A Stereoselective Strigolactone Biosynthesis Catalyzed by a 2-Oxoglutarate-Dependent Dioxygenase in Sorghum.

Seeds of root parasitic plants, Striga, Orobanche and Phelipanche spp., are induced to germinate by strigolactones (SLs) exudated from host roots. In Striga-resistant cultivars of Sorghum bicolor, the loss-of-function of the Low Germination Stimulant 1 (LGS1) gene changes the major SL from 5-deoxystrigol (5DS) to orobanchol, which has an opposite C-ring stereochemistry. The biosynthetic pathway of 5DS catalyzed by LGS1 has not been fully elucidated. Since other unknown regulators, in addition to LGS1 encoding a sulfotransferase, appear to be necessary for the stereoselective biosynthesis of 5DS, we examined Sobic.005G213500 (Sb3500), encoding a 2-oxoglutarate-dependent dioxygenase, as a candidate regulator, which is co-expressed with LGS1 and located 5'-upstream of LGS1 in the sorghum genome. When LGS1 was expressed with known SL biosynthetic enzyme genes including the cytochrome P450 SbMAX1a in Nicotiana benthamiana leaves, 5DS and its diastereomer 4-deoxyorobanchol (4DO) were produced in approximately equal amounts, while the production of 5DS was significantly larger than that of 4DO when Sb3500 was also co-expressed. We also confirmed the stereoselective 5DS production in an in vitro feeding experiment using synthetic chemicals with recombinant proteins expressed in Escherichia coli and yeast. This finding demonstrates that Sb3500 is a stereoselective regulator in the conversion of the SL precursor carlactone to 5DS, catalyzed by LGS1 and SbMAX1a, providing a detailed understanding of how different SLs are produced to combat parasitic weed infestations.

Identification of a Prunus MAX1 homolog as a unique strigol synthase.

Strigol is the first identified and one of the most important strigolactones (SLs), but the biosynthetic pathway remains elusive. We functionally identified a strigol synthase (cytochrome P450 711A enzyme) in the Prunus genus through rapid gene screening in a set of SL-producing microbial consortia, and confirmed its unique catalytic activity (catalyzing multistep oxidation) through substrate feeding experiments and mutant analysis. We also reconstructed the biosynthetic pathway of strigol in Nicotiana benthamiana and reported the total biosynthesis of strigol in the Escherichia coli-yeast consortium, from the simple sugar xylose, which paves the way for large-scale production of strigol. As proof of concept, strigol and orobanchol were detected in Prunus persica root extrudes. This demonstrated a successful prediction of metabolites produced in plants through gene function identification, highlighting the importance of deciphering the sequence-function correlation of plant biosynthetic enzymes to more accurately predicate plant metabolites without metabolic analysis. This finding revealed the evolutionary and functional diversity of CYP711A (MAX1) in SL biosynthesis, which can synthesize different stereo-configurations of SLs (strigol- or orobanchol-type). This work again emphasizes the importance of microbial bioproduction platform as an efficient and handy tool to functionally identify plant metabolism.

Heterologous expression of the lectin CmRlec from Cordyceps militaris (Cordycipitaceae, Ascomycota) in Escherichia coli.

Ascomycete lectins may play an important role in their life cycle. In this report, we mined a ricin B-type lectin, named CmRlec, from the Cordyceps militaris genome by homology search. Furthermore, we succeeded in the soluble expression of CmRlec using ß-glucuronidase as a solubilization tag and demonstrated that this lectin is a novel chitin-recognizing lectin.

Strigolactone biosynthesis catalyzed by cytochrome P450 and sulfotransferase in sorghum.

Root parasitic plants such as Striga, Orobanche, and Phelipanche spp. cause serious damage to crop production world-wide. Deletion of the Low Germination Stimulant 1 (LGS1) gene gives a Striga-resistance trait in sorghum (Sorghum bicolor). The LGS1 gene encodes a sulfotransferase-like protein, but its function has not been elucidated. Since the profile of strigolactones (SLs) that induce seed germination in root parasitic plants is altered in the lgs1 mutant, LGS1 is thought to be an SL biosynthetic enzyme. In order to clarify the enzymatic function of LGS1, we looked for candidate SL substrates that accumulate in the lgs1 mutants and performed in vivo and in vitro metabolism experiments. We found the SL precursor 18-hydroxycarlactonoic acid (18-OH-CLA) is a substrate for LGS1. CYP711A cytochrome P450 enzymes (SbMAX1 proteins) in sorghum produce 18-OH-CLA. When LGS1 and SbMAX1 coding sequences were co-expressed in Nicotiana benthamiana with the upstream SL biosynthesis genes from sorghum, the canonical SLs 5-deoxystrigol and 4-deoxyorobanchol were produced. This finding showed that LGS1 in sorghum uses a sulfo group to catalyze leaving of a hydroxyl group and cyclization of 18-OH-CLA. A similar SL biosynthetic pathway has not been found in other plant species.

Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis.

Strigolactones (SLs) are a class of plant hormones which regulate shoot branching and function as host recognition signals for symbionts and parasites in the rhizosphere. However, steps in SL biosynthesis after carlactone (CL) formation remain elusive. This study elucidated the common and diverse functions of MAX1 homologs which catalyze CL oxidation. We have reported previously that ArabidopsisMAX1 converts CL to carlactonoic acid (CLA), whereas a rice MAX1 homolog has been shown to catalyze the conversion of CL to 4-deoxyorobanchol (4DO). To determine which reaction is conserved in the plant kingdom, we investigated the enzymatic function of MAX1 homologs in Arabidopsis, rice, maize, tomato, poplar and Selaginella moellendorffii. The conversion of CL to CLA was found to be a common reaction catalyzed by MAX1 homologs, and MAX1s can be classified into three types: A1-type, converting CL to CLA; A2-type, converting CL to 4DO via CLA; and A3-type, converting CL to CLA and 4DO to orobanchol. CLA was detected in root exudates from poplar and Selaginella, but not ubiquitously in other plants examined in this study, suggesting its role as a species-specific signal in the rhizosphere. This study provides new insights into the roles of MAX1 in endogenous and rhizosphere signaling.

Δ4-dn-iso-OPDA, a bioactive plant hormone of Marchantia polymorpha.

Significant progress has been recently made in our understanding of the evolution of jasmonates biosynthesis and signaling. The bioactive jasmonate activating COI1-JAZ co-receptor differs in bryophytes and vascular plants. Dinor-iso-12-oxo-phytodienoic acid (dn-iso-OPDA) is the bioactive hormone in bryophytes and lycophytes. However, further studies showed that the full activation of hormone signaling in Marchantia polymorpha requires additional unidentified hormones. Δ4-dn-OPDAs were previously identified as novel bioactive jasmonates in M. polymorpha. In this paper, we describe the major bioactive isomer of Δ4-dn-OPDAs as Δ4-dn-iso-OPDA through chemical synthesis, receptor binding assay, and biological activity in M. polymorpha. In addition, we disclosed that Δ4-dn-cis-OPDA is a biosynthetic precursor of Δ4-dn-iso-OPDA. We demonstrated that in planta cis-to-iso conversion of Δ4-dn-cis-OPDA occurs in the biosynthesis of Δ4-dn-iso-OPDA, defining a key biosynthetic step in the chemical evolution of hormone structure. We predict that these findings will facilitate further understanding of the molecular evolution of plant hormone signaling.

DIENELACTONE HYDROLASE LIKE PROTEIN1 negatively regulates the KAI2-ligand pathway in Marchantia polymorpha.

Karrikins are smoke-derived butenolides that induce seed germination and photomorphogenesis in a wide range of plants.1,2,3 KARRIKIN INSENSITIVE2 (KAI2), a paralog of a strigolactone receptor, perceives karrikins or their metabolized products in Arabidopsis thaliana.4,5,6,7 Furthermore, KAI2 is thought to perceive an unidentified plant hormone, called KAI2 ligand (KL).8,9 KL signal is transduced via the interaction between KAI2, MORE AXILLARY GROWTH2 (MAX2), and SUPPRESSOR of MORE AXILLARY GROWTH2 1 LIKE family proteins (SMXLs), followed by the degradation of SMXLs.4,7,10,11,12,13,14 This signaling pathway is conserved both in A. thaliana and the bryophyte Marchantia polymorpha.14 Although the KL signaling pathway is well characterized, the KL metabolism pathways remain poorly understood. Here, we show that DIENELACTONE HYDROLASE LIKE PROTEIN1 (DLP1) is a negative regulator of the KL pathway in M. polymorpha. The KL signal induces DLP1 expression. DLP1 overexpression lines phenocopied the Mpkai2a and Mpmax2 mutants, while dlp1 mutants phenocopied the Mpsmxl mutants. Mutations in the KL signaling genes largely suppressed these phenotypes, indicating that DLP1 acts upstream of the KL signaling pathway, although DLP1 also has KL pathway-independent functions. DLP1 exhibited enzymatic activity toward a potential substrate, suggesting the possibility that DLP1 works through KL inactivation. Investigation of DLP1 homologs in A. thaliana revealed that they do not play a major role in the KL pathway, suggesting different mechanisms for the KL signal regulation. Our findings provide new insights into the regulation of the KL signal in M. polymorpha and the evolution of the KL pathway in land plants.

Production and stably maintenance of strigolactone by transient expression of biosynthetic enzymes in Nicotiana benthamiana.

Strigolactones (SLs) are phytohormones that play an essential role in plant-microbe interactions. The instability of SLs makes it challenging to use them for application to agriculture. In this study, we successfully produced a large amount of the 4-deoxyorobanchol (4DO), one of SLs, in the leaves of Nicotiana benthamiana, using a transient expression system to express SL biosynthetic enzymes. Using this system, the yield of 4DO was 2.1 ± 0.3 µg/gFM (fresh mass). Treatment of leaves at 80°C for 16 h killed Agrobacterium and approximately half amount of 4DO was left in the leaves (1.0 µg/gFM (calculated based on the original FM) ± 0.3). Interestingly, incubation of dried leaves at room temperature for 1 month maintained an almost equal amount of 4DO (0.9 ± 0.2 µg/gFM) in the leaves. These results suggest that high accumulation of 4DO with stability for long periods can be achieved in plant leaves.

Canonical strigolactones are not the major determinant of tillering but important rhizospheric signals in rice.

Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga-resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed-germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.

An ancestral function of strigolactones as symbiotic rhizosphere signals.

In flowering plants, strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), an SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating its origin in the common ancestor of land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.