An improved axenic system for studying pre-infection development of the parasitic plant Orobanche ramosa

BACKGROUND AND AIMS: Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control early stages of host infection in Orobanche are poorly understood, partly due to the lack of experimentally tractable in vitro systems that allow the efficient application of molecular tools. Here an improved axenic system for the analysis of pre-infection stages in O. ramosa in the absence of the host plant is described. METHODS: An optimized protocol for seed disinfection, based on formaldehyde, was developed. Orobanche ramosa seeds were conditioned in Petri dishes with filter paper, stimulated by addition of the synthetic strigol analogue GR24, and the percentage of germination as well as attachment-organ formation was determined. KEY RESULTS: Treatment of O. ramosa seeds with tobacco-root exudate or with GR24 resulted in highly reproducible germination rates around 70 %. A conditioning period of 8 d was both necessary and sufficient to allow optimal germination in response to GR24. Conditioned seeds that were dehydrated for several months remained fully responsive to GR24 without the need of a new conditioning period. Treatments as short as 5 min with GR24 were sufficient to fully and irreversibly induce the seed germination response. Approximately half of the germinated seeds initiated attachment-organ development. Similar rates of attachment organ induction were also detected in the rare cases of seeds that had germinated spontaneously on water. CONCLUSIONS: The results suggest that the conditioning period produces persistent changes in the seeds required for responsiveness to external stimulants. The rapid action of GR24 suggests that it may act via a receptor-mediated signalling mechanism. While germination in O. ramosa is induced by exogenous stimuli, attachment organ differentiation appears to be triggered by unknown endogenous signals. The new in vitro culture system will have useful applications for the molecular analysis of early stages of parasitic development in Orobanche.     

IAA production during germination of Orobanche spp. seeds

Broomrapes (Orobanche spp.) are parasitic plants, whose growth and development fully depend on the nutritional connection established between the parasite and the roots of the respective host plant. Phytohormones are known to play a role in establishing the specific Orobanche-host plant interaction. The first step in the interaction is seed germination triggered by a germination stimulant secreted by the host-plant roots. We quantified indole-3-acetic acid (IAA) and abscisic acid (ABA) during the seed germination of tobacco broomrape (Orobanche ramosa) and sunflower broomrape (O. cumana). IAA was mainly released from Orobanche seeds in host-parasite interactions as compared to non-host-parasite interactions. Moreover, germinating seeds of O. ramosa released IAA as early as 24 h after the seeds were exposed to the germination stimulant, even before development of the germ tube. ABA levels remained unchanged during the germination of the parasites' seeds. The results presented here show that IAA production is probably part of a mechanism triggering germination upon the induction by the host factor, thus resulting in seed germination.     

Isolation of mannose 6-phosphate reductase cDNA,changes in enzyme activity and mannitol content in broomrape (Orobanche ramosa) parasitic on tomato roots

We are interested in developing a control strategy efficient at the early stages of subterranean development of Orobanche in the inhibition of mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), the key enzyme of mannitol production in the parasite. We examined M6PR gene expression during pre-conditioning, germination, procaulome growth, underground shoot development and emergence of Orobanche ramosa L. attached to tomato roots, the enzyme activity at each of the above stages and the level of stored mannitol in the parasite. A 1120-pb length cDNA isolated by 3' and 5'RACE was identified as a M6PR sequence by cDNA expression in E. coli and M6PR activity measurement. Only one M6PR gene was detected in O. ramosa following southern blot analysis. M6PR expression, analysed by RT-PCR, was constant from the pre-conditioned seed to the emergence of broomrape, i.e. M6PR expression is constitutive in Orobanche . M6PR activity was also detected in pre-conditioned seeds and attachment to tomato roots resulted in a two-fold increase in enzyme activity during tubercle enlargement and crown root formation. Hexose and mannitol accumulation was strongly enhanced in the attached parasite, with accumulation primarily in the shoot. These results support the prospect of utilizing M6PR inhibitors as early applied herbicides to control this parasite in the early stages of its development.     

Variability of interactions between barrel medic (Medicago truncatula) genotypes and Orobanche species

A number of broomrapes ( Orobanche spp.) are major threats for grain legumes in the Mediterranean area. Previous studies have shown very high levels of resistance to Orobanche crenata in Medicago truncatula with little variation among accessions hampering, therefore, its use for genetic analysis. In order to identify alternative systems for ulterior genetic and genomic analysis, we studied early stages of the interaction between M. truncatula accessions and a range of Orobanche species. We found significant differences in the induction of germination on Orobanche aegyptiaca , Orobanche foetida var. broteri , Orobanche minor , Orobanche nana and Orobanche ramosa seeds. Accessions also varied in the number of O. aegyptiaca , O. crenata , O. foetida var. broteri , O. nana and O. ramosa attachments supported. Applications of the synthetic germination stimulant GR24 increased the germination level of Orobanche cumana and O. minor . No attachments were observed for any of these species because of physical barriers typical of host resistance. On the contrary, increase of O. nana germination by GR24 led to an increase in number of attachments albeit the normal development of the nodules was stopped in a later stage. The genetic variation observed for induction of germination and subsequent attachment will be useful for isolating and characterising genes involved in early stages of Orobanche –host plant interaction and for the study of the biosynthetic pathways of production of germination stimulants.     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56–69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P<0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.     

Peroxiredoxins from the plant parasitic root-knot nematode, Meloidogyne incognita, are required for successful development within the host

Root-knot nematodes, Meloidogyne spp., are sedentary biotrophic parasites which are able to infest > 2000 plant species. After root invasion they settle sedentarily inside the vascular cylinder and maintain a compatible interaction for up to 8 weeks. Plant cells respond to pathogen attacks by producing reactive oxygen species (ROS). These ROS, in particular hydroperoxides, are important regulators of host-parasite interactions and partly govern the success or failure of disease. ROS producing and ROS scavenging enzymes from both the pathogen and the host finely tune the redox state at the host-pathogen interface. We have analysed the gene structure and organization of peroxiredoxins (prx) in Meloidogyne incognita and analysed their role in the establishment of the nematode in its host. Meloidogyne incognita has seven prx genes that can be grouped with other nematode prx into three clades. Clade B prx genes are more actively transcribed in parasitic stages compared with free-living pre-parasitic juveniles. We confirmed in vitro the activity of one of these, Mi-prx2.1, on hydrogen peroxide and butylhydroperoxide. We showed by ultrastructural immunocytochemistry the expression of clade B PRX proteins in the hypodermis and pseudocoelum beneath the tissues directly in contact with the environment, both in free-living and parasitic stages. Finally, knock-down of clade B prx genes led to a significant reduction in the ability of the nematodes to complete their life cycle in the host. The expression of clade B PRX proteins in the tissues in close contact with plant cells during parasitism and the impaired development of nematodes inside the host after clade B prx knock-down suggest an important role for these genes during infection.     

A new method for in-situ monitoring of the underground development of Orobanche cumana in sunflower (Helianthus annuus) with a mini-rhizotron

AIMS: To develop an in-situ, non-destructive method for observation and monitoring of the underground developmental stages of the root parasite Orobanche cumana. SCOPE: The parasitic weed Orobanche causes severe damage to vegetables and field crops. Most of the damage caused to the crops occurs during the underground, unobservable parasitism stage. Sunflower (Helianthus annuus 'Adi') plants were planted in soil that was artificially inoculated with O. cumana seeds. Clear Plexiglas mini-rhizotron plastic observation tubes were inserted into the soil. Seed germination, early stage of penetration, and formation of tubercles and spikes were observed non-destructively and were monitored throughout the growing season by mean of a mini-rhizotron camera. Use of this technology enabled the complete individual parasite life cycle from the very early development (including germination) to Orobanche shoot to be monitored. In addition, the effect of the systemic herbicide Cadre (imazapic) on the development of O. cumana was inspected and quantified. CONCLUSIONS: This novel methodology facilitates the in-situ study of major aspects of the host-parasite interaction and of parasite suppression, such as parasitism dynamics, parasite growth rate, and the effect of chemical treatments on the parasite.     

Stimulation of Orobanche ramosa seed germination by fusicoccin derivatives: a structure-activity relationship study

A structure-activity relationship study was conducted assaying 25 natural analogues and derivatives of fusicoccin (FC), and cotylenol, the aglycone of cotylenins, for their ability to stimulate the seed germination of the parasitic species Orobanche ramosa. Some of the compounds tested proved to be highly active, being 8,9-isopropylidene of the corresponding FC aglycone and the dideacetyl derivative the most active FC derivatives. In both groups of glucosides and aglycones (including cotylenol), the most important structural feature to impart activity appears to be the presence of the primary hydroxy group at C-19. Furthermore, the functionalities and the conformation of the carbotricyclic ring proved to play a significant role. The dideacetyl derivative of FC, being easily and rapidly obtainable in high yield starting by FC, could be of interest for its practical application as a stimulant of Orobanche ramosa seed germination, inducing the "suicidal germination", an interesting approach for parasitic plant management.     

<Emphasis Type="Italic">Xenopus laevis</Emphasis> peroxiredoxins: Gene expression during development and characterization of the enzymes

Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1–6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10–63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1–6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1–6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.     

Potential role of winter rape weeds in the extension of broomrape in Poitou-Charentes

In the Poitou-Charentes district, among the 82 species of winter rape weeds identified, 22 displayed a strong affinity for this crop (Brassica napus L.). In fields, 50% of these weeds were parasitized by Orobanche ramosa, playing the role of host plants. Greenhouse co-cultures (weed/Orobanche ramosa) showed that weeds non-parasitized in fields could be attacked by broomrape, developing a more or less complete cycle. In vitro co-cultures (weed/Orobanche ramosa) revealed that root exudates of non-parasitized weeds, in fields or in greenhouse co-cultures, could induce Orobanche ramosa seed germination, but not attachment. These weeds could play the role of false hosts.     

Genetic relationships among Orobanche species as revealed by RAPD analysis

RAPD markers were used to study variation among 20 taxa in the genus OROBANCHE: O. alba, O. amethystea, O. arenaria, O. ballotae, O. cernua, O. clausonis, O. cumana, O. crenata, O. densiflora, O. foetida, O. foetida var. broteri, O. gracilis, O. haenseleri, O. hederae, O. latisquama, O. mutelii, O. nana, O. ramosa, O. rapum-genistae and O. santolinae. A total of 202 amplification products generated with five arbitrary RAPD primers was obtained and species-specific markers were identified. The estimated Jaccard's differences between the species varied between 0 and 0.864. The pattern of interspecific variation obtained is in general agreement with previous taxonomic studies based on morphology, and the partition into two different sections (Trionychon and Orobanche) is generally clear. However, the position in the dendrogram of O. clausonis did not fit this classification since it clustered with members of section TRIONYCHON: Within this section, O. arenaria was relatively isolated from the other members of the section: O. mutelii, O. nana and O. ramosa. Within section Orobanche, all O. ramosa populations showed a similar amplification pattern, whereas differences among O. crenata populations growing on different hosts were found. Orobanche foetida and O. densiflora clustered together, supporting the morphological and cytological similarities and the host preferences of these species.     

Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%–80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.     

Sunflower sesquiterpene lactone models induce Orobanche cumana seed germination

Six sunflower sesquiterpene lactone models which share structural features of the lactone rings of strigol and its synthetic analogues, the GR family, with different conformational flexibilities were tested as Orobanche cumana germination stimulants. Among them, parthenolide and 3,5-dihydroxydehydrocostus-lactone significantly increased O. cumana germination, presenting higher activity than GR-24, used as a standard in the germination bioassay. The effect of these two compounds is species-specific, showing no germination stimulant activity on other Orobanche spp. tested (O. crenata, O. ramosa and O. aegyptiaca). Data presented are discussed in terms of a structure-activity relationship.     

Infection of tomato roots using callii of broomrape

Parasitic weeds like members of the genera Orobanche and Striga cause heavy damage to grain, vegetables and other field crops. The germinating seedlings of the parasites attack the roots of their hosts, following a series of recognition events that control germination, seedling growth and attachment to the host root. An in vitro system has been developed that allows the production of partially differentiated callii of Orobanche aegyptiaca Pers. Protrusions of subcultures of these callii behaved like radicles of germinating seeds. Using this system normal infection of tomato roots was obtained in vitro. The infection process included haustorium formation and the development of young Orobanche plants under aseptic conditions. The system developed should now permit a study of biochemical events during infection of the host by Orobanche .     

Molecular Studies on Orobanche

The optimization of polymerase chain reaction (PCR) for random amplified polymorphic DNA (RAPD) analysis in Orobanche was investigated and the results were applied to analysis of O. aegyptiaca, O. oxyloba, O. ramosa and three O. crenata collections gathered from different locations in Egypt, Genotypes were compared using 20 random primers. The polymorphisms detected were only between the tested species, while identical banding patterns were generated in collections of the same species.
Genetic similarity among species was estimated on the basis of the percentage of common bands between species and a dendogram was constructed using the unweighted pair grouping method.
Two primers were chosen as specific PCR primers to differentiate between the studied Orobanche species. The results of this study show that RAPDs can be used as a method for clarifying relationships between species of Orobanche .     

Tomato strigolactones are derived from carotenoids and their biosynthesis is promoted by phosphate starvation

* Strigolactones are rhizosphere signalling compounds that mediate host location in arbuscular mycorrhizal (AM) fungi and parasitic plants. Here, the regulation of the biosynthesis of strigolactones is studied in tomato (Solanum lycopersicum). * Strigolactone production under phosphate starvation, in the presence of the carotenoid biosynthesis inhibitor fluridone and in the abscisic acid (ABA) mutant notabilis were assessed using a germination bioassay with seeds of Orobanche ramosa; a hyphal branching assay with Gigaspora spp; and by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. * The root exudates of tomato cv. MoneyMaker induced O. ramosa seed germination and hyphal branching in AM fungi. Phosphate starvation markedly increased, and fluridone strongly decreased, this activity. Exudates of notabilis induced approx. 40% less germination than the wild-type. The LC-MS/MS analysis confirmed that the biological activity and changes therein were due to the presence of several strigolactones; orobanchol, solanacol and two or three didehydro-orobanchol isomers. * These results show that the AM branching factors and parasitic plant germination stimulants in tomato root exudate are strigolactones and that they are biosynthetically derived from carotenoids. The dual activity of these signalling compounds in attracting beneficial AM fungi and detrimental parasitic plants is further strengthened by environmental conditions such as phosphate availability.     

The microaerophilic respiration of Campylobacter mucosalis

A model is proposed for the respiratory adaptation to falling oxygen concentration during growth of the microaerophilic bacterium Campylobacter mucosalis. During the early stages of growth, the oxidation of formate is a two-stage branched process involving the production of H2O2 followed by its peroxidatic removal. In later stages of growth, at lower oxygen concentrations, the predominant electron flow is linear to a membrane-bound cytochrome-c oxidase which reduces O2 directly to H2O. Several components of this model have been investigated. H2O2 was produced during formate oxidation and accumulated when electron transfer to the cytochrome-c peroxidase was inhibited. A cytochrome c-553, of the Class 1 types, was purified and shown to be the specific electron donor to both the peroxidase and the membrane-bound oxidase. The levels of this cytochrome c and of the peroxidase were higher in cells harvested early in growth. In later stages of growth, the activity of the membrane-bound oxidase increased. Proton pumping across the membrane was detected with either H2O2 or oxygen as terminal electron acceptor. The novel energy-conserving role of H2O2 in this catalase-negative bacterium is discussed in relation to its microaerophilic nature.     

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.     

DEVELOPMENTAL ANATOMY IN ROOTS OF IPOMOEA PURPUREA. I. RADICLE AND PRIMARY ROOT

The developmental anatomy of the primary root of Ipomoea purpurea was studied at several growth stages, beginning with the radicle. The radicle is generally composed of three superimposed tiers of initials, which produce the vascular cylinder, cortex, and columella; and a peripheral band of lateral rootcap-epidermal initials. The radicular cortex contains 16–19 immature laticifers; none of the tissue regions in the radicle contains mature cells. Following germination and during the first 2–3 cm growth of the primary root the apical meristem and its derivative tissues undergo a series of modifications. Root apical diameter decreases as cells in lateral portions of the rootcap elongate; meanwhile, the columella enlarges vertically. The relationship between cortical and columellar initials changes as fewer mitoses occur in the former while the latter remain active. In longer roots the columellar initials are directly in contact with the vascular initials. Cortical size diminishes during early root growth as cortical laticifers and their associated cells cease to be produced by the outer cortical initials and ground meristem. Early procambium, at the level of vascular pattern initiation, decreases in diameter by cellular reorientation, and the vascular cylinder decreases in overall diameter although the tetrarch pattern remains unchanged.