The transport of metabolites in the plants is an indispensable phenomenon for the dynamic functioning of numerous cellular processes ranging from growth, development, survival, defense, maintaining homeostasis, and coordination in the system (Larsen et al., 2017a; Erb and Kliebenstein, 2020). Specifically, the vast number of secondary metabolites (SMs) produced by plants serve crucial purposes including their conclusively proven function as defense signaling molecules for protecting against several pathogens and additionally as bioactive compounds for medicinal usage (Isah, 2019). Until recently SM transporters are being overlooked for the improvement of various biotechnological processes such as enhanced production of bioactive compounds that would provide ample supply for herbal formulations at the industrial level. Although, a few studies (Rischer et al., 2013; Gupta et al., 2017; Rastegari et al., 2019) have attempted to examine as well as regulate the functions of several key enzymes from SM biosynthetic pathways for improving the yield of bioactive compounds, but the success rate remains limited. However, in the recent past, the engineering of SM transporters has been emerging as the most viable alternative to the conventional approaches used so far for improved metabolite production (Lv et al., 2016). Despite the abundant presence of transporters in the plants (Larsen et al., 2017a; Tang et al., 2020) (approximately 10% of the Arabidopsis thaliana genome encodes transporter proteins), only a handful are having their function and substrate specificity well deciphered by experimental evidence, especially the ones involved in the transport of SMs. Thus, it is challenging to find the appropriate candidates for transporter engineering to accomplish the long-term goal of systemic metabolic engineering for the betterment of plants as well as for medicinal benefits.

Considering this, better knowledge of SM transporters, particularly their diversity, functionality, regulatory parameters, approaches for their identification, and characterization is a pre-requisite for future research. In this review article, we attempt to provide a perspective and create a foundation in the field of systemic metabolic engineering by utilizing the most feasible approach of engineering the potential transporters by modulating their expression. Here, we are discussing the existing data as well as updating the understanding of plant SM transporters. Few of the previously published reviews (Shitan et al., 2014a; Lv et al., 2016; Shitan, 2016; Gani et al., 2021) have mostly dealt with various SM transporters and their potential role in metabolic engineering in plants. However, meager information is available on the diverse functionalities of transporters and the factors linked to their regulation. Herein, we extracted relevant information for more than 60 different transporters from various important plants to obtain a comprehensive picture and to develop a better understanding of plant SM transporters. The present review also reflects on the latest advancement of knowledge in the area, which could be translated for systemic metabolic engineering in plants.

Plants are the producers of a significant number of specialized metabolites, which are also commonly known as SMs. SMs are small organic compounds that can be further categorized into terpenoids, alkaloids, flavonoids, and other phenolic compounds (Razdan, 2018). These metabolites not only play the role of bioactive compounds for therapeutic purposes but also naturally help combat the biotic and abiotic stress conditions by acting as defense molecules for the plants (Naik and Al-Khayri, 2016). The majority of SMs are synthesized, stored, and functionally activated at different locations in the plant system hence their mobilization from source to sink (site of synthesis to site of accumulation or activation) is necessary. In contrast to primary metabolites, the source-sink relationship is highly dynamic for the partitioning of SMs in the plants. The biosynthesis of main primary metabolites takes place exclusively in the leaves via the process of photosynthesis, therefore leaves are the major source tissue from where the one-way transport of metabolites is directed toward various sink tissues such as tuber, seed, stem, fruit, and roots (Griffiths et al., 2016). On the contrary, the biosynthesis of SM pool is not restricted to a specific tissue and many metabolites ultimately travel using various transporters from their source tissues to respective sink tissues either for accumulation or functional activation. Additionally, few specialized SMs are also involved in the transporter-mediated long-distance translocation via xylem and/or phloem pathways (Nour-Eldin and Halkier, 2013). For instance, in Nicotiana tabacum, the transport of nicotine from roots to leaves is mediated by the xylem pathway (Shoji et al., 2009) while in A. thaliana glucosinolates translocate from leaves to seeds, rosette to roots, and roots to rosette possibly via xylem and phloem mediated pathways (Andersen et al., 2013). For such types of long-distance mobilization of metabolites it may be possible that multiple transport events are needed, which might involve transporters localized at the plasma membrane and vacuolar membrane of roots and leaves (Yazaki et al., 2007). Therefore, it is believed that the transport of the SM pool is supposedly a two-way process because some metabolites travel upward while others move in the downward direction due to their diverse functional requirements at different locations in the plant. Further, various tissues can act as a source or a sink depending on the expression level of substrate biosynthetic pathway and transporter genes (Jorgensen et al., 2015). The distribution of SMs at the intercellular and intracellular level is considered as a fundamental part of growth, development, and mode of cell-environment communication (Schulz, 2011) yet, there exists a huge gap in understanding the mechanisms of transport. Plant metabolites are known to travel via several modes such as simple diffusion, membrane vesicle-mediated, and substrate-specific transporters (Shitan, 2016). Several of the plant SMs get transported by importer or exporter proteins, possibly located on the vacuolar and/or plasma membrane of the cell.

With the availability of biological resources, the development of experimental approaches, and combinatorial use of various tools (Table 1) such as bioinformatics, genomics, transcriptomics, proteomics, forward or reverse genetics, expression in the heterologous host, and transporter activity assays, it has become relatively easy to predict and identify potential transporters in the plants (Barbier-Brygoo et al., 2001; Kell et al., 2015; Lv et al., 2016; Larsen et al., 2017a; Tang et al., 2020). Many of these tools/techniques have also been repeatedly sought-after for the discovery and characterization of several plant SM transporters. Still, their functional validation and substrate specificity determination remains immensely challenging. In the recent past, genome-wide studies alone or in combination with RNA-Seq analysis followed by qRT-PCR validation have been successfully implemented to predict/identify potential transporters (Wen et al., 2020; Sun et al., 2021). In addition, co-expression analysis using known biosynthesis pathway genes also assists in identifying co-regulatory transporters (Biala et al., 2017; Larsen et al., 2017b; Yan L. et al., 2021). Further, several web servers such as WoLF PSORT, Cell-PLoc 2.0, and TMHMM have been commonly used for the prediction of subcellular localization and transmembrane topology (Wen et al., 2020; Mackon et al., 2021). For the comprehensive functional characterization of transporters, experimental validations using virus-induced gene silencing (VIGS), RNA interference (RNAi), and the study of mutant libraries have been of great use (Larsen et al., 2017a; Tang et al., 2020). However, the recently emerged genome editing tools like transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindrome repeats (CRISPR) have efficiently modified the transporters of various crops including Zea mays (Zhang et al., 2017) and Oryza sativa (Zhou et al., 2015; Nieves-Cordones et al., 2017) but their applicability in the study of SM transporters still awaits further research. The expression of transporters in the heterologous host systems such as mutant yeast strains and Xenopus oocyte cells has also been extremely valuable to study the actual functioning in the intracellular environment (Tang et al., 2020). However, these do not necessarily act as the most efficient hosts (Jørgensen et al., 2017; Dastmalchi et al., 2019) because expression levels of the transporter might get affected due to codon usage bias (Wu et al., 2021). Therefore, the Agrobacterium-mediated transformation of Nicotiana benthamiana leaves (Wu et al., 2021) and N. tabacum Bright Yellow 2 (BY-2) cells (Hakkinen et al., 2018) has emerged as the most convenient, compatible, and quick tool which has been successfully used to study the expression of various plant transporters (Francisco et al., 2013; Khare et al., 2017; Demurtas et al., 2019). Interestingly, a recent study has reported that yeasts can be specifically engineered with required transport machinery and co-factors to regulate the biosynthesis and accumulation of medicinal compounds (Srinivasan and Smolke, 2021).

The present review provides the current perspective of a large number of SM transporters particularly discovered in the past two decades from various important plant species (Supplementary Table 1). Herein, the classification, structural, mechanistic, and functional aspects of four major transporter families namely ATP Binding Cassette (ABC), multidrug and toxic compound extrusion (MATE), purine uptake permease (PUP), and nitrate and peptide transporter family (NPF) have been discussed to better comprehend their role in the transport of specific plant SMs (Table 2; Figure 1).

The availability of a specific substrate in the required quantity is of the prime consideration for a transporter to be fully functional. However, the activity of transporters is not entirely dependent just on the substrate but also distinctly coordinated and regulated by several elements such as cellular and external environmental conditions, plant growth/developmental stages, nutrients, stress stimulus, tissue/organ specificity, phytohormones, elicitors, ionic gradients, etc. which are controlled at multiple levels (Figures 2, 3; Tang et al., 2020; Dhara and Raichaudhuri, 2021). Perhaps, the complexity of transport regulation also arises due to cross-talks with other metabolic pathways (Lynch et al., 2021) and multifold control points at the transcriptional, post-transcriptional, translational, and post-translational levels (Barbier-Brygoo et al., 2001).

In order to achieve the elevated availability of the substrate, the upregulation of biosynthetic pathway genes is one of the ways which can probably induce the respective transporters in a substrate-dependent manner. Thus, keen attention has been paid to perform co-expression studies between various transporters such as NtMATE1, NtMATE2 (Shoji et al., 2009), Nt-JAT1 (Morita et al., 2009), SmABCC1, SmABCG46, SmABCG40, SmABCG4 (Yan L. et al., 2021), and crucial genes from biosynthesis pathways of their substrates which have consistently shown positive co-regulatory mechanisms. In addition to altering the substrate biosynthesis pathway, the exogenous supply of substrate or substrate analogs also considerably affects the activity of transporters. For instance, the addition of sclareol to the suspension cultures of N. plumbaginifolia induces the expression of NpPDR1 transporter, which exports sclareol to the leaf surface and a comparable outcome was also obtained when suspension cells were treated with sclareolide, a structural analog of sclareol (Jasiński et al., 2001; Grec et al., 2003). Similarly, the exogenous treatment of medicarpin precursors namely 4-coumarate and liquiritigenin to the roots of M. truncatula seedlings significantly increases the mRNA accumulation of MtABCG10 transporter (Biala et al., 2017).

Additionally, the dependency of co-transport compounds such as glutathione has also been reported for the transport of several flavonoids including malvidin 3-O-glucoside and cyanidin 3-O-glucoside by VvABCC1 (Francisco et al., 2013) and AtABCC2 (Behrens et al., 2019) transporters, respectively. On the other hand, nutrients are also found to affect the functioning of various transporters, and ABCG37 of A. thaliana, which transports scopoletin is one such example that gets highly activated when the plant is grown under iron-deficient conditions. The Fe deficiency probably promotes the synthesis of phenolic compounds including scopoletin, which is possibly due to the upregulation of some of its biosynthetic enzymes from the phenylpropanoid pathway that eventually led to an increase in the mRNA transcript of the transporter (Fourcroy et al., 2014). Cellular pH also control the functioning of those transporters, which utilize proton gradient as the energy source such as NtMATE1/2 significantly get upregulated upon acidification of cytoplasm (Shoji et al., 2009). Further, expression of the SM transporters is mostly restricted to the specific plant tissues and/or developmental stages owing to their functional relevance. The AaPDR3 transporter of A. annua which specifically expresses in T-shaped trichomes of old leaves and roots represents tissue-specific expression (Fu et al., 2017). In addition, AaPDR3 expression was also found to be developmentally regulated with a concomitant increase with that of the leaf age. Whereas, the CrTPT2 transporter of C. roseus is negatively regulated with leaf age and therefore the expression is limited to younger leaves only (Yu and De Luca, 2013). Similarly, spatial expression pattern has also been observed for other plant SM transporters including NtNUP1 (Hildreth et al., 2011), BUP1 (Dastmalchi et al., 2019), and CjABCB1/2 (Shitan et al., 2003, 2013) which were reported to majorly express specifically in root tips, latex, and rhizomes, respectively.

The role of chemical elicitors such as JA, MeJ, and salicylic acid (SA) has been well established in the enhanced production of several SMs including alkaloids, polyphenols, and terpenoids (De Geyter et al., 2012; Ho et al., 2020) which presumably also impact the activity of their respective transporters. For instance, BY-2 cells of N. tabacum displayed upregulation of nicotine-specific Nt-JAT1 transporter upon elicitation with MeJ. cDNA-amplified fragment length polymorphism-based transcript profiling indicates that the elicitation primarily induces the production of various nicotine biosynthesis enzymes. Further investigation by qRT-PCR in BY-2 cells and expression studies in tobacco seedlings also support the transcript-based data of enhanced expression of crucial biosynthesis genes, which is in coherence with strong induction of Nt-JAT1 transporter. Thus, the study precisely demonstrates the co-regulation of Nt-JAT1 with nicotine biosynthesis genes (Morita et al., 2009). On the other hand, expression of AtABCG40 transporter from A. thaliana, which is assumed to play role in transporting sclareol due to its functional similarity with other sclareol transporters, was observed to be positively enhanced upon elicitor treatments. AtABCG40 displayed 260-fold induction at 12 h in response to SA while MeJ induces up to 40-fold after 6 h of treatment (Campbell et al., 2003). Similarly, fungal oligosaccharide when applied to the roots of M. truncatula could elevate the mRNA transcripts of MtABCG10, which plays an important role in defense response by modulating medicarpin biosynthesis in the roots of the plant (Banasiak et al., 2013). In addition, various plant growth regulators such as cytokinins, auxins, brassinosteroids, ethylene, and gibberellins are also reported to have a notable impact on the biosynthesis and accumulation of various nutrients or metabolites which in turn, might affect the functioning of corresponding transporters (Rawat et al., 2013; Jamwal et al., 2018). One such example is cytokinin-mediated substantial suppression of macronutrient (ammonium, nitrate, sulfate, and phosphate) transporters (Sakakibara et al., 2006). As reviewed by Jamwal et al. (2018) phytohormones are capable of significantly stimulating the SM production in the plant cell, for example, IBA, ethylene, kinetin, and GA₃ enhances the biosynthesis of terpenoids, phenolics, glucosinolates, and steroidal lactones, respectively (Jamwal et al., 2018). As a result, it can be proposed that an increased metabolite pool should probably activate their transporters but only limited reports are available which state phytohormone mediated regulation of plant SM transporters. For example, auxins have been shown to enhance the expression of strigolactone biosynthesis genes of P. hybrida, which is possibly affecting the transporter function too. The transcript levels of PhPDR1, a strigolactone transporter were found to be increased in response to 1-naphthaleneacetic acid (NAA) treatment (Kretzschmar et al., 2012). Whereas the significance of ethylene in regulating the activity of AtABCG40 transporter was observed when pathogen-induced expression of AtABCG40 transporter was significantly upregulated to 16-fold at 3 h after being treated with ethylene (Campbell et al., 2003). Moreover, the role of biotic stresses in transporter regulation is also evidently highlighted by significant upregulation of the glucosinolate transporters GTR1 and GTR2 of B. rapa in response to herbivory (Touw et al., 2020). Likewise, expression of NpPDR1 is also found to be induced following infection with fungal (Botrytis cinerea) and bacterial (Pseudomonas syringae) pathogens via JA signaling pathway (Stukkens et al., 2005). The Solanum tuberosum leaves when infected with Phytophthora infestans showed upregulation of the StPDR1/2/3/4 transporters, out of these, StPDR2 is proposed to be involved in the secretion of antifungal diterpene sclareol, which suggests the probable function of StPDRs in biotic stress responses. In addition, the cell suspension of S. tuberosum also showed upregulation of StPDR2 in response to the chemical treatment, which is certainly an indication of its role in abiotic stress tolerance (Ruocco et al., 2011). Similarly, abiotic factors such as metal stresses (Al, Mn, and Zn) are also reported to regulate the expression of CcMATE34, CcMATE45, and CcMATE4 transporters of Cajanus cajan in a tissue-specific manner (Dong et al., 2019).

The presence of cis-regulatory elements (CRE) which are responsive toward various regulatory factors such as hormones, environmental conditions, biotic and abiotic stresses can control the expression of their respective genes at transcriptional levels. The common CREs have been identified in the promoter region of various potential ABC transporter genes of Capsicum and Brassica species (Yan et al., 2017; Lopez-Ortiz et al., 2019). CaABCC and CaABCG transporters of Capsicum contain light, heat, low temperature, and drought-responsive CREs (CCAATBOX1 and LTRECOREATCOR15) (Lopez-Ortiz et al., 2019). While, BraABC transporters from Brassica are found to have CREs that are responsive to various phytohormones including ABA, SA, GA (WRKY71OS and GT1CONSENSUS), pathogens (WRKY71OS), and wounding (WBOXNTERF3) (Yan et al., 2017). Additionally, the credit for enhanced transcription of FaTT12-1 transporter of strawberry in response to red light irradiation has also been given to the occurrence of corresponding light-responsive CREs (Chen et al., 2018). Likewise, the promoter region of sclareol transporter NpPDR1 also possesses regulatory sequences, namely SB1 (sclareol box) (CACTAACACAAAGTAA) and SB3 (TTATGAACAGTAATTA) that are inducible in response to sclareolide, a close analog of sclareol. The mutation (nucleotide substitution) in the sequence of SB1 decreased the sclareolide mediated expression of NpPDR1 by twofold while mutated SB3 almost completely diminished the transporter activity due to promoter inactivation. Further, it has also been observed that NpPDR1 which is otherwise positively induced becomes unresponsive toward MeJ upon mutating SB3, which suggests the role of SB3 as the transcriptional regulator in MeJ mediated induction of NpPDR1 (Grec et al., 2003). Similarly, the role of transcription factors (TF) has also been well studied in context to the regulation of plant SMs. For example, the heterologous expression of A. thaliana MYB TF TT2 in the M. truncatula is reported to upregulate the MtMATE1 which transports proanthocyanidin precursor epicatechin 3′-O-glucoside (E3′G) into the vacuole and further massive accumulation of E3′G in hairy roots confirms TF induced uptake by MtMATE1 transporter (Zhao and Dixon, 2009). Similarly, the LAP1-MYB type TF when constitutively expressed in M. truncatula, led to significant upregulation of MtMATE2 transporter, which was primarily due to TF induced over production of anthocyanins (Zhao et al., 2011). Another transporter SlMTP77 was also found to be upregulated in transgenic tomato lines overexpressing ANT1-MYB TF. In vegetative tissues, ANT1 lines caused strong purple pigmentation, which is due to increased biosynthesis of anthocyanidin that enhanced the activity of SlMTP77 (Mathews et al., 2003). Additionally, a genome-wide study in S. lycopersicum has examined the regulatory gene networks, which showed the importance of TFs and chromatin regulators as the master regulators for transcriptional control. The study has also identified a strong positive association of MYB28 TF and SQUAMOSA BINDING PROTEIN TF (SlySBP15) with potential MATE transporters of tomato (Santos et al., 2017). Furthermore, the transcriptional profile of VvMATE1 transporter in seed berries of V. vinifera was found to be positively regulated with the increase in expression of TFs namely VvMYBPA1 and VvMYBPA2 (Perez-Diaz et al., 2014). Several other TFs such as AaERF1/AaERF2 of A. annua (Yu et al., 2012), TSAR1/TSAR2 of M. truncatula (Mertens et al., 2016), and ORCA2/ORCA3 of C. roseus (Sun and Peebles, 2016) have been reported to play a role in regulating biosynthesis and accumulation of SM flux (Liu et al., 2017).

Like any other plant protein, the activity of transporters is also anticipated to be optimized and controlled by various post-translational modifications (PTM) such as phosphorylation, ubiquitination, acetylation, methylation, and glycosylation, etc. The role of phosphorylation and ubiquitination have been studied in various plant transporter proteins including potassium/nitrate/ammonium transporters and inorganic phosphate transporter (PHT1;4/PT2), respectively (Tang et al., 2020). In contrast, the activity of the NtPDR1 transporter, which exports sclareol and capsidiol out of the cell is presumed to be affected by its glycosylation at Asn 738 site (Pierman et al., 2017). However, more research is required to experimentally validate the significance of PTMs in context to the regulation of SM transporters. Yet, it seems reasonable to expect a similar mechanism by PTMs for SM transporters as already reported for various other plant proteins belonging to the same transporter families. For instance, the regulatory mechanisms of the ABCB1 transporter of A. thaliana are unveiled by its comparative study with a functionally characterized mammalian ABCC7 transporter which ultimately deciphered a conserved mechanism of regulation i.e., protein phosphorylation at serine residues by PINOID kinase. The addition of phosphate group occurs at S634 residue on the linker regulatory domain, which joins the NBD folds of the ABCB1 and such chemical modification is reported to modulate the function of the transporter. Besides, its negative regulation has also been observed when phosphorylation occurs at a different non-linker location (Aryal et al., 2015). Another important report from an auxin transporter ABCB19 of A. thaliana, also demonstrates phosphorylation mediated negative regulation by PHOTOTROPIN1 kinase (Christie et al., 2011). Additionally, the chemical modification of SMs might also enhance their potential as a substrate which tends to have a profound effect on their uptake by respective transporters (Le Roy et al., 2016). Various plant SM transporters exclusively utilize modified substrates or prefer them with higher affinity. Specifically, the flavonoids undertake various chemical modifications including glycosylation, acylation, methylation, or hydroxylation, to get recognized by their transporters (Zhao and Dixon, 2010). As reported for the AtABCC2 transporter, the cyanidin 3-O-glucoside anthocyanins are the exclusive substrates over their aglycon variants (Behrens et al., 2019). Furthermore, AM1 and AM3 transporters from V. vinifera selectively utilize acylated anthocyanins as substrate (Gomez et al., 2009), whereas MtMATE2 prefers malonylated flavonoids over the non-malonylated form (Zhao et al., 2011).

A large number of plant SMs such as terpenoids, phenolics, alkaloids, tannins, and many others possess antioxidant potential (Wink, 2015; Rehman and Khan, 2017). While several other SMs also function as redox modulatory compounds such as organic isothiocyanates from the Brassicaceae family and polysulfanes from Allium genus (Jacob et al., 2011). As observed for primary metabolites such as sugars, the transport activity is well known to be regulated by the redox status of the cell (Griffiths et al., 2016). Therefore, it can be postulated that the altered redox status of a cell might also possibly have an impact on SM accumulation and transport. However, unlike primary metabolites, this hypothesis lacks scientific validation for redox-regulated transport of SMs.

Secondary metabolites are extremely diverse from a structural as well as functional standpoint hence modulation in their transport should have significant effects on a complex array of plant processes. Engineering SM transporters by modulating their expression/activity will pave the way for enhancing metabolite production, tissue/organ-specific accumulation, biotic and abiotic stress tolerance, reducing anti-nutritional compounds, identification of transporters with novel functions, and their characterization (Figure 4; Nour-Eldin and Halkier, 2013; Jorgensen et al., 2015; Lv et al., 2016). The fine-tuning of transporter expression needs to be executed in accordance with the regulatory factors so that overall plant growth and other basic functions linked to SMs are not hampered. Further, the development of an efficient system for transporter engineering also remains challenging due to limitations involved in the functional characterization and substrate specificity determination for most transporters (Larsen et al., 2017a). Although due to ligand specificity, few of the transporters might mimic enzymes if they recognize only a particular SM, yet in most cases, transporters can bind with multiple substrates. Therefore, modulating the expression of a specific transporter will also possibly disturb the unknown plant processes hence the selection of an appropriate transporter whose functional specificities have very well been determined is the key requisite. So far, limited reports are available for the implementation of transporter engineering by altering the accumulation and transport of important SMs. However, it is indeed a promising approach for enhanced production of desirable SMs that is plausibly attainable if the organ-specific sequestration inside the cells or export and accumulation into the culture medium can be regulated systematically to amend the control of metabolite flux (Kell et al., 2015). The below discussed examples highlight prospects of transporter engineering in context to various plant SMs.

A recent study in C. roseus demonstrated that a fivefold higher accumulation of catharanthine was achieved in hairy roots by overexpression of the CrTPT2 transporter, which was otherwise unattainable by conventional strategies (Wang et al., 2019). This directed the translocation of catharanthine metabolite in a regulated manner from its synthesis site hairy roots to the vacuole or intercellular space for storage. Therefore, the possibility of negative feedback inhibition was also prevented that might have happened due to excess accumulation at the synthesis site and eventually promoted enhanced yield of catharanthine in the sink tissue. In contrast, another transporter of C. roseus CrNPF2.9 upon downregulation showed significantly reduced transport of strictosidine from leaf vacuole to cytosol, which led to organ-specific accumulation of strictosidine only in the leaf vacuole (Payne et al., 2017). Further, modulation in transporter expression also has an impact on the response of plants toward various biotic stresses because it ultimately affects the accumulation of various SMs that are important components of the plant immune system (Isah, 2019). For example, the MtABCG10 transporter of M. truncatula transports precursors for the synthesis of medicarpin in the hairy roots of the plant (Biala et al., 2017). Medicarpin is the major phytoalexin of Medicago, which is known to participate in defense responses, particularly against fungal pathogens. The RNAi-mediated silencing of the MtABCG10 gene resulted in a compromised defense response against root infecting fungi Fusarium oxysporum (Banasiak et al., 2013), thus it can be postulated that knockdown of transporter expression caused reduced translocation of isoflavone medicarpin precursors hence disease resistance was found to be hampered. A similar response was also observed in N. plumbaginifolia where silencing of NpPDR1 transporter made the plants more sensitive toward various fungal and oomycete pathogens (Bultreys et al., 2009). On the other hand, Nb-ABCG1/2 transporters of N. benthamiana are essential in providing resistance against a pathogen P. infestans due to their involvement in the transport of defense compound capsidiol to the site of pathogen attack (Shibata et al., 2016). Thus, it can be expected that the engineering of SM transporters having a vital role in defense mechanisms holds great potential toward the production of disease-resistant crops. Similarly, many transporters are also known to play important role in abiotic stress tolerance such as drought resistance. The members of ABCG subfamily have been known to transport wax toward the surface of the plant, which eventually reduces the water loss due to its lipophilic properties (Shitan et al., 2013). Another commonly used drought tolerance mechanism by plants is the utilization of ABA phytohormone to attain stomatal closure under water deficit conditions. Thus, the contribution of transporters in regulating ABA influx/efflux is worth exploring and one such example is AtABCG25 exporter of A. thaliana, which participates in the intercellular distribution of ABA, whereas for the import of extracellular ABA, plasma membrane-localized AtABCG40 transporter is needed which is reported to act against drought stress (Kang et al., 2010; Dahuja et al., 2021). Studies also suggest that the biosynthesis and transport of SMs get affected under drought stress. Specifically, to fight against drought-induced oxidative stress, defense-related SMs such as phenolics and terpenes are needed to be increased in the cell (Kumar et al., 2021). Therefore, regulating the transport and accumulation of SMs linked with drought stress tolerance also seems to be achievable by transporter engineering approaches. Although much information is not available but some of the recent studies in cassava (Yan Y. et al., 2021) and grapevine (Tu et al., 2020) have proposed that enhanced lignin accumulation is also positively linked with drought resistance, which opens up the possibility of modulating the lignin transporters to increase its deposition in the cell wall. Altogether, drought tolerance using transporter modulation should have a positive impact on reducing crop losses in the agricultural sector.

The significance of transporter modulation in removal and/or reduction of the anti-nutritional metabolites from valuable plant parts has been reported in A. thaliana (Nour-Eldin et al., 2012) and Brassica species (Nour-Eldin et al., 2017). This strategy will surely increase the nutritional value of the crops by preventing/reducing the accumulation of toxic SMs in the edible plant parts. The expression of AtGTR1 and AtGTR2 transporters of A. thaliana was significantly reduced by generating their double mutants via DNA mutagenesis (Nour-Eldin et al., 2012), which led to the negligible accumulation of glucosinolates in seeds while their source tissues such as leaves and silique wall have shown more than 10-fold higher accumulation. Further, Nour-Eldin et al. (2017) have attempted to translate a similar strategy from A. thaliana to even more challenging and complex polyploid Brassica species, which are globally utilized as oilseed crops but suffer from low nutritional content due to the accumulation of glucosinolates in their seeds. Downregulation of GTR transporters from B. rapa (BrGTR2) and Brassica juncea (BjGTR2) was achieved by inserting early stop codons in their genes via mutagenesis and this facilitated a more than 60% reduction in the accumulation of seed glucosinolates (Nour-Eldin et al., 2017). The reduction of glucosinolates from edible seeds has also been attempted previously using transgenic approaches (Augustine et al., 2013) and interspecific crossing (Burton et al., 2003) only with limited success. Therefore, the remarkable outcomes of modulating Brassica GTR transporters have certainly validated the potential of transporter engineering in agriculturally important plants. These genetic manipulations are preferred for reducing glucosinolate contents over classical post-harvest processing of crops. From an agrobiotechnology perspective, post-harvest techniques are quite demanding in terms of cost and time, and on the other side, these might also affect protein digestibility and solubility in the meal. It has also been proposed that B. juncea with low levels of seed glucosinolates might also be suitable for arid regions (Burton et al., 2003). The identification of GTRs opens the avenue for many other crops where the distribution of anti-nutritional compounds is under the control of transport proteins.

Besides engineering the expression of the gene, modulation in the activity of the transporter protein by other means can also aid in the functional characterization and substrate specificity analysis. If the preliminary information of unknown transporters can be obtained by bioinformatic tools then their further identification can be performed either by providing exogenous substrates/analogs to upregulate or the chemical inhibitors to downregulate the activity of the transporters that will certainly facilitate functional validation. For instance, the expression studies of C. japonica CjABCB1 in the Xenopus oocyte system revealed that treatment with specific chemical agents has significantly inhibited the transporter activity (Shitan et al., 2003). At the same time, uptake of the proposed substrate berberine was also drastically diminished, which indicated that CjABCB1 is a berberine-specific transporter that enables its translocation into the rhizome. Similarly, the substrate specificity determination for NtNUP1 transporter of N. tabacum was conducted by expressing cDNA of NUP1 gene in S. pombe and the transformed cells were analyzed for the uptake of radiolabeled substrates. Further, the uptake assays demonstrate that ¹⁴C-nicotine accumulation was significantly high in the case of NUP1 expressing cells relative to control cells (expressing empty vector) (Hildreth et al., 2011). As previously discussed in sections “ATP Binding Cassette Transporters,” “Multidrug and Toxic Compound Extrusion Transporters,” “Purine Uptake Permease Transporters,” “Nitrate and Peptide Transporter Family Transporters,” several proteins have also been discovered for their transport abilities by studying the effects of their altered expression levels. One such example is AaPDR3 transporter whose overexpression has shown an increase in the β-caryophyllene content while downregulation led to its reduced accumulation, which has identified AaPDR3 as a transporter for β-caryophyllene (Fu et al., 2017).

Metabolic engineering of plants has been given a lot of importance in recent years due to its immense potentials. Various strategies have been deciphered to increase the yield of SMs and their accumulation. In the majority of earlier research efforts, the increase in metabolite yield was achieved through the modulation of the biosynthetic pathway genes. However, advancement of knowledge in the area opens possibilities of regulation of potential transporter genes for precise metabolic engineering. The transporters of plant SMs are the key players that have an impact not just at the site of metabolite synthesis and accumulation but systemically affect the plant metabolic system in one way or the other. In the recent past, several SM transporters have been identified in plants but their functional validation and substrate specificity determination is a huge challenge and equally critical for the metabolic engineering of plants. The present review emphasizes the knowledge gain in the area concerning SM transporters and their modulation. The transporter engineering, particularly for plant stress (biotic and abiotic) tolerance, SM accumulation, and removal of anti-nutritional compounds will be of great future potential. Further, the leverage of this technology can be extended to many medicinal, agricultural, aromatic, and other economically important plants for their optimum utilization and value addition. However, for efficient transporter engineering, more emphasis is required to identify the possible involvement of multiple transporters and/or their cross-talks for the long-distance mobilization of SMs. In addition, more and more transporters associated with secretion and storage should be annotated to get better insight into their functionality and possible applications. Similarly, the workings of the two-way transport system and the source-sink relationship linked to the distribution of SM pool need to be investigated in detail. Overall, it is realized that transport engineering is a dynamic and fairly complex process and emerges as a critical tool for systemic metabolic engineering in plants. However, it is also equally important to address the associated challenges using modern experimental tools and approaches to make it more effective and harness its fullest potential.

PN wrote the original draft. PP conceptualized the idea, supervised, and critically reviewed the manuscript. Both authors significantly contributed to finalizing the manuscript and approved it for submission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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