Suberin biosynthesis is initiated with fatty acid synthesis. In plants, C16:0, C18:0 and C18:1 fatty acids are synthesized in plastids by the fatty acid synthase complex. These compounds enter the endoplasmic reticulum (ER) after the CoA group is added. C16:0-CoA, C18:0-CoA and C18:1-CoA form VLCFAs by fatty acid elongation complex (FAE) in the ER. The β-ketoacyl-CoA synthase (KCS) enzymes perform essential functions in this process, catalyzing the rate-limiting step and determining product length. KCS2/DAISY and KCS20 are essential enzymes for suberin polyaliphatic monomer biosynthesis in Arabidopsis thaliana (Lee et al., 2009; Franke et al., 2009). The atkcs2/daisy mutant displayed notable changes in suberin composition with decreased C22 and enriched C16, C18 and C20 derivatives. In addition, the atkcs20kcs2 double mutant exhibited abnormal endodermal suberin lamellae. Chemical analysis revealed significantly reduced C22 and C24 VLCFA levels, while C20 VLCFA derivatives accumulated excessively (Lee et al., 2009). Similar results have been reported in in potato (Solanum tuberosum). StKCS6 silencing in the tuber periderm reduced the levels of VLCFAs exceeding C28 in length (Serra et al., 2009a). All these indicated that the KCS family is closely related to the elongation of fatty acid which act as monomers of suberin lamella ( Figure 1 ).

Compared to unmodified fatty acids, ω-hydroxy acids and α, ω-diacids are crucial for suberin polyester extension (Serra and Geldner, 2022). Cytochrome P450 monooxygenases are essential enzymes in ω-hydroxylate fatty acid biosynthesis in plants. The atcyp86a1/horst mutants exhibited significantly reduced levels of ω-hydroxy acids in roots compared with the wild type (Hofer et al., 2008). Similarly, CYP86B1/RALPH has been identified as a critical oxidase for suberin monomer ω-hydroxy acid and α,ω-dicarboxylic acid biosynthesis in Arabidopsis, with atcyp86b1/ralph mutants demonstrating a substantial reduction in C22 and C24 ω-hydroxy acid and α,ω-dicarboxylic acid contents (Molina et al., 2009; Compagnon et al., 2009). In potato, CYP86A33 silencing led to a 60% decrease in aliphatic suberin, particularly affecting C18:1 ω- hydroxyacid (70%) and α,ω-dicarboxylic acids (90%) levels (Serra et al., 2009b). In addition to CYP86s, CYP94s have also been reported to be closely related to fatty acid ω-hydroxylation process. The atcyp94b1 mutant exhibited marked reduction in ω-hydroxy acid and α,ω-dicarboxylic acid levels. Conversely, heterologous AoCYP94B1 overexpression in Arabidopsis resulted in enhanced accumulation of C18 octadecanol C16 ω-hydroxy acids, and C16 α,ω-dicarboxylic acids compared with the wild type (Krishnamurthy et al., 2020). Similar outcomes were observed with AoCYP94B3 (Krishnamurthy et al., 2021).

Primary alcohols and glycerol constitute essential components of suberin polyaliphatic monomers. The fatty acyl reductases facilitate the reduction in α-carboxylic groups of VLCFA-CoA to generate suberin monomer primary alcohols. Arabidopsis mutants atfar1, atfar4, and atfar5 showed modified suberin composition in the root and seed coat, with decreased levels of C22, C20 and C18 primary alcohols, respectively (Domergue et al., 2010).

The glycerol-3-phosphate acyltransferase (GPAT) transfers the acyl-CoA to glycerol-3 phosphate. GPAT5 demonstrates broad substrate specificity with the CoA-derivatives of C16–C22 fatty acids, ω-hydroxy acids, and α,ω-dicarboxylic acids. It is an essential role in suberin synthesis is evidenced by the atgpat5 mutant, which showed a 50% reduction in aliphatic suberin in roots and significantly decreased the suberin-related dicarboxylic acid and ω-hydroxy acid contents of seed coats (Yang et al., 2012; Beisson et al., 2007).

Ferulic acid, synthesized by phenylpropanoid pathway, is the primary aromatic monomer of suberin (Graça, 2015). Its significance in suberin formation was demonstrated through phenylpropanoid pathway inhibition studies, which showed blocked suberin deposition that can be restored by exogenous ferulic acid application (Andersen et al., 2021). Aliphatic suberin feruloyl transferase (ASFT) mediates the transfer of feruloyl-CoA acyl groups to ω-hydroxy acids and primary alcohols, a crucial step in suberin monomer biosynthesis. Arabidopsis atasft mutants demonstrated near-complete absence of ferulic acid in their polyphenolic domain affecting aliphatic suberin monomer levels (Molina et al., 2009; Gou et al., 2009). Furthermore, ENHANCED SUBERIN1 (ESB1) is involved in suberin synthesis, as evidenced by the Arabidopsis mutant atesb1 showing significantly increased root suberin content (Baxter et al., 2009).

While numerous enzymes involved in suberin monomer synthesis or modification are located in the ER lumen or at the cytosolic face of the ER, suberin lamellae formation typically occurs on the plasma membrane surface or cell wall. The transport of suberin monomers from the ER to the plasma membrane and their transmembrane transport are essential steps in suberin lamellae formation (Serra and Geldner, 2022). The mechanisms underlying these processes remain incompletely understood, and several hypotheses exist regarding them. Lipid-like suberin monomers synthesized at the ER may transfer directly through the ER-plasma membrane contact site. Vesicle trafficking by the Golgi and trans-Golgi networks represents another potential mechanism, given its established role in cuticular wax delivery (Mcfarlane et al., 2014). Additionally, earlier studies documented plasma membrane invaginations containing extracellular tubules or vesicles. These structures were initially proposed as intermediates in lipid-like substance transport, including suberin monomers (Scott and Peterson, 1979; Peterson and Ma, 2001). This concept remained dormant until recent research identified similar structures called extracellular vesicular-tubular (EVs), which demonstrate a strong correlation with suberin lamellae formation. The accumulation of large numbers of EVs in suberizing cells were observed through electron-microscopy (De Bellis et al., 2022).

After suberin monomers reach the plasma membrane, they are transported across the membrane to the apoplast. Several ABC transporters of the G-clade (ABCG) have been implicated in this process. The Arabidopsis DSO/ABCG11 transporter influences cutin metabolism in reproductive organs and suberin content of roots (Panikashvili et al., 2010). In addition, three Arabidopsis transporters (ABCG2, ABCG6, and ABCG20) participate in suberin metabolism in roots and seed coats (Yadav et al., 2014; Fedi et al., 2017). The triple mutant of these genes exhibited changes in suberin lamellae structure, composition, and properties in the root and seed coat (Yadav et al., 2014). Further research confirmed AtABCG1’s role in suberin transport, as the atabcg1 mutant showed decreased root suberin content, particularly in VLCFAs, primary alcohols, and dicarboxylic acids (Shanmugarajah et al., 2019). Furthermore, homologs of these genes, such as StABCG1 and OsABCG5/RCN1, facilitate suberin precursor transport (Landgraf et al., 2014; Shiono et al., 2014). Beyond ABCG transporters, the LIPID TRANSFER PROTEIN (LTP) superfamily contributes to cuticular wax deposition and pollen wall formation (Chen et al., 2022; Gao et al., 2021; Edqvist et al., 2018). Evidence indicates that LTPs participate in suberin monomers transport. AtLTPI-4 contributes to suberin formation in Arabidopsis crown galls, as the atltpi4 mutant showed significantly reduced suberin accumulation. Protein expression in epidermal cells increased C24 and C26 VLCFA levels (Deeken et al., 2016). In addition, Arabidopsis LTPG15, expressed in the root endodermis and seed coat, facilitates suberin monomer transport (Lee and Suh, 2018). Collectively, both ABCG transporters and LTPs may transport suberin monomers across the membrane to the apoplast where suberin lamellae form.

Suberin deposition exhibits cell and tissue specificity and responds to various environmental stresses, reflecting strict transcriptional regulation of suberin biosynthesis-related genes. The MYB family regulates suberin biosynthesis and deposition across species and tissues (Cesarino, 2022). AtMYB41 activates aliphatic suberin synthesis and deposition. AtMYB41 overexpression enhanced the transcription of suberin biosynthesis genes in leaves and induced suberin deposition on leaf cell walls, forming suberin-lamellae-like structures (Kosma et al., 2014). Subsequently, additional MYB transcription factors involved in suberin biosynthesis regulation were identified and functionally validated. MdMYB93 demonstrated a significant role in regulating suberin deposition in russeted apple (Malus domestica) skins (Legay et al., 2016). MYB9 and MYB107 regulate suberin deposition in seed coats and fruit skins (Lashbrooke et al., 2016). Shukla et al. identified four MYB transcription factors (MYB41, MYB53, MYB92, and MYB93) that individually respond to developmental and exogenous signals and promote endodermal suberin formation (Shukla et al., 2021). In potato wound-healing tissues, StMYB102 and StMYB74 function as regulators of wound suberin biosynthesis and deposition (Wahrenburg et al., 2021).

The NAC and WRKY families serve as transcriptional regulators of suberin biosynthesis. ANAC046 expression occurs primarily in the root endodermis and periderm, with wound-induced expression in leaves. ANAC046 overexpression enhanced the expression of suberin biosynthesis genes in the roots and leaves, increasing root wax and suberin accumulation. This indicated that ANAC046 functions as a key transcription factor promoting suberin biosynthesis in Arabidopsis roots (Mahmood et al., 2019). StNAC103 and StNAC101 act as suberin biosynthesis repressors in potato, evidenced by increased suberin and wax in RNAi-mediated mutants (Soler et al., 2020). WRKY33 functions as an upstream regulator of CYP94B1 in Arabidopsis, with atwrky33 mutants showing reduced suberin and salt-sensitive phenotypes (Krishnamurthy et al., 2020). Krishnamurthy et al. identified AtWRKY9 as a suberin biosynthesis regulator through its control of AtCYP94B31 and AtCYP86B1 expression in Arabidopsis (Krishnamurthy et al., 2021).

Various hormones, including abscisic acid (ABA) (Shiono et al., 2022; Shiono and Matsuura, 2024), ethylene (Liu et al., 2021), auxin (Ursache et al., 2021), and gibberellin (Binenbaum et al., 2023) participate in regulating suberin biosynthesis.

Suberin lamellae serve as a natural barrier that restricts Na⁺ transport from roots to shoots through the bypass flow. Its thickness and location directly influence the effectiveness of salt exclusion in plants. Three rice (Oryza sativa) varieties cultivated under varying salt concentrations demonstrated increased root suberization and upregulation of suberin synthesis genes in response to salt stress. The formation of both the endodermal and exodermal suberin lamellae advanced toward the root tip, indicating accelerated root suberization under salt stress. The variety exhibiting the highest degree of root suberization displayed the lowest Na⁺ accumulation in shoots (Krishnamurthy et al., 2009, 2011). Similar observations were documented in Avicennia officinalis, where salt treatment increased root suberin lamellae thickness and significantly reduced Na⁺ transport to aboveground parts by the xylem (Krishnamurthy et al., 2014). Subsequent research using corn (Zea mays), olive (Olea europaea), and grape (Vitis vinifera) also showed salt stress induced thickening of the suberin lamellae (Rossi et al., 2015; Shen et al., 2015; Wang et al., 2023). These findings demonstrate that salt stress promotes suberin deposition and thickening, effectively inhibiting Na⁺ absorption and transportation, thereby establishing an inverse relationship between Na⁺ permeability and root suberization.

In addition to the change of the structure, suberin composition undergoes changes under salt stress. NaCl treatment induced a 22% increase in total suberin content in Arabidopsis after 100 mM NaCl exposure, with significant increases in dicarboxylic fatty acids and modest increases in 18:0 ferulate and 20:0 and 22:0 coumarates (De Silva et al., 2021). In Chenopodium album, salt stress significantly increased saturated and unsaturated VLCFAs with chain lengths of C20–C26 (Ivanova et al., 2016). Moreover, increasing saturation and length of fatty acids represents an adaptation strategy against salt stress in halophytes (Sui et al., 2018). These findings indicate that salt stress enhances VLCFA components in plant suberin, potentially strengthening the hydrophobic barrier, limiting Na⁺ flux, and maintaining membrane stability.

The structural changes in suberin lamellae structure under salt stress indicate the regulation of suberin synthesis and transport-related gene expression. Transcriptome analyses reveal the salt stress-induced transcription of biosynthesis genes (Wei et al., 2020; Yang et al., 2018). In rice, an increased transcript levels of suberin biosynthesis gene was detectable as early as 30 minutes after NaCl treatment (Krishnamurthy et al., 2009). Analysis of tissue-specific differential induction revealed that the expression of suberin-related genes in roots correlates most strongly with salt stress. In quinoa (Chenopodium quinoa), CqGPAT5a and CqGPAT5b demonstrated high expression in roots with rapid induction under high salt stress (Wang et al., 2025b). Similarly, VvKCS11 exhibits root-specific high expression and strong salt stress induction in grape (Yang et al., 2020). Salt treatment increases the expression of CYP94B3 and CYP86B1, key suberin precursor synthesis genes, in the roots of both Arabidopsis and medicinal plants (Krishnamurthy et al., 2021).

Studies using suberin-related gene mutants further demonstrate suberin’s significance in plant salt tolerance. In wheat (Triticum aestivum), TaGPAT6 overexpression enhanced suberin deposition in the seed coat and root tip outer layers, improving salt tolerance through reduced Na⁺ accumulation. Conversely, tagpat6 mutants exhibited decreased suberin deposition, enabling Na⁺ accumulation and resulting in salt sensitivity (Wang et al., 2025a). The Arabidopsis mutant cyp86a1 displayed salt sensitivity with increased Na⁺ and decreased K⁺ accumulation (Wang et al., 2020). Furthermore atmyb107 and atmyb9 mutants showed a significantly reduced seed suberin monomer content, leading to decreased germination rates under salt stress (Lashbrooke et al., 2016). Similarly, ABCG23 mutation reduced C24 ω-hydroxy fatty acids and 1, ω-dicarboxylic acids in the mutant seed coats, diminishing germination rates under salt stress (Kim et al., 2025). These mutant studies demonstrate that altered suberin composition or lamellae structure affects the ion and water absorption, often resulting in salt-sensitive phenotypes (De Silva et al., 2021).