A Systematic Investigation of Lipid Transfer Proteins Involved in Male Fertility and Other Biological Processes in Maize

Plant lipid transfer proteins (LTPs) play essential roles in various biological processes, including anther and pollen development, vegetative organ development, seed development and germination, and stress response, but the research progress varies greatly among Arabidopsis, rice and maize. Here, we presented a preliminary introduction and characterization of the whole 65 LTP genes in maize, and performed a phylogenetic tree and gene ontology analysis of the LTP family members in maize. We compared the research progresses of the reported LTP genes involved in male fertility and other biological processes in Arabidopsis and rice, and thus provided some implications for their maize orthologs, which will provide useful clues for the investigation of LTP transporters in maize. We predicted the functions of LTP genes based on bioinformatic analyses of their spatiotemporal expression patterns by using RNA-seq and qRT-PCR assays. Finally, we discussed the advances and challenges in substrate identification of plant LTPs, and presented the future research directions of LTPs in plants. This study provides a basic framework for functional research and the potential application of LTPs in multiple plants, especially for male sterility research and application in maize.


Introduction
Lipid transfer proteins (LTPs), also named non-specific LTPs (nsLTPs), defined by their ability to facilitate transfer of non-specific phospholipids and fatty acids between membranes in vitro, widely exist in all land plants but not in algae and other organisms. Therefore, LTPs are considered as key proteins for plant survival on and colonization of land [1,2]. LTPs are small, basic proteins characterized by eight conserved cysteine residues, low molecular weight (usually between 6.5 to 10 kDa), and a high content of four or five a-helices [3]. The LTPs are stabilized by four conserved disulfide bridges formed by an eight-Cys motif (8CM) with the general form C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C, resulting in a central hydrophobic cavity suitable for the binding of various lipids [4]. According to the sequence similarity, the LTPs can be classified into 10 types, including five major types (LTP1, LTP2, LTPc, LTPd, and LTPg) and five minor types (LTPe, LTPf, LTPh, LTPj, and LTPk) [4]. Most LTP proteins include an N-terminal signal peptide and an LTP domain, and some also possess a glycophosphatidylinositol (GPI) motif (exit in LTPg), and the signal peptide and GPI motif are required for LTP subcellular localization and biological function [4,5]. Recent studies have shown that many intracellular LTPs localize on different organelles, forming a shuttle, bridge or tube that links donor and acceptor compartments to transport specific substrates [6,7]. LTPs were characterized as mediators ment. Other characteristics of the 65 ZmLTP genes, including the corresponding gene models in the B73 reference genome (B73v3 and B73v4), genome physical locus, topology, theoretical pI using Expasy (https://web.expasy.org/protparam/, accessed on 25 October 2022), and subcellular localization prediction using Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/, accessed on 25 October 2022), are listed in Table S1. In a word, the chromosome locations, spatial expression patterns, and other characteristics are useful to explore the function and evolution of maize LTPs in the future.  Furthermore, the RNA-seq data revealing spatial expression patterns of maize LTP genes are retrieved from MaizeGDB (Table 1). Notably, ZmLTP1.5, ZmLTP2.9, ZmLTPc1 (Zmms44), ZmLTPc2 (Zmnthr3), ZmLTPd9, and ZmLTPx1 are specially expressed in tassel, the ZmLTPg20 is specially expressed in anther, ZmLTPx2 is specially expressed in endosperm, and ZmLTPd7 and ZmLTPg12 are specially expressed in roots (Table 1), which will provide useful clues to explore their biological functions in different organs' development. Other characteristics of the 65 ZmLTP genes, including the corresponding gene models in the B73 reference genome (B73v3 and B73v4), genome physical locus, topology, theoretical pI using Expasy (https://web.expasy.org/protparam/, accessed on 25 October 2022), and subcellular localization prediction using Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/ bioinf/Cell-PLoc-2/, accessed on 25 October 2022), are listed in Table S1. In a word, the chromosome locations, spatial expression patterns, and other characteristics are useful to explore the function and evolution of maize LTPs in the future.  ZmLTPd14 Notes: 1 . Gene ID was based on Zm-B73-REFERENCE-NAM-5.0; 2 . The expression information of maize LTP genes was based on B73 RefGen_v3; 1 and 2 retrieved from MaizeGDB (www.maizegdb.org, accessed on 10 October 2022). "+" and "−" indicate whether or not the gene was expressed in the corresponding tissue, and "+" was highlighted with gray background.

Phylogenetic Analysis and Classification of LTPs in Maize
To analyze the phylogenetic relationship of the LTP gene family, the 65, 80, and 84 LTP protein sequences in maize, rice, and Arabidopsis were used to construct the phylogenetic tree by using the maximum likelihood method and the MEGA7 program. All the identified LTPs can be divided into three groups (namely group 1 to 3) in the phylogenetic tree ( Figure 2). Group 1 can be further classified into six clades (Clades 1-1 to 1-6), and contains 91 LTPs, including 28 in maize, 31 in rice, and 32 in Arabidopsis. Among them, OsLTPg25 (previously named as OsC6) [16], OsLTPg28 (previously named as OsLTP47) [31], AtLTPg3, and AtLTPg4 [17] were reported to be involved in pollen or anther development. Group 2 contains 89 LTPs, including 25 in maize, 35 in rice, and 29 in Arabidopsis, which can be further classified into five clades (Clades 2-1 to 2-5). The largest number of LTPs related to male sterility were reported in group 2, such as AtLTPc3, AtLTPc1 [14], and their orthologs OsLTPc1 (previously named as OsC4) [39,40], ZmLTPc1 (previously named as Zmms44) [32], and ZmLTPc2 (previously named as MZm3-3/athr3) [34]. In addition, OsLTPd11 (previously named as OsDIL/OsLTP6) [41,42], AtLTP1.7 (previously named as AtLTP12) [43], and AtLTP1.8 (previously named as AtLTP5) [44,45] were reported to be involved in anther development. Group 3 can be further classified into three clades (Clades 3-1 to 3-3), and contains 91 LTPs, including 28 in maize, 31 in rice, and 32 in Arabidopsis. ZmLTPx2, ZmLTPg11 [33], and their orthologs OsLTPg29 (also named as OsLTPL94 or OsEPAD1) [5,15] were reported to participate in anther and pollen wall development. Interestingly, there are only 19 Arabidopsis LTP genes in the clade 3-2 ( Figure 2), indicating that these genes appear after the divergency of dicotyledons and monocotyledons during plant evolution. In general, the phylogenetic analysis results provide not only the phylogenetic relationship of the LTP genes, but also give some helpful information for exploring the functions of unknown LTP genes in maize, rice, and Arabidopsis.

Gene Ontology Analysis of LTPs in Maize
The Gene Ontology (GO) database has the highest annotation ratio compared with other analysis databases, such as CDD (Conserved Domain Database), KOG (Ortholog Group), or NR (Non-redundant database) [46]. In order to comprehend the molecular functions and putative pathways involving maize LTPs, GO enrichment analysis of the functional significance was performed. The 65 ZmLTPs were defined in 25 significant GO terms (Table S2). The analysis showed that all the ZmLTPs were separated into two main categories (biological processes and molecular functions), which included 20 and 5 significant GO terms, respectively ( Figure 3A). For the enriched biological processes, the 20 GO terms can be grouped into two classes, including biotic and abiotic stress response (10 GO terms: 0009627, 0009814, 0006955, 0045087, 0002376, 0098542, 0043207, 0051707, 0009607, and 0009605), and lipid and other substance transport and localization (10 GO terms: 0006869, 0071702, 0044765, 0006810, 0010876, 0033036, 1902578, 0051234, 0051179, and 005174). Notably, about 11 ZmLTPs were shown to participate in lipid and other substance transport and localization, which is in concordance with the molecular role of LTP in transporting hydrophobic molecules in vitro, suggesting that ZmLTPs play an important role in membrane components translocation. Five ZmLTPs were exhibited to participate in biotic and abiotic stress response ( Figure 3A and Table S2). This highlights the putative association of ZmLTPs in stress tolerance behavior of maize. In case of molecular functions, the 12 ZmLTPs were shown to participate in "lipid binding" (GO: 0008289) and five ZmLTPs were shown to be involved in binding of fatty acid, monocarboxylic acid, carboxylic acid and organic acid (GO: 0005504, 0033293, 0031406 and 0043177). In a word, the GO analysis indicated that ZmLTPs may be involved in diverse biological processes, such as lipid binding, lipid transport and localization, and biotic and abiotic stress response.  (65), rice (80), and Arabidopsis (84). Group 1 can be further classified into six clades (Clades 1-1 to 1-6), which contains 91 LTPs, including 28 in maize, 31 in rice and 32 in Arabidopsis. Group 2 can be further classified into five clades (Clades 2-1 to 2-5), which contain 89 LTPs, including 25 in maize, 35 in rice, and 29 in Arabidopsis. Group 3 can be further classified into three clades (Clades 3-1 to 3-3), and contains 91 LTPs, including 28 in maize, 31 in rice, and 32 in Arabidopsis. Gray, yellow, and violet background represent the LTPs in Arabidopsis, rice, and maize; and gray, yellow, and violet asterisks represent the male fertile genes in Arabidopsis, rice, and maize, respectively. The dark spots on the branches of the tree indicate the evolutionary distance; the larger dark spot means the closer evolutionary distance of LTPs. (65), rice (80), and Arabidopsis (84). Group 1 can be further classified into six clades (Clades 1-1 to 1-6), which contains 91 LTPs, including 28 in maize, 31 in rice and 32 in Arabidopsis. Group 2 can be further classified into five clades (Clades 2-1 to 2-5), which contain 89 LTPs, including 25 in maize, 35 in rice, and 29 in Arabidopsis. Group 3 can be further classified into three clades (Clades 3-1 to 3-3), and contains 91 LTPs, including 28 in maize, 31 in rice, and 32 in Arabidopsis. Gray, yellow, and violet background represent the LTPs in Arabidopsis, rice, and maize; and gray, yellow, and violet asterisks represent the male fertile genes in Arabidopsis, rice, and maize, respectively. The dark spots on the branches of the tree indicate the evolutionary distance; the larger dark spot means the closer evolutionary distance of LTPs.

Functions of LTP Transporters in Rice and Arabidopsis and Their Implications in Maize
Until now, at least 25 and 11 LTP genes have been characterized in Arabidopsis and rice, respectively, but only seven have been reported in maize ( Table 2). Considering the functional conservation of orthologous genes among multiple plant species, the functions of rice and Arabidopsis LTPs will provide important clues for exploring that of maize orthologs. Increasing evidence indicates that plant LTP transporters play important roles in anther and pollen development, vegetative and female organ development, seed development and germination, and biotic and abiotic stress response during plant biological processes (Table 2, Figure 4).  reductase [48,49], ZmMs33 encoding a glycerol-3-phosphate acyltransferase [50][51][52][53], ZmMs30 encoding a GDSL lipase [54], and ZmPKSB encoding a polyketide synthase [55]. Then, the lipid precursors are transported into the pollen surface and anther outer wall by ABCGs such as ZmABCG26 and ZmMs13/ZmABCG2a [48,56,57], LTPs such as Zmms44 [32], and other transporters [47]. Loss functions of the anther-specific or highexpressed LTP genes often lead to defective anther cuticle and pollen wall formation and thus male sterility in plants [58].

Anther and Pollen Development
Plant lipids, including fatty acids and their derivatives, work as the essential building blocks of anther cuticle and pollen wall formation, and genic male sterility (GMS) would arise if the lipid metabolism is disrupted during anther and pollen development in plants [47]. Anther cuticles and pollen walls are composed of complex substances, including cutin, wax, and sporopollenin [47]. The precursors of sporopollenin, cutin, and wax are synthesized in anther tapetum, and catalyzed by a series of lipid metabolism-related GMS gene-encoding enzymes, such as ZmMs25/ZmFAR1 encoding a plastid-localized fatty acyl reductase [48,49], ZmMs33 encoding a glycerol-3-phosphate acyltransferase [50][51][52][53], ZmMs30 encoding a GDSL lipase [54], and ZmPKSB encoding a polyketide synthase [55]. Then, the lipid precursors are transported into the pollen surface and anther outer wall by ABCGs such as ZmABCG26 and ZmMs13/ZmABCG2a [48,56,57], LTPs such as Zmms44 [32], and other transporters [47]. Loss functions of the anther-specific or high-expressed LTP genes often lead to defective anther cuticle and pollen wall formation and thus male sterility in plants [58].  To date, at least 15 LTP transporters have been reported to be involved in anther and/or pollen development in Arabidopsis, rice, and maize (Table 2 and Figure 4A). The type III LTPs AtLTPc1 (At5g07230), and AtLTPc3 (At5g62080) are required for the exine precursors transport from the tapetal ER to the microspore surface, and also act as the part of components of pollen exine [14]. The mutants of atltpg3 and atltpg4 showed deformed or collapsed pollen grains, indicating the essential roles of AtLTPg3 and AtLTPg4 in pollen development [17]. Zmms44, the ortholog of AtLTPc1 and AtLTPc3, is the only cloned dominant male sterility gene in maize, functions as a signal peptide, and facilitates the secretion of protein from tapetal cells into the locule [32]. OsC4, an ortholog of AtLTPc1 and AtLTPc3 in rice, along with OsC6 are related to tapetal PCD of anther [16,39,40], and OsC6 is reported to be localized in anther extracellular space [16]. In tapetal cytoplasm, OsC6-loaded pollen wall constituents (PWCs) are recruited by OsLTP47, which is plasma membrane-localized, and PWCs were transferred to OsLTP47; then, OsLTP47 recruits OsC6 in anther locules to the tapetal cell membrane and transfers PWCs to OsC6 for further transports into pollen exine [31]. Further, OsC6 is also involved in anther cuticle formation by translocation of cutin precursors from tapetum to the anther outer wall [16]. These data indicate that the orthologous LTP genes play similar roles in different plants, and LTPs may play relay roles in lipid transport during anther and pollen development.
Another example, OsLTPL94/OsEPAD1, determines pollen exine patterning as a microspore membrane recruiting protein, and OsLTPL94/OsEPAD1 is not only secreted by pollen mother cells (PMCs) but also derived from the tapetum [5,15]. ZmLTPg11 and ZmLTPx2, specifically expressed in the microsporocytes, transported the same substrates with their orthologs of OsLTPL94/OsEPAD1 and TaMs1 in rice and wheat; additionally, the pollen development is not effective in the zmltpg11/zmltpx2 double mutant [33]. These findings suggest that some of the orthologous LTPs, such as OsLTPL94/OsEPAD1, TaMs1, ZmLTPg11, and ZmLTPx2, may play conserved roles in lipid transport essential for pollen wall (exine) development.
Moreover, the function of LTP genes in male fertility might be influenced by abiotic stress. For example, OsDIL/OsLTP6 is related to drought stress tolerance, and OsDILoverexpressing transgenic plants showed fewer defective anther sacs and less severe tapetal defects when treated with drought at the reproductive stage in rice [41,42]. AtLTP12 determines site proline biosynthesis, which can restore the fertility of proline-deficient microspores [43]. However, transgenic plants with gain of function for AtLTP5, the homolog of AtLTP12, shows abnormal vegetative and pollen tube growth, and AtLTP5 is mainly expressed in root, shoots, leaves, and pistil of Arabidopsis [44,45]. MZm3-3 (ZmLTPc2) plays an important role in pollen coat formation, but the mechanism needs to be further studied [34]. Together, LTP transporters may play conserved and divergent roles in lipid transport for anther and pollen development and male fertility in multiple plants.
Transcriptional regulatory pathways that control male fertility through lipid metabolism have been well studied and reviewed in Arabidopsis, rice, and maize [47,58]. Many LTP genes are regulated by transcription factors (TFs) essential to male fertility in plants (Figure 4A). Previous studies suggest that OsGAMYB, an important component of gibberellin (GA) signaling, regulates early anther development [72]. OsGAMYB directly regulates the expression of LTP genes, such as OsC6 and OsC4 [73]. OsTDR and OsPTC1 act downstream of OsGAMYB, and OsPTC1 associates with OsTDR to regulate expression of their downstream genes [74]. For example, OsC6 and OsC4 work at the downstream of OsPTC1, and OsTDR1 directly regulates the expression of OsC6 [75,76]. AtAMS, the orthologs of OsTDR in Arabidopsis, directly regulates At5g62080 (AtLTPc3), At1g66850 (AtLTP2.5), and At3g51590 (AtLTP1.7) [77]. OsEAT1 interacts with the OsTDR protein, and directly regulates the expression of OsLTPL94, which plays a key role in tapetum and microspore development [15,78]. Taken together, TFs may transcriptionally regulate the expression of LTP genes involved in male fertility directly or indirectly as well as synergistically.

Vegetative and Female Organ Development
Another important function of LTP transporters is related to vegetative and seed development (Table 2 and Figure 4B, C). Several LTP transporters are reported to be involved in vegetative organ development ( Figure 4B). For example, AtLTP1, a regulator of ethylene response and signaling, takes part in the export of lipids to the plant surface [21,25,59]. AtLTP2, as the homolog of AtLTP1, plays a major structural role to maintain the adhesion integrating the mainly hydrophobic cuticle and the hydrophilic underlying cell wall [60]. AtLTP1 to AtLTP6 play multifunction roles in plant growth and reproduction [45]. In atltpg2 and atltpg1 atltpg2 double mutants, the composition of cuticular wax in the stems and siliques was significantly altered [23]. OsPSD1/OsPTD1, the ortholog of AtLTP6 in rice, regulates cell development, plant height, and sensitivity to temperature conditions [22,24].
Some homologous or orthologous LTPs may play similar roles in plant vegetative development between monocots and dicots. AtLTPG23 and AtLTPG26 are related to suberin biosynthesis [3]. AtLSR1 regulates leaf senescence [63].
Additionally, some LTP genes are involved in seed development or seed germination ( Figure 4C). For example, OsLTPL36 is essential for seed quality, seed development, and germination in rice [20]. As the ortholog of OsLTPL36, ZmBETL-9 is transcribed in the outer surface of the developing endosperm [35]. AtEND1 is widely expressed in root, leaf, stem, flower, and seed, and plays important roles in gametophytic tissues and developing endosperm in Arabidopsis [62]. AtLTPG5 and AtLTPG6 are involved in cuticle development and seed coat suberization [17]. In summary, LTP transporters play indispensable roles in lipid secretion and transportation in different organs in plants, and thus are critical for plant growth and development.

Biotic and Abiotic Stress Response
Stress resistance is an important characteristic for crop breeding. However, the mechanism of LTPs in the induction of stress resistance is not fully understood [10]. According to the stress factors and functional models, we classified all the reported LTP transportersmediated stress response into two groups: abiotic stress and biotic stress ( Table 2 and Figure 4D).
Abiotic stress factors include hormones, cold, and salt, etc. For example, enhancing abscisic acid (ABA), salicylic acid, and 16-hydroxypalmitic acid could induce the expression of OsLTP5 in rice [26]. Zm-LTP, the ortholog of OsLTP5 in maize, binds to calmodulin (CaM) in a Ca 2+ -independent manner to modulate its lipid-binding ability [36]. The mutant of atazi1 is hypersensitive to salt stress, while AtAZI1-overexpressing plants are markedly more tolerant [29,38]. OsLTPL159 is involved in cold tolerance at the early seedling stage in rice [30]. ZmLTP3 enhances plant salt tolerance and drought resistance [37]. For signal transport, AtDIR1 and AtDIR1-like are required for the transmission of a mobile signal during systemic acquired resistance [65,79]. For biotic stress, the AtLTP3 contributes to disease susceptibility in Arabidopsis by enhancing ABA biosynthesis and the atltp3 mutant seeds showed impaired germination under salt and osmotic treatments [19,66,67]. In addition, the double mutant of atltp3/atltp4 showed that the susceptibility to Pseudomonas is reduced and the ABA biosynthesis genes are down-regulated [45,66]. AtDRN1 is involved in response to the avirulent bacterial phytopathogen Pst DC3000 [69]. AtLTPG1 could alter the composition of cuticular lipids, increase plastoglobules, and enhance susceptibility to infection by the fungal pathogen Alternaria brassicicola [27]. OsLTP1, the first identified LTP gene of rice, plays important roles in structural barriers and organ protection against mechanical disruption and pathogen attack [70,71]. OsLTP110 could inhibit the germination of Pyricularia oryzae spores in vitro, and overexpression of OsLTP110 could bring a substantial resistance to biotic stresses [68]. In summary, some LTP transporters play critical roles during plant response to abiotic and biotic stresses, which will be helpful to improve crop stress resistance by manipulating the related LTP genes in the future.

Functional Prediction of LTP Genes Based on Bioinformatics Analysis
As the biological functions of genes are related to their spatiotemporal expression patterns, bioinformatics analyses will provide useful information for exploring the function of maize LTP genes. They are a useful way to predict unknown gene function by using transcriptomic analyses based on RNA-seq data [47,58,80,81]. In order to predict the functions of maize LTP genes involved in anther and pollen development, we used the RNA-seq data of W23 (from stages S2 to S12), B73, Oh43, and Zheng58 (from stages S5 to S12) developing anthers and qPCR analysis of maize anther from stage S5 to S13 to analyze the expression patterns of all maize LTP genes during anther development ( Figure 5).
Based on the RNA-seq data, all the maize LTP genes can be classified into two clusters (I and II). The cluster I contains two subclusters (I-1 and I-2). The subcluster I-1 includes eight LTP genes, which are expressed at early development stages (from stage S2) of maize anthers. Among them, ZmLTPg11 and ZmLTPg4 are orthologous to male-sterile genes OsLTPg29 and AtLTPG4 [5,15,17], suggesting these two genes may be required for anther development. The subcluster I-2 contains 11 LTP genes, with the expression peaks at middle development stages (from S6 to S10) of maize anthers. There are five anther or tassel specific expression genes (ZmLTPc1/Zmms44, ZmLTPc2/Zmanthr3, ZmLTP2.9, ZmLTP1.5, and ZmLTPd9) in subcluster I-2 based on the information in MaizeGDB (www.maizegdb.org), and two of them (ZmLTPc1/Zmms44 and ZmLTPc2/Zmanthr3) have been reported to be essential for pollen development. It is reasonable to predict the other three LTP genes may also be required for anther and pollen development, which should be proved with experimental data in the future.
The cluster II contains four subclusters (II-1 to II-4). Subcluster II-1 includes five genes with high expression at late development stages (from S10 to S13) of maize anthers. Among them, ZmLTPg20 is anther-specific expression gene, and ZmLTPg15 is orthologous to the male-sterile gene AtLTPG3 [17]. The subcluster II-2 and II-3 contain five LTP genes respectively, which are expressed lower than cluster I or subcluster II-1 in developing anther. Among them, ZmLTPg26 and ZmLTPg14 are orthologs of male-sterile genes OsLTP47 and AtLTPG4 [17,31], respectively. Subcluster II-4 includes 29 genes with low expression in W23 anthers, whereas some of them show expression peaks at certain anther stages in different inbred lines, such as ZmLTP2.5 with high expression at stages S7-S9 in B73, Oh43, and Zheng58 anthers, but undetectable expression in W23 anther. This finding indicates that some gene expression might be related to their genetic backgrounds. Notably, five LTP genes (ZmLTPx2, ZmLTPx1, ZmLTPd16, ZmLTPd15, and ZmLTP1.8-1) are orthologous to the male-sterile genes OsLTPg29, OsC6, OsLTP6, and AtLTP5/AtLTP12 [5,15,16,[41][42][43][44][45], respectively, and ZmLTPx1 is also a tassel-specific expression gene in maize, implying that these LTP genes may be involved in anther development and male fertility. Furthermore, the expression patterns of anther-or tassel-specific expression genes and the orthologs of GMS genes in Arabidopsis and rice are confirmed by qRT-PCR analysis ( Figure 5B). These findings indicate that these LTP genes may be required for maize anther development and male fertility, which need to be proved in the future.

Substrate Identification Strategies of Plant LTP Transporters
As previously reported, lipid metabolism plays important roles in plant reproductive development, including anther cuticle and pollen wall development [82], and the LTP transporters play roles in various physiological processes in plants. However, the exact substrates of most LTP transporters are still unclear. Here, we summarize several approaches to identify the substrates of LTP transporters (Table 3), which will be helpful for exploring the molecular mechanism of maize LTP transporters in the future. . These genes are clustered into two clusters. Genes in cluster I and II were clustered into two and four sub-clusters, respectively. The blue font indicates anther-or tassel-specific expression genes. (B) qPCR analysis of 16 LTP genes, which are tassel-specific expression, GMS or orthologs of rice and Arabidopsis GMS genes from stages 5 to 13 (S5-S13). Data are means SD, n = 3.

Figure 5. Expression analysis of LTP genes in maize. (A) Expression analysis of LTP genes in maize
based on RNA-seq data in maize inbred lines W23, B73, Oh43, and Zheng58. These genes are clustered into two clusters. Genes in cluster I and II were clustered into two and four sub-clusters, respectively. The blue font indicates anther-or tassel-specific expression genes. (B) qPCR analysis of 16 LTP genes, which are tassel-specific expression, GMS or orthologs of rice and Arabidopsis GMS genes from stages 5 to 13 (S5-S13). Data are means SD, n = 3. Cuticular wax Substance analysis by using GC-MS system [21] 10 AtLTP2 Cuticular wax Substance analysis by using GC-MS system [60] 11 AtLTP3 Fatty acid Substance analysis by using GC-MS system [19] 12 AtLTPG1 Cuticular wax Substance analysis by using GC-MS system [27] 13 AtLTPG2 Cuticular wax Substance analysis by using GC-MS system [23] 14 AtLTPG4 Cuticular wax Substance analysis by using GC-MS system [17] 15 AtLTPG6 Cuticular wax Substance analysis by using GC-MS system [17] 16 OsLTPL36 Fatty acid Substance analysis by using GC-MS system [20] 17 OpsLTP1 Fatty acid Substance analysis by using GC-MS system [67] 18 OsLTP5 Fatty acid Substance analysis by using GC-MS system [26] 19 AtLTPG15 Suberin monomer Substance analysis by using GC-MS system [61] 20 OsLTP47 Fatty acid Substance analysis by using GC-MS system [31] The most effective approach to identify the substrates of LTP transporters is a transport assay by PIP lipid strips and membrane lipid strips or isotope labelling, etc. However, those transport assays must be based on the overexpression of the LTP protein in the proper expression systems, such as the prokaryotic expression system. TaMs1 and its homologous genes OsLTPg29/OsLTPL94/OsEPAD1 and ZmLTPg11 in rice and maize, respectively, were confirmed to transport phospholipids in anthers by protein-lipid overlay assay by PIP lipid strips and membrane lipid strips [33,83]. AtDIR1 and AtAZI1 transport azelaic acid and the phosphorylated sugar derivative glycerol-3-phosphate by using isotope labelling of 14C-containing products measured by the TLC method [38]. Moreover, the substrates of some LTP transporters, such as OpsLTP1 and AtLTP3, have been determined by using the quantification of total lipids by spectrophotometric methods [67], and of Lc-LTP2 transporters FAs (C12-C22) and lysolipids by molecular modeling, 2-p-toluidinonaphthalene-6sulphonate (TNS) displacement, and liposome leakage experiments [84].
The GC-MS system is a convenient and approach to study the potential substrates of LTP transporters by using mutant and wild-type plant tissues, such as anthers, roots and leaves. The substrates of at least 12 LTP transporters have been predicted by using this approach in Arabidopsis, rice, and maize (Table 3). One advantage of this approach is that mixtures such as the content of cytosol in plants can be directly employed, and subsequently the isolated compounds can be used as a direct proof. Thus, this is a very powerful approach to identify the substrate of LTP transporters, although the conclusion is not very convincing. For example, OsLTP47 is reported to be essential for transporting the anther sporopollenin precursors, and fatty acid content was reduced in osltp47 mutant lines compared to WT due to the lipidic analysis of the wild-type and mutant mature anthers by using GC-MS [31]. In a word, the substrate identification of plant LTP transporters has made great progress based on different strategies, while the translocated substrates and detailed transport mechanism of the majority of LTP proteins remain unexplored, which need to be investigated in the future.

Discussion
LTP transporters play pivotal roles in multiple biological processes, such as abiotic and biotic stress response, plant signal transduction, and biosynthesis of lipid polymer sporopollenin and protective water-impermeable barriers in different plant organs [1,12,17,28,85]. Compared with the relatively comprehensive and systematic in-depth studies of LTP genes in Arabidopsis and rice, the functional mechanisms of maize LTP genes remain largely unknown. Here, we presented a preliminary introduction and characterization of the whole 65 LTP genes in maize. There are at least four significant points compared with previous studies [13]. First, we analyzed the basic characteristics and spatial expression patterns of all the 65 ZmLTP genes, which were identified based on the up-to-date maize B73 reference genome information, and seven genes were different from the previous study. Moreover, we performed a phylogenetic and gene ontology analysis of the LTP transporters in maize, which provides a basic framework for future research on maize LTP genes. Second, we summarized the research progress of the LTP transporters involved in diverse biological processes in model plants Arabidopsis and rice, such as anther and pollen development, vegetative organ development, seed development and germination, and biotic and abiotic stress response, which provides a mode to study the unknown LTP transporters in maize based on the functional conservation of LTP orthologs during plant evolution, together with gene ontology analysis. Third, we predicted the potential functions of maize LTP genes involved in anther development by using transcriptomic analysis based on RNA-seq and qRT-PCR assays. These findings provide useful clues for functional investigation of LTP transporters in maize. Finally, we summarized the advances and challenges in substrate identification of plant LTP transporters, and presented the future research directions and potential applications of LTP proteins in crop molecular breeding.

Phylogenetic and GO Analysis of Maize LTP Genes
Multiple alignments of maize, rice, and Arabidopsis LTP amino acid sequences were performed by ClustalW of MEGA7. Phylogenetic trees were constructed with MEGA7 using the maximum likelihood method, and bootstrap values were based on 500 replicates. The GO analysis was performed by GENE ONTOLOGY (http://geneontology.org/, accessed on 2 November 2022).

RNA Extraction, qPCR, and RNA-Seq Analyses
Total RNA of B73 anthers at stages S5 to S13 were isolated by using a Trizol reagent (Invitrogen, Waltham, MA, USA). The reverse transcription (RT) reaction was operated according to the protocol of the RT system (TransGen, Beijing, China).
RNA-sequencing (RNA-seq) analysis of maize anthers from stages S2 to S13 for W23, and S5 to S13 for B73, oh43, and Zheng58 were carried out as described by Jiang et al. [81].
For quantitative real time-PCR (qPCR), three technical replicates and three biological replicates were performed on each sample, and data were analyzed by using the 2 -DDCt method; each data point is the mean from three replicates ± SD. ZmUbi2 (Zm00001d05383) were used as the internal controls. The primers of qPCR analysis are shown in Table S3.

Conclusions
In conclusion, the findings presented here will shed light on our understanding of the critical roles of maize LTPs involved in various biological processes, including the transport and localization of lipidic precursors for anther development and male fertility, vegetative organ and seed development, and signal transport for biotic and abiotic stress response and resistance. Notably, the predicted biological functions of maize LTPs can be verified by using reverse genetics such as CRISPR/Cas9 or RNAi mutagenesis analysis [81,86,87]. With the advances of LTP gene cloning and functional characterization, this study will be an excellent gene resource for improvement of the grain yield, seed quality, and stress tolerance in maize via manipulating the related LTPs, such as through the multiple-control sterility system [54,88], dominant male sterility system [89,90], and other genetic strategies [91,92]. Therefore, this study provides a basic framework for functional research and the potential application of LTPs in multiple plants, especially for male sterility research and application in maize.