Comprehensive Overview of the Brassinosteroid Biosynthesis Pathways: Substrates, Products, Inhibitors, and Connections

Brassinosteroids (BRs) as a class of steroid plant hormones participate in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, flowering, and seed germination, as well as in responding to environmental stresses. During four decades of research, the BR biosynthetic pathways have been well studied with forward- and reverse genetics approaches. The free BRs contain 27, 28, and 29 carbons within their skeletal structure: (1): 5α-cholestane or 26-nor-24α-methyl-5α-cholestane for C27-BRs; (2) 24α-methyl-5α-cholestane, 24β-methyl-5α-cholestane or 24-methylene-5α-cholestane for C28-BRs; (3) 24α-ethyl-5α-cholestane, 24(Z)-ethylidene-5α-cholestane, 25-methyl-5α-campestane or 24-methylene-25-methyl-5α-cholestane for C29-BRs, as well as different kinds and orientations of oxygenated functions in A- and B-ring. These alkyl substituents are also common structural features of sterols. BRs are derived from sterols carrying the same side chain. The C27-BRs without substituent at C-24 are biosynthesized from cholesterol. The C28-BRs carrying either an α-methyl, β-methyl, or methylene group are derived from campesterol, 24-epicampesterol or 24-methylenecholesterol, respectively. The C29-BRs with an α-ethyl group are produced from sitosterol. Furthermore, the C29 BRs carrying methylene at C-24 and an additional methyl group at C-25 are derived from 24-methylene-25-methylcholesterol. Generally, BRs are biosynthesized via cycloartenol and cycloartanol dependent pathways. Till now, more than 17 compounds were characterized as inhibitors of the BR biosynthesis. For nine of the inhibitors (e.g., brassinazole and YCZ-18) a specific target reaction within the BR biosynthetic pathway has been identified. Therefore, the review highlights comprehensively recent advances in our understanding of the BR biosynthesis, sterol precursors, and dependencies between the C27-C28 and C28-C29 pathways.


INTRODUCTION
Brassinosteroids (BRs) represent the sixth class of plant hormones. Since the discovery of brassinolide (BL) in 1979, about 70 naturally occurring compounds from this group have been reported as free molecules or conjugates with glucose and fatty acids. BRs are structurally very similar to androgens, estrogens, corticoids, and ecdysteroids. Their presence was reported both in lower and higher plants, especially in angiosperms; and also in all plant organs, including roots, stems, leaves, flowers, anthers, pollen, seeds, and grain (Bajguz and Tretyn, 2003;Yokota et al., 2017;Bajguz, 2019;Zullo and Bajguz, 2019). BRs play an essential role in the development and growth of plants. They elicit a broad spectrum of morphological and physiological responses as well as a tolerance against abiotic and biotic stress (Bajguz and Hayat, 2009;Bajguz and Piotrowska-Niczyporuk, 2014;Wei and Li, 2016;Wendeborn et al., 2017;Ahanger et al., 2018;Siddiqui et al., 2018;Nolan et al., 2020).

CHEMICAL STRUCTURE OF BRs
Based on the total number of carbons, BRs are divided into C 27 , C 28 , and C 29 -type. The basic structure of C 27 -BRs is a 5acholestane skeleton, C 28 -BRs: 5a-ergostane, and C 29 -BRs: 5astigmastane ( Figure S1). Differences in the structure of these hormones are due to the type and orientation of oxygenated functions in the A-and B-ring, as well as the number and position of functional groups in the side chain of the molecule. These modifications arise during oxidation and reduction reactions. Based on the cholesterol (CR) side chain, BRs are divided by different substituents into C-23, C-24, C-25, 23-oxo, 24S-methyl, 24R-methyl, 24-methylene, 24S-ethyl, 24ethylidene, 24-methylene-25-methyl, 24-methyl-25-methyl; without substituent at C-23, without substituent at C-24, and without substituents at C-23, C-24. BRs can also conjugate with glucose and fatty acids (Fujioka and Yokota, 2003;Bajguz, 2007;Piotrowska and Bajguz, 2011;Wendeborn et al., 2017;Zullo and Bajguz, 2019).

BRs BIOSYNTHETIC PATHWAYS
Three pathways of the BR biosynthesis leading to the production of C 27 -, C 28 -, or C 29 -type of BRs are currently known (Figure 1; Figure S1). Early steps of their synthesis are common for each type and may occur via mevalonate (MVA) or non-MVA pathway, while later steps differentiate the BR biosynthesis pathways (cycloartenol-and cycloartanol-dependent). So far, most of the reactions, enzymes, and genes were discovered and characterized by the C 28 -BR biosynthesis pathway (mostly in Arabidopsis thaliana, from which the majority of genes in this pathway were isolated). Their biosynthesis includes two major stages: biosynthesis of campesterol and 22a-hydroxycampesterol. The direct substrate of C 27 -BRs viz. cholesterol (CR) is finally converted to 28-norBL, whereas the biosynthesis of C 29 -BRs is initiated from b-sitosterol and leads to 28-homoBL. However, not all indirect compounds of these two pathways have been identified (Figure 1; Figure S1) (Fujioka et al., 2002;Kwon and Choe, 2005;Fujita et al., 2006;Ohnishi et al., 2006b;Chung and Choe, 2013;Roh et al., 2017;Kim et al., 2018;Rozhon et al., 2019).

Early Steps of BRs Biosynthesis
Biosynthesis of isopentenyl pyrophosphate (IPP), which is an indirect compound in the CR synthesis pathway, can occur via two pathways: non-MVA in lower plants and MVA in the most of higher plants. The initial compounds in non-MVA pathway are D-glyceraldehyde 3-phosphate and pyruvate, which are transformed into the 1-deoxy-D-xylulose 5-phosphate (DOXP) by the DOXP synthase. Then, DOXP is converted to 2-C-methyl-D-erythritol 4-phosphate (MEP) by the DOXP reductoisomerase. The next step leads to the formation of 4-(cytidine 5'-diphospho)-2-C methyl-D-erythritol (CDP-ME) from MEP. The reaction is catalyzed by the CDP-ME synthase. Then, CDP-ME is converted to CDP-methyl-D-erythritol-2-phosphate by the CDP-ME kinase, and the obtained compound is transformed to 2-C-methyl-Derythitol-2,4-cyclodiphosphate by the ME cyclodiphosphate synthase. Finally, with the action of reductases and dehydratases, the IPP is synthesized (Figure 1; Figure S1) (Lichtenthaler, 2000). In the MVA pathway, three molecules of acetyl-CoA are combined to produce 3-hydroxy-3-methyloglutaryl-CoA by the HMG-CoA synthase. The obtained compound is reduced to MVA by the HMG-CoA reductase. IPP is synthesized from MVA through the two indirect phosphorylation intermediates, such as MVA-phosphate and MVA-pyrophosphate (MVA-PP) (Miziorko, 2011). Enzymes involved in these reactions are the MVA kinase, phospho-MVA kinase, and MVA-PP decarboxylase, respectively (Figure 1; Figure S1) (Wang et al., 2017).
Biochemical changes of IPP via geranyl pyrophosphate and farnesyl pyrophosphate lead to the synthesis of squalene, which is oxidized to squalene-2,3-oxide via squalene epoxidase and the latter is converted to cycloartenol by the cycloartenol synthase. Cycloartenol is a key compound for the BR biosynthesis because it constitutes a substrate for multistep reactions in few pathways, which lead to the synthesis of C 27 -, C 28 -, and C 29 -BRs. Conversion of cycloartenol via cycloartanol and in a series of subsequent reactions to cholesterol/cholestanol and/or 6oxocholestanol leads to the synthesis of C 27 -BRs (Figure 1; Figure S1) (Wang et al., 2017).
Cycloartenol may also be a substrate of the C-24 methylation reaction, which is catalyzed by the sterol C-24 methyltransferase (SMT1), and leads to 24-methylenecycloartanol. The next few reactions are catalyzed by C-4 sterol methyl oxidase (SMO1), cycloprophylsterol isomerase, obtusifoliol 14a-demethylase (CYP51), and sterol C-14 reductase, leading to the synthesis of 4a-methylergostatrienol. Indirect products of these reactions are cycloeucalenol and obtusifoliol. In next step, the sterol C-14 reductase which is encoded by the FACKEL/HYDRA2 gene catalyzes the reduction of 4a-methylergostatrienol to 4amethylergostadienol, which is converted in the subsequent reaction to 24-methylenelophenol by the sterol 8,7 isomerase   Schneider, 2002;Sonawane et al., 2016;Wang et al., 2017). 24-methylenelophenol is a substrate of two independent pathways of sterol biosynthesis. The first leads to the biosynthesis of isofucosterol/b-sitosterol that are precursors of the C 29 -BR biosynthesis (Xin et al., 2016); the second pathway, 24-converts methylenelophenol to campesterol, which is a substrate of the C 28 -BR biosynthesis (Figure 1; Figure S1) (Choe, 2006;Sonawane et al., 2016).

Biosynthesis of C 28 -BRs
24-methylenelophenol may also be converted to episterol, which is the first characteristic metabolite in the C 28 -BR biosynthesis.
The reaction is catalyzed by the C-4 sterol methyl oxidase 2 (SMO2) (Figure 1; Figure S1). Then, episterol is converted to 5dehydroepisterol by the sterol C-5(6) desaturase encoded by the DWF7 gene (also known as STE1), which is then converted to 24-methyleneCR (catalyzed by 7-dehydrocholesterol reductase encoded by the DWF5 gene) (Choe, 2006;Ohnishi, 2018). Further stages of the C 28 -BR biosynthesis may proceed through two parallel pathways, called the late and early C-22 oxidation pathway. Reduction of 24-methyleneCR to campesterol initiates the late C-22 oxidation pathway and is catalyzed by the C-24(25)-sterol reductase in a two-step reaction in which 24-methyl-desmosterol is an intermediate (Dockter et al., 2014). The enzyme (also known as sterol side-chain reductase 1), which catalyzes the production of campesterol, is encoded by the DWF1 gene. Campesterol is then transformed in the 5,4 isomerization reaction to (24R)-ergostan-4-en-3b-one. The latter is then converted through the DET2-mediated 5areduction to (24R)-5a-ergostan-3-one, which is transformed into campestanol (CN). In the parallel, early C-22 oxidation pathway, C-22a hydroxylation of 24-methyleneCR leads to the synthesis of 22-hydroxy-24-methyleneCR. The reaction of C-22a hydroxylation is catalyzed by the C-22a hydroxylase, which is encoded by the DWF4 gene. The enzyme belongs to the P450 cytochrome family (Fujiyama et al., 2019). The next reactions are analogous to the late C-22 oxidation pathway and result in the synthesis of 22-hydroxy forms of the corresponding compounds. However, an essential difference between the C-22 oxidation subpathways is the synthesis of 6-deoxocathasterone (6-deoxoCT) from (22S,24R)-22-hydroxy-5a-ergostan-3-one, without synthesis of campestanol (CN) (CN-independent pathway of BRs biosynthesis) as a result of the early C-22 oxidation. On the other hand, in each stage of the late C-22 oxidation pathway, the compound can be hydroxylated by the C-22a hydroxylase into hydroxygenated forms of early C-22 pathway (Choe, 2004;Ohnishi, 2018). Moreover, biochemical changes of 22-hydroxymethyleneCR can lead to the synthesis of 6deoxodolichosterone, which may be further converted into dolichosterone (DS), and dolicholide (DL), as well as to castasterone (CS) and BL (Roh et al., 2017).
Campestanol may be a substrate of the BR biosynthesis in a parallel manner, both in late C6 oxidation pathway (when hydroxylation of carbon atoms in the A-ring and both C-22 and C-23 positions of the side chain occurs before oxidation of C6) and early C-6 oxidation pathway (when hydroxylation takes place after oxidation of C6) (Figure 1; Figure S1) . In A. thaliana both the early and late C6 oxidation pathways are functional ; however, the late C6 oxidation pathway plays a prominent role during photomorphogenesis, whereas the parallel early C6 oxidation dominates during skotomorphogenesis . Generally, the late C6 pathway is more prominent in plants (e.g., in potato, it is the only type of the C 28 -BR biosynthesis). The late C6 pathway begins with hydroxylation of CN into the 6-deoxoCT by the 22ahydroxylase. 6-deoxoCT may also be synthesized directly from 22-hydroxy5a-ergostan-3-one (the CN-independent pathway). Then, 6-deoxoCT is hydroxylated through the C-23 hydroxylase (encoded by the CPD gene) to the 6-deoxoteasterone, which is then C-3 oxidized into the 3-dehydro-6-deoxoteasterone (6-deoxo-3-DT) through the CYP90D C3-oxidase. In the next step, 6-deoxo-3-DT is converted to 6-deoxoTY. This reaction is catalyzed by the D11 CYP724B1 enzyme. Then, 6-deoxoTY is hydroxylated to 6-deoxoCS by the 2a-hydroxylase encoded by the DDWF1 gene. 6-deoxoCS is converted to castasterone (CS) (BR-6-oxidase1 and BR-6-oxidase2 catalyze the reaction). Then, CS is converted to BL via Baeyer-Villiger oxidation by the BR-6-oxidase2 (CYP85A2) (Choe, 2004;Choe, 2006;Vriet et al., 2013;Nakano and Asami, 2014;Ohnishi, 2018).

Links Between C 27 -C 28 and C 28 -C 29 Pathways
The C 27 -BRs biosynthetic pathway links with the C 28 pathway through the following reactions: 28-norTE ! TE, 28-nor-3-DT ! 3-DT, 28-norTY ! TY, and 28-norCS ! CS. On the other hand, C 29 -BRs conversion to C 28 -BRs occurs through the following reactions: 28-homoTE ! TE, 28-homoTY ! TY, 28-homoCS ! CS, 28-homoDS ! CS, and 28-homoDS ! DS ! CS. Therefore, it is suggested that the biosynthetic connection of C 27 -and C 29 -BRs with C 28 -BRs occurs mainly between the end products of the pathways. Five pathways are biosynthetically connected to produce CS, an active BR, in plants (Figure 1; Figure S1). Direct substrates to synthesize CS are: 28-norCS, DS, 6-deoxoCS by 6a-hydroxyCS, 28-homoDS, and 28-homoCS. Thus, it is most conceivable that all the biosynthetic pathways of BRs in plants are funneled into CS to carry out the relevant biological activities. It is known that the early C-6 oxidation pathways of C 27 -, C 28 -, and C 29 -BRs are commonly interrupted in plant tissues (Fujita et al., 2006;Joo et al., 2012;Joo et al., 2015;Kim et al., 2018). A recent study of barley (Hordeum vulgare) BR mutants indicated that the accumulation of 28-homoCS is inversely correlated with the accumulation of CS: mutants deficient in the biosynthesis of CS accumulate the highest concentrations of 28-homoCS, on the other hand, the BRinsensitive line, in which the highest concentration of CS was observed, accumulates the lowest concentration of 28-homoCS (Gruszka et al., 2016).
The first reported BR inhibitor, i.e., KM-01 was isolated from a microbial culture medium. It inhibited BR activity in a rice lamina inclination test. Despite the unclear site of action, KM-01 exhibits highly potent activity (Kim et al., 1994;Kim et al., 1995;Kim et al., 1998). However, brassinazole (Brz) represents the first specific BR biosynthesis inhibitor, which blocks the conversion of campestanol to 6-deoxoCT, 6-deoxoCT to 6-deoxoTE, 6oxocampestanol to CT, and CT to TE in the BR biosynthetic pathways. Brz2001 is a modified form of Brz containing an allyl moiety instead of the methyl group. Both inhibitors block the  (Asami and Yoshida, 1999;Asami et al., 2000;Asami et al., 2001;Asami et al., 2003b). Brz and Brz2001 can induce morphological changes, including dwarfism, altered leaf color, and curling in de-etiolated barley (Sekimata et al., 2001). Brz decreased the level of BRs in the barley leaves, but not in roots. The inhibition effect of Brz on plant growth is reversed by exogenous BR (Bajguz and Asami, 2004;Bajguz and Asami, 2005;Bajguz et al., 2019). Propiconazole, a triazole compound, also affects similar to Brz (Hartwig et al., 2012). Another triazole-type BR biosynthesis inhibitors, YCZ-18, and yucaizol, bind to the CYP90D1 enzyme and inhibit the BR-induced cell elongation. However, only BL negates the inhibitory effect of YCZ-18 or yucaizol. Therefore, it was suggested that they function differently from Brz (Oh et al., 2015a;Oh et al., 2015b). Fenarimol is known for inhibiting cytochrome P450 monoxides involved in 14a-demethylation during the biosynthesis of ergosterol. Simultaneously, it inhibits the conversion of CT to TE, and evokes the phenotype of BR-deficient mutants with short hypocotyls, de-etiolate darkgrown seedlings, and dark green downward curled leaves of light-grown A. thaliana (Wang et al., 2001;Oh et al., 2015a). Plants treated with triadimefon show reduced elongation of stems and petioles, dark green and thicker leaves, delayed senescence, and increased expression levels of the CPD gene. The phenotypes could be recovered with CT, TE, TY, CS, and BL (Asami et al., 2003a). On the other hand, imazalil causes severe hypocotyl shortening in A. thaliana, which could be reversed by the application of 24-epibrassinolide (Werbrouck et al., 2003). Seedlings of A. thaliana treated with spironolactone showed dark, downward curled leaves, and shortened hypocotyls, which could be reversed by BL application . Although A. thaliana mutants viz. cpd, det2-1, or cbb1 treated with brassinopride enhanced apical hook formation, the normal phenotype was recovered by BL (Gendron et al., 2008). Voriconazole, fluconazole, and fenproprimorph ( Figure 2) inhibit cycloeucalenol-obtusifoliol isomerase and have a reductive impact on BRs level (Rozhon et al., 2013;Rozhon et al., 2019).

AUTHOR CONTRIBUTIONS
AB and MC prepared a draft of figures and text. AB and DG corrected and finalized the review.