ATP-Binding Cassette G Transporters and Their Multiple Roles Especially for Male Fertility in Arabidopsis, Rice and Maize

ATP-binding cassette subfamily G (ABCG) transporters are extensive in plants and play essential roles in various processes influencing plant fitness, but the research progress varies greatly among Arabidopsis, rice and maize. In this review, we present a consolidated nomenclature and characterization of the whole 51 ABCG transporters in maize, perform a phylogenetic analysis and classification of the ABCG subfamily members in maize, and summarize the latest research advances in ABCG transporters for these three plant species. ABCG transporters are involved in diverse processes in Arabidopsis and rice, such as anther and pollen development, vegetative and female organ development, abiotic and biotic stress response, and phytohormone transport, which provide useful clues for the functional investigation of ABCG transporters in maize. Finally, we discuss the current challenges and future perspectives for the identification and mechanism analysis of substrates for plant ABCG transporters. This review provides a basic framework for functional research and the potential application of ABCG transporters in multiple plants, including maize.


Introduction
ATP-binding cassette (ABC) transporter, one of the largest proteins family, widely exists in bacteria, animals, and plants [1]. ABC transporters are involved in countless cellular processes, including the transmembrane transport of various unrelated molecules [2]. ABC proteins possess nucleotide-binding domain (NBD) which contain several highly conserved motifs, including the Walker A and B sequences, the ABC signature motif, the H loop and the Q loop; and transmembrane domains (TMDs), each composed of several hydrophobic α-helices [1,3]. ABC proteins can be full-size (with two TMDs and two NBDs) or half-size (with one TMD and one NBD), in which case they may function as homodimer or heterodimer [2]. The two NBDs cooperate to bind and hydrolyze ATP, providing the energy for transport, and the TMDs are involved in substrate recognition and translocation across the lipid bilayer [3]. In plants, ABC proteins can be divided into eight subfamilies, including ABCA to ABCG, and ABCI. Among them, the ABCG subfamily is the largest one in plants, and there are 43 and 54 members in Arabidopsis and rice, respectively [3]. The outstanding diversification of ABCGs is thought to be associated with plant adaptation to land environment [4]. In Arabidopsis and rice, ABCG transporters have been widely studied and linked with anther and pollen development, cuticle formation, defense response, hormone transport, and seed germination [2]. The substrates of ABCG transporters, including lipids, hormones, and heavy metal ions, etc., have been unambiguously identified using isotope labeling experiments in vivo, or indirectly by imprecise measurements 2 of 21 using the GC-MS system [5]. However, the actual substrates of most ABCG transporters remain uncharacterized.
It has been reported that 54 ABCG members exist in maize [6], but only three ABCG transporters (ZmMS2/ZmABCG26, ZmMS13 and ZmGL13) have been characterized and reported to be required for male fertility or leaf cuticle formation in maize [7][8][9][10]. Notably, the current nomenclature of the maize ABCG subfamily does not comply with the standard naming system used in Arabidopsis and rice [3,6], and thus results in a lot of inconveniences and problems in maize ABCG research.
Here, we present a consolidated nomenclature system for the maize ABCG subfamily, and focus on functional research progress of the reported ABCG genes in Arabidopsis and rice, and their implications for maize orthologs. The functions of maize ABCG genes can be predicted by homologous and bioinformatic analyses. Furthermore, we summarize the current progress and challenges in the identification of ABCG substrates, and present the future research directions and potential applications for ABCG proteins in plants.

The Nomenclature and Characterization of the Maize ABCG Subfamily
A consolidated nomenclature for maize ABCG transporters will provide much needed clarity and a framework for future research. Based on BLAST searches against the maize B73 reference genome (B73 RGV4.0) in the MaizeGDB database (https://maizegdb.org/ (accessed on 8 May 2022)), a total of 51 ABCG genes were identified in maize, and named as ZmABCG1 to ZmABCG51 according to their domain organizations (including 31 half-size ABCGs with one NBD-TMD and 20 full-size ABCGs with two NBD-TMD) and chromosome locations in maize (Table 1). Among them, 34 ZmABCGs have synonyms in MaizeGDB, including different members with the same name, such as two members (Zm00001d028689 and Zm00001d013960) named ABCG2, four members (Zm00001d032601, Zm00001d002871, Zm00001d048621 and Zm00001d020811) named ABCG11, etc., which will lead to confusion in the functional research of maize ABCG transporters in the future. Thus, it is desirable to create a unified nomenclature before the confusing and repeated ABCG names proliferate further.
Notably, the total number (51) of maize ABCG genes is different from that (54) in the previous report [6], which may have resulted from an alternative splicing of three ABCG genes, including ZmABCG5, ZmABCG7 and ZmABCG12 (Table S1). Furthermore, the spatial expression patterns of maize ABCG genes, based on RNA-seq data analyses in MaizeGDB, are shown in Table 1. These will provide useful information for investigating their biological functions. Other characteristics of the 51 ZmABCG genes, including the corresponding gene models in B73 reference genome (B73v3), genome physical locus, topology, and subcellular localization prediction using Cell-PLoc 2.0 (http://www.csbio. sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 20 April 2022)), are listed in Table S1. All of the 51 ZmABCG genes are distributed across the 10 chromosomes of maize, varying in numbers from eight on chromosome 8, to two on chromosome 5 (Table 1, Figure 1). The chromosome location information of ABCGs will be helpful in exploring their function and evolution in the future. and subcellular localization prediction using Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 20 April 2022)), are listed in Table S1. All of the 51 ZmABCG genes are distributed across the 10 chromosomes of maize, varying in numbers from eight on chromosome 8, to two on chromosome 5 (Table 1, Figure 1). The chromosome location information of ABCGs will be helpful in exploring their function and evolution in the future.

Multiple Functions of ABCG Transporters in Arabidopsis, Rice and Maize
To date, at least 30 ABCG genes in Arabidopsis and 11 ABCG genes in rice have been functionally characterized, but only three ABCG genes have been identified in maize. These reported ABCG transporters play essential roles in various biological processes, such as anther and pollen development, vegetive and female organ development, biotic and abiotic stress response, and phytohormone transport and signaling (

Multiple Functions of ABCG Transporters in Arabidopsis, Rice and Maize
To date, at least 30 ABCG genes in Arabidopsis and 11 ABCG genes in rice have been functionally characterized, but only three ABCG genes have been identified in maize. These reported ABCG transporters play essential roles in various biological processes, such as anther and pollen development, vegetive and female organ development, biotic and abiotic stress response, and phytohormone transport and signaling (Table 2; Figure 3).    OsSTR2 Os07g0191600 -

Anther and Pollen Development
ABCG transporters are involved in the translocation of various cuticular wax and cutin monomers, sporopollenin precursors, and tryphine components, from the tapetum where they are generated, to the anther outer surface and locule for anther cuticle and pollen wall formation, respectively ( Figure 3A). Loss-function mutations of some ABCG transporters often lead to defective anther cuticle and pollen wall formation and thus lead to male sterility in plants.
To date, at least 12 ABCG genes have been reported to be required for anther/pollen development and male fertility in multiple plant species (Table 2). For example, Arabidopsis AtABCG11 acts as both homodimer or heterodimer with other ABCGs (e.g., AtABCG5, 9, 12 and 14), and plays multiple roles in flower cuticle formation, root suberin metabolism, and proper vascular development. The abcg11 mutant displays dwarfism, male sterility, post-genital organ fusions, and reduced cutin load in flowers [17,25,26]. OsABCG26 and ZmMS13, orthologs of AtABCG11 in rice and maize, are essential for anther cuticle and pollen exine formation, especially for the translocation of the cuticular wax and cutin monomers generated in the tapetum across anther wall layers for anther cuticle formation [7,18,19]. AtABCG26 and its orthologs OsABCG15 in rice and ZmMS2 in maize, play similar roles in controlling pollen exine development and male fertility by mainly transferring sporopollenin precursors from the tapetum onto the developing microspore surface, although loss-of-function mutations of OsABCG15 and ZmMs2 also lead to defective anther cuticle formation [8,10,15,16]. Arabidopsis AtABCG1 and AtABCG16, and their rice ortholog OsABCG3, are required for the transport of nexine and intine precursors for male gametophyte development in the post-meiotic stages, and for pollen tube growth; the atabcg1atabcg16 double mutant and osabcg3 mutant show defective nexine and intine formation, and thus male fertility [21][22][23][24]. Furthermore, AtABCG9 and AtABCG31 are involved in the specific transport of steryl glycosides from the tapetum for pollen coat (tryphine) deposition [11]. Most recently, Liu et al. has reported that AP1/2β adaptins mediated exocytosis of tapetum-specific ABCG transporters (such as AtABCG9 and AtABCG16) from the trans-Golgi network (TGN) to the plasma membrane, and are required for pollen development in Arabidopsis [70]. AtABCG28 is specifically expressed in mature pollen grains and pollen tubes, and is critical for localizing polyamines [precursors of reactive oxygen species (ROS)] at the growing pollen tube tip in Arabidopsis [31]. Together, ABCG transporters may play conserved and divergent roles in transferring various precursors essential for anther development and male fertility in monocots (rice and maize) and dicots (Arabidopsis).

Vegetative and Female Organ Development
Additionally, several ABCG transporters are reported to be involved in the formation of diffusion barriers, such as cuticle, suberin, and lignin, during the vegetative and female organ development in plants (Table 2, Figure 3B). For example, AtABCG5, a half-size transporter of cutin and wax precursors, is required for the dense cuticle layer formation in young seedlings, forming homodimer or heterodimers with AtABCG11, and possibly with AtABCG12 and other ABCG transporters [33]. AtABCG11, but not AtABCG12, forms a homodimer to transport wax and cutin precursors, and AtABCG11 forms a heterodimer with AtABCG12 to transport wax precursors from the cuticle layer in Arabidopsis [26]. AtABCG13 is required for the secretion of flower cuticular lipids, particularly in petals and carpels [37], similar to the full-size AtABCG32 transporter, which exports particular cutin precursors from the epidermal cell in leaves and flowers [43]. AtABCG27 and AtABCG33 may be involved in cellulose synthesis, based on expression analysis using Arabidopsis cell suspensions during tracheary element differentiation [40]. Furthermore, the orthologs of Arabidopsis AtABCG32, rice OsABCG31 [45] and maize ZmGL13 [9] are also essential for leaf cutin and cuticular wax formation. Notably, four half-size Arabidopsis ABCG transporters, AtABCG1, AtABCG2, AtABCG6, and AtABCG20 [21,27], as well as their rice ortholog OsABCG5/RCN1 [34] are responsible for the transport of suberin monomers in roots and seed coats, and OsABCG5/RCN1 is also essential for shoot branching, by promoting the outgrowth of lateral shoots [35]. AtABCG29 acts as a monolignol (p-coumaryl alcohol) transporter involved in lignin biosynthesis [42]. Collectively, ABCG transporters play indispensable roles in the formation of various diffusion barriers in plants, which are critical for plant growth and the development against various stresses.

Biotic and Abiotic Stress Response
Another important function of ABCG transporters is in the protection against biotic and abiotic stresses (Table 2, Figure 3C). Many ABCG transporters are involved in pathogen and other biotic stress responses by the secretion of defense molecules. For example, AtABCG34 mediated the secretion of camalexin to defend against the necrotrophic pathogens Alternaria brassicicola and Botrytis cinerea [47], and AtABCG36/PEN3 was reported to enhance resistance against several non-adapted pathogens [58]. AtABCG36/PEN3 and AtABCG40/PDR12 mediate camalexin secretion for the resistance against Botrytis cinerea [63]. The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice [64]. Increasing reports reveal that ABCG transporters perform important physiological functions in plant drought stress response by regulating stomatal closure, such as OsABCG5/RCN1 [50], AtABCG17 and AtABCG18 [67], AtABCG21 and AtABCG22 [39,68], AtABCG25 [69], and AtABCG40 [62], while OsABCG9 plays a critical role in the transportation of epicuticular wax and is essential for rice drought response [53]. Overexpression of the AtABCG19 transporter confers kanamycin resistance to transgenic plants, which may be related to zinc homeostasis in plants [51,52]. Furthermore, AtABCG35, AtABCG36 and their rice ortholog OsABCG36, as well as OsABCG43, are involved in cadmium tolerance and other heavy metal stresses [54][55][56]60]. Together, ABCG transporters play critical roles in various biotic and abiotic stress responses in plants by transporting different substrates.

Hormone Transport and Signaling
In addition, ABCG transporters are also involved in phytohormone transport, which is very important for plant growth and development, including seed germination, shoot development, root formation, stress response, and other physiological processes [71]. To date, at least four types of hormones transported by 14 transporters have been identified in Arabidopsis and rice (Table 2, Figure 3D). Among them, Arabidopsis AtABCG14 is essential for the long-distance translocation of cytokinins from root to shoot [65], and its ortholog, OsABCG18, plays a similar role in rice and promotes grain yield [66]. Several ABCGs have been identified as ABA transporters that are required for the long-distance translocation of ABA in Arabidopsis and rice, such as AtABCG17 and AtABCG18 [67], AtABCG22 [68], AtABCG25 [69], AtABCG30, AtABCG31, and AtABCG40 [30,62], and OsABCG5 [50]. While auxin and its precursors (IBA) are reported to be transported by ABCG transporters, including AtABCG1 and AtABCG16 [20], AtABCG36 [59], and AtABCG37 [49], the ABCG transporters AtABCG1 and AtABCG16 are also involved in subcellular distribution in the metabolism and signaling of Jasmonates [29].
In summary, some ABCG transporters play different roles during plant growth and development, as they may function as homodimers and/or heterodimers with different ABCGs and thus transport various substrates.

Functional Predictions of ABCG Genes in Maize
Compared with Arabidopsis and rice, less ABCG genes have been functionally identified in maize (Table 2). Considering the functional conservation of orthologs during plant evolution, maize ABCG genes might also play similar roles with their orthologs, as with Arabidopsis and rice. Moreover, the spatiotemporal expression patterns of genes are often associated with their biological functions. Therefore, homologous and bioinformatics analyses will provide useful information for exploring the function of maize ABCG genes.

Functional Prediction of ABCGs Based on Homologous Analysis
Multiple studies have shown that some ABCG orthologs of Arabidopsis, rice, and maize play both conserved and divergent roles in regulating anther development and male fertility. For example, ZmABCG27/ZmMS2 and its orthologs OsABCG15 and AtABCG26, are all required for the translocation of sporopollenin lipidic precursors from the tapetum to the locules for pollen exine development in maize, rice, and Arabidopsis [8,10,[13][14][15]19]. ZmABCG13/ZmMS13 is essential for anther cuticle and pollen exine formation, consistent with its orthologs AtABCG11 and OsABCG26 in Arabidopsis and rice [7,17,19]. Here, we list the maize orthologs of all identified ABCG genes in Arabidopsis and rice (Table 2), which will provide an important clue for the functional characterization of maize ABCG genes by reverse genetic strategies, such as CRISPR/Cas9 or RNAi. For example, given that AtABCG9 and AtABCG31 are essential for the deposition of steryl glycosides on the pollen coat, and thus for pollen fitness [11], and OsABCG3 and its orthologs AtABCG1/16 are required for pollen wall (nexine and intine layers) formation [18,21,22,24], their orthologs in maize might also be involved in pollen wall development, which needs to be confirmed in the future via reverse genetic strategies. Therefore, it is feasible to predict the functions of more unknown ABCG genes in maize using homologous analysis.

Functional Prediction of ABCGs Based on Bioinformatic Analysis
The expression analysis based on RNA sequencing (RNA-seq) data provides a new window to predict gene function. Based on anther RNA-seq data, there are 62 putative maize genic male-sterility (GMS) genes, 125 putative lipid metabolic GMS genes, and 112 putative sugar metabolic GMS genes which have been predicted in maize [72][73][74]. Additionally, some of these predicted GMS genes have been verified through CRISPR/Cas9 mutagenesis in maize [8,75]. Thus, bioinformatic analysis, such as RNA-seq data analysis, provides an effective way to predict the functions of unknown genes.
Here, we carried out transcriptomic analyses of all maize ABCG members based on the RNA-seq data from four maize inbred lines W23, B73, Oh43, and Zheng58 developing anthers ( Figure 4A). Based on the RNA-seq data analyses, the 51 maize ABCG genes can be classified into two clusters (I and II). Cluster I can be further divided into four subclusters: I-1 to I-4. Subcluster I-1 consists of six ABCG genes, and half of them (ZmABCG22, ZmABCG8 and ZmABCG24) show peak expressions at middle anther stages (S8 to S9-10), and half of them (ZmABCG10, ZmABCG11 and ZmABCG34) display peak expressions at late anther stages (S11 or S12), in agreement with the qPCR results ( Figure 4B1-B6). Interestingly, the rice ortholog (OsABCG3) of ZmABCG8 and ZmABCG24, and Arabidopsis ortholog (AtABCG31) of ZmABCG34 have been reported as being required for pollen wall development and male fertility [11,21], indicating that these ABCGs are most likely involved in anther and pollen development in maize. Subcluster I-2 and I-3 include eight and 26 ABCG members, respectively, with relatively low expression during different anther developmental stages. Notably, ZmABCG26 and ZmABCG14 in subcluster 1-3, show an anther-specific expression pattern based on the RNA-seq data retrieved from the MaizeGDB website (http://www.maizegdb.org/ (accessed on 20 April 2022)), and they are orthologs of AtABCG28 required for pollen tube growth and male fertility in Arabidopsis [28], indicating that ZmABCG26 and ZmABCG14 might also be required for pollen tube growth in maize. This needs to be proven by reverse genetics. Subcluster I-4 includes three ABCG genes with multiple expression peaks in developing anthers ( Figure 4B7-B9), including ZmABCG2 which is orthologous to AtABCG1/16 [21,22], suggesting its potential roles in pollen wall development. Cluster II covers eight ABCG genes with relatively high expression during anther development [7,8,10] (Figure 4B10-B15), including four orthologs of the known GMS genes in Arabidopsis and rice, such as ZmABCG27/ZmMS2 orthologous to AtABCG26 and OsABCG15 [8,10,[13][14][15]19], ZmABCG13/ZmMS13, ZmABCG3 and ZmABCG6 orthologous to AtABCG11 and OsABCG26 [7,17,19], and two of them (ZmABCG27 and ZmABCG13), but not ZmABCG3, have been confirmed to be required for male fertility in maize [7,8,10], implying that these orthologous ABCG genes play both conserved and diversified roles during anther development among different plants.

Substrate Identification of Plant ABCG Transporters
Although ABCG transporters take part in various physiological processes in plants, the exact substrates of most ABCG transporters are still unclear. The functional insights

Substrate Identification of Plant ABCG Transporters
Although ABCG transporters take part in various physiological processes in plants, the exact substrates of most ABCG transporters are still unclear. The functional insights into plant ABCG transporters are mainly gained from reverse genetics (e.g., CRISPR/Cas9 mutagenesis) and metabolite analysis. However, the phenotypic effects resulting from knockout studies might be pleiotropic, thus hindering the identification of the actual substrates.
The most powerful approach to identify the substrates of ABCG transporters is to use a transport assay which demonstrates translocation activity across a membrane in a strictly ATP-dependent manner. The prerequisite for the transport assay is the overexpression of the ABCG protein in a proper expression system, such as tobacco (N. benthamiana) protoplast system and yeast strains (YMM12 and BY-2) cell lines [30,44,49]. To date, substrates of ten Arabidopsis ABCG transporters have been identified by employing radioactivity labeled compounds, i.e., by using isotope labeling experiments in vivo (Table 3). For example, Arabidopsis AtABCG11 and AtABCG32 are reported to export cutin precursors (e.g.,10,16-diOH, C16:0-2-glycerol and W-OH C16:0) for plant cuticle formation, based on the export assay using the protoplast system of N. benthamiana [44]. Four AtABCG transporters collaboratively deliver ABA from the endosperm to the embryo for controlling seed germination: AtABCG25 and AtABCG31 export ABA from the endosperm, whereas AtABCG30 and AtABCG40 import ABA into the embryo [30]. However, many isotope labeling compounds are not commercially available and these experiments are time consuming, which hinders their extensive utilization and efficiency.
An alternative approach is the substrate analysis of ABCG mutant and wild-type plant tissues using the GC-MS system. Here, a rational guess about the substrate or substrate class can be made by using the metabolite analysis of specific plant tissues, such as anthers, roots and leaves. The substrates of at least 15 ABCG transporters in Arabidopsis, rice and maize have been predicted based on this approach (Table 3). One advantage of this approach is that mixtures, such as the content of the plant's cytosol, can be directly employed, and subsequently, the isolated compounds can be used as direct proof. Thus, this is a very powerful approach in identifying the substrate of ABCG transporters, although the conclusion is not very convincing. For example, ZmMS13 and ZmMS2 encoding ZmABCG13 and ZmABCG27, respectively, are reported to be essential for the transport of anther cuticle and sporopollenin precursors, partly due to the lipidomic analysis of the wild-type and mutant mature anthers, using GC-MS [7,10].
Besides the two approaches mentioned above, other direct or indirect transport assays have also been reported to have been used to identify the substrates of ABCG transporters. For example, the lipidic substrates of AtABCG1 and ZmMS13/ZmABCG13 were indicated by the ATPase assay of purified protein in vitro [7,27], Cytokinin was identified as the substrate of OsABCG18 based on an export assay with a heterologous expression of Os-ABCG18 in the protoplast system of N. benthamiana [66], and AtABCG28 was required for the apical accumulation of reactive oxygen species in growing pollen tubes based on the immunostaining of polyamines in the growing tip of pollen tubes [31] (Table 3).
Notably, although the substrate identification of plant ABCG transporters has gained some encouraging progress, the translocated substrates and the detailed transport mechanisms of the majority of ABCG proteins remain unclear, which need to be investigated in the future.

Conclusions and Perspective
ABCG transporters, as one of the largest subfamilies of ABC transporters, play critical roles in various processes influencing plant fitness, especially in plant reproductive and vegetative organ development, hormone transport and stress response. Compared with the unified named ABCG transporters in Arabidopsis and rice [3], the ABCG transporters in maize lack a standard naming system, leading to confusion in the functional research of maize ABCGs. In this review, we firstly established a consolidated nomenclature and chromosome location map of the 51 ABCG transporters in the maize genome (Table 1 and Figure 1), providing a basic framework for future research. Based on the phylogenetic analysis, the whole ABCG subfamily members in Arabidopsis, rice and maize can be divided into two subgroups and eight clades (Figure 2). These results will provide useful clues for exploring the functions of unknown ABCG genes in maize. Secondly, we summarized the latest research progress of ABCG transporters present in Arabidopsis, rice, and maize. The functions of the reported ABCG transporters are involved in diverse processes, such as anther and pollen development, vegetative and female organ development, abiotic and biotic stress response, and phytohormone transport (Table 2 and Figure 3). Thirdly, compared with the plentiful in-depth studies of ABCGs in Arabidopsis and rice, less ABCG transporters in maize have been functionally characterized to date. We thus provide two methods for the functional prediction of ABCG transporters in maize, namely the homologous and bioinformatic analyses, based on the functional conservation of ABCG orthologs during plant evolution and spatiotemporal expression patterns of ABCG genes in maize (Table 1 and Figure 4). Finally, we summarize and discuss the current approaches and challenges in the substrate identification of plant ABCG transporters, including the transport assays, by employing radioactivity compounds in vivo, metabolite analysis, using the GC-MS system, and other direct or indirect transport assays (Table 3), although the actual substrates of most plant ABCG transporters remain largely unknown.
In summary, given the tremendous progress in defining the critical roles of plant (mainly in Arabidopsis and rice) ABCG transporters involved in various biological processes influencing male fertility and plant fitness, it is plausible that the corresponding ABCG orthologs might also play similar roles in other crops, including maize. This can be verified by the use of reverse genetics (e.g., CRISPR/Cas9 mutagenesis analysis), and thus deepen our understanding of the functional mechanism of ABCG transporters in multiple plants. The derived mutant lines with elite characters, such as male sterility or higher fitness, have potential applications in hybrid crop breeding and seed production in the future.

Conflicts of Interest:
The authors declare no conflict of interest.