The Plant Fatty Acyl Reductases

Fatty acyl reductase (FAR) is a crucial enzyme that catalyzes the NADPH-dependent reduction of fatty acyl-CoA or acyl-ACP substrates to primary fatty alcohols, which in turn acts as intermediate metabolites or metabolic end products to participate in the formation of plant extracellular lipid protective barriers (e.g., cuticular wax, sporopollenin, suberin, and taproot wax). FARs are widely present across plant evolution processes and play conserved roles during lipid synthesis. In this review, we provide a comprehensive view of FAR family enzymes, including phylogenetic analysis, conserved structural domains, substrate specificity, subcellular localization, tissue-specific expression patterns, their varied functions in lipid biosynthesis, and the regulation mechanism of FAR activity. Finally, we pose several questions to be addressed, such as the roles of FARs in tryphine, the interactions between transcription factors (TFs) and FARs in various environments, and the identification of post-transcriptional, translational, and post-translational regulators.


Introduction
Long-chain (LC), or very-long-chain (VLC), primary fatty alcohols are important derivatives of long-chain fatty acids (LCFAs), or very-long-chain fatty acids (VLCFAs). They are primarily involved in the formation of the following four extracellular lipid-phenolic protective layers in the plant kingdom: cuticle coatings in aerial surfaces of land plants, sporopollenin found in the outer walls of pollen spore coatings, suberin which exists in the extracellular walls of various external and internal tissue layers, and suberin-associated waxes in mature taproots [1]. With one exception, primary fatty alcohols are present in the seeds of the jojoba plant (Simmondsia chinensis) in the form of wax esters as a lipid energy reserve for postgerminative development [2,3]. In early 1971, Kolattukudy put forward the conjecture that fatty acyl-CoA reductase and aldehyde reductase synergistically catalyze the synthesis of primary fatty alcohols [4]. Up to now, this two-step process via an aldehyde intermediate has not been confirmed in plants. However, it was found that the reduction of fatty acyl-CoAs to primary fatty alcohols can be performed by a single alcohol-forming FAR without releasing the intermediate fatty aldehyde [5,6]. The first FAR gene cloned and characterized came from jojoba [6]. Subsequently, related FAR genes have been cloned from other plant species, including Arabidopsis thaliana [7][8][9][10][11], Physcomitrella patens [12], rice (Oryza sativa) [13], wheat (Triticum aestivum) [14][15][16][17][18], maize (Zea mays) [19], Aegilops tauschii [20], Brachypodium distachyon [21,22], Brassica napus [23], and cotton (Gossypium hirsutum) [24].
Alcohol-forming FARs in plants can be divided into two categories according to their subcellular localization: microsomal-localized FAR, and plastid-localized FAR, which take acyl-CoA, acyl-ACP, or both [11,25] as substrates. Microsomal-localized FAR is usually in charge of oil production in seeds and the accumulation of wax and suberin, whereas plastidassociated FAR is primarily involved in the biosynthesis of sporopollenin. Each member of the FAR enzyme family is restricted to a unique lipid metabolic pathway due to differences in substrate specificity, tissue-specific expression pattern, and subcellular localization. In addition, lipid metabolism pathways are incredibly complex biological processes in which many enzymes participate, and the regulatory network is even more intricate. Hence, an in-depth exploration of the function and regulation of FARs has far-reaching and immense significance to the genetic improvement of crops. Future research should also focus on new biological functions of FAR genes in lipid synthesis and their regulatory molecular mechanisms at different scales, including post-transcriptional, translation, and post-translational levels. Herein, we present a concise review of the latest research into the FAR family of enzymes and further emphasize newly emerging questions that must be addressed to deepen our comprehension of these crucial enzymes.

Phylogenetic Analysis
FAR is reported to be a small plant gene family [1]. To provide some clues about the function of this gene family, five model species with annotated genomes from the evolution of terrestrial plants were selected. These included one bryophyte (P. patens), one pteridophyta (Diphasiastrum complanatum), one gymnosperm (Ginkgo biloba), one dicotyledon (A. thaliana), and one monocotyledon (Z. mays) ( Figure 1A). Then the protein sequences of eight AtFARs were used as templates to perform BLASTPs against all of the genes annotated in the remaining four representative genomes. Phylogenetic analysis using multiple alignments of protein sequences from the five model species of FARs, and the characterized FARs with known functions (Table 1), were inferred using the neighbor-joining method [26]. The consequent neighbor-joining tree showed that all proteins could be clustered into three distinct clades (represented by red, green and yellow, respectively) ( Figure 1B). All of the FAR members of the yellow clade originated from monocotyledons, while all of the FAR members of the green clade descended from dicotyledons. Their function is responsible for the biosynthesis of suberin or cuticular wax. The red clade includes the FAR members from the above five model species and rice. Some of the FARs, whose functions have been characterized are required for spore (pollen) outer wall development, including PpMS2-1 from P. patens, OsDPW [13] from rice, ZmMs25 [19] from maize, and AtMS2/FAR2 [8] from Arabidopsis. These findings suggest that the sporopollenin synthesis-associated FARs existed in both early divergent land plants and the Angiosperms, and the function may be conserved across terrestrial plants. Further analysis with denser sampling and more sophisticated evolution models is helpful to decipher the evolution of FAR.  FARs with different colors represent distinct functions. Royal purple is associated with cuticular wax, mauve is associated with suberin, red is associated with sporopollenin, blue is associated with storage wax, and black represents an unknown function. The phylogenetic analysis was conducted by MEGA11.0 software using the neighbor-joining method. The tree is drawn proportionally, and the branch length is the same as the evolutionary distance unit used to infer the phylogenetic tree.  Royal purple is associated with cuticular wax, mauve is associated with suberin, red is associated with sporopollenin, blue is associated with storage wax, and black represents an unknown function. The phylogenetic analysis was conducted by MEGA11.0 software using the neighbor-joining method. The tree is drawn proportionally, and the branch length is the same as the evolutionary distance unit used to infer the phylogenetic tree.

Structural Domains
Plant FARs are composed of about 500 amino acids in which microsomal-localized FARs contain core enzyme structure composed of NAD_binding_4 domain and sterile domain, whereas plastid-localized FARs contain an N-terminal extension (plastid transit peptide) in addition to core enzyme structure ( Figure 2A) [8,11,13,19]. Multiple sequence alignment was carried out for the amino acid sequences of FARs of the five model species mentioned above and the results showed that the NAD_binding_4 domain, of all FARs, contained the NAD(P) H-binding motif (GXXGXX(G/A)) and the active site motif (YXXXK) ( Figure 2B), indicating that these two motifs were highly conserved during the evolution of terrestrial plants. A study discovered that constructs containing MS2 fragments with deletion of the NAD_binding_4, or FAR_C domain, or even with deletion of the GXXGXX(G/A) or YXXXK motif, were unable to rescue the phenotype of defective pollen exine in ms2 mutant [8]. Tyrosine (Y) and lysine (K) residues in the YXXXK active site motif were predicted to play direct roles in the enzyme activity based on kinetic studies with other reductases [31,32]. Site-specific mutations of the two amino acid residues of FAR5 resulted in the inability to produce primary fatty alcohols in yeast [27]. Moreover, subsequent research showed that the mutation of the four amino acid residues (GXXGXX(G/A) and YXXXK, residues underlined) in the above two conserved motifs had a significant impact on the enzymatic activity and substrate selection of ZmMS25 in vitro [19].

Substrate Specificity
FARs possess distinct substrate specificities regarding acyl chain saturation and chain length. FAR isoform divergence in substrate specificity is directly connected to their diversity in function and varying subcellular localizations. The physiological properties of the final biosynthetic product are frequently dependent on the substrate specificity of FARs (Table 1). In a fascinating example, the preference of FAR enzymes expressed in pheromone glands for fatty acyl substrates containing cis or trans double bonds leads to reproductive segregation between the two races of European corn borer moth [33].

Substrate Specificity
FARs possess distinct substrate specificities regarding acyl chain saturation and chain length. FAR isoform divergence in substrate specificity is directly connected to their diversity in function and varying subcellular localizations. The physiological properties of the final biosynthetic product are frequently dependent on the substrate specificity o FARs (Table 1). In a fascinating example, the preference of FAR enzymes expressed in pheromone glands for fatty acyl substrates containing cis or trans double bonds leads to reproductive segregation between the two races of European corn borer moth [33].

Subcellular Localization and Expression Pattern
In the plant kingdom, FAR proteins are confined to only two subcellular compartments (i.e., plastid and ER) ( Table 1). Several pollen development-associated FARs are known to localize to the plastid envelope, including AtMS2/FAR2 [8], OsDPW [13], and ZmMs25 [19], whereas those wax and suberin-associated FAR enzymes are reported to localize in the ER where wax and suberin biosynthesis occurs.
The expression pattern of a gene is closely related to its function (Table 1). AtFAR1, AtFAR4, and AtFAR5 are mainly expressed in tissues where the suberin deposits [10]. AtFAR3/CER4 is highly expressed in aerial organs of the plant, which is consistent with its roles in wax biosynthesis [7], in addition, the FARs from other plants also display similar expression patterns, such as Ae.tFAR3, Ae.tFAR4, and Ae.tFAR6 from Ae. tauschii [20], GhFAR3.1A and GhFAR3.1D from cotton [24], and TaFARs from Triticum aestivum [14][15][16][17]. AtMS2/FAR2 expression is restricted to flowers, which is consistent with its roles in pollen exine development [8]. In addition to Arabidopsis, PpMS2-1 from P. patens exhibits a sporophyte-specific expression pattern [12]. ZmMs25 is expressed specifically in anther, which is in agreement with its roles in anther and pollen development in maize [19]. Rice OsDPW is mainly expressed in the tapetum and microspores [13]. Further study of the expression pattern of FAR in diverse plant species is required to clarify the function of FAR during lipid metabolism comprehensively.

Cuticular Wax Synthesis-Associated FARs
Cuticular wax is a complex mixture of VCLFAs and their derivatives ranging from C20 to C60 synthesized in the ER ( Figure 3A), They include primary alcohols, fatty aldehydes, alkanes, and esters, and may also contain cyclic compounds, such as terpenoids and sterols [38,39] on the aerial surface of all terrestrial plants which plays a vital role in protecting them from the attack of diverse biotic and abiotic stress factors, such as drought, UV-B radiation, mechanical damage, and even bacterial and fungal pathogens [38,[40][41][42]. Changes in the cuticular wax primary alcohol composition significantly impact the crystal structure and hydrophobic properties of the epidermis [43,44]. Cuticular wax primary alcohols can also act as signal molecules and play an important role in pathogen and host recognition [45]. In addition, triacontanol (C30-OH) acts as a growth regulator, enhancing plant photosynthesis and increasing dry matter accumulation [46].
AtFAR3/CER4 plays a dominant role in the accumulation of cuticular wax-associated primary alcohols of Arabidopsis [7]. Intuitively, the atcer4 mutant shows a stem "glossy" phenotype, suggesting that the absence of primary alcohols has a significant impact on the assembly and arrangement of epidermal wax crystals [7,34]. Interestingly, the mutation of AtFAR3/CER4 results in the almost complete deletion of VLC monounsaturated primary alcohols in the stems in comparison to the wild type, and co-expressing AtFAR3/CER4 with AtCER17/ADS4 in yeast produced VLC monounsaturated (n-6) primary alcohols, indicating VLC monounsaturated acyl-CoAs are also the substrates of AtFAR3/CER4 [37].
pathogens [38,[40][41][42]. Changes in the cuticular wax primary alcohol composition significantly impact the crystal structure and hydrophobic properties of the epidermis [43,44]. Cuticular wax primary alcohols can also act as signal molecules and play an important role in pathogen and host recognition [45]. In addition, triacontanol (C30-OH) acts as a growth regulator, enhancing plant photosynthesis and increasing dry matter accumulation [46]. Acyl-ACPs synthesized de novo in the plastid are reduced by FAR to produce fatty alcohols. This product could then be exported to the anther locule by an unknown mechanism where it polymerizes at the surface of the microspore. (C) ER-Localized FARs are involved in the suberin and suberin-associated wax production. De novo fatty acid synthesis occurs in the plastid. Fatty acyl elongation occurs via the FAE complex producing VLCFAs. FARs catalyze acyl reduction to produce suberin monomer primary alcohols and α, ω-diols. Coumaric, caffeic, and ferulic acids produced by the phenylpropanoid pathway are linked to fatty alcohols by BAHD-type acyltransferases to produce alkyl hydroxycinnamates (AHCs). Abbreviations: PM, plasma membrane; CW, cell wall.
AtFAR3/CER4 plays a dominant role in the accumulation of cuticular wax-associated primary alcohols of Arabidopsis [7]. Intuitively, the atcer4 mutant shows a stem "glossy" phenotype, suggesting that the absence of primary alcohols has a significant impact on the assembly and arrangement of epidermal wax crystals [7,34]. Interestingly, the mutation of AtFAR3/CER4 results in the almost complete deletion of VLC monounsaturated primary alcohols in the stems in comparison to the wild type, and co- FARs are involved in sporopollenin biosynthesis. Acyl-ACPs synthesized de novo in the plastid are reduced by FAR to produce fatty alcohols. This product could then be exported to the anther locule by an unknown mechanism where it polymerizes at the surface of the microspore. (C) ER-Localized FARs are involved in the suberin and suberin-associated wax production. De novo fatty acid synthesis occurs in the plastid. Fatty acyl elongation occurs via the FAE complex producing VLCFAs. FARs catalyze acyl reduction to produce suberin monomer primary alcohols and α, ω-diols. Coumaric, caffeic, and ferulic acids produced by the phenylpropanoid pathway are linked to fatty alcohols by BAHD-type acyltransferases to produce alkyl hydroxycinnamates (AHCs). Abbreviations: PM, plasma membrane; CW, cell wall.
The primary alcohols and esters generated by the alcohol-forming pathway only account for 15-25% of the total wax in Arabidopsis inflorescence stems and rosette leaves. In contrast, the alcohols take a predominant role in leaf epidermal wax in some important crops, such as in corn and barley where primary alcohols account for about 70-80% of the wax components [48][49][50]. Therefore, an accurate interpretation of each FAR's function in synthesizing cuticular wax primary alcohols among different crop species is crucial for reconstructing plant cuticular wax layers in some important crops.

Sporopollenin Synthesis-Associated FARs
Sporopollenin, a complex polymer consisting of polyhydroxylated aliphatic compounds and phenolics, has extreme stability and recalcitrance, thus ensuring the integrity of the pollen when it is subjected to various external physical and chemical pres-sures such as hydrostatic, chemical reagents, and non-oxidative chemical and biological degradation [51][52][53]. De novo synthesis of fatty acids occurs in tapetal plastids, where they are reduced to LC primary alcohols by FAR proteins (Figure 3B).
To date, sporopollenin synthesis-associated FAR genes were studied in several plant species such as Arabidopsis, rice, and maize [8,13,19]. AtMS2/FAR2 from Arabidopsis is first identified as essential for sporopollenin synthesis [8,54]. OsDPW from rice [13] and ZmMs25 from maize [19] are also required for sporopollenin biosynthesis, suggesting that the metabolic pathway of sporopollenin is conserved among angiosperms. Interestingly, unlike the Arabidopsis atms2 mutant, the anther cuticle of the rice dpw mutant is also defective, which indicates that the functions of related genes and/or enzymes have diversified during evolution [13]. In addition to angiosperms, sporopollenin is also widely found in Chlorophyta, Bryophyta, Pteridophyta, Marchantia polymorpha, and even fungi [55]. Moreover, PpMS2-1, a putative moss homolog of AtMS2/FAR2, participates in the development of the outer wall of the spore since its mutant phenotype is remarkably similar to that of defective microspore exine in Arabidopsis [12]. These findings indicate that the underlying mechanism of sporopollenin biosynthesis is highly conserved during the land plant evolutionary process. Moreover, during the process of evolution from lower plants to higher plants, the composition of the spore outer wall (pollen outer wall) becomes more complex [12].

FARs Involved in Suberin and Suberin-Associated Waxes Biosynthesis
Suberin is a hydrophobic heteropolymer composed of phenolics, glycerol, and various fatty acid derivatives that mainly act as a protective barrier for controlling the flow of water, solutes, and gases, protecting plants from various abiotic stresses and pathogenic infections [56][57][58][59][60]. Its aliphatic portion is a polyester composed mainly of ω-Hydroxyl fatty acids, α, ωdicarboxylic acid with chain lengths ranging from C16 to C28, FAs, and primary fatty alcohols [61] ( Figure 3C).
In Arabidopsis, AtFAR1, AtFAR4, and AtFAR5 are reported to be involved in the accumulation of suberin-associated primary alcohols, and the total fatty alcohol load in suberin is reduced by 70-80% in atfar1 atfar4 atfar5 triple mutant lines [10,60]. In B. distachyon, the mutation of BdFAR4 leads to a significant reduction in the content of C20:0-and C22:0-OH compared with the wild type [22].
In the periderm of underground storage organs, suberin is found in association with waxes. These suberin-associated waxes are composed of linear aliphatic with shorter chain lengths than cuticular wax and have been found in diverse plant species such as potato (Solanum tuberosum) [62], Camelina (Camelina sativa) [63] and Arabidopsis [64,65]. Alkyl hydroxycinnamates (AHCs), which are formed by esterification of C18:0 to C22:0 primary fatty alcohol with coumaric acid, caffeic acid, or ferulic acid, are the main component of suberin-associated waxes [65] ( Figure 3C). The biosynthesis of AHCs of suberin-associated root waxes includes the following steps: the biosynthesis of hydroxycinnamate, the reduction of fatty acyl-chains, and the transfer of CoA-activated hydroxycinnamate derivatives onto hydroxylated aliphatic [66]. In Arabidopsis, three FARs (AtFAR1, AtFAR4, and AtFAR5) required for primary alcohol synthesis in suberin are also involved in the production of fatty alcohols in suberin-associated taproot waxes [60,65,67]. The contents of soluble fatty alcohols and AHCs in root waxes of atfar1 atfar4 atfar5 triple mutant lines are reduced by more than 80% [60,67]. Apart from Arabidopsis, AHC synthesis-associated FAR is rarely reported in plant species. However, AHCs are widely present in angiosperms [62,68], gymnosperms [69,70], and possibly even in P. patens [71], suggesting that some enzymes might play similar roles as AtFARs in catalyzing the production of AHCs.

Regulation of FAR Genes
Extracellular lipid protective barriers are crucial in the tolerance to various environmental stresses. Many of the FAR genes involved in extracellular lipid metabolism are induced by various abiotic or biotic stresses including drought, salt, cold, wounding, and the infection of fungi. For example, the transcriptional levels of three suberin-associated genes, AtFAR1, AtFAR4, and AtFAR5, are gradually up-regulated after wounding and salt treatment [10]. The transcripts of several wax-associated genes including TaFARs (i.e., TaFAR1-TaFAR8) and BdFARs (BdFAR1-BdFAR4) are also induced by abiotic stress treatment such as cold, drought, and/or high salt [14][15][16][17]21,22]. The transcripts of TaFAR6, TaFAR7, and TaFAR8 are also induced by powdery mildew (Blumeria graminis) infection [17]. Thus far, some MYB transcription factors have been identified to regulate the expression levels of wax-associated FARs under abiotic stresses (Table 2). For example, AtMYB94 was dramatically induced by salt stress and drought stress, and it can activate the expression of AtFAR3/CER4 through direct promoter binding [72]. PtoMYB142 from Populus tomentosa contributes to drought tolerance by directly binding to the promoter of the wax biosynthesis gene PtoCER4 and regulating its expression [73].
In addition, some TFs were identified to play positive roles in regulating the expression levels of suberin-associated genes ( Table 2). In the seed coat, AtMYB107 interacts strongly with the AtFAR1 promoter, and its mutation significantly reduces the expression of AtFAR1, AtFAR4, and AtFAR5 [74]. In the cell wall of Arabidopsis leaf epidermal cells, AtMYB41 overexpression increases the abundance of AtFAR1, AtFAR4, and AtFAR5 transcripts and leads to the ectopic deposition of suberin monomer C18-C22 primary alcohols [75]. BdMYB41 from B. detachyon, which is closely related to AtMYB41, directly interacts with the promoter region of BdFAR4 [22]. During wound suberization, AchnMYB41, AchnMYB107, and Achn-MYC2 from kiwifruit activate AchnFAR to enhance primary fatty alcohol accumulation [30]. Some highly conserved MYBs are found to regulate the sporopollenin synthesis-associated FAR genes (Table 2). AtMYB103 (also called MYB80 and MS188) and its direct upstream regulator AtAMS are identified to be essential for the expression of AtMS2/FAR2 in pollen walls [76,77]. TaTDRL and TaMYB103 are homologs of AtAMS and AtMYB103, respectively. Both can directly bind to the promoter to synergistically activate the expression of TaTAA1a [78]. OsMYB80 and ZmMYB84, as homologs of AtMYB103, directly activate the expression of OsDPW and ZmMs25, respectively [19,79]. Thus far, most studies have focused on the roles of MYBs, whereas only one study showed that Arabidopsis SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) indirectly regulates AtCER4 expression by affecting other unknown TFs [80].

ZmMYB84
Zea mays ZmMs25 1 Sporopollenin biosynthesis [19] 1 Direct regulation through promoter binding; 2 Indirect regulation through other transcription factors; 3 No experimental data exists to confirm whether it is direct regulation or indirect regulation.

Conclusions and Perspectives
Fatty acyl reductases target acyl-CoAs or acyl-ACPs to provide fatty alcohol substrates for lipid synthesis processes which is vital for the normal growth and development of plants.
Herein, a brief cluster analysis was first conducted on the related FAR proteins and their conserved structural domains, tissue-specific expression patterns, subcellular localization, and unique roles in different lipid metabolic pathways. These were then summarized in this review ( Figure 3). Lastly, this review also described the mechanisms by which FAR is regulated. Although the progress made in recent decades has significantly advanced our understanding of the FAR gene family, particularly the conservation of function and regulation, several questions remain unanswered.

1.
The pollen wall is a complex multi-layer structure wrapped on the outer surface of pollen ( Figure 3B). Aliphatic alcohols not only exist in the exine in the form of sporopollenin but also in the cavities of the pollen exine in the form of tryphine [83]. Tryphine is composed of complex lipids, wax esters, flavonoids, hydroxycinnamoyl spermidine metabolites, and proteins [84,85]. Little is known about the formation of tryphine. Therefore, it is of great interest to investigate whether any specific alcohol-forming FARs are involved in tryphine production.

2.
In Arabidopsis, AtFAR1, AtFAR4, and AtFAR5 display different specificity towards substrates with different chain lengths, which are mainly responsible for the synthesis of C22:0-OH, C20:0-OH, and C18:0-OH, respectively. Interestingly, recent studies showed that the levels of LC suberin monomers including C18:0-OH positively correlate with environmental factors such as precipitation, evapotranspiration, temperature, and UV index, whereas those of VLC suberin monomers, including C20:0-OH and C22:0-OH, display the opposite trend [86]. This indicated that AtFAR1, AtFAR4, and AtFAR5 are differentially regulated by various environmental cues. Understanding the regulatory mechanism of AtFAR1, AtFAR4, and AtFAR5 in response to different environmental conditions will provide new insights into plants' abilities to adapt to different environmental factors.

3.
Thus far, regulatory mechanisms of FARs have been comprehensively studied at the transcriptional level, but little is known about how FARs are regulated at the post-transcriptional level, the translational level, and the post-translational level.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available from the authors on request.

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