Planteose as a storage carbohydrate required for early stage of germination of Orobanche minor and its metabolism as a possible target for selective control.

Root parasitic weeds in Orobanchaceae cause serious damage to worldwide agriculture. Germination of the parasites requires host-derived germination stimulants, such as strigolactones, as indicators of host roots within reach of the parasite's radicles. This unique germination process was focused on to identify metabolic pathways required for germination, and to design a selective control strategy. A metabolomic analysis of germinating seeds of clover broomrape, Orobanche minor, was conducted to identify its distinctive metabolites. Consequently, a galactosyl-sucrose trisaccharide, planteose (α-d-galactopyranosyl-(1→6)-ß-d-fructofuranosyl-(2→1)-α-d-glucopyranoside), was identified as a metabolite that decreased promptly after reception of the germination stimulant. To investigate the importance of planteose metabolism, the effects of several glycosidase inhibitors were examined, and nojirimycin bisulfite (NJ) was found to alter the sugar metabolism and to selectively inhibit the germination of O. minor. Planteose consumption was similar in NJ-treated seeds and non-treated germinating seeds; however, NJ-treated seeds showed lower consumption of sucrose, a possible intermediate of planteose metabolism, resulting in significantly less glucose and fructose. This inhibitory effect was recovered by adding glucose. These results suggest that planteose is a storage carbohydrate required for early stage of germination of O. minor, and NJ inhibits germination by blocking the supply of essential glucose from planteose and sucrose. Additionally, NJ selectively inhibited radicle elongation of germinated seeds of Orobanchaceae plants (Striga hermonthica and Phtheirospermum japonicum). Thus, NJ will be a promising tool to develop specific herbicides to the parasites, especially broomrapes, and to improve our understanding of the molecular mechanisms of this unique germination.

Strigolactones as germination stimulants for root parasitic plants.

Witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) are the two most devastating root parasitic plants belonging to the family Orobanchaceae and are causing enormous crop losses throughout the world. Seeds of these root parasites will not germinate unless they are exposed to chemical stimuli, 'germination stimulants' produced by and released from plant roots. Most of the germination stimulants identified so far are strigolactones (SLs), which also function as host recognition signals for arbuscular mycorrhizal fungi and a novel class of plant hormones inhibiting shoot branching. In this review, we focus on SLs as germination stimulants for root parasitic plants. In addition, we discuss how quantitative and qualitative differences in SL exudation among sorghum cultivars influence their susceptibility to Striga.

Unique phytochrome responses of the holoparasitic plant Orobanche minor.

Holoparasitic plants such as Orobanche spp. have lost their photosynthetic ability, so photoresponses to optimize photosynthesis are not necessary in these plants. Photoresponses are also involved in the regulation of plant development but the photoresponses of holoparasites have not been characterized in detail. In this study, the phytochrome (phy)-related photoresponse of Orobanche minor was investigated. Its photoreceptor, phytochrome A (OmphyA), was also characterized. Light effects on germination, shoot elongation, anthocyanin biosynthesis, and OmphyA expression and subcellular localization were analyzed. Red light (R):far-red light (FR) reversible inhibition of O. minor seed germination demonstrated that phy-mediated responses are retained in this holoparasite. Shoot elongation was inhibited by FR but not by R. This pattern is unique among known patterns of plant photoresponses. Additionally, molecular analysis showed that OmphyA is able to respond to the light signals. Interestingly, the unique pattern of photoresponses in O. minor seems to have been modified for adaptation to its parasitic life cycle. We hypothesize that this alteration has resulted from the loss or alteration of some phy-signaling components. Elucidation of altered components in phy signaling in this parasite will provide useful information not only about its physiological characteristics but also about general plant photoreception systems.

Fabacyl acetate, a germination stimulant for root parasitic plants from Pisum sativum.

A germination stimulant, fabacyl acetate, was purified from root exudates of pea (Pisum sativum L.) and its structure was determined as ent-2'-epi-4a,8a-epoxyorobanchyl acetate [(3aR,4R,4aR,8bS,E)-4a,8a-epoxy-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-decahydro-2H-indeno[1,2-b]furan-4-yl acetate], by 1D and 2D NMR spectroscopic, ESI- and EI-MS spectrometric, X-ray crystallographic analyses, and by comparing the (1)H NMR spectroscopic data and relative retention times (RR(t)) in LC-MS and GC-MS with those of synthetic standards prepared from (+)-orobanchol and (+)-2'-epiorobanchol. The (1)H NMR spectroscopic data and RR(t) of fabacyl acetate were identical with those of an isomer prepared from (+)-2'-epiorobanchol except for the opposite sign in CD spectra. This is the first natural ent-strigolactone containing an epoxide group. Fabacyl acetate was previously detected in root exudates of other Fabaceae plants including faba bean (Vicia faba L.) and alfalfa (Medicago sativa L.).

7-Oxoorobanchyl acetate and 7-Oxoorobanchol as germination stimulants for root parasitic plants from flax (Linum usitatissimum).

Germination stimulants for root parasitic plants produced by flax (Linum usitatissimum L.) were purified and characterized. The root exudate of flax contained at least 8 active fractions, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) analyses suggested that there were 6 strigolactones. Two of them were identified as orobanchol and orobanchyl acetate by comparing NMR and GC-MS and LC-MS/MS data with those of synthetic standards. One of the two novel strigolactones was purified and determined as 7-oxoorobanchyl acetate [((3aS,4S,8bS,E)-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2,7-dioxo-3,3a,4,5,6,7,8,8b-octahydro-2H-indeno[1,2-b]furan-4-yl acetate) by 1D and 2D NMR spectroscopic, and ESI- and EI-MS spectrometric analyses. The other one was also purified and identified as 7-oxoorobanchol. The remaining two compounds could not been characterized due to their scarcity.

Isolation and identification of alectrol as (+)-orobanchyl acetate, a germination stimulant for root parasitic plants.

Alectrol, a germination stimulant for root parasitic plants, was purified from root exudates of red clover (Trifolium pratense L.) and identified as a strigolactone, (+)-orobanchyl acetate [(3aS,4S,8bS,E)-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-octahydro-2H-indeno[1,2-b]furan-4-yl acetate], by 1D and 2D NMR spectroscopy and ESI- and EI-MS spectrometry. Orobanchyl acetate afforded an [M-42](+) ion in EI-MS and thus had been recognized as an isomer of strigol. Orobanchyl acetate was detected in root exudates of soybean (Glycine max L.) and cowpea (Vigina unguiculata L.) along with orobanchol.

Brz220 interacts with DWF4, a cytochrome P450 monooxygenase in brassinosteroid biosynthesis, and exerts biological activity.

Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.

Progesterone: its occurrence in plants and involvement in plant growth.

Progesterone is a mammalian gonadal hormone. In the current study, we identified and quantified progesterone in a range of higher plants by using GC-MS and examined its effects on the vegetative growth of plants. The growth of Arabidopsis (Arabidopsis thaliana) seedlings was promoted by progesterone at low concentrations but suppressed at higher concentrations under both light and dark growth conditions. The growth of the gibberellin-deficient mutant lh of pea (Pisum sativum) was also promoted by progesterone. An earlier study demonstrated that progesterone binds to MEMBRANE STEROID BINDING PROTEIN 1 (MSBP1) of Arabidopsis. In this work, we cloned the homologous genes of Arabidopsis, MSBP2 and STEROID BINDING PROTEIN (SBP), as well as of rice (Oryza sativa), OsMSBP1, OsMSBP2 and OsSBP and examined their expression in plant tissues. All of these genes, except OsMSBP1, were expressed abundantly in plant tissues. The roles of progesterone in plant growth were also discussed.

2'-epi-orobanchol and solanacol, two unique strigolactones, germination stimulants for root parasitic weeds, produced by tobacco.

Germination stimulants for root holoparasitic weeds broomrapes ( Orobanche and Phelipanche spp.) produced by tobacco ( Nicotiana tabacum L.) were purified and characterized. The root exudates of tobacco contained at least five different stimulants, and LC-MS/MS analyses revealed that four of them were strigolactones; a tetradehydrostrigol isomer, a didehydrostrigol isomer, and two strigol isomers. The two isomers of strigol were identified as (+)-orobanchol and its 2'-epimer by comparison of NMR and GC- and LC-MS data with those of synthetic standards. The structure of the tetradehydrostrigol isomer, the major stimulant of the bright yellow tobacco cultivars, was determined as 4-alpha-hydroxy-5,8-dimethyl-GR24 [( E)-4-alpha-hydroxy-5,8-dimethyl-3-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-3a,4-dihydro-3 H-indeno[1,2- b]furan-2(8b H)-one] and named solanacol. 2'-Epi-orobanchol and solanacol are the first natural strigolactones having a 2'-epi stereochemistry and a benzene ring, respectively.

Cloning of a cryptochrome homologue from the holoparasitic plant Orobanche minor Sm.

Orobanche minor is a non-photosynthetic root holoparasitic plant. Although it is known that photosynthesis-related genes are inactivated or have been eliminated from the plastid genomes of holoparasites, little is known about the alterations in their genes involved in the signaling networks by which light regulates photosynthesis. Cryptochromes (crys), which are blue-light receptors, appear to control both photosynthesis-related and non-photosynthetic responses to light in higher plants. Because we are interested in to what extent a cry-mediated light signaling network remains in the holoparasites, we cloned CRY homologous cDNA from O. minor (OmCRY1) and used real-time RT-PCR to compare its expression under natural daylight and darkness. We found that the OmCRY1 has a high degree of homology with CRY1 s from photosynthetic plants. Expression of the OmCRY1 gene was higher in plants grown in the dark than that in the plants grown under natural daylight. This is the first report of the gene expression of a blue-light receptor in non-photosynthetic plants.

Production of clover broomrape seed germination stimulants by red clover root requires nitrate but is inhibited by phosphate and ammonium.

The effect of nutrients (nitrate, ammonium, urea, phosphate and potassium) on the production and/or exudation of germination stimulants for clover broomrape (Orobanche minor Sm.), a root holoparasite, by its host red clover (Trifolium pratense L.) was examined using hydroponically grown material. Potassium (K2SO4) concentrations up to 100 mg l-1 (based on K) did not affect the production of germination stimulants by red clover while, in contrast, phosphate (NaH2PO4) was highly inhibitory even at concentrations as low as 1 mg l-1 (based on P). Nitrate (NaNO3) markedly promoted stimulant production in a dose-dependent manner from 2 to 50 mg l-1 (based on N). Ammonium [(NH4)2SO4] had no effect at 2 mg l-1 (based on N) but was inhibitory at higher concentrations. Ammonium is known to be a seed germination inhibitor of root parasites, indicating that ammonium has a dual inhibitory action. Urea had no effect at 2 mg l-1 (based on N) but was promotive at higher concentrations. These results provide a basis for the inhibitory effects of nitrogen fertilizer on infection by root parasitic weeds, broomrapes and witchweeds, and explain why these parasites prevail in areas where there is lower phosphorus availability in soils.

Fluridone and norflurazon, carotenoid-biosynthesis inhibitors, promote seed conditioning and germination of the holoparasite Orobanche minor.

Fluridone and norflurazon, two carotenoid-biosynthesis inhibitors, shortened the conditioning period required by seeds of Orobanche minor in order to respond to the germination stimulant strigol. Neither fluridone nor norflurazon alone induced seed germination of O. minor, they promoted strigol-induced germination. In addition, these compounds restored the conditioning and germination of seeds at a supraoptimal temperature (30 degrees C) as well as in the light. Gibberellic acid (GA(3)) showed similar promotive and protective effects on the conditioning and germination of O. minor seeds. Although fluridone and norflurazon are known to prevent abscisic acid (ABA)-biosynthesis, and stresses such as supraoptimal temperatures have been reported to induce ABA accumulation in plants, the amount of ABA in the seeds or that released from the seeds into the conditioning media was not affected by the fluridone treatment and by exposure to the supraoptimal temperature. These results indicate that the promotive and protective effects of fluridone and norflurazon on the conditioning and germination of O. minor seeds would be attributed to other perturbations rather than the inhibition of ABA-biosynthesis.

A specific and potent inhibitor of brassinosteroid biosynthesis possessing a dioxolane ring.

Screening for brassinosteroid biosynthesis inhibitors was performed to find azole derivatives that induced dwarfism, to resemble brassinosteroid-deficient mutants in Arabidopsis, and which could be rescued by brassinosteroid. Through this screening experiment, propiconazole fungicide was selected as a likely inhibitor of brassinosteroid biosynthesis and, thus, propiconazole derivatives with optimized activity and selectivity were synthesized. The biological activity of these compounds was evaluated by examining cress stem elongation. Among the compounds tested, 2RS,4RS-1-[2-(4-trifluoromethylphenyl)-4-n-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole (12) showed the most potent capability to retard cress stem elongation in the light. The compound-induced hypocotyl dwarfism was restored by the coapplication of 10 nM brassinolide but not by 1 microM gibberellin. These results suggest that 12 should affect brassinosteroid biosynthesis. The potency and specificity of 12 were greater than those of brassinazole, a previously reported brassinosteroid biosynthesis inhibitor.

Analysis of strigolactones, germination stimulants for striga and orobanche, by high-performance liquid chromatography/tandem mass spectrometry.

A simple and rapid analytical method for strigolactones, germination stimulants for the root parasitic weeds witchweed (Striga spp.) and broomrape (Orobanche spp.), has been developed using high-performance liquid chromatography connected to tandem mass spectrometry (LC/MS/MS). The natural strigolactones (strigol, sorgolactone, orobanchol, and alectrol) were clearly separated and identified by LC/MS/MS. As low as 0.1 pg/microL of strigol and 0.5 pg/microL of sorgolactone could be quantified, whereas 1 pg/microL was needed for the quantification of orobanchol (S/N > 10). Using this method, it was found that red clover produces orobanchol and alectrol but not strigol. The roots of red clover seedlings were found to produce 13, 70, 58, and 65 pg of orobanchol/plant 1, 2, 3, and 4 weeks after germination, respectively.

Strigolactones: structures and biological activities.

Strigolactones released from plant roots induce seed germination of root parasitic weeds, witchweeds (Striga spp.) and broomrapes (Orobanche spp.), and hyphal branching of symbiotic arbuscular mycorrhizal (AM) fungi. In addition to these functions in the rhizosphere, strigolactones have recently been shown to be a novel class of plant hormones regulating shoot outgrowth. The natural strigolactones identified so far have the common C-D ring moiety, which is thought to be the essential structure for exhibiting biological activity. The introduction of substitutions on the A-B ring moiety of 5-deoxystrigol, the basic strigolactone, affords various strigolactones, e.g. hydroxylation on C-4, C-5 and C-9 leads to orobanchol, strigol and sorgomol respectively. Then, acetylation and probably other derivatisations of these hydroxy-strigolactones would occur. Although the C-2'-(R) stereochemistry was thought to be an important structural feature for potent germination stimulation activity, 2'-epi-strigolactones were found in root exudates of tobacco, rice, pea and other plant species, indicating that at least some plants produce both epimers.

Strigolactones, host recognition signals for root parasitic plants and arbuscular mycorrhizal fungi, from Fabaceae plants.

Both root parasitic plants and arbuscular mycorrhizal (AM) fungi take advantage of strigolactones, released from plant roots as signal molecules in the initial communication with host plants, in order to commence parasitism and mutualism, respectively. In this study, strigolactones in root exudates from 12 Fabaceae plants, including hydroponically grown white lupin (Lupinus albus), a nonhost of AM fungi, were characterized by comparing retention times of germination stimulants on reverse-phase high-performance liquid chromatography (HPLC) with those of standards and by using tandem mass spectrometry (LC/MS/MS). All the plant species examined were found to exude known strigolactones, such as orobanchol, orobanchyl acetate, and 5-deoxystrigol, suggesting that these strigolactones are widely distributed in the Fabaceae. It should be noted that even the nonmycotrophic L. albus exuded orobanchol, orobanchyl acetate, 5-deoxystrigol, and novel germination stimulants. By contrast to the mycotrophic Fabaceae plant Trifolium pratense, in which phosphorus deficiency promoted strigolactone exudation, neither phosphorus nor nitrogen deficiency increased exudation of these strigolactones in L. albus. Therefore, the regulation of strigolactone production and/or exudation seems to be closely related to the nutrient acquisition strategy of the plants.

Phosphorus deficiency in red clover promotes exudation of orobanchol, the signal for mycorrhizal symbionts and germination stimulant for root parasites.

Plant derived sesquiterpene strigolactones, which have previously been characterized as germination stimulants for root parasitic plants, have recently been identified as the branching factors which induce hyphal branching morphogenesis, a critical step in host recognition by arbuscular mycorrhizal (AM) fungi. We show here that, in red clover plants (Trifolium pratense L.), which is known as a host for both AM fungi and the root holoparasitic plant Orobanche minor Sm., reduced supply of phosphorus (P) but not of other elements examined (N, K, Mg, Ca) in the culture medium significantly promotes the release of a strigolactone, orobanchol, by the roots of this plant. In red clover plants, the level of orobanchol exudation appeared to be regulated by P availability and was in good agreement with germination stimulation activity of the root exudates. This implies that under P deficiency, plant roots attract not only symbiotic fungi but also root parasitic plants through the release of strigolactones. This is the first report demonstrating that nutrient availability influences both symbiotic and parasitic interactions in the rhizosphere.

Resistance of red clover (Trifolium pratense) to the root parasitic plant Orobanche minor is activated by salicylate but not by jasmonate.

BACKGROUND AND AIMS: Obligate root holoparasites of the genus Orobanche attack dicotyledonous crops and cause severe losses in many parts of the world. Chemical induction of plant defence systems such as systemic acquired resistance was proposed to be an available strategy to control the root parasite, but the detailed mechanisms involved have not been clarified. The aim of this study was to elucidate the effects of salicylic acid (SA), jasmonic acid (JA) and their analogues on resistance of red clover to Orobanche parasitism. METHODS: Roots of red clover grown in plastic chambers were applied with SA, S-methyl benzo[1,2,3]thiadiazole-7-carbothioate (BTH), methyl jasmonate (MeJA) and n-propyl dihydrojasmonate (PDJ), and then were inoculated with O. minor seeds. Attachments of the parasite were observed after 5 weeks. KEY RESULTS: SA and BTH, inducers of SA-mediated defences, significantly reduced the number of established parasites by more than 75 %. By contrast, MeJA and PDJ, inducers of JA-mediated defences, did not affect parasitism. The reduction in the number of established parasites by SA and BTH was due to the inhibited elongation of O. minor radicles and the activation of defence responses in the host root including lignification of the endodermis. CONCLUSIONS: These results suggest that SA-induced resistance, but not JA-induced resistance, is effective in inhibiting Orobanche parasitism and that the resistance is expressed by the host root both externally and internally.

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.

Strigolactones released from plant roots induce hyphal branching of symbiotic arbuscular mycorrhizal (AM) fungi and germination of root parasitic weeds, Striga and Orobanche spp. We already demonstrated that, in red clover plants (Trifolium pratense L.), a host for both AM fungi and the root holoparasitic plant Orobanche minor Sm., reduced supply of phosphorus (P) but not of other elements examined (N, K, Ca, Mg) in the culture medium significantly promoted the secretion of a strigolactone, orobanchol, by the roots of this plant. Here we show that in the case of sorghum [Sorghum bicolor (L.) Moench], a host of both the root hemiparasitic plant Striga hermonthica and AM fungi, N deficiency as well as P deficiency markedly enhanced the secretion of a strigolactone, 5-deoxystrigol. The 5-deoxystrigol content in sorghum root tissues also increased under both N deficiency and P deficiency, comparable to the increase in the root exudates. These results suggest that strigolactones may be rapidly released after their production in the roots. Unlike the situation in the roots, neither N nor P deficiency affected the low content of 5-deoxystrigol in sorghum shoot tissues.

Roles of brassinosteroids and related mRNAs in pea seed growth and germination.

The levels of endogenous brassinosteroids (BRs) and the expression of the biosynthesis/metabolism/perception genes involved have been investigated during the development and germination of pea (Pisum sativum) seeds. When seeds were rapidly growing, the level of biologically active BRs (brassinolide [BL] and castasterone [CS]) and the transcript levels of two BR C-6 oxidases (CYP85A1 and CYP85A6) reached a maximum, suggesting the significance of BL and CS in seed development. In the early stages of germination, CS, but not BL, appeared and its level increased in the growing tissues in which the transcript level of CYP85A1 and CYP85A6 was high, suggesting the significance of CS in seed germination and early seedling growth of pea. 6-Deoxocathasterone (6-deoxoCT) was the quantitatively major BR in mature seeds. At the early stage of germination, the level of 6-deoxoCT was specifically decreased, whereas the levels of downstream intermediates were increased. It seems that 6-deoxoCT is the major storage BR and is utilized during germination and early growth stages. The level of the mRNAs of BR biosynthesis and perception genes fluctuated during seed development. In mature seeds, most of mRNAs were present, but the level was generally lower compared with immature seeds. However, CYP90A9 mRNA rapidly increased during seed development and reached the maximum in mature seeds. The mRNAs stored in mature pea seeds seem to be utilized when seeds germinate. However, it was found that de novo transcription of mRNAs also starts as early as during seed imbibition.