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. 2021 Jan 13;185(3):902–913. doi: 10.1093/plphys/kiaa113

Identification and characterization of sorgomol synthase in sorghum strigolactone biosynthesis

Takatoshi Wakabayashi 1, Shunsuke Ishiwa 1, Kasumi Shida 1, Noriko Motonami 1, Hideyuki Suzuki 2, Hirosato Takikawa 3, Masaharu Mizutani 1, Yukihiro Sugimoto 1,✉,2
PMCID: PMC8133691  PMID: 33793911

Abstract

Strigolactones (SLs), first identified as germination stimulants for root parasitic weeds, act as endogenous phytohormones regulating shoot branching and as root-derived signal molecules mediating symbiotic communications in the rhizosphere. Canonical SLs typically have an ABCD ring system and can be classified into orobanchol- and strigol-type based on the C-ring stereochemistry. Their simplest structures are 4-deoxyorobanchol (4DO) and 5-deoxystrigol (5DS), respectively. Diverse canonical SLs are chemically modified with one or more hydroxy or acetoxy groups introduced into the A- and/or B-ring of these simplest structures, but the biochemical mechanisms behind this structural diversity remain largely unexplored. Sorgomol in sorghum (Sorghum bicolor [L.] Moench) is a strigol-type SL with a hydroxy group at C-9 of 5DS. In this study, we characterized sorgomol synthase. Microsomal fractions prepared from a high-sorgomol-producing cultivar of sorghum, Sudax, were shown to convert 5DS to sorgomol. A comparative transcriptome analysis identified SbCYP728B subfamily as candidate genes encoding sorgomol synthase. Recombinant SbCYP728B35 catalyzed the conversion of 5DS to sorgomol in vitro. Substrate specificity revealed that the C-8bS configuration in the C-ring of 5DS stereoisomers was essential for this reaction. The overexpression of SbCYP728B35 in Lotus japonicus hairy roots, which produce 5DS as an endogenous SL, also resulted in the conversion of 5DS to sorgomol. Furthermore, SbCYP728B35 expression was not detected in nonsorgomol-producing cultivar, Abu70, suggesting that this gene is responsible for sorgomol production in sorghum. Identification of the mechanism modifying parental 5DS of strigol-type SLs provides insights on how plants biosynthesize diverse SLs.

A cytochrome P450 monooxygenase catalyzes the production of sorgomol in sorghum and drives structural diversity of strigolactones.

Introduction

Strigolactones (SLs) are carotenoid-derived plant metabolites that function as signaling molecules, both endogenously as phytohormones and exogenously in the rhizosphere. SLs regulate various aspects of plant architecture and development, including the inhibition of shoot branching (Gomez-Roldan et al., 2008; Umehara et al., 2008; Zwanenburg and Blanco-Ania, 2018). These compounds also promote the formation of mutually-beneficial symbioses between plants and arbuscular mycorrhizal fungi, which provide phosphate to the plants (Akiyama et al., 2005). The seeds of root parasitic weeds belonging to the Orobanchaceae family, such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.), ensure their germination close to a suitable host using SLs as a germination cue (Cook et al., 1966; Samejima et al., 2016).

The SLs are classified into two types, canonical and noncanonical. The core structure of canonical SLs consists of a tricycle lactone ring (ABC ring) system linked to a methyl butenolide (D-ring) with C-2′R configuration via an enol–ether bridge (Figure 1). On the contrary, noncanonical SLs such as heliolactone (Ueno et al., 2014), avenaol (Kim et al., 2014), zealactone (Charnikhova et al., 2017; Xie et al., 2017), and lotuslactone (Xie et al., 2019) possess an unclosed BC ring. Canonical SLs are divided into the orobanchol- and strigol-type, based on the stereochemistry of the BC-junction. Of these, 4-deoxyorobanchol (4DO) and 5-deoxystrigol (5DS) have the simplest orobanchol- and strigol-type SL structures, respectively (Figure 1). They also share the same planar structure but the former and the latter display C-8bR and C-8bS stereochemistry in the C-ring, respectively. The structural diversity of canonical SLs arises from a chemical modification of the A- and B-rings, mostly consisting of hydroxy and/or acetoxy substituent(s).

Figure 1.

Figure 1

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Proposed pathway for SL biosynthesis. Enzymes catalyzing the reaction shown with bold arrows were identified in this study, whereas those indicated with thin solid arrows were reported in previous studies. Black dashed arrow indicates the putative pathway catalyzed by an unknown enzyme in sorghum. Ga, Gossypium arboreum; Os, Oryza sativa; Sb, Sorghum bicolor; Sl, Solanum lycopersicum; Vu, Vigna unguiculata.

The biosynthesis of carlactone (CL), an intermediate compound in SL biosynthesis, involves the isomerization of all-trans-β-carotene into 9-cis-β-carotene by DWARF 27 (D27), followed by the sequential oxidative cleavage by carotenoid cleavage dioxygenase 7 (CCD7) and CCD8 (Alder et al., 2012). Downstream of these enzymes, Arabidopsis (Arabidopsis thaliana) MORE AXILLARY GROWTH 1 (MAX1), which encodes cytochrome P450 (CYP) CYP711A1, oxidizes CL at C19 to produce carlactonoic acid (CLA; Abe et al., 2014). This conversion has been demonstrated in CYP711As from many plant species and in both canonical and noncanonical SL biosynthesis (Iseki et al., 2018; Yoneyama et al., 2018; Zhang et al., 2018; Mori et al., 2020b). Several candidate biosynthetic pathways for monohydroxylated canonical SLs have been proposed by feeding experiments with SL biosynthetic precursors (Iseki et al., 2018; Ueno et al., 2018; Zhang et al., 2018); the genes involved in the modulation of these pathways have been identified to some extent (Figure 1). For the biosynthesis of orobanchol-type SLs, it was demonstrated that rice (Oryza sativa) OsCYP711A2/Os900 catalyzes the conversion of CL to 4DO via CLA and that OsCYP711A3/Os1400 introduces a hydroxy group at C-4 of 4DO to produce orobanchol (Zhang et al., 2014). By contrast, we also identified an alternative orobanchol biosynthesis pathway catalyzed by CYP722C, a subfamily found specifically in dicots (Wakabayashi et al., 2019). VuCYP722C in cowpea (Vigna unguiculata) and SlCYP722C in tomato (Solanum lycopersicum) have been found to catalyze the direct conversion of CLA to orobanchol (Wakabayashi et al., 2019). On the contrary, for strigol-type SLs, GaCYP722C from cotton (Gossypium arboreum) catalyzes the in vitro conversion of CLA to 5DS (Wakabayashi et al., 2020) and CYP722C in Lotus japonicus is involved in the synthesis of 5DS (Mori et al., 2020a). However, the enzyme catalyzing the conversion of 5DS to monohydroxylated SLs has yet to be identified.

Sorghum (Sorghum bicolor [L.] Moench), a staple crop in Africa, is a representative plant producing strigol-type SLs. It was reported that a mutation at LOW GERMINATION STIMULANT 1 (LGS1) in sorghum altered the SL profile secreted from roots: the major SL in lgs1 mutants is orobanchol, while that in wild type (WT) is 5DS (Gobena et al., 2017; Bellis et al., 2020). LGS1 encodes a putative sulfotransferase and is likely to be involved in the generation of 5DS, but its biochemical function remains unclear. We previously found that sorghum produces different strigol-type SLs depending on the cultivars. We identified Sudax as a high-sorgomol-producing cultivar and Abu70 as a cultivar producing 5DS but not sorgomol, previously designated as NM16 and NM01, respectively (Motonami et al., 2013). We also demonstrated that Sudax converted exogenously-administered 5DS to sorgomol and that this conversion was inhibited by the addition of a CYP inhibitor uniconazole-P, suggesting that CYP catalyzes the introduction of a hydroxy group at C-9 of 5DS (Motonami et al., 2013). However, the gene responsible for this reaction has not been identified.

In this study, we identified the enzyme sorgomol synthase, which catalyzes the hydroxylation of 5DS to form sorgomol. Microsomal fractions containing membrane-bound CYPs prepared from Sudax roots catalyzed the conversion of 5DS to sorgomol; this conversion was inhibited by uniconazole-P, suggesting the influence of CYP in this conversion. We conducted transcriptome analysis of root tissue from Sudax grown in phosphate-rich (Pi-rich) and -deficient (Pi-deficient) conditions, which are known to significantly influence SL production (Yoneyama et al., 2007), and selected a candidate CYP, SbCYP728B35. We confirmed the in vitro conversion of 5DS to sorgomol using recombinant SbCYP728B35 expressed in Escherichia coli. The stereospecificity of substrates was analyzed using 5DS stereoisomers. We also constructed hairy roots of L. japonicus, which is a high 5DS-producing plant, overexpressing SbCYP728B35 to confirm the function of sorgomol production in planta. Finally, we demonstrated the importance of the SbCYP728B35 gene for the production of sorgomol by analyzing the gene expression in a nonsorgomol-producing cultivar, Abu70.

Results

Sorgomol synthase is a member of CYP but not MAX1 homologs

Previous feeding experiments suggested that a CYP enzyme was involved in the conversion of 5DS to sorgomol (Motonami et al., 2013). Based on this knowledge, the microsomal fraction containing membrane-bound CYPs was prepared from Sudax roots grown under Pi-deficient conditions, which induce SL production (Yoneyama et al., 2007; Motonami et al., 2013), followed by in vitro enzyme assay using 5DS as a substrate. As a result, sorgomol was detected in the reaction mixture of the microsomal fraction with 5DS, and the conversion of 5DS to sorgomol was inhibited by adding uniconazole-P at a concentration of 100 μM (Supplemental Figure S1). These results confirm the role of CYP-dependent monooxygenation in sorgomol biosynthesis from 5DS.

Based on the report of orobanchol biosynthesis catalyzed by rice CYP711As (Zhang et al., 2014), we started by examining the enzymatic function of sorghum CYP711As. We identified four SbCYP711A genes, namely SbCYP711A13 (Sobic.010G170400), SbCYP711A18 (Sobic.003G269600), SbCYP711A19 (Sobic.004G095500), and SbCYP711A31 (Sobic.003G269500), using the amino acid homology search in the Phytozome v12.1 database (S. bicolor v3.1.1; https://phytozome.jgi.doe.gov/pz/portal.html; Nelson, 2009; McCormick et al., 2018). In the roots of Sudax, the expression of all CYP711A genes, as well as SbD27 (Sobic.005G168200), SbCCD7 (Sobic.006G170300), and SbCCD8 (Sobic.003G293600; Mohemed et al., 2018) were upregulated under Pi-deficient conditions (Figure 2A) with a concomitant increase in the production of 5DS and sorgomol (Supplemental Figure S2), as shown by known expression patterns of genes involved in SL biosynthesis (Wakabayashi et al., 2019). To characterize the enzymatic function of these SbCYP711As, recombinant proteins were expressed in E. coli strain DH5α and subjected to in vitro enzyme analysis using either CL, CLA, or 5DS as a substrate. Among the candidate proteins, only recombinant SbCYP711A18 successfully catalyzed the conversion of CL to CLA (Supplemental Figure S3). However, none of the recombinant proteins converted CLA to 5DS or 5DS to hydroxylated 5DS, suggesting that SbCYP711A18 has a conserved CYP711A function in converting CL to CLA, as reported in other plants (Yoneyama et al., 2018). Other enzymes may be involved in the conversion of CLA to 5DS, and CYPs other than SbCYP711As may catalyze hydroxylation of 5DS.

Figure 2.

Figure 2

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The role of SbCYP728B35 in sorgomol synthesis. A, The relative expression of genes involved in SL biosynthesis in Sudax roots grown under Pi-rich (+P) and Pi-deficient (–P) conditions. Error bars represent means ± SE (n = 3 biologically independent plants). Asterisks indicate a significant difference between +P and –P conditions (*P < 0.05 by the Student’s t test). B, In vitro enzyme assay of recombinant SbCYP728B1 and SbCYP728B35. Sorgomol was detected in a reaction mixture of recombinant SbCYP728B1 or SbCYP728B35 with 5DS as a substrate by LC-MS/MS analysis. Multiple reaction monitoring (MRM) transition at m/z 364.1 > 317.1 in positive ionization mode was applied for sorgomol detection. LC Method I was used to separate SLs. C, Sorgomol synthase activities of recombinant SbCYP728B1 and SbCYP728B35. The activity was calculated as the amount of sorgomol produced per minute per nmol recombinant CYP protein. Error bars represent means ± SE (n = 3). Asterisk indicates a significant difference between the activity of SbCYP728B1 and SbCYP728B35 (*P < 0.05 by the Student’s t test).

Sorghum CYP728B35 effectively converts 5DS to sorgomol in vitro

To screen CYP genes involved in the synthesis of sorgomol from 5DS, we performed a comparative transcriptome analysis by RNA-sequencing using Sudax roots grown under Pi-rich and Pi-deficient conditions. Among the genes annotated as CYPs in the sorghum genome database (McCormick et al., 2018), 67 genes displayed a 1.5-fold increase in fragments per kilobase of transcript per million mapped reads (FPKM) values in response to Pi-deficiency and were ranked in descending order according to the degree of expression changes, starting with SbCYP728B1 (Sobic.002G336100), the most upregulated gene (Supplemental Data Set S1). In a previous study, the unknown gene VuCYP728B (Vigun08g197300) in cowpea was coexpressed with SL biosynthesis genes (Wakabayashi et al., 2019), suggesting that SbCYP728B1 and members of its subfamily are likely to be involved in SL biosynthesis. Three genes, SbCYP728B1, SbCYP728B31 (Sobic.002G320200), and SbCYP728B35 (Sobic.008G122800), were recorded in the database as belonging to the CYP728B subfamily. The expression patterns of these genes were re-evaluated by reverse transcription-quantitative PCR (RT-qPCR). The expression of SbCYP728B1 and SbCYP728B35 was significantly upregulated under Pi-deficient conditions; however, that of SbCYP728B31 was below the detection limit (Figure 2A). Therefore, SbCYP728B1 and SbCYP728B35 were selected for further functional analysis.

Recombinant SbCYP728B1 and SbCYP728B35 proteins were subjected to in vitro enzyme assays using 5DS as a substrate. LC-MS/MS analysis detected sorgomol in both reaction mixtures, with the recombinant SbCYP728B35 protein showing a greater conversion rate of 5DS to sorgomol with 770-fold greater sorgomol synthase activity than that of SbCYP728B1 (Figure 2B and C). Furthermore, a kinetic analysis of recombinant SbCYP728B35 activity as sorgomol synthase revealed that its catalysis of 5DS hydroxylation at C-9 was carried out with high affinity and moderate efficiency (Km = 0.63 μM and kcat = 0.70 min−1; Supplemental Figure S4 and Supplemental Table S1). The low Km value of recombinant CYP728B35 for 5DS indicates a high affinity of the enzyme to the substrate, comparable to that of orobanchol synthase (OsCYP711A3/Os1400) to 4DO, the Km value of which was reported to be 0.74 μM (Zhang et al., 2014).

Substrate specificity of SbCYP728B35

Sorgomol synthase activity of recombinant SbCYP728B1 and SbCYP728B35 was examined toward all 5DS stereoisomers (5DS, ent-5DS, 2ʹ-epi-5DS, and ent-2ʹ-epi-5DS) as substrates (Figure 3A). The conversion rates of 5DS stereoisomers to the corresponding sorgomol isomers were evaluated. Recombinant SbCYP728B35 showed similar activity toward 5DS and 2ʹ-epi-5DS, while ent-5DS and ent-2ʹ-epi-5DS were poor substrates with very limited conversion rates (Figure 3B). The substrate specificity of recombinant SbCYP728B1 was different from that of SbCYP728B35: recombinant SbCYP728B1 showed the greatest activity toward 2ʹ-epi-5DS, with an activity 45-fold greater than that toward 5DS. The activity toward ent-2ʹ-epi-5DS was the second greatest, while ent-5DS was not a preferred substrate, as for SbCYP728B35 (Supplemental Figure S5).

Figure 3.

Figure 3

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Substrate specificity of SbCYP728B35 toward 5DS stereoisomers. A, Structural composition of different 5DS stereoisomers. B, Substrate specificity evaluated based on the conversion efficiency of 5DS stereoisomers to corresponding sorgomol isomers. Relative activity toward each substrate was normalized to activity against 5DS. Error bars represent means ± SE (n = 3). Different letters indicate significant differences (P < 0.05 by the Tukey–Kramer test). C, Conversion of 5DS stereoisomers in Sudax plants. Each 5DS stereoisomer and fluridone (FL) at a concentration of 40 nM and 1 μM, respectively, were exogenously administered simultaneously to hydroponic culture media of Sudax. Sorgomol stereoisomers were detected by LC–MS/MS analysis with MRM transition at m/z 364.1 > 317.1 in positive ionization mode. LC Method II was used to separate SLs.

We also examined the conversion of each 5DS stereoisomer in sorghum plants. The 5DS and 2ʹ-epi-5DS administered to a hydroponic culture of Sudax were metabolized to sorgomol and 2ʹ-epi-sorgomol, respectively. By contrast, ent-5DS was barely metabolized to ent-sorgomol, and the conversion of ent-2ʹ-epi-5DS to ent-2ʹ-epi-sorgomol was not observed (Figure 3C). These results of substrate recognition and metabolism in sorghum plants were consistent with the results of recombinant SbCYP728B35 substrate specificity from our in vitro assays.

In planta conversion of 5DS to sorgomol by SbCYP728B35

Considering that recombinant SbCYP728B35 showed significantly higher enzymatic activity, the results of the kinetic analysis, and the similarity between substrate specificity of the recombinant protein and the conversion of 5DS stereoisomers in sorghum plants, SbCYP728B35 is most likely the gene responsible for sorgomol synthesis in this plant. Therefore, to demonstrate the function of SbCYP728B35 in planta, SbCYP728B35 cDNA was overexpressed in L. japonicus (SbCYP728B35-OE) by Agrobacterium rhizogenes-mediated hairy root transformation. The root culture of L. japonicus produced a high amount of 5DS with an undetectable amount of sorgomol (Sugimoto and Ueyama, 2008). In control WT lines, 5DS was detected, but sorgomol was below the detection limit. However, in SbCYP728B35-OE lines, both 5DS and sorgomol were detected. The contents of 5DS were greater in control lines than in SbCYP728B35-OE lines, as the latter used the 5DS produced by L. japonicus as a substrate and converted it to sorgomol (Figure 4). This result demonstrates that SbCYP728B35 acts as a sorgomol synthase gene in planta.

Figure 4.

Figure 4

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SL analysis of culture media in the L. japonicus SbCYP728B35-OE lines. A and B, LC–MS/MS analyses of culture media of WT and SbCYP728B35-OE lines (#9 and #44). MRM transition at m/z 331.1 > 97.0 and m/z 364.1 > 317.1 in positive ionization mode were selected for detection of 5DS (A) and sorgomol (B), respectively. LC Method II was used to separate SLs. C, Quantification of 5DS and sorgomol in culture media of WT and SbCYP728B35-OE lines. Error bars represent means ± SE (n = 3). n.d., not detected. Asterisks indicate a significant difference between WT and SbCYP728B35-OE lines (*P < 0.05 by the Student’s t test).

Expression of SL biosynthesis genes in a nonsorgomol-producing sorghum cultivar

We analyzed the expressions of SL biosynthesis-related genes in the roots of a nonsorgomol-producing cultivar, Abu70 (Motonami et al., 2013). LC-MS/MS analysis revealed that 5DS and sorgomol were detected in the hydroponic culture media of Sudax, whereas sorgomol was undetectable in Abu70, regardless of phosphate concentration in the growth media, although 5DS content increased under Pi-deficient conditions (Supplemental Figure S2). In the roots of Abu70, expression of SbD27, SbCCD7, SbCCD8, SbCYP711As, and SbCYP728B1 was upregulated in response to Pi-deficiency; however, the expression of SbCYP728B35 was not detected (Figure 5). Moreover, amplification of the SbCYP728B35 sequence in Abu70 was not confirmed by PCR using either Abu70 cDNA or genomic DNA as a template, with the same primers used for cloning of this gene in Sudax. The lack of SbCYP728B35 expression is consistent with negligible production of sorgomol in this cultivar, suggesting its role in sorgomol production in sorghum.

Figure 5.

Figure 5

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Expression of SL biosynthesis-related genes in a nonsorgomol-producing cultivar of sorghum, Abu70. Error bars represent means ± SE (n = 3 biologically independent plants). Asterisks indicate a significant difference between +P and –P conditions (*P < 0.05 by the Student’s t test).

Discussion

In this study, we identified the gene SbCYP728B35, which encodes sorgomol synthase. This enzyme catalyzes the introduction of a hydroxy group into 5DS at C-9 in vitro and in planta. The present study reveals the genetic basis behind the structural diversity in strigol-type SLs.

The CYP728B subfamily is widely conserved in plants, irrespective of sorgomol production (Nelson and Werck-Reichhart, 2011; Wang and Bouwmeester, 2018). Homologs of SbCYP728B35 exhibiting sorgomol synthase activity may be functional in sorgomol-producing plants. Indeed, this subfamily is also found in cotton (G. arboreum; Iseki et al., 2018) in the form of gene Ga01G0851, as reported in the Cotton FGD database (Zhu et al., 2017). On the contrary, in nonsorgomol-producing plants, we previously reported that VuCYP728B was coexpressed with genes for SL biosynthesis and SL receptor (VuCCD7, VuCCD8, VuCYP722C, and VuD14) in roots of cowpea (Wakabayashi et al., 2019). It has recently been reported that the mutations in rice OsSMAX1, a negative regulator of SL biosynthesis, caused upregulation of the expression of OsCYP728B1 (LOC_Os07g33620), OsCCD7, and OsCCD8 (Choi et al., 2020). Therefore, the CYP728B subfamily may also be involved in SL biosynthesis in nonsorgomol producing plants, particularly in the oxidation of carbon(s) in the A- or B-ring of canonical SLs, or the A-ring of noncanonical SLs. Furthermore, the gene TwCYP728B70 from Tripterygium wilfordii has recently been reported to catalyze the oxidation of C-18 from methyl to the acid moiety of dehydroabietic acid in the biosynthesis of triptolide, a useful plant specialized metabolite (Tu et al., 2020). This suggests that the CYP728B subfamily is involved not only in SL biosynthesis but also in various specialized plant metabolic pathways.

The in vitro experiments on substrate specificity using recombinant SbCYP728B35 showed that 5DS and 2ʹ-epi-5DS were converted to sorgomol and 2ʹ-epi-sorgomol, respectively, with comparable levels of efficiency. Their enantiomers were barely converted to the corresponding hydroxylated compounds. These substrate preferences were consistent with the conversion of exogenously-administered 5DS stereoisomers in Sudax (Figure 3). The common structural feature in 5DS and 2ʹ-epi-5DS, different from their enantiomers, is the C-8bS configuration of the C-ring. The recombinant enzyme exclusively recognizes this configuration. By contrast, the C-2ʹ configuration in the D-ring was not important for substrate recognition, as both 5DS and the unnatural-type 2ʹ-epi-5DS were available as substrates. This substrate tolerance would not normally impact sorgomol production in plants, as all naturally-occurring SLs possess a C-2ʹR configuration in the D-ring.

We also found that the expression of SbCYP728B35 was not detectable in the nonsorgomol-producing cultivar, Abu70 (Figure 5). On the contrary, the expression of SbCYP728B1 was upregulated in response to Pi-deficiency in both Sudax and Abu70 (Figures 2A and 5). The cDNA sequence of SbCYP728B1 in Abu70 was confirmed to be identical to that in Sudax, suggesting that SbCYP728B35 likely plays a pivotal role in sorgomol production in sorghum and that the contribution of SbCYP728B1 is negligible. Also, SbCYP728B1 may be involved in other aspects of plant specialized metabolism in sorghum, as for TwCYP728B70 in T. wilfordii.

Among the CYP711A subfamily of sorghum, the functions of CYP711A enzymes other than SbCYP711A18 remain to be determined. However, differences in the conversion activity of CL to CLA between members of its subfamily were in good agreement with previous results in maize (Zea mays): ZmMAX1a and ZmMAX1c, which are closely related to SbCYP711A13 and A19, respectively, showed very weak activities, whereas ZmMAX1b, which is closely related to SbCYP711A18, showed high activity (Yoneyama et al., 2018). In contrast to rice OsCYP711As, sorghum SbCYP711As did not catalyze the generation of canonical SL from CL through CLA. Given our result that exogenously-administered CLA was converted to 5DS in sorghum plants (Iseki et al., 2018), it may be that enzymes other than CYP711As catalyze this specific conversion. We recently identified GaCYP722C from cotton (G. arborem), which catalyzes the conversion of CLA to 5DS in vitro (Wakabayashi et al., 2020). The CYP722 family is found in monocot and dicot genomes. Dicot plants have both CYP722A and CYP722C, or only CYP722A as found in Arabidopsis; gramineous plants have only CYP722B (Nelson and Werck-Reichhart, 2011). The functions of genes in the CYP722B subfamily are still unknown, but they may be involved in the generation of canonical SLs. Furthermore, while the enzymatic function of LGS1 remains unknown, it is known to play an important role in BC-ring formation, as mutations at LGS1 alter the stereochemistry of SLs present in root exudates (Gobena et al., 2017; Bellis et al., 2020). It is believed that LGS1 regulates the stereospecific formation of the BC-ring or is directly involved in ring formation. Identification of the BC-ring-forming enzyme will clarify the entire SL biosynthetic pathway in sorghum.

In conclusion, we elucidated some of the drivers behind the structural diversity of SLs in plants. Our findings showed that the CYP728B subfamily plays a crucial role in derivatization of the parent canonical SL, 5DS. Diverse SLs secreted from roots into the rhizosphere may function less as phytohormones and more as communication signals in plant–plant and plant–microorganism interactions (Wakabayashi et al., 2019). We successfully generated a transgenic line of L. japonicus to produce sorgomol by overexpressing the gene SbCYP728B35 (Figure 4), indicating that the SL profile produced by plants can be artificially modified. The high production of a specific SL can help us to assess the effects of individual SLs on plant growth and development and to further understand their functions in the rhizosphere. We believe that these findings will contribute to a better understanding of the mechanisms and drivers of SL diversity in plants.

Materials and methods

Plant materials

Seeds of sorghum (S. bicolor [L.] Moench), cultivar Sudax (NM16) with high sorgomol production, and cultivar Abu70 (NM01) with high 5DS production but negligible source of sorgomol (nonsorgomol-producing cultivar; Motonami et al., 2013) were supplied by Kaneko Seeds, Gunma, Japan; and Prof. Abdel Gabar Babiker, Sudan University of Science and Technology, Sudan; respectively. Seeds of L. japonicus, ecotype Miyakojima MG20, were provided by the Biological Resource Center in Lotus and Glycine, Miyazaki University, Japan.

Chemicals

The CL, CLA, 5DS, and sorgomol stereoisomers were prepared as described in previous research (Nomura et al., 2013; Abe et al., 2014; Iseki et al., 2018).

Sorghum hydroponic culture and SL extraction from culture media

Sorghum seedlings were grown hydroponically with 0.4-strength Long Ashton nutrient solution for 2 w, then kept under different phosphate conditions in 0.4-strength Long Ashton nutrient solution (“Pi-rich conditions”) or tap water (“Pi-deficient conditions”) as described in a previous study (Motonami et al., 2013). Feeding experiments with each 5DS stereoisomer at a concentration of 40 nM were performed using Sudax, as described in a previous report (Iseki et al., 2018). Sorghum roots were washed with tap water and stored at −80°C until further testing. The SLs were extracted from culture media and subjected to purification procedures using a previously established protocol (Iseki et al., 2018).

SL analyses

The SLs were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using an LC-MS/MS system (Waters) consisting of an Acquity Ultra Performance liquid chromatograph (UPLC) and an Acquity quadrupole tandem mass spectrometer (TQ Detector). Data acquisition and analyses were performed on the MassLynx 4.1 software (Waters). The analytical conditions have been reported previously (Iseki et al., 2018). Separations were performed using a two-gradient program: 50%–100% (v/v) MeOH in H2O for 0–20 min (LC Method I) and 50%–70% (v/v) MeOH in H2O for 0–20 min (LC Method II).

Preparation for the microsomal fraction from sorghum roots

Roots (ca. 15 g fresh weight) of Sudax grown under Pi-deficient conditions were suspended in 75 mL of a buffer solution containing 50 mM potassium phosphate (pH 7.25), 20% (v/v) glycerol, 2 mM EDTA, 2 mM dithiothreitol, and 3.75 g polyvinylpolypyrrolidone. The roots were ground in a mortar with a pestle. The obtained homogenate was then filtered through four layers of gauze, the filtrates were centrifuged at 7,650 g for 10 min, and the supernatants recentrifuged at 100,000 g for 60 min. The pellet was suspended in the above buffer to provide the microsomal fraction. All procedures were conducted at 0°C–4°C. This microsomal fraction was used for the enzyme assays.

RNA-sequencing analysis

Total RNA was extracted from roots of Sudax grown under Pi-rich and Pi-deficient conditions using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol. The hydroponic culture conditions are described above and the RNA extracted from one individual in each culture condition was used for RNA-seq analysis (without replication). RNA quality was assessed using a BioAnalyzer 2100 (Agilent Technologies). A 10-μg aliquot of total RNA was used for constructing a cDNA library using the Illumina TruSeq Prep Kit v2 according to the manufacturer’s protocol (Illumina). The resulting cDNA library was sequenced using HiSeq1000 (Illumina) with 100 bp paired-end (PE) reads. Using HISAT2 (Kim et al., 2015), fastq files were mapped to the sorghum reference genome (McCormick et al., 2018). The SAM files obtained were sorted and converted into BAM files using Samtools (Li et al., 2009). The FPKM values were quantified for each sample to measure the expression levels of Sudax genes using StringTie (Pertea et al., 2015; Pertea et al., 2016). The FPKM values between the Pi-rich and Pi-deficient samples were then compared (Supplemental Data Set S1).

RT-qPCR analysis

Primer sets listed in Supplemental Table S2 were used to perform the RT-qPCR analysis. Total RNA was extracted from three independent lines of powdered sorghum roots using the RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s protocol. The cDNA templates were prepared using the ReverTra Ace qPCR RT Mix with genomic DNA (gDNA) remover (Toyobo), and amplified using a LighCycler Nano (Roche) with GeneAce SYBR qPCR Mix α No ROX (Nippon Gene). Gene expression levels were normalized against the values obtained for the sorghum SbActin1 gene (Mohemed et al., 2018). Data acquisition and analysis were performed using the software of the LightCycler Nano and normalized gene expression levels were calculated based on the 2−ΔΔCT method.

Expression of CYP proteins in E. coli

The bacterial expression vector pCWori+ was used to express recombinant CYP proteins in E. coli (Barnes et al., 1991). For the heterologous expression of sorghum CYP cDNAs, codons unfavorable to E. coli were replaced by modifying the nucleotide sequence of cDNAs to enrich A/T residues without changing the amino acid sequence using gene synthesis (Eurofins Genomics). These newly-synthesized cDNAs, which incorporated the restriction sites for Nde I at the 5ʹ end and Sal I at the 3ʹ end, were ligated into the Nde I/Sal I site of the pCWori+ vector. Escherichia coli strain DH5α was transformed with the constructs. The recombinant CYP proteins were expressed as described previously (Asami et al., 2001) with modifications. Transformed cells were grown overnight in Luria-Bertani medium supplemented with 100 μg mL−1 ampicillin and then incubated in 50 mL Terrific Broth medium containing 100 μg mL−1 ampicillin, 0.2% (w/v) glucose, and 0.5 mM γ-aminolevulinic acid. The medium was shaken at 225 rpm at 37°C. After 3 h of incubation, 0.1 mM isopropyl β-D-thiogalactoside and 1 μg mL−1 chloramphenicol were added. The culture was shaken continuously at 150 rpm for 72 h at 25°C. The cultured cells were harvested by centrifugation at 2,330 g for 30 min. After centrifugation, spheroplasts were sonicated in a buffer solution containing 20 mM potassium phosphate (pH 7.25), 20% (w/v) glycerol, 1 mM EDTA, and 0.1 mM dithiothreitol, and subjected to centrifugation at 100,000 g for 1 h. After centrifugation, the pellets were sonicated in the same buffer to provide microsomal fractions, which were used in the enzyme reactions.

In vitro enzyme assay

The reaction mixture consisted of 50 mM potassium phosphate (pH 7.25), 25 μL microsomal fraction (from Sudax roots or CYP-expressing E. coli), 1 unit/mL Arabidopsis NADPH–CYP reductase (Mizutani and Ohta, 1998), 2.5 mM NADPH, and 25 μM substrate; for a total volume of 250 μL. Reactions were initiated by the addition of NADPH and performed at 30°C for 60 min. The reaction products were extracted twice using an equivalent volume of ethyl acetate. The organic phase was collected and evaporated. The residues were dissolved in acetonitrile and analyzed by LC-MS/MS.

Biochemical analysis of recombinant SbCYP728Bs

To evaluate sorgomol synthase activity, the active recombinant proteins SbCYP728B1 and SbCYP728B35 were quantified based on the carbon monoxide (CO)-difference spectra using an extinction coefficient (Σ = 91 mM−1 cm−1 (Omura and Sato, 1964; Supplemental Figure S6). The in vitro enzyme assay was conducted as described above.

For the kinetic analysis of SbCYP728B35, kinetics parameters of 1.3 pmol recombinant SbCYP728B35 protein were determined in triplicate assays. The activity was assayed using 5DS at concentrations ranging from 0.1 to 10 μM. The reaction was performed at 30°C for 30 min. The extraction and LC–MS/MS analysis of the reaction product was performed as described above. Kinetic parameters were determined through a nonlinear regression using ANEMONA (Hernández and Ruiz, 1998).

Transformation of L. japonicus

The full-length SbCYP728B35 cDNA was amplified by PCR using PrimeSTAR HS DNA polymerase (Takara Bio) and primers listed in Supplemental Table S2. A binary vector pSbCYP728B35-OE-HR for SbCYP728B35 overexpression was constructed from the binary vector pBINPLUS (van Engelen et al., 1995; Lee et al., 2019): the cDNA of SbCYP728B35 was inserted under the control of cauliflower mosaic virus 35S (CaMV35S) promoter and NOS terminator in the T-DNA region. The binary vector pSbCYP728B35-OE-HR was electroporated into A. rhizogenes ATCC15834.

Agrobacterium rhizogenes-mediated hairy root transformation was performed according to a previous report (Kumagai and Kouchi, 2003) with some modifications. Lotus japonicus ecotype Miyakojima MG20 seedlings were aseptically grown on an agar plate, then excised with a razor blade at the lower part of the hypocotyl. The excised face was treated with an A. rhizogenes suspension in a petri dish. Seedlings with cotyledons were placed horizontally onto agar plates with B5 medium for a week. The plants were then transferred onto another plate containing B5 medium with 250 μg mL−1 cefotaxime and allowed to grow for another 2 w. Hairy roots were excised from the plants and transferred onto fresh B5 medium with 250 μg mL−1 cefotaxime and 50 μg mL−1 kanamycin. The transgenic hairy roots were selected using genomic PCR, targeting SbCYP728B35 using the primers mentioned above. The transgenic lines were subsequently transferred to liquid B5 medium containing 5 μM indole-3-butyric acid for propagation. After the roots had sufficiently grown, they were transferred onto a fresh liquid B5 medium, then removed 24 h later, and SLs were extracted from the culture media.

Accession numbers

The raw RNA-sequencing data from this study were submitted to the DNA Databank of Japan (DDBJ) Read Archive under accession number DRA007444.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Conversion of 5DS to sorgomol by microsomal fraction prepared from Sudax roots grown under Pi-deficient conditions.

Supplemental Figure S2. SL contents in root exudates of sorghum cultivars, Sudax, and Abu70.

Supplemental Figure S3. In vitro enzyme assay using recombinant SbCYP711A subfamily.

Supplemental Figure S4. Michaelis–Menten kinetics of recombinant SbCYP728B35 incubated with 5DS as a substrate.

Supplemental Figure S5. Substrate specificity of recombinant SbCYP728B1 toward 5DS stereoisomers.

Supplemental Figure S6. CO-difference spectra of recombinant SbCYP728B1 and SbCYP728B35 expressed in E. coli.

Supplemental Table S1. Enzyme kinetic parameters of recombinant SbCYP728B35.

Supplemental Table S2. Primers used for this study.

Supplemental Data Set S1. Upregulated sorghum CYP genes in response to phosphate deficiency.

Funding

This work was supported by JST/JICA SATREPS (JPMJSA1607 to Y.S.) and JSPS KAKENHI (25292065 to Y.S).

Conflict of interest statement. The authors declare no conflict of interest.

Supplementary Material

kiaa113_Supplementary_Data
Click here for additional data file. (891.9KB, zip)

 

M.M. and Y.S. designed research; T.W., S.I., K.S., N.M., and H.S. performed research; H.T. contributed supplying synthetic compounds; T.W., S.I., H.S., and Y.S. analyzed data; and T.W., M.M., and Y.S. wrote the article. All authors approved the final version of the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys) is: Yukihiro Sugimoto (yukihiro@kobe-u.ac.jp).

References

  1. Abe S, Sado A, Tanaka K, Kisugi T, Asami K, Ota S, Kim H Il, Yoneyama K, Xie X, Ohnishi T, et al. (2014) Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro. Proc Natl Acad Sci USA 111: 18084–18089 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akiyama K, Matsuzaki K, Hayashi H (2005) Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435: 824–827 [DOI] [PubMed] [Google Scholar]
  3. Alder A, Jamil M, Marzorati M, Bruno M, Vermathen M, Bigler P, Ghisla S, Bouwmeester H, Beyer P, Al-Babili S (2012) The path from β-carotene to carlactone, a strigolactone-like plant hormone. Science 335: 1348–1351 [DOI] [PubMed] [Google Scholar]
  4. Asami T, Mizutani M, Fujioka S, Goda H, Min YK, Shimada Y, Nakano T, Takatsuto S, Matsuyama T, Nagata N, et al. (2001) Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta. J Biol Chem 276: 25687–25691 [DOI] [PubMed] [Google Scholar]
  5. Barnes HJ, Arlotto MP, Waterman MR (1991) Expression and enzymatic activity of recombinant cytochrome P450 17α-hydroxylase in Escherichia coli. Proc Natl Acad Sci USA 88: 5597–5601 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bellis ES, Kelly EA, Lorts CM, Gao H, DeLeo VL, Rouhan G,, Budden A, Bhaskara GB, Hu Z, Muscarella R, et al. (2020) Genomics of sorghum local adaptation to a parasitic plant. Proc Natl Acad Sci USA 117: 4243–4251 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charnikhova TV, Gaus K, Lumbroso A, Sanders M, Vincken JP, De Mesmaeker A, Ruyter-Spira CP, Screpanti C, Bouwmeester HJ (2017) Zealactones. Novel natural strigolactones from maize. Phytochemistry 137: 123–131 [DOI] [PubMed] [Google Scholar]
  8. Choi J, Lee T, Cho J, Servante EK, Pucker B, Summers W, Bowden S, Rahimi M, An K, An G, et al. (2020) The negative regulator SMAX1 controls mycorrhizal symbiosis and strigolactone biosynthesis in rice. Nat Commun 11: 2114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cook CE, Whichard LP, Turner B, Wall ME, Egley GH (1966) Germination of witchweed (Striga lutea Lour.): isolation and properties of a potent stimulant. Science 154: 1189–1190 [DOI] [PubMed] [Google Scholar]
  10. Gobena D, Shimels M, Rich PJ, Ruyter-Spira C, Bouwmeester H, Kanuganti S, Mengiste T, Ejeta G (2017) Mutation in sorghum LOW GERMINATION STIMULANT 1 alters strigolactones and causes Striga resistance. Proc Natl Acad Sci USA 114: 4471–4476 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun EA, Pillot JP, Letisse F, Matusova R, Danoun S, Portais JC, et al. (2008) Strigolactone inhibition of shoot branching. Nature 455: 189–194 [DOI] [PubMed] [Google Scholar]
  12. Hernández A, Ruiz MT (1998) An EXCEL template for calculation of enzyme kinetic parameters by non-linear regression. Bioinformatics 14: 227–228 [DOI] [PubMed] [Google Scholar]
  13. Iseki M, Shida K, Kuwabara K, Wakabayashi T, Mizutani M, Takikawa H, Sugimoto Y (2018) Evidence for species-dependent biosynthetic pathways for converting carlactone to strigolactones in plants. J Exp Bot 69: 2305–2318 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12: 357–360 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim H Il, Kisugi T, Khetkam P, Xie X, Yoneyema K, Uchida K, Yokota T, Nomura T, McErlean CSP, Yoneyama K (2014) Avenaol, a germination stimulant for root parasitic plants from Avena strigosa. Phytochemistry 103: 85–88 [DOI] [PubMed] [Google Scholar]
  16. Kumagai H, Kouchi H (2003) Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Mol Plant Microbe Interact 16: 663–668 [DOI] [PubMed] [Google Scholar]
  17. Lee HJ, Nakayasu M, Akiyama R, Kobayashi M, Miyachi H, Sugimoto Y, Umemoto N, Saito K, Muranaka T, Mizutani M (2019) Identification of a 3β-hydroxysteroid dehydrogenase/3-ketosteroid reductase involved in α-tomatine biosynthesis in tomato. Plant Cell Physiol 60: 1304–1315 [DOI] [PubMed] [Google Scholar]
  18. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25: 2078–2079 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McCormick RF, Truong SK, Sreedasyam A, Jenkins J, Shu S, Sims D, Kennedy M, Amirebrahimi M, Weers BD, McKinley B, et al. (2018) The Sorghum bicolor reference genome: improved assembly, gene annotations, a transcriptome atlas, and signatures of genome organization. Plant J 93: 338–354 [DOI] [PubMed] [Google Scholar]
  20. Mizutani M, Ohta D (1998) Two isoforms of NADPH:cytochrome P450 reductase in Arabidopsis thaliana. Gene structure, heterologous expression in insect cells, and differential regulation. Plant Physiol 116: 357–367 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mohemed N, Charnikhova T, Fradin EF, Rienstra J, Babiker AGT, Bouwmeester HJ (2018) Genetic variation in Sorghum bicolor strigolactones and their role in resistance against Striga hermonthica. J Exp Bot 69: 2415–2430 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mori N, Nomura T, Akiyama K (2020a) Identification of two oxygenase genes involved in the respective biosynthetic pathways of canonical and non-canonical strigolactones in Lotus japonicus. Planta 251: 40. [DOI] [PubMed] [Google Scholar]
  23. Mori N, Sado A, Xie X, Yoneyama K, Asami K, Seto Y, Nomura T, Yamaguchi S, Yoneyama K, Akiyama K (2020b) Chemical identification of 18-hydroxycarlactonoic acid as an LjMAX1 product and in planta conversion of its methyl ester to canonical and non-canonical strigolactones in Lotus japonicus. Phytochemistry 174: 112349. [DOI] [PubMed] [Google Scholar]
  24. Motonami N, Ueno K, Nakashima H, Nomura S, Mizutani M, Takikawa H, Sugimoto Y (2013) The bioconversion of 5-deoxystrigol to sorgomol by the sorghum, Sorghum bicolor (L.) Moench. Phytochemistry 93: 41–48 [DOI] [PubMed] [Google Scholar]
  25. Nelson D, Werck-Reichhart D (2011) A P450-centric view of plant evolution. Plant J 66: 194–211 [DOI] [PubMed] [Google Scholar]
  26. Nelson DR (2009) The cytochrome P450 homepage. Hum Genomics 4: 59–65 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nomura S, Nakashima H, Mizutani M, Takikawa H, Sugimoto Y (2013) Structural requirements of strigolactones for germination induction and inhibition of Striga gesnerioides seeds. Plant Cell Rep 32: 829–838 [DOI] [PubMed] [Google Scholar]
  28. Omura T, Sato R (1964) The carbon monoxide-biding pigment of liver microsomes. J Biol Chem 239: 2370–2378 [PubMed] [Google Scholar]
  29. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL (2016) Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc 11: 1650–1667 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pertea M, Pertea GM, Antonescu CM, Chang T-C,, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33: 290–295 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Samejima H, Babiker AG, Mustafa A, Sugimoto Y (2016) Identification of Striga hermonthica-resistant upland rice varieties in Sudan and their resistance phenotypes. Front Plant Sci 7: 1–12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sugimoto Y, Ueyama T (2008) Production of (+)-5-deoxystrigol by Lotus japonicus root culture. Phytochemistry 69: 212–217 [DOI] [PubMed] [Google Scholar]
  33. Tu L, Su P, Zhang Z, Gao L, Wang J, Hu T, Zhou J, Zhang Y, Zhao Y, Liu Y, et al. (2020) Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis. Nat Commun 11: 971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ueno K, Furumoto T, Umeda S, Mizutani M, Takikawa H, Batchvarova R, Sugimoto Y (2014) Heliolactone, a non-sesquiterpene lactone germination stimulant for root parasitic weeds from sunflower. Phytochemistry 108: 122–128 [DOI] [PubMed] [Google Scholar]
  35. Ueno K, Nakashima H, Mizutani M, Takikawa H, Sugimoto Y (2018) Bioconversion of 5-deoxystrigol stereoisomers to monohydroxylated strigolactones by plants. J Pestic Sci 43: 198–206 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya N, Magome H, Kamiya Y, Shirasu K, Yoneyama K, et al. (2008) Inhibition of shoot branching by new terpenoid plant hormones. Nature 455: 195–200 [DOI] [PubMed] [Google Scholar]
  37. Wakabayashi T, Hamana M, Mori A, Akiyama R, Ueno K, Osakabe K, Osakabe Y, Suzuki H, Takikawa H, Mizutani M, et al. (2019) Direct conversion of carlactonoic acid to orobanchol by cytochrome P450 CYP722C in strigolactone biosynthesis. Sci Adv 5: eaax9067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wakabayashi T, Shida K, Kitano Y, Takikawa H, Mizutani M, Sugimoto Y (2020) CYP722C from Gossypium arboreum catalyzes the conversion of carlactonoic acid to 5-deoxystrigol. Planta 251: 97. [DOI] [PubMed] [Google Scholar]
  39. Wang Y, Bouwmeester HJ (2018) Structural diversity in the strigolactones. J Exp Bot 69: 2219–2230 [DOI] [PubMed] [Google Scholar]
  40. Xie X, Kisugi T, Yoneyama K, Nomura T, Akiyama K, Uchida K, Yokota T, McErlean CSP, Yoneyama K (2017) Methyl zealactonoate, a novel germination stimulant for root parasitic weeds produced by maize. J Pestic Sci 42: 58–61 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Xie X, Mori N, Yoneyama K, Nomura T, Uchida K, Yoneyama K, Akiyama K (2019) Lotuslactone, a non-canonical strigolactone from Lotus japonicus. Phytochemistry 157: 200–205 [DOI] [PubMed] [Google Scholar]
  42. Yoneyama K, Mori N, Sato T, Yoda A, Xie X, Okamoto M, Iwanaga M, Ohnishi T, Nishiwaki H, Asami T, et al. (2018) Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis. New Phytol 218: 1522–1533 [DOI] [PubMed] [Google Scholar]
  43. Yoneyama K, Xie X, Kusumoto D, Sekimoto H, Sugimoto Y, Takeuchi Y, Yoneyama K (2007) Nitrogen deficiency as well as phosphorus deficiency in sorghum promotes the production and exudation of 5-deoxystrigol, the host recognition signal for arbuscular mycorrhizal fungi and root parasites. Planta 227: 125–132 [DOI] [PubMed] [Google Scholar]
  44. van Engelen FA, Molthoff JW, Conner AJ, Nap J-P, Pereira A, Stiekema WJ (1995) pBINPLUS: An improved plant transformation vector based on pBIN19. Transgenic Res 4: 288–290 [DOI] [PubMed] [Google Scholar]
  45. Zhang Y, Cheng X, Wang Y, Díez-Simón C, Flokova K, Bimbo A, Bouwmeester HJ, Ruyter-Spira C (2018) The tomato MAX1 homolog, SlMAX1, is involved in the biosynthesis of tomato strigolactones from carlactone. New Phytol 219: 297–309 [DOI] [PubMed] [Google Scholar]
  46. Zhang Y, van Dijk ADJ, Scaffidi A, Flematti GR, Hofmann M, Charnikhova T, Verstappen F, Hepworth J, van der Krol S, Leyser O, et al. (2014) Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis. Nat Chem Biol 10: 1028–1033 [DOI] [PubMed] [Google Scholar]
  47. Zhu T, Liang C, Meng Z, Sun G, Meng Z, Guo S, Zhang R (2017) CottonFGD: an integrated functional genomics database for cotton. BMC Plant Biol 17: 101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zwanenburg B, Blanco-Ania D (2018) Strigolactones: new plant hormones in the spotlight. J Exp Bot 69: 2205–2218 [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

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