiller Number and Grain Yield in Rice. Plant Physiol. 2019, 180, 1031–1045. [Google Scholar] [CrossRef]
  21. Hunt, E.; Gattolin, S.; Newbury, H.J.; Bale, J.S.; Tseng, H.-M.; Barrett, D.A.; Pritchard, J. A mutation in amino acid permease AAP6 reduces the amino acid content of the Arabidopsis sieve elements but leaves aphid herbivores unaffected. J. Exp. Bot. 2010, 61, 55–64. [Google Scholar] [CrossRef] [PubMed]
  22. Okumoto, S.; Schmidt, R.; Tegeder, M.; Fischer, W.N.; Rentsch, D.; Frommer, W.B.; Koch, W. High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis. J. Biol. Chem. 2002, 277, 45338–45346. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Peng, B.; Kong, H.; Li, Y.; Wang, L.; Zhong, M.; Sun, L.; Gao, G.; Zhang, Q.; Luo, L.; Wang, G.; et al. OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice. Nat. Commun. 2014, 5, 4847. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Liu, S.; Wang, D.; Mei, Y.; Xia, T.; Xu, W.; Zhang, Y.; You, X.; Zhang, X.; Li, L.; Wang, N.N. Overexpression of GmAAP6a enhances tolerance to low nitrogen and improves seed nitrogen status by optimizing amino acid partitioning in soybean. Plant Biotechnol. J. 2020, 18, 1749–1762. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Schmidt, R.; Stransky, H.; Koch, W. The amino acid permease AAP8 is important for early seed development in Arabidopsis thaliana. Planta 2007, 226, 805–813. [Google Scholar] [CrossRef]
  26. Couturier, J.; de Fay, E.; Fitz, M.; Wipf, D.; Blaudez, D.; Chalot, M. PtAAP11, a high affinity amino acid transporter specifically expressed in differentiating xylem cells of poplar. J. Exp. Bot. 2010, 61, 1671–1682. [Google Scholar] [CrossRef] [Green Version]
  27. Perchlik, M.; Foster, J.; Tegeder, M. Different and overlapping functions of Arabidopsis LHT6 and AAP1 transporters in root amino acid uptake. J. Exp. Bot. 2014, 65, 5193–5204. [Google Scholar] [CrossRef] [Green Version]
  28. Hirner, A.; Ladwig, F.; Stransky, H.; Okumoto, S.; Keinath, M.; Harms, A.; Frommer, W.B.; Koch, W. Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll. Plant Cell 2006, 18, 1931–1946. [Google Scholar] [CrossRef] [Green Version]
  29. Zhang, R.; Zhu, J.; Cao, H.-Z.; Xie, X.-L.; Huang, J.-J.; Chen, X.-H.; Luo, Z.-Y. Isolation and characterization of LHT-type plant amino acid transporter gene from Panax ginseng Meyer. J. Ginseng Res. 2013, 37, 361–370. [Google Scholar] [CrossRef] [Green Version]
  30. Ugartechea-Chirino, Y.; Swarup, R.; Swarup, K.; Peret, B.; Whitworth, M.; Bennett, M.; Bougourd, S. The AUX1 LAX family of auxin influx carriers is required for the establishment of embryonic root cell organization in Arabidopsis thaliana. Ann. Bot. 2010, 105, 277–289. [Google Scholar] [CrossRef] [Green Version]
  31. Marchant, A.; Kargul, J.; May, S.T.; Muller, P.; Delbarre, A.; Perrot-Rechenmann, C.; Bennett, M.J. AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J. 1999, 18, 2066–2073. [Google Scholar] [CrossRef] [PubMed]
  32. Adamowski, M.; Friml, J. PIN-Dependent Auxin Transport: Action, Regulation, and Evolution. Plant Cell 2015, 27, 20–32. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Han, X.; Xu, X.; Fang, D.D.; Zhang, T.; Guo, W. Cloning and expression analysis of novel Aux/IAA family genes in Gossypium hirsutum. Gene 2012, 503, 83–91. [Google Scholar] [CrossRef] [PubMed]
  34. Meyer, A.; Eskandari, S.; Grallath, S.; Rentsch, D. AtGAT1, a high affinity transporter for gamma-aminobutyric acid in Arabidopsis thaliana. J. Biol. Chem. 2006, 281, 7197–7204. [Google Scholar] [CrossRef] [Green Version]
  35. Grallath, S.; Weimar, T.; Meyer, A.; Gumy, C.; Suter-Grotemeyer, M.; Neuhaus, J.M.; Rentsch, D. The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns. Plant Physiol. 2005, 137, 117–126. [Google Scholar] [CrossRef] [Green Version]
  36. Fujiwara, T.; Mitsuya, S.; Miyake, H.; Hattori, T.; Takabe, T. Characterization of a novel glycinebetaine/proline transporter gene expressed in the mestome sheath and lateral root cap cells in barley. Planta 2010, 232, 133–143. [Google Scholar] [CrossRef]
  37. Guo, N.; Xue, D.; Zhang, W.; Zhao, J.-M.; Xue, C.-C.; Yan, Q.; Xue, J.-Y.; Wang, H.-T.; Zhang, Y.-M.; Xing, H. Overexpression of GmProT1 and GmProT2 increases tolerance to drought and salt stresses in transgenic Arabidopsis. J. Integr. Agric. 2016, 15, 1727–1743. [Google Scholar] [CrossRef]
  38. Ruan, L.; Wei, K.; Li, J.; He, M.; Wu, L.; Aktar, S.; Wang, L.; Cheng, H. Responses of tea plants (Camellia sinensis) with different low-nitrogen tolerances during recovery from nitrogen deficiency. J. Sci. Food Agric. 2022, 102, 1405–1414. [Google Scholar] [CrossRef]
  39. Ma, H.; Cao, X.; Shi, S.; Li, S.; Gao, J.; Ma, Y.; Zhao, Q.; Chen, Q. Genome-wide survey and expression analysis of the amino acid transporter superfamily in potato (Solanum tuberosum L.). Plant Physiol. Biochem. 2016, 107, 164–177. [Google Scholar] [CrossRef]
  40. Wu, M.; Wu, S.; Chen, Z.; Dong, Q.; Yan, H.; Xiang, Y. Genome-wide survey and expression analysis of the amino acid transporter gene family in poplar. Tree Genet. Genomes 2015, 11, 83. [Google Scholar] [CrossRef]
  41. Qiao, X.; Li, Q.; Yin, H.; Qi, K.; Li, L.; Wang, R.; Zhang, S.; Paterson, A.H. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019, 20, 38. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Xie, T.; Chen, C.; Li, C.; Liu, J.; Liu, C.; He, Y. Genome-wide investigation of WRKY gene family in pineapple: Evolution and expression profiles during development and stress. BMC Genom. 2018, 19, 490. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Chaw, S.-M.; Liu, Y.-C.; Wu, Y.-W.; Wang, H.-Y.; Lin, C.-Y.I.; Wu, C.-S.; Ke, H.-M.; Chang, L.-Y.; Hsu, C.-Y.; Yang, H.-T.; et al. Stout camphor tree genome fills gaps in understanding of flowering plant genome evolution. Nat. Plants 2019, 5, 63–73. [Google Scholar] [CrossRef] [Green Version]
  44. Kong, H.; Landherr, L.L.; Frohlich, M.W.; Leebens-Mack, J.; Ma, H.; dePamphilis, C.W. Patterns of gene duplication in the plant SKP1 gene family in angiosperms: Evidence for multiple mechanisms of rapid gene birth. Plant J. 2007, 50, 873–885. [Google Scholar] [CrossRef] [PubMed]
  45. Li, W.H.; Yang, J.; Gu, X. Expression divergence between duplicate genes. Trends Genet. 2005, 21, 602–607. [Google Scholar] [CrossRef]
  46. Tao, C.; Jin, X.; Zhu, L.; Xie, Q.; Wang, X.; Li, H. Genome-wide investigation and expression profiling of APX gene family in Gossypium hirsutum provide new insights in redox homeostasis maintenance during different fiber development stages. Mol. Genet. Genom. 2018, 293, 685–697. [Google Scholar] [CrossRef] [Green Version]
  47. Foster, J.; Lee, Y.-H.; Tegeder, M. Distinct expression of members of the LHT amino acid transporter family in flowers indicates specific roles in plant reproduction. Sex. Plant Reprod. 2008, 21, 143–152. [Google Scholar] [CrossRef]
  48. Heil, M. Nectar: Generation, regulation, and ecological functions. Trends Plant Sci. 2011, 16, 191–200. [Google Scholar] [CrossRef]
  49. Zhou, Y.; Li, M.; Zhao, F.; Zha, H.; Yang, L.; Lu, Y.; Wang, G.; Shi, J.; Chen, J. Floral Nectary Morphology and Proteomic Analysis of Nectar of Liriodendron tulipifera Linn. Front. Plant Sci. 2016, 7, 826. [Google Scholar] [CrossRef] [Green Version]
  50. Liu, H.; Ma, J.; Li, H. Transcriptomic and microstructural analyses in Liriodendron tulipifera Linn. reveal candidate genes involved in nectary development and nectar secretion. BMC Plant Biol. 2019, 19, 531. [Google Scholar] [CrossRef] [Green Version]
  51. Gerdakaneh, M.; Mozafari, A.-A.; Sioseh-mardah, A.; Sarabi, B. Effects of different amino acids on somatic embryogenesis of strawberry (Fragaria x ananassa Duch.). Acta Physiol. Plant. 2011, 33, 1847–1852. [Google Scholar] [CrossRef]
  52. Chen, T.; Yang, D.; Fan, R.; Zheng, R.; Lu, Y.; Cheng, T.; Shi, J.; Chen, J. Gamma-Aminobutyric acid a novel candidate for rapid induction in somatic embryogenesis of Liriodendron hybrid. Plant Growth Regul. 2022, 96, 293–302. [Google Scholar] [CrossRef]
  53. Hu, L.; Wang, P.; Long, X.; Wu, W.; Zhang, J.; Pan, Y.; Cheng, T.; Shi, J.; Chen, J. The PIN gene family in relic plant L. chinense: Genome-wide identification and gene expression profiling in different organizations and abiotic stress responses. Plant Physiol. Biochem. 2021, 162, 634–646. [Google Scholar] [CrossRef] [PubMed]
  54. Ueda, A.; Shi, W.M.; Sanmiya, K.; Shono, M.; Takabe, T. Functional analysis of salt-inducible proline transporter of barley roots. Plant Cell Physiol. 2001, 42, 1282–1289. [Google Scholar] [CrossRef] [Green Version]
  55. Pan, X.; Hu, M.; Wang, Z.; Guan, L.; Jiang, X.; Bai, W.; Wu, H.; Lei, K. Identification, systematic evolution and expression analyses of the AAAP gene family in Capsicum annuum. BMC Genom. 2021, 22, 463. [Google Scholar] [CrossRef]
  56. Wu, W.; Zhu, S.; Zhu, L.; Wang, D.; Liu, Y.; Liu, S.; Zhang, J.; Hao, Z.; Lu, Y.; Cheng, T.; et al. Characterization of the Liriodendron chinense MYB Gene Family and Its Role in Abiotic Stress Response. Front. Plant Sci. 2021, 12, 641280. [Google Scholar] [CrossRef] [PubMed]
  57. Wu, W.; Zhu, S.; Xu, L.; Zhu, L.; Wang, D.; Liu, Y.; Liu, S.; Hao, Z.; Lu, Y.; Yang, L.; et al. Genome-wide identification of the Liriodendron chinense WRKY gene family and its diverse roles in response to multiple abiotic stress. BMC Plant Biol. 2022, 22, 25. [Google Scholar] [CrossRef]
  58. Yao, Y.; Zhao, H.; Sun, L.; Wu, W.; Li, C.; Wu, Q. Genome-wide identification of MAPK gene family members in Fagopyrum tataricum and their expression during development and stress responses. BMC Genom. 2022, 23, 96. [Google Scholar] [CrossRef]
  59. Li, M.; Hou, L.; Liu, S.; Zhang, C.; Yang, W.; Pang, X.; Li, Y. Genome-wide identification and expression analysis of NAC transcription factors in Ziziphus jujuba Mill. reveal their putative regulatory effects on tissue senescence and abiotic stress responses. Ind. Crops Prod. 2021, 173, 114093. [Google Scholar] [CrossRef]
  60. Wang, Z.; Zhu, J.; Yuan, W.; Wang, Y.; Hu, P.; Jiao, C.; Xia, H.; Wang, D.; Cai, Q.; Li, J.; et al. Genome-wide characterization of bZIP transcription factors and their expression patterns in response to drought and salinity stress in Jatropha curcas. Int. J. Biol. Macromol. 2021, 181, 1207–1223. [Google Scholar] [CrossRef]
  61. Wang, P.; Dong, Y.; Zhu, L.; Hao, Z.; Hu, L.; Hu, X.; Wang, G.; Cheng, T.; Shi, J.; Chen, J. The role of gamma-aminobutyric acid in aluminum stress tolerance in a woody plant, Liriodendron chinense × tulipifera. Hortic. Res. 2021, 8, 80. [Google Scholar] [CrossRef] [PubMed]
  62. Xu, B.; Long, Y.; Feng, X.; Zhu, X.; Sai, N.; Chirkova, L.; Betts, A.; Herrmann, J.; Edwards, E.J.; Okamoto, M.; et al. GABA signalling modulates stomatal opening to enhance plant water use efficiency and drought resilience. Nat. Commun. 2021, 12, 1952. [Google Scholar] [CrossRef] [PubMed]
  63. UniPorT. Available online: https://www.uniprot.org/ (accessed on 12 February 2022).
  64. JGI. Available online: https://phytozome.jgi.doe.gov/pz/portal.html (accessed on 13 February 2022).
  65. pfam. Available online: http://pfam.xfam.org (accessed on 15 February 2022).
  66. NCBI-CDD. Available online: http://www.ncbi.nlm.nih.gov/cdd/ (accessed on 16 February 2022).
  67. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.E.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021, 49, D412–D419. [Google Scholar] [CrossRef] [PubMed]
  68. ExPASy. Available online: https://web.expasy.org/protparam/ (accessed on 17 January 2022).
  69. Cell-PLoc 2.0. Available online: http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 17 February 2022).
  70. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  71. Lam-Tung, N.; Schmidt, H.A.; von Haeseler, A.; Bui Quang, M. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [Google Scholar]
  72. iTOL. Available online: https://itol.embl.de/tree/2182103287321642042781 (accessed on 16 April 2022).
  73. meme. Available online: http://meme-suite.org/tools/meme (accessed on 20 February 2022).
  74. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  75. ΔG prediction server v1.0. Available online: https://dgpred.cbr.su.se/index.php?p=home (accessed on 16 April 2022).
  76. Wang, Y.; Tang, H.; DeBarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-h.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, 1–49. [Google Scholar] [CrossRef] [Green Version]
  77. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A toolkit incorporating gamma-series methods and sliding window strategies. Genom. Proteom. Bioinform. 2010, 8, 77–80. [Google Scholar] [CrossRef] [Green Version]
  78. Xu, C.; Cai, X.; Chen, Q.; Zhou, H.; Cai, Y.; Ben, A. Factors Affecting Synonymous Codon Usage Bias in Chloroplast Genome of Oncidium Gower Ramsey. Evol. Bioinform. 2011, 7, 271–278. [Google Scholar] [CrossRef] [Green Version]
  79. PlantCARE. Available online: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 2 March 2022).
  80. Willems, E.; Leyns, L.; Vandesompele, J. Standardization of real-time PCR gene expression data from independent biological replicates. Anal. Biochem. 2008, 379, 127–129. [Google Scholar] [CrossRef]
  81. Yang, R.-F.; Shu, J.-H.; Li, Z.-Y.; Liu, J.-L.; Zhang, Y. Molecular cloning and functional analysis of bovine mammary gland matrix attachment regions. Yi Chuan Hered. 2006, 28, 31–35. [Google Scholar]
Ijms 23 04765 g001 550
Figure 1. Phylogenetic tree of L. chinense (Lc), O. sativa (Os), A. thaliana (At), A. trichopoda (Atr), S. bicolor (Sb), P. trichocarpa (Pt), Z. mays (Zm), and V. vinifera (Vv) AAAP proteins. Multiple sequence alignment of full-length proteins was performed by muscle and the phylogenetic tree using the maximum-likelihood estimation with IQtree2.13 and 1000 bootstrap replications: the optimal model was JTT+F+R7. The tree was divided into eight subgroups, marked by different-colored backgrounds.
Figure 1. Phylogenetic tree of L. chinense (Lc), O. sativa (Os), A. thaliana (At), A. trichopoda (Atr), S. bicolor (Sb), P. trichocarpa (Pt), Z. mays (Zm), and V. vinifera (Vv) AAAP proteins. Multiple sequence alignment of full-length proteins was performed by muscle and the phylogenetic tree using the maximum-likelihood estimation with IQtree2.13 and 1000 bootstrap replications: the optimal model was JTT+F+R7. The tree was divided into eight subgroups, marked by different-colored backgrounds.
Ijms 23 04765 g001
Ijms 23 04765 g002 550
Figure 2. Phylogenetic relationships of the AAAP gene family in L. chinense. Multiple sequence alignment of full-length proteins was performed by muscle and the phylogenetic tree using the maximum-likelihood estimation with IQtree2.13 and 1000 bootstrap replications: the optimal model was VT+F+G4. The tree was divided into 8 subgroups, marked by different-colored backgrounds.
Figure 2. Phylogenetic relationships of the AAAP gene family in L. chinense. Multiple sequence alignment of full-length proteins was performed by muscle and the phylogenetic tree using the maximum-likelihood estimation with IQtree2.13 and 1000 bootstrap replications: the optimal model was VT+F+G4. The tree was divided into 8 subgroups, marked by different-colored backgrounds.
Ijms 23 04765 g002
Ijms 23 04765 g003 550
Figure 3. Phylogenetic relationship and gene structures of LcAAAPs. Phylogenetic tree of 52 LcAAAPs proteins. Maximum-Likelihood tree was constructed using IQtree. Bootstrap support values from 1000 reiterations are indicated at each node. The 52 LcAAAPs in the tree were divided into eight subfamilies. Gene structure was indicated by green and yellow rectangles, respectively.
Figure 3. Phylogenetic relationship and gene structures of LcAAAPs. Phylogenetic tree of 52 LcAAAPs proteins. Maximum-Likelihood tree was constructed using IQtree. Bootstrap support values from 1000 reiterations are indicated at each node. The 52 LcAAAPs in the tree were divided into eight subfamilies. Gene structure was indicated by green and yellow rectangles, respectively.
Ijms 23 04765 g003
Ijms 23 04765 g004 550
Figure 4. Phylogenetic relationship and conserved motifs of LcAAAPs. Phylogenetic tree of 52 LcAAAPs proteins. Conserved motifs of LcAAAPs proteins. Each colored box represents a specific motif in the protein identified using the MEME motif search tool. The order of the motifs corresponds to their position within individual protein sequences.
Figure 4. Phylogenetic relationship and conserved motifs of LcAAAPs. Phylogenetic tree of 52 LcAAAPs proteins. Conserved motifs of LcAAAPs proteins. Each colored box represents a specific motif in the protein identified using the MEME motif search tool. The order of the motifs corresponds to their position within individual protein sequences.
Ijms 23 04765 g004
Ijms 23 04765 g005a 550Ijms 23 04765 g005b 550
Figure 5. Prediction results of the transmembrane domain of LcATL1a, LcLHT2, LcAPP4b, LcATL11, LcAAP17, LcATL9, and LcPorT1 proteins. The grey area represents the putative transmembrane helix.
Figure 5. Prediction results of the transmembrane domain of LcATL1a, LcLHT2, LcAPP4b, LcATL11, LcAAP17, LcATL9, and LcPorT1 proteins. The grey area represents the putative transmembrane helix.
Ijms 23 04765 g005aIjms 23 04765 g005b
Ijms 23 04765 g006 550
Figure 6. Distribution and segmental duplication of LcAAAP genes in L. chinense. The different colors in the panel show the chromosomes using a circle. Yellow lines connect homologous genes; chromosome numbers are marked outside of the circle.
Figure 6. Distribution and segmental duplication of LcAAAP genes in L. chinense. The different colors in the panel show the chromosomes using a circle. Yellow lines connect homologous genes; chromosome numbers are marked outside of the circle.
Ijms 23 04765 g006
Ijms 23 04765 g007 550
Figure 7. Genome-wide synteny analysis of LcAAAP gene family among L. chinense and three other species. Synteny analysis of LcAAAP genes between L. chinense, Arabidopsis, rice, and grape. Gray lines in the background indicate the collinear blocks between L. chinense, Arabidopsis, rice, and grape genomes, while the blue lines highlight the syntenic AAAP gene pairs. Red open triangles represent homologous genes.
Figure 7. Genome-wide synteny analysis of LcAAAP gene family among L. chinense and three other species. Synteny analysis of LcAAAP genes between L. chinense, Arabidopsis, rice, and grape. Gray lines in the background indicate the collinear blocks between L. chinense, Arabidopsis, rice, and grape genomes, while the blue lines highlight the syntenic AAAP gene pairs. Red open triangles represent homologous genes.
Ijms 23 04765 g007
Ijms 23 04765 g008a 550Ijms 23 04765 g008b 550
Figure 8. The number of cis-elements in LcAAAP promoters. The motif types of hormones and stress responsiveness are shown in green and purple lines in LcAAAP gene families. Red represents the quantity. The bar chart on the right represents the number of response elements contained in each gene, and different colors represent different types of elements.
Figure 8. The number of cis-elements in LcAAAP promoters. The motif types of hormones and stress responsiveness are shown in green and purple lines in LcAAAP gene families. Red represents the quantity. The bar chart on the right represents the number of response elements contained in each gene, and different colors represent different types of elements.
Ijms 23 04765 g008aIjms 23 04765 g008b
Ijms 23 04765 g009 550
Figure 9. The LcAAAP genes expression profiles in different organs. The heatmap shows the mean of three biological replicates. Transcripts per million (TPM) was used to indicate the gene expression level. (a–c): The expression level of LcAAAP genes in different organs, nectary development, and somatic embryogenesis of hybrid Liriodendron; Lc1-4 and Lt1-4 represent the developmental process of the middle part of the petals of L. chinense and L. tulipifera, respectively. PEM: embryogenic callus; ES1: 10 days after liquid culture; ES2: 2 days after screening; ES3: ABA 1 day of treatment; ES4: ABA treatment for three days; ES5: globular embryo; ES6: heart-shaped embryo; ES7: torpedo embryo; ES8: immature cotyledon embryo; ES9: mature cotyledon embryo; PL: plantlet.
Figure 9. The LcAAAP genes expression profiles in different organs. The heatmap shows the mean of three biological replicates. Transcripts per million (TPM) was used to indicate the gene expression level. (a–c): The expression level of LcAAAP genes in different organs, nectary development, and somatic embryogenesis of hybrid Liriodendron; Lc1-4 and Lt1-4 represent the developmental process of the middle part of the petals of L. chinense and L. tulipifera, respectively. PEM: embryogenic callus; ES1: 10 days after liquid culture; ES2: 2 days after screening; ES3: ABA 1 day of treatment; ES4: ABA treatment for three days; ES5: globular embryo; ES6: heart-shaped embryo; ES7: torpedo embryo; ES8: immature cotyledon embryo; ES9: mature cotyledon embryo; PL: plantlet.
Ijms 23 04765 g009
Ijms 23 04765 g010 550
Figure 10. The LcAAAP genes expression profiles in different stress. The heatmap shows the mean of three biological replicates. Transcripts per million (TPM) was used to indicate the gene expression level. Expression patterns of LcAAAP genes in leaves of hybrid Liriodendron under heat (40 °C), low temperature (4 °C), and drought stress. Stars of different colors represent different gene expression patterns, black represents up-regulation and then down-regulation, green represents down-regulation, blue represents down-regulation and then up-regulation, and red represents up-regulation.
Figure 10. The LcAAAP genes expression profiles in different stress. The heatmap shows the mean of three biological replicates. Transcripts per million (TPM) was used to indicate the gene expression level. Expression patterns of LcAAAP genes in leaves of hybrid Liriodendron under heat (40 °C), low temperature (4 °C), and drought stress. Stars of different colors represent different gene expression patterns, black represents up-regulation and then down-regulation, green represents down-regulation, blue represents down-regulation and then up-regulation, and red represents up-regulation.
Ijms 23 04765 g010
Ijms 23 04765 g011 550
Figure 11. Prediction network of protein interactions for LcAAAP, LcWRKY, and LcMYB gene families. Pink ovals represent AAAP gene family members, dark green hexagons represent MYB/WRKY transcription factors, and dark blue dashed lines represent predicted correlations.
Figure 11. Prediction network of protein interactions for LcAAAP, LcWRKY, and LcMYB gene families. Pink ovals represent AAAP gene family members, dark green hexagons represent MYB/WRKY transcription factors, and dark blue dashed lines represent predicted correlations.
Ijms 23 04765 g011
Ijms 23 04765 g012 550
Figure 12. Correlation analysis of LcAAAP/LcWRKY/LcMYB genes under drought, low temperature, and heat stress. The red arrows indicate that these genes were significantly upregulated under the three stressors.
Figure 12. Correlation analysis of LcAAAP/LcWRKY/LcMYB genes under drought, low temperature, and heat stress. The red arrows indicate that these genes were significantly upregulated under the three stressors.
Ijms 23 04765 g012
Ijms 23 04765 g013 550
Figure 13. Gene expression of LcGAT1 in hybrid Liriodendron under Al toxicity and GABA treatment. Mock represents the ultrapure water treatment; Al represents the aluminum stress treatment; GABA represents exogenously applied GABA treatment; Al+GABA represents Al stress treatment and then exogenously applied GABA treatment.
Figure 13. Gene expression of LcGAT1 in hybrid Liriodendron under Al toxicity and GABA treatment. Mock represents the ultrapure water treatment; Al represents the aluminum stress treatment; GABA represents exogenously applied GABA treatment; Al+GABA represents Al stress treatment and then exogenously applied GABA treatment.
Ijms 23 04765 g013
Ijms 23 04765 g014 550
Figure 14. Gene expression of LcGAT2 and 4 in hybrid Liriodendron under Al toxicity and GABA treatment. Mock represents the ultrapure water treatment; Al represents the aluminum stress treatment; GABA represents exogenously applied GABA treatment; Al+GABA represents Al stress treatment and then exogenously applied GABA treatment.
Figure 14. Gene expression of LcGAT2 and 4 in hybrid Liriodendron under Al toxicity and GABA treatment. Mock represents the ultrapure water treatment; Al represents the aluminum stress treatment; GABA represents exogenously applied GABA treatment; Al+GABA represents Al stress treatment and then exogenously applied GABA treatment.
Ijms 23 04765 g014
Ijms 23 04765 g015 550
Figure 15. Gene expression of LcPorT in hybrid Liriodendron under Al toxicity and GABA treatment. Mock represents the ultrapure water treatment; Al represents the aluminum stress treatment; GABA represents exogenously applied GABA treatment; Al+GABA represents Al stress treatment and then exogenously applied GABA treatment.
Figure 15. Gene expression of LcPorT in hybrid Liriodendron under Al toxicity and GABA treatment. Mock represents the ultrapure water treatment; Al represents the aluminum stress treatment; GABA represents exogenously applied GABA treatment; Al+GABA represents Al stress treatment and then exogenously applied GABA treatment.
Ijms 23 04765 g015
Table
Table 1. The information of 52 LcAAAP genes in L. chinense.
Table 1. The information of 52 LcAAAP genes in L. chinense.
Gene IDGene NameLocusLocationORF (bp)Amino Acid LengthMW (KDa) pISubcellular Localization
Lchi00592LcANT1chr1108060548137745849.917.98Cell membrane. Chloroplast.
Lchi00907LcAPP2chr198782372118539443.239.15Cell membrane. Golgi apparatus.
Lchi29437LcGAT1achr164003591109536439.339.65Cell membrane
Lchi29438LcGAT1cchr163956981110436739.969.54Cell membrane
Lchi29446LcGAT1bchr163673692108035938.929.7Cell membrane
Lchi01730LcAPP4bchr272696256262287397.329.5Cell membrane
Lchi01732LcAPP9chr272772418155151656.639.23Cell membrane
Lchi02902LcATL15chr293230749120640143.999.03Cell membrane. Golgi apparatus.
Lchi02903LcATL12achr293204648158752858.459Cell membrane
Lchi02904LcATL12bchr293165315130543447.187.98Cell membrane. Golgi apparatus.
Lchi08727LcLAX5achr216848262139246352.348.95Cell membrane
Lchi25677LcLAX2chr245173338141947253.499.09Cell membrane
Lchi10219LcLHT1chr367706260165355060.348.74Cell membrane
Lchi10341LcPorT1chr375189713107435740.219.9Cell membrane
Lchi22116LcGAT2chr3904020135645149.538.87Cell membrane. Golgi apparatus.
Lchi02035LcAPP4achr484533298145248353.249.02Cell membrane
Lchi04257LcPorT2chr43186704111937240.729.36Cell membrane
Lchi04338LcAUX1achr4642668142547453.438.68Cell membrane
Lchi09378LcANT2chr448992807139846550.566.41Cell membrane
Lchi10104LcAPP17chr47882185986728831.326.3Cell membrane
Lchi16175LcLHT3chr480850467128442747.478.73Cell membrane. Chloroplast. Golgi apparatus.
Lchi16222LcAPP6achr479369615137445750.529.01Golgi apparatus.
Lchi16226LcAPP6bchr479186487134144649.259.39Golgi apparatus.
Lchi16229LcAPP6dchr479108510166855562.666.44Cell membrane. Chloroplast.
Lchi29396LcATL8bchr411575526268589497.755.56Cell membrane
Lchi33844LcLHT6chr428079397165355060.59.49Cell membrane
Lchi05928LcLHT4chr874276594120940244.788.94Cell membrane. Golgi apparatus.
Lchi14454LcATL11chr1029873111165054961.757.53Cell membrane. Cytoplasm. Mitochondrion. Nucleus.
Lchi13365LcGAT4chr1132708594124241345.049.34Cell membrane
Lchi23105LcAPP4cchr1128298237123941245.758.7Cell membrane
Lchi20114LcLAX5bchr1255555547129643148.969.12Cell membrane
Lchi18276LcAPP1achr138635305153651156.198.83Cell membrane
Lchi18277LcAPP1bchr138656388150350055.158.91Cell membrane
Lchi03159LcATL9chr146263787195731834.889.19Cell membrane
Lchi10700LcATL1achr14486919402850949103.816.41Cell membrane
Lchi20962LcATL5achr1417705266144648151.798.84Cell membrane. Chloroplast
Lchi32419LcATL1bchr1436109607169856561.588.49Chloroplast.
Lchi19559LcLHT2chr1522627745260186696.388.97Cell membrane
Lchi13826LcAVT6chr1620708277139246349.397.01Cell membrane. Chloroplast. Cytoplasm. Golgi apparatus.
Lchi13928LcLHT8chr1627398496147048954.179.56Cell membrane
Lchi12202LcATL5cchr1713443833138946249.687.62Cell membrane. Golgi apparatus.
Lchi16887LcAPP7cchr172623000120340044.086.41Cell membrane
Lchi16888LcAPP7achr17265480733301109123.627.78Cell membrane. Cytoplasm.
Lchi16889LcAPP7bchr172708322138346051.99.16Cell membrane
Lchi32115LcATL5bchr1753273477118539442.315.94Cell membrane
Lchi25213LcAUX1bchr182575200142547453.498.68Cell membrane
Lchi30378LcATL7cchr1856630104132344047.126.14Cell membrane. Chloroplast. Cytoplasm. Golgi apparatus.
Lchi30379LcATL7achr1856674114132644147.668.24Cell membrane. Chloroplast. Golgi apparatus.
Lchi34889LcAPP6cContig177310550111637141.419.85Cell membrane
Lchi31213LcATL7bContig2824600473132644147.648.56Cell membrane. Chloroplast. Golgi apparatus.
Lchi31214LcATL7dContig2824620344132344047.16.14Cell membrane. Chloroplast. Cytoplasm. Golgi apparatus.
Lchi28875LcATL8aContig5091166972143447751.379.13Golgi apparatus.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Hu, L.; Fan, R.; Wang, P.; Hao, Z.; Yang, D.; Lu, Y.; Shi, J.; Chen, J. Identification, Phylogenetic and Expression Analyses of the AAAP Gene Family in Liriodendron chinense Reveal Their Putative Functions in Response to Organ and Multiple Abiotic Stresses. Int. J. Mol. Sci. 2022, 23, 4765. https://doi.org/10.3390/ijms23094765

AMA Style

Hu L, Fan R, Wang P, Hao Z, Yang D, Lu Y, Shi J, Chen J. Identification, Phylogenetic and Expression Analyses of the AAAP Gene Family in Liriodendron chinense Reveal Their Putative Functions in Response to Organ and Multiple Abiotic Stresses. International Journal of Molecular Sciences. 2022; 23(9):4765. https://doi.org/10.3390/ijms23094765

Chicago/Turabian Style

Hu, Lingfeng, Ruifang Fan, Pengkai Wang, Zhaodong Hao, Dingjie Yang, Ye Lu, Jisen Shi, and Jinhui Chen. 2022. "Identification, Phylogenetic and Expression Analyses of the AAAP Gene Family in Liriodendron chinense Reveal Their Putative Functions in Response to Organ and Multiple Abiotic Stresses" International Journal of Molecular Sciences 23, no. 9: 4765. https://doi.org/10.3390/ijms23094765

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop