Whispers in the dark: Signals regulating underground plant-plant interactions.

Plants are able to actively detect and respond to the presence in neighboring plants, in order to optimize their physiology to promote survival and reproduction despite the presence of competing organisms. A key but still poorly understood mechanism for neighbor detection is through the perception of root exudates. In this review, we explore recent findings on the role of root exudates in plant-plant interactions, focusing both on general interactions and also the highly specialized example of root parasite-host plant interactions.

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.

Extraction and Quantification of Plant Hormones and RNA from Pea Axillary Buds.

The quantification of plant hormones and related gene expression is essential to improve the understanding of the molecular regulation of plant growth and development. However, plant hormone quantification is still challenging due to extremely low endogenous levels and high chemical diversity. In this study, we present a convenient extraction protocol that enables the simultaneous extraction of both phytohormones and RNA from the same sample in a small quantity (approximately 10 mg). Using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS), this protocol provides a method to quantify 13 phytohormones and their derivatives from four classes (cytokinin, auxin, abscisic acid, and gibberellin) at the speed of 14 min per sample.

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.

Plant Growth Suppressive Activity of (R)-3-(7'-Aryl-9'-hydroxyprop-8'-yl)coumarin, Structural Isomer of Z-2-Hydroxybenzylidene-γ-butyrolactone-type Lignan.

3-(7'-Aryl-9'-hydroxyprop-8'-yl)coumarin, which is a structural isomer of a Z-2-hydroxybenzylidene-γ-butyrolactone-type lignan, was stereoselectively synthesized and subjected to plant growth regulation examination. (R)-4'-Methoxyphenyl derivative 3 showed stereospecific plant growth suppressive activity. The significance of the presence of hydroxy group at the 9'-position for the activity was clarified. The effect of the substituent at the 7'-aryl group was also shown. The 3'-methoxy, 4'-methoxy, and 4'-trifluoromethyl derivatives 10, 3, and 22 led to the most significant growth suppression of Italian ryegrass roots. The 2'-methoxy derivative 9 and 4'-methoxy derivative 3 provided the most growth suppressive activity against lettuce shoots and roots, respectively.

Environmental strigolactone drives early growth responses to neighboring plants and soil volume in pea.

There has been a dramatic recent increase in the understanding of the mechanisms by which plants detect their neighbors,1 including by touch,2 reflected light,3 volatile organic chemicals, and root exudates.4,5 The importance of root exudates remains ill-defined because of confounding experimental variables6,7 and difficulties disentangling neighbor detection in shoot and roots.8-10 There is evidence that root exudates allow distinction between kin and non-kin neighbors,11-13 but identification of specific exudates that function in neighbor detection and/or kin recognition remain elusive.1 Strigolactones (SLs), which are exuded into the soil in significant quantities in flowering plants to promote recruitment of arbuscular mycorrhizal fungi (AMF),14 seem intuitive candidates to act as plant-plant signals, since they also act as hormones in plants,15-17 with dramatic effects on shoot growth18,19 and milder effects on root development.20 Here, using pea, we test whether SLs act as either cues or signals for neighbor detection. We show that peas detect neighbors early in the life cycle through their root systems, resulting in strong changes in shoot biomass and branching, and that this requires SL biosynthesis. We demonstrate that uptake and detection of SLs exuded by neighboring plants are needed for this early neighbor detection, and that plants that cannot exude SLs are outcompeted by neighboring plants and fail to adjust growth to their soil volume. We conclude that plants both exude SLs as signals to modulate neighbor growth and detect environmental SLs as a cue for neighbor presence; collectively, this allows plants to proactively adjust their shoot growth according to neighbor density.

Supra-organismal regulation of strigolactone exudation and plant development in response to rhizospheric cues in rice.

Plants have evolved elaborate mechanisms to detect neighboring plants, which typically involve the perception of "cues" inadvertently produced by the neighbor.1 Strigolactones are hormonal signaling molecules2,3 that are also exuded into the rhizosphere by most flowering plant species to promote arbuscular mycorrhizal symbioses.4 Since flowering plants have an endogenous perception system for strigolactones,5 strigolactones are obvious candidates to act as a cue for neighbor presence, but have not been shown to act as such. To test this hypothesis in rice plants, we quantified two major strigolactones of rice plants, orobanchol and 4-deoxyorobanchol, in root exudates by using LC-MS/MS (MRM) and examined feedback regulation of strigolactone biosynthesis and changes in shoot branching phenotypes in rice plants grown at different densities in hydroponics and soil culture. We show that the presence of neighboring plants, or greater root volume, results in rapidly induced changes in strigolactone biosynthesis, sensitivity, and exudation and the subsequent longer-term changes in shoot architecture. These changes require intact strigolactone biosynthesis in neighboring plants and intact strigolactone signaling in focal plants. These results suggest that strigolactone biosynthesis and exudation in rice plants are driven by supra-organismal environmental strigolactone levels. Strigolactones thus act as a cue for neighbor presence in rice plants, but also seem to act as a more general root density-sensing mechanism in flowering plants that integrates soil volume and neighbor density and allows plants to adapt to the limitations of the rhizosphere.

Ancestral sequence reconstruction of the CYP711 family reveals functional divergence in strigolactone biosynthetic enzymes associated with gene duplication events in monocot grasses.

The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several diversifying oxidation reactions on the structure. Evidence was obtained for functional divergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone diversification in early land plants, which may have extended to the diversification of strigol-type SLs.

Germination Stimulant Activity of Isothiocyanates on Phelipanche spp.

The root parasitic weed broomrapes, Phelipanche spp., cause severe damage to agriculture all over the world. They have a special host-dependent lifecycle and their seeds can germinate only when they receive chemical signals released from host roots. Our previous study demonstrated that 2-phenylethyl isothiocyanate is an active germination stimulant for P. ramosa in root exudates of oilseed rape. In the present study, 21 commercially available ITCs were examined for P. ramosa seed germination stimulation, and some important structural features of ITCs for exhibiting P. ramosa seed germination stimulation have been uncovered. Structural optimization of ITC for germination stimulation resulted in ITCs that are highly active to P. ramosa. Interestingly, these ITCs induced germination of P. aegyptiaca but not Orobanche minor or Striga hermonthica. P. aegyptiaca seeds collected from mature plants parasitizing different hosts responded to these ITCs with different levels of sensitivity. ITCs have the potential to be used as inducers of suicidal germination of Phelipanche seeds.

Structure-activity relationship of the aromatic moiety of 6-substituted 5,6-dihydro-2H-pyran-2-one to find the novel compound showing higher plant growth inhibitory activity.

In the course of our research on the structure-activity relationship of 5,6-dihydro-2H-pyran-2-one, (S)-6-[(R)-2-hydroxy-6-(4-fluorophenyl)hexyl]-5,6-dihydro-2H-pyran-2-one was found to show 2-3-fold more potent plant growth inhibitory activity against Italian ryegrass shoots (IC50 = 95 µm) and roots (IC50 = 17 µm) than compound bearing unsubstituted phenyl group. The small and electron withdrawing atom at 4-position of the benzene ring caused the higher activity.

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.

Strigolactones, how are they synthesized to regulate plant growth and development?

Strigolactones (SLs) are multifunctional plant metabolites working not only as allelochemicals in the rhizosphere, but also as a novel class of hormones regulating growth and development in planta. To date, more than 30 SLs have been characterized, but the reason why plants produce structurally diverse SLs and the details of their biosynthetic pathway remain elusive. Recent studies using transcriptomics and reverse genetic techniques have paved the way to clarify the entire biosynthetic pathway of structurally diverse SLs. In this review, we discuss how various SLs are synthesized and what SL structural diversity means for plant growth and development.

Evaluation and Quantification of Natural Strigolactones from Root Exudates.

Strigolactones (SLs) in the root exudates can be detected by germination assays with root parasitic weed seeds, but precise and accurate evaluation and quantification are possible only by chemical analysis with the liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here we describe methods for root exudate collection, sample preparation, and LC-MS/MS analysis of SLs.

A Rapid Method for Quantifying RNA and Phytohormones From a Small Amount of Plant Tissue.

Phytohormones are involved in most plant physiological processes and the quantification of endogenous phytohormone levels and related gene expressions is an important approach to studying phytohormone functions. However, the quantification of phytohormones is still challenging due to their extremely low endogenous level in plant tissues and their high chemical diversity. Therefore, developing a method to simultaneously quantify phytohormone levels and RNA would strongly facilitate comparative analyses of phytohormones and gene expression. The present work reports a convenient extraction protocol enabling multivariate analysis of phytohormones and RNA from small amounts of plant material (around 10 mg). This high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method demonstrates quantification of phytohormones and their related metabolites from four plant hormone classes: cytokinin, auxin, abscisic acid, and gibberellin. The UPLC-MS/MS method can quantify thirteen phytohormones and their metabolites simultaneously in 14 min. To validate the developed method, we determined the dynamic profiles of phytohormones and gene expressions in small axillary shoot buds in garden pea. This new method is applicable to quantification analysis of gene expression and multiple phytohormone classes in small amounts of plant materials. The results obtained using this method in axillary buds provide a basis for understanding the phytohormone functions in shoot branching regulation.

Do Phosphate and Cytokinin Interact to Regulate Strigolactone Biosynthesis or Act Independently?

Strigolactones (SLs) are essential host recognition signals for both root-parasitic plants and arbuscular mycorrhizal (AM) fungi in the rhizosphere, and in planta SLs or their metabolites function as a novel class of plant hormones that regulate various aspects of plant growth through crosstalk with other hormones. Although nutrient availability is one of the important factors influencing SL production and exudation, and phosphate (Pi) deficiency significantly promotes SL production and exudation in host plants of AM fungi, how nutrient availability modulates SL production and exudation remains elusive. Cytokinin (CK), a canonical plant hormone, has extensively been studied as a shoot branching promoter and its biosynthesis is also influenced by mineral nutrients, especially nitrate, indicating that CK might be another key factor that affect SL production and exudation. In the present study, we show that CKs (t-zeatin, benzyladenine, kinetin, and CPPU) applied to hydroponic culture media significantly suppressed the SL levels in both the root exudates and the root tissues of rice plants grown under Pi deficiency. In a split-root system, CK suppressed SL production locally, while Pi affected SL production systemically, suggesting that Pi and CK act on SL production independently in rice plants.

Hydroxyl carlactone derivatives are predominant strigolactones in Arabidopsis.

Strigolactones (SLs) regulate important aspects of plant growth and stress responses. Many diverse types of SL occur in plants, but a complete picture of biosynthesis remains unclear. In Arabidopsis thaliana, we have demonstrated that MAX1, a cytochrome P450 monooxygenase, converts carlactone (CL) into carlactonoic acid (CLA) and that LBO, a 2-oxoglutarate-dependent dioxygenase, can convert methyl carlactonoate (MeCLA) into a metabolite called [MeCLA + 16 Da]. In the present study, feeding experiments with deuterated MeCLAs revealed that [MeCLA + 16 Da] is hydroxymethyl carlactonoate (1'-HO-MeCLA). Importantly, this LBO metabolite was detected in plants. Interestingly, other related compounds, methyl 4-hydroxycarlactonoate (4-HO-MeCLA) and methyl 16-hydroxycarlactonoate (16-HO-MeCLA), were also found to accumulate in lbo mutants. 3-HO-, 4-HO-, and 16-HO-CL were detected in plants, but their expected corresponding metabolites, HO-CLAs, were absent in max1 mutants. These results suggest that HO-CL derivatives may be predominant SLs in Arabidopsis, produced through MAX1 and LBO.

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.

Strigolactones (SLs) are a group of plant apocarotenoids that act as rhizosphere signaling molecules for both arbuscular mycorrhizal fungi and root parasitic plants. They also regulate plant architecture as phytohormones. The model legume Lotus japonicus (synonym of Lotus corniculatus) produces canonical 5-deoxystrigol (5DS) and non-canonical lotuslactone (LL). The biosynthesis pathways of the two SLs remain elusive. In this study, we characterized the L. japonicus MAX1 homolog, LjMAX1, found in the Lotus japonicus genome assembly build 2.5. The L. japonicus max1 LORE1 insertion mutant was deficient in 5DS and LL production. A recombinant LjMAX1 protein expressed in yeast microsomes converted carlactone (CL) to 18-hydroxycarlactonoic acid (18-OH-CLA) via carlactonoic acid (CLA). Identity of 18-OH-CLA was confirmed by comparison of the methyl ester derivative of the MAX1 product with chemically synthesized methyl 18-hydroycarlactonoate (18-OH-MeCLA) using LC-MS/MS. (11R)-CL was detected as an endogenous compound in the root of L. japonicus.13C-labeled CL, CLA, and 18-OH-MeCLA were converted to [13C]-5DS and LL in plant feeding experiments using L. japonicus WT. These results showed that LjMAX1 is the crucial enzyme in the biosynthesis of Lotus SLs and that 18-hydroxylated carlactonoates are possible precursors for SL biosynthesis in L. japonicus.

How Do Strigolactones Ameliorate Nutrient Deficiencies in Plants?

Strigolactones (SLs), a group of plant secondary metabolites, play an important role as a host recognition signal for symbiotic arbuscular mycorrhizal (AM) fungi in the rhizosphere. SLs promote symbioses with other beneficial microbes, including root nodule bacteria. Root parasitic weeds also take advantage of SLs as a clue to locate living host roots. In plants, SLs function as plant hormones regulating various growth and developmental processes including shoot and root architectures. Plants under nutrient deficiencies, especially that of phosphate, promote SL production and exudation to attract symbionts and to optimize shoot and root architecture.

Regulation of biosynthesis, perception, and functions of strigolactones for promoting arbuscular mycorrhizal symbiosis and managing root parasitic weeds.

Strigolactones (SLs) are carotenoid-derived plant secondary metabolites that play important roles in various aspects of plant growth and development as plant hormones, and in rhizosphere communications with symbiotic microbes and also root parasitic weeds. Therefore, sophisticated regulation of the biosynthesis, perception and functions of SLs is expected to promote symbiosis of beneficial microbes including arbuscular mycorrhizal (AM) fungi and also to retard parasitism by devastating root parasitic weeds. We have developed SL mimics with different skeletons, SL biosynthesis inhibitors acting at different biosynthetic steps, SL perception inhibitors that covalently bind to the SL receptor D14, and SL function inhibitors that bind to the serine residue at the catalytic site. In greenhouse pot tests, TIS108, an azole-type SL biosynthesis inhibitor effectively reduced numbers of attached root parasites Orobanche minor and Striga hermonthica without affecting their host plants; tomato and rice, respectively. AM colonization resulted in weak but distinctly enhanced plant resistance to pathogens. SL mimics can be used to promote AM symbiosis and to reduce the application rate of systemic-acquired resistance inducers which are generally phytotoxic to horticultural crops. © 2019 Society of Chemical Industry.