Abscisic acid influences tillering by modulation of strigolactones in barley.

Strigolactones (SLs) represent a class of plant hormones that are involved in inhibiting shoot branching and in promoting abiotic stress responses. There is evidence that the biosynthetic pathways of SLs and abscisic acid (ABA) are functionally connected. However, little is known about the mechanisms underlying the interaction of SLs and ABA, and the relevance of this interaction for shoot architecture. Based on sequence homology, four genes (HvD27, HvMAX1, HvCCD7, and HvCCD8) involved in SL biosynthesis were identified in barley and functionally verified by complementation of Arabidopsis mutants or by virus-induced gene silencing. To investigate the influence of ABA on SLs, two transgenic lines accumulating ABA as a result of RNAi-mediated down-regulation of HvABA 8'-hydroxylase 1 and 3 were employed. LC-MS/MS analysis confirmed higher ABA levels in root and stem base tissues in these transgenic lines. Both lines showed enhanced tiller formation and lower concentrations of 5-deoxystrigol in root exudates, which was detected for the first time as a naturally occurring SL in barley. Lower expression levels of HvD27, HvMAX1, HvCCD7, and HvCCD8 indicated that ABA suppresses SL biosynthesis, leading to enhanced tiller formation in barley.

The interaction of strigolactones with abscisic acid during the drought response in rice.

Both strigolactones (SLs) and abscisic acid (ABA) biosynthetically originate from carotenoids. Considering their common origin, the interaction of these two hormones at the biosynthetic and/or regulatory level may be anticipated. Here we show that, in rice, drought simultaneously induces SL production in the root, and ABA production and the expression of SL biosynthetic genes in the shoot. Under control conditions, the ABA concentration was higher in shoots of the SL biosynthetic rice mutants dwarf10 (d10) and d17 than in wild-type plants, while a similar trend was observed for the SL perception mutant d3. These differences were enhanced under drought. However, drought did not result in an increase in leaf ABA content in the rice mutant line d27, carrying a mutation in the gene encoding the first committed enzyme in SL biosynthesis, to the same extent as in the other SL mutants and the wild type. Accordingly, d10, d17, and d3 lines were more drought tolerant than wild-type plants, whereas d27 displayed decreased tolerance. Finally, overexpression of OsD27 in rice resulted in increased levels of ABA when compared with wild-type plants. We conclude that the SL and ABA pathways are connected with each other through D27, which plays a crucial role in determining ABA and SL content in rice.

Genetic variation in Sorghum bicolor strigolactones and their role in resistance against Striga hermonthica.

Sorghum is an important food, feed, and industrial crop worldwide. Parasitic weeds of the genus Striga constitute a major constraint to sorghum production, particularly in the drier parts of the world. In this study we analysed the Striga germination stimulants, strigolactones, in the root exudates of 36 sorghum genotypes and assessed Striga germination and infection. Low germination-stimulating activity and low Striga infection correlated with the exudation of low amounts of 5-deoxystrigol and high amounts of orobanchol, whereas susceptibility to Striga and high germination-stimulating activity correlated with high concentrations of 5-deoxystrigol and low concentrations of orobanchol. Marker analysis suggested that similar genetics to those previously described for the resistant sorghum variety SRN39 and the susceptible variety Shanqui Red underlie these differences. This study shows that the strigolactone profile in the root exudate of sorghum has a large impact on the level of Striga infection. High concentrations of 5-deoxystrigol result in high infection, while high concentrations of orobanchol result in low infection. This knowledge should help to optimize the use of low germination stimulant-based resistance to Striga by the selection of sorghum genotypes with strigolactone profiles that favour normal growth and development, but reduce the risk of Striga infection.

Zealactones. Novel natural strigolactones from maize.

In the root exudate and root extracts of maize hybrid cv NK Falkone seven putative strigolactones were detected using UPLC-TQ-MS-MS. All seven compounds displayed MS-MS-fragmentation common for strigolactones and particularly the presence of a fragment of m/z 97 Da, which may indicate the presence of the so-called D-ring, suggests they are strigolactones. The levels of all these putative strigolactones increased upon phosphate starvation and decreased upon fluridone (carotenoid biosynthesis inhibitor) treatment, both of which are a common response for strigolactones. All seven compounds were subsequently isolated with prep-HPLC-MS. They all exhibited Striga hermonthica seed germination inducing activity just as the synthetic strigolactone analog GR24. The structure of two of the seven compounds was elucidated by NMR spectroscopy as: methyl (2E,3E)-4-(3,3-dimethyl-5-oxo-2-(prop-1-en-2-yl)tetrahydrofuran-2-yl)-2-(((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)methylene)but-3-enoate (two diastereomers 1a and 1b). Strigolactones (1a/b) are closely related to the methyl ester of carlactonoic acid (MeCLA) and heliolactone. However, they contain a unique 4,4-dimethyltetrahydrofuran-2-one motif as the "A-ring" instead of the classical (di)methylcyclohexene. Because these compounds were isolated from maize (Zea mays) we called them "zealactone 1a and 1b". The implications of this discovery for our view on strigolactones and their biosynthesis are discussed.

Evaluation of field resistance to Striga hermonthica (Del.) Benth. in Sorghum bicolor (L.) Moench. The relationship with strigolactones.

BACKGROUND: Significant losses in sorghum biomass and grain yield occur in sub-Saharan Africa owing to infection by the root-parasitic weed Striga hermonthica (Del.) Benth. One strategy to avoid these losses is to adopt resistant crop varieties. For further delineation of the role of germination stimulants in resistance, we conducted a field experiment employing six sorghum genotypes, in eastern Sudan, and in parallel analysed the strigolactone levels in the root exudates of these genotypes under controlled conditions in Wageningen. RESULTS: The root exudates of these genotypes displayed large differences in strigolactone composition and Striga-germination-inducing activity. Korokollow, Fakimustahi and Wadfahel exuded the highest amounts of 5-deoxystrigol. Fakimustahi was by far the highest sorgomol producer, and Wadbaco and SRN39 produced the highest amount of orobanchol. The concentration of 5-deoxystrigol in the root exudate showed a significant positive correlation with in vitro Striga germination and was positively associated with Striga infection in the field experiments, whereas orobanchol was negatively associated with Striga infection in the field experiments. CONCLUSION: For the first time a close association is reported between strigolactone levels analysed under laboratory conditions and Striga infection in the field in sorghum genotypes. These genotypes may be used for further study of this resistance mechanism and for the introgression of the low germination trait in other sorghum varieties to breed for a strigolactone composition with low stimulant activity. The use of such improved varieties in combination with other Striga management tools could possibly alleviate the current Striga problem on the African continent. © 2016 Society of Chemical Industry.

Characterization of Low-Strigolactone Germplasm in Pea (Pisum sativum L.) Resistant to Crenate Broomrape (Orobanche crenata Forsk.).

Crenate broomrape (Orobanche crenata Forsk.) is a devastating parasitic weed threatening the cultivation of legumes around the Mediterranean and in the Middle East. So far, only moderate levels of resistance were reported to occur in pea (Pisum sativum L.) natural germplasm, and most commercial cultivars are prone to severe infestation. Here, we describe the selection of a pea line highly resistant to O. crenata, following the screening of local genetic resources. Time series observations show that delayed emergence of the parasite is an important parameter associated with broomrape resistance. High performance liquid chromatography connected to tandem mass spectrometry analysis and in vitro broomrape germination bioassays suggest that the resistance mechanism might involve the reduced secretion of strigolactones, plant hormones exuded by roots and acting as signaling molecules for the germination of parasitic weeds. Two years of replicated trials in noninfested fields indicate that the resistance is devoid of pleiotropic effects on yield, in contrast to pea experimental mutants impaired in strigolactone biosynthesis and, thus, is suitable for use in breeding programs.

Rhizobium Lipo-chitooligosaccharide Signaling Triggers Accumulation of Cytokinins in Medicago truncatula Roots.

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Osmotic stress represses strigolactone biosynthesis in Lotus japonicus roots: exploring the interaction between strigolactones and ABA under abiotic stress.

MAIN CONCLUSION: Strigolactone changes and cross talk with ABA unveil a picture of root-specific hormonal dynamics under stress. Strigolactones (SLs) are carotenoid-derived hormones influencing diverse aspects of development and communication with (micro)organisms, and proposed as mediators of environmental stimuli in resource allocation processes; to contribute to adaptive adjustments, therefore, their pathway must be responsive to environmental cues. To investigate the relationship between SLs and abiotic stress in Lotus japonicus, we compared wild-type and SL-depleted plants, and studied SL metabolism in roots stressed osmotically and/or phosphate starved. SL-depleted plants showed increased stomatal conductance, both under normal and stress conditions, and impaired resistance to drought associated with slower stomatal closure in response to abscisic acid (ABA). This confirms that SLs contribute to drought resistance in species other than Arabidopsis. However, we also observed that osmotic stress rapidly and strongly decreased SL concentration in tissues and exudates of wild-type Lotus roots, by acting on the transcription of biosynthetic and transporter-encoding genes and independently of phosphate abundance. Pre-treatment with exogenous SLs inhibited the osmotic stress-induced ABA increase in wild-type roots and down-regulated the transcription of the ABA biosynthetic gene LjNCED2. We propose that a transcriptionally regulated, early SL decrease under osmotic stress is needed (but not sufficient) to allow the physiological increase of ABA in roots. This work shows that SL metabolism and effects on ABA are seemingly opposite in roots and shoots under stress.

Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis.

Strigolactones (SLs) are a class of phytohormones and rhizosphere signaling compounds with high structural diversity. Three enzymes, carotenoid isomerase DWARF27 and carotenoid cleavage dioxygenases CCD7 and CCD8, were previously shown to convert all-trans-ß-carotene to carlactone (CL), the SL precursor. However, how CL is metabolized to SLs has remained elusive. Here, by reconstituting the SL biosynthetic pathway in Nicotiana benthamiana, we show that a rice homolog of Arabidopsis More Axillary Growth 1 (MAX1), encodes a cytochrome P450 CYP711 subfamily member that acts as a CL oxidase to stereoselectively convert CL into ent-2'-epi-5-deoxystrigol (B-C lactone ring formation), the presumed precursor of rice SLs. A protein encoded by a second rice MAX1 homolog then catalyzes the conversion of ent-2'-epi-5-deoxystrigol to orobanchol. We therefore report that two members of CYP711 enzymes can catalyze two distinct steps in SL biosynthesis, identifying the first enzymes involved in B-C ring closure and a subsequent structural diversification step of SLs.

Differential activity of Striga hermonthica seed germination stimulants and Gigaspora rosea hyphal branching factors in rice and their contribution to underground communication.

Strigolactones (SLs) trigger germination of parasitic plant seeds and hyphal branching of symbiotic arbuscular mycorrhizal (AM) fungi. There is extensive structural variation in SLs and plants usually produce blends of different SLs. The structural variation among natural SLs has been shown to impact their biological activity as hyphal branching and parasitic plant seed germination stimulants. In this study, rice root exudates were fractioned by HPLC. The resulting fractions were analyzed by MRM-LC-MS to investigate the presence of SLs and tested using bioassays to assess their Striga hermonthica seed germination and Gigaspora rosea hyphal branching stimulatory activities. A substantial number of active fractions were revealed often with very different effect on seed germination and hyphal branching. Fractions containing (-)-orobanchol and ent-2'-epi-5-deoxystrigol contributed little to the induction of S. hermonthica seed germination but strongly stimulated AM fungal hyphal branching. Three SLs in one fraction, putative methoxy-5-deoxystrigol isomers, had moderate seed germination and hyphal branching inducing activity. Two fractions contained strong germination stimulants but displayed only modest hyphal branching activity. We provide evidence that these stimulants are likely SLs although no SL-representative masses could be detected using MRM-LC-MS. Our results show that seed germination and hyphal branching are induced to very different extents by the various SLs (or other stimulants) present in rice root exudates. We propose that the development of rice varieties with different SL composition is a promising strategy to reduce parasitic plant infestation while maintaining symbiosis with AM fungi.

OsJAR1 is required for JA-regulated floret opening and anther dehiscence in rice.

Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.

Striga hermonthica MAX2 restores branching but not the Very Low Fluence Response in the Arabidopsis thaliana max2 mutant.

Seed germination of Striga spp. (witchweeds), one of the world's most destructive parasitic weeds, cannot be induced by light but is specifically induced by strigolactones. It is not known whether Striga uses the same components for strigolactone signaling as host plants, whether it has endogenous strigolactone biosynthesis and whether there is post-germination strigolactone signaling in Striga. Strigolactones could not be detected in in vitro grown Striga, while for host-grown Striga, the strigolactone profile is dominated by a subset of the strigolactones present in the host. Branching of in vitro grown Striga is affected by strigolactone biosynthesis inhibitors. ShMAX2, the Striga ortholog of Arabidopsis MORE AXILLARY BRANCHING 2 (AtMAX2) - which mediates strigolactone signaling - complements several of the Arabidopsis max2-1 phenotypes, including the root and shoot phenotype, the High Irradiance Response and the response to strigolactones. Seed germination of max2-1 complemented with ShMAX2 showed no complementation of the Very Low Fluence Response phenotype of max2-1. Results provide indirect evidence for ShMAX2 functions in Striga. A putative role of ShMAX2 in strigolactone-dependent seed germination of Striga is discussed.

Natural variation of rice strigolactone biosynthesis is associated with the deletion of two MAX1 orthologs.

Rice (Oryza sativa) cultivar Azucena--belonging to the Japonica subspecies--exudes high strigolactone (SL) levels and induces high germination of the root parasitic plant Striga hermonthica. Consistent with the fact that SLs also inhibit shoot branching, Azucena is a low-tillering variety. In contrast, Bala, an Indica cultivar, is a low-SL producer, stimulates less Striga germination, and is highly tillered. Using a Bala × Azucena F6 population, a major quantitative trait loci--qSLB1.1--for the exudation of SL, tillering, and induction of Striga germination was detected on chromosome 1. Sequence analysis of the corresponding locus revealed a rearrangement of a 51- to 59-kbp stretch between 28.9 and 29 Mbp in the Bala genome, resulting in the deletion of two cytochrome P450 genes--SLB1 and SLB2--with high homology to the Arabidopsis SL biosynthesis gene, MAX1. Both rice genes rescue the Arabidopsis max1-1 highly branched mutant phenotype and increase the production of the SL, ent-2'-epi-5-deoxystrigol, when overexpressed in Bala. Furthermore, analysis of this region in 367 cultivars of the publicly available Rice Diversity Panel population shows that the rearrangement at this locus is a recurrent natural trait associated with the Indica/Japonica divide in rice.

New strigolactone mimics: structure-activity relationship and mode of action as germinating stimulants for parasitic weeds.

Strigolactones (SLs) are new plant hormones with varies important bio-functions. This Letter deals with germination of seeds of parasitic weeds. Natural SLs have a too complex structure for synthesis. Therefore, there is an active search for SL analogues and mimics with a simpler structure with retention of activity. SL analogues all contain the D-ring connected with an enone moiety through an enol ether unit. A new mechanism for the hydrolysis SL analogues involving bidentate bound water and an α,ß-hydrolase with a Ser-His-Asp catalytic triad has been proposed. Newly discovered SL mimics only have the D-ring with an appropriate leaving group at C-5. A mode of action for SL mimics was proposed for which now supporting evidence is provided. As predicted an extra methyl group at C-4 of the D-ring blocks the germination of seeds of parasitic weeds.

Carotenoid cleavage dioxygenase 7 modulates plant growth, reproduction, senescence, and determinate nodulation in the model legume Lotus japonicus.

Strigolactones (SLs) are newly identified hormones that regulate multiple aspects of plant development, infection by parasitic weeds, and mutualistic symbiosis in the roots. In this study, the role of SLs was studied for the first time in the model plant Lotus japonicus using transgenic lines silenced for carotenoid cleavage dioxygenase 7 (LjCCD7), the orthologue of Arabidopsis More Axillary Growth 3. Transgenic LjCCD7-silenced plants displayed reduced height due to shorter internodes, and more branched shoots and roots than the controls, and an increase in total plant biomass, while their root:shoot ratio remained unchanged. Moreover, these lines had longer primary roots, delayed senescence, and reduced flower/pod numbers from the third round of flower and pod setting onwards. Only a mild reduction in determinate nodule numbers and hardly any impact on the colonization by arbuscular mycorrhizal fungi were observed. The results show that the impairment of CCD7 activity in L. japonicus leads to a phenotype linked to SL functions, but with specific features possibly due to the peculiar developmental pattern of this plant species. It is believed that the data also link determinate nodulation, plant reproduction, and senescence to CCD7 function for the first time.

Tomato strigolactones: a more detailed look.

Strigolactones are plant signaling molecules that induce germination of parasitic plant seeds, initiate host plant - arbuscular mycorrhizal fungus symbiosis and act as plant hormones controlling shoot branching and root architecture. To date four unique strigolactones (e.g., orobanchol, didehydroorobanchol isomers 1 and 2 and the aromatic strigolactone solanacol) have been reported in the root exudates and extracts of tomato (Solanum lycopersicum). Here we report on the presence of several additional strigolactones in tomato root exudates and extracts, orobanchyl acetate, two 7-hydroxyorobanchol isomers, 7-oxoorobanchol and two additional didehydroorobanchol isomers and discuss their possible biological relevance.

The tomato CAROTENOID CLEAVAGE DIOXYGENASE8 (SlCCD8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis.

Strigolactones are plant hormones that regulate both above- and belowground plant architecture. Strigolactones were initially identified as rhizosphere signaling molecules. In the present work, the tomato (Solanum lycopersicum) CAROTENOID CLEAVAGE DIOXYGENASE 8 (SlCCD8) was cloned and its role in rhizosphere signaling and plant physiology assessed by generating knock-down lines. Transgenic SlCCD8 plants were generated by RNAi-mediated silencing. Lines with different levels of strigolactone reduction--confirmed by UPLC-MS/MS--were selected and their phenotypes investigated. Lines exhibiting reduced SlCCD8 levels displayed increased shoot branching, reduced plant height, increased number of nodes and excessive adventitious root development. In addition, these lines exhibited reproductive phenotypes such as smaller flowers, fruits, as well as fewer and smaller seeds per fruit. Furthermore, we show that strigolactone loading to the xylem sap is possibly restricted to orobanchol. Infestation by Phelipanche ramosa was reduced by 90% in lines with a relatively mild reduction in strigolactone biosynthesis and secretion while arbuscular mycorrhizal symbiosis, apical dominance and fruit yield were only mildly affected. This demonstrates that reduction of strigolactone biosynthesis could be a suitable tool in parasitic weed management. Furthermore, our results suggest that strigolactones are involved in even more physiological processes than so far assumed.

Genetic variation in strigolactone production and tillering in rice and its effect on Striga hermonthica infection.

Tillering in cereals is a complex process in the regulation of which also signals from the roots in the form of strigolactones play an important role. The strigolactones are signalling molecules that are secreted into the rhizosphere where they act as germination stimulants for root parasitic plants and hyphal branching factors for arbuscular mycorrhizal fungi. On the other hand, they are also transported from the roots to the shoot where they inhibit tillering or branching. In the present study, the genetic variation in strigolactone production and tillering phenotype was studied in twenty rice varieties collected from all over the world and correlated with S. hermonthica infection. Rice cultivars like IAC 165, IAC 1246, Gangweondo and Kinko produced high amounts of the strigolactones orobanchol, 2'-epi-5-deoxystrigol and three methoxy-5-deoxystrigol isomers and displayed low amounts of tillers. These varieties induced high S. hermonthica germination, attachment, emergence as well as dry biomass. In contrast, rice cultivars such as Super Basmati, TN 1, Anakila and Agee displayed high tillering in combination with low production of the aforementioned strigolactones. These varieties induced only low S. hermonthica germination, attachment, emergence and dry biomass. Statistical analysis across all the varieties confirmed a positive correlation between strigolactone production and S. hermonthica infection and a negative relationship with tillering. These results show that genetic variation in tillering capacity is the result of genetic variation in strigolactone production and hence could be a helpful tool in selecting rice cultivars that are less susceptible to S. hermonthica infection.