Sterol biosynthesis in heterotrophic plant parasites

Cycloartenol derivatives are present in the non-photosynthetic parasitic plants Cuscuta europaea (dodder), Cuscuta epithymum and Orobanche lutea (broomrape). C.europaea and O.lutea are capable of biosynthesizing their own sterols. There is therefore no direct link, in a chlorophyll-containing phylum, between the cycloartenol pathway to sterols and photosynthesis.     

The resting metabolism of dodder seeds

Extracts of mature seeds of Cuscuta reflexa were examined for any deficiency in key enzymes. The activities of malate dehydrogenase, β-amylase and fructose 1,6-diphosphate aldolase exceeded 5.0 μmol substrate/min/g, while those of starch phosphorylase, α-amylase, acid phosphatase, phosphogluconate dehydrogenase (decarboxylating), aspartate aminotransferase, glucose 6-phosphate dehydrogenase, fructose 1,6-diphosphatase and alanine aminotransferase fell within the range 1 to 5 μmol/min/g and hexokinase, isocitrate dehydrogenase and alkaline phosphatase were below 1 μmol substrate/min/g seed powder. No activity of the following were found: acid invertase, alkaline invertase, phytase and glutamate dehydrogenase. Some of these observations were made also for seeds of Cuscuta campestris and Cuscuta indicora.     

Cladogenesis and reticulation in <Emphasis Type="Italic">Cuscuta</Emphasis> sect. <Emphasis Type="Italic">Denticulatae</Emphasis> (Convolvulaceae)

As traditionally circumscribed, Cuscuta sect. Denticulatae is a group of three parasitic plant species native to the deserts of Western USA (Cuscuta denticulata, Cuscuta nevadensis) and the central region of Baja California, Mexico (Cuscuta veatchii). Molecular phylogenetic studies confirmed the monophyly of this group and suggested that the disjunct C. veatchii is a hybrid between the other two species. However, the limited sampling left the possibility of alternative biological and methodological explanations. We expanded our sampling to multiple individuals of all the species collected from across their entire geographical ranges. Sequence data from the nuclear and plastid regions were used to reconstruct the phylogeny and find out if the topological conflict was maintained. We obtained karyotype information from multiple individuals, investigated the morphological variation of the group thorough morphometric analyses, and compiled data on ecology, host range, and geographical distribution. Our results confirmed that C. veatchii is an allotetraploid. Furthermore, we found previously unknown autotetraploid population of C. denticulata, and we describe a new hybrid species, Cuscuta psorothamnensis. We suggest that this newly discovered natural hybrid is resulting from an independent (and probably more recent) hybridization event between the same diploid parental species as those of C. veatchii. All the polyploids showed host shift associated with hybridization and/or polyploidy and are found growing on hosts that are rarely or never frequented by their diploid progenitors. The great potential of this group as a model to study host shift in parasitic plants associated with recurrent allopolyploidy is discussed.     

Photocontrol of Hook Opening in Cuscuta gronovii Willd

Hook opening in seedlings of Cuscuta gronovii Willd. occurred only after prolonged exposures to blue, red, or far red light. Prolonged far red exposure was less effective than prolonged exposure to red or blue light. Brief far red irradiation inhibited the inductive effect of red light. The far red inhibition was in turn reversed by brief red irradiation. These effects suggest the involvement of two photosystems in the control of hook opening in Cuscuta gronovii Willd.: a phytochrome-mediated system and a separate high energy requirement.     

Utilizzazione Di Afidi Per Il Prelevamento Della Linfa in « Cuscuta Epithymum »

Abstract

Use of Aphids for sampling the cell sap of Cuscuta epithymum. — A variety of Myzus persicae that feeds specifically on Cuscuta epithymum has been employed in a study of the translocation of nutrilites from the host, Trifolium repens, to the parasite, C. epithymum. If the host was exposed to an atmosphere containing C¹⁴,O₂, the Aphids feeding on the Cuscuta filaments became labelled very rapidly and to considerable extent. The analysis of the distribution of radioactivity in the Cuscuta filaments and the Aphids revealed that extensive degradation of the compounds and randomization of the label occurred in the insects.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

Über das Vorkommen eines cytokininartigen Faktors inCuscuta reflexa

Zusammenfassung InCuscuta reflexa-Extrakten konnte mit Hilfe verschiedener biologischer Teste ein cytokininartiger Faktor nachgewiesen werden. Die Bedeutung dieses Faktors für das Verhältnis zwischen derCuscuta und ihren Wirtspflanzen wird diskutiert.

---

On the existence of a cytokinin-like factor in cuscuta reflexa

Summary In extracts ofCuscuta reflexa Roxb. a cytokinin like factor (CAF = Cuscuta active factor) was found. It was shown that activity of this factor is similar to that of kinetin in all essential points. In tobacco-stem-tissue tests a promotion of growth by CAF was observed. In chlorophyll-preservation tests CAF produced a strong inhibition of chlorophyll dissimilation. Moreover in tests with¹⁴C-labelled glycine a migration of the glycine and other amino acids due to CAF was found.The occurrence of the observed cytokinin-like factor inCuscuta reflexa is discussed with respect to the parasite-host relations ofCuscuta.

     

Seed formation and pollination system in Cuscuta obtusiflora: First record of preanthesis cleistogamy in parasitic plants and some functional inferences

With the purpose of increasing the currently available data on reproductive biology about Neotropical Cuscuta species in general, and mode of reproduction and seed ontogeny in Cuscuta obtusiflora H.B. and K. in particular, both developing flowers and seeds have been analysed by means of standard photomicroscopy together with histological techniques, stereomicroscopy, fluorescence microscopy and field observation.     

Calcium signaling during the plant-plant interaction of parasitic Cuscuta reflexa with its hosts

The plant parasite Cuscuta reflexa induces various responses in compatible and incompatible host plants. The visual reactions of both types of host plants including obvious morphological changes require the recognition of Cuscuta ssp. A consequently initiated signaling cascade is triggered which leads to a tolerance of the infection or, in the case of some incompatible host plants, to resistance. Calcium (Ca²⁺) release is the major second messenger during signal transduction. Therefore, we have studied Ca²⁺ spiking in tomato and tobacco during infection with C. reflexa. In our recently published study¹ Ca²⁺ signals were monitored as bioluminescence in aequorin-expressing tomato plants after the onset of C. reflexa infestation. Signals at the attachment sites were observed from 30 to 48 h after infection. In an assay with leaf disks of aequorin-expressing tomato which were treated with different C. reflexa plant extracts it turned out that the substance that induced Ca²⁺ release in the host plant was closely linked to the parasite''s haustoria.Key words: cuscut, odder, calcium signaling, plant parasitismThe genera Cuscuta, also known as dodder, includes 170 parasitic species with a worldwide distribution. Members of Cuscuta ssp. belong to the 1% of angiospermic plants that live as holoparasites and depend on nutrients, water and carbohydrates from other host plants.² Cuscuta spp. lack roots or leaves but possess specific penetrating organs, the so called haustoria, which are fully developed 5–6 days after the first contact, when an interaction between parasite and host is established.As for all dicotyledonous plants, the typical Cuscuta spp. life cycle starts with the germination of seeds. At the stage of a rootless seedling, Cuscuta ssp. has just a few days to find and successfully invade a host plant. Although Cuscuta ssp. seedlings appear to coil indiscriminately around any vertical elongated object, they seem to have an efficient “sense of smell” to recognize potential “victims” and are therefore able to infest host plants more rapidly and efficiently.³ As soon as a host is reachable, Cuscuta ssp. starts to wind around the host shoot and initiates the attachment process as well as the development of haustoria.^2,4 Already at this initial phase of infection (12–48 h post attachment) the host plant senses the parasite and initiates an onset of several signals which are only partially known. Amongst the several induced genes are for example those encoding AGPs (Arabinogalactan Proteins), proteins promoting the parasite''s adherence.⁵ Also proteins are produced which might be important for nutrient and water uptake⁶ or which modify the host cell wall.⁷In this addendum article, we focus on signals which occur in host plants within the early infection stage prior to a vascular bundle connection and refer to our article about Ca²⁺ signalling in C. reflexa infected tomato plants.⁸ Besides phytohormones or other initial signalling molecules, such cellular calcium signals might be involved in controlling the expression of important genes for developmental or resistance related processes.In our approach, Cuscuta reflexa shoots of ∼25 cm length were wrapped around transgenic constitutively aeqourin-expressing tomato (Solanum lycopersicum) and tobacco (Nicotiana benthamiana) plants. With a highly sensitive ccd-camera we then monitored the two interacting organisms. The Ca²⁺-signals which are released by the host-plant could be detected as light-emission. The first cytosolic calcium signals were observed 24–48 h after the parasite attachment when the haustoria formation was already initiated. Light, indicating a cytosolic calcium influx was clearly visible directly where the parasite started to penetrate host tissue via its haustoria (Fig. 1) and often appeared several times within 1–6 h. As the light signals per recorded picture were collected for 10 min it is not clear if the duration of such cytosolic calcium influx comprises 10 ms or 10 min. An additional experiment in our study was the usage of a Cuscuta reflexa haustorium extract which was applied to aequorin expressing tomato leaf discs. Here it turned out that the Ca²⁺-ion influx happened steadily and slowly, because signals were only visible when summed up from 2 h recording. The finding that both boiled haustoria extract and control extract, made from Cuscuta reflexa shoots without haustoria, are inactive, suggests that a protein which is expressed during the infection process might be the direct or indirect trigger of such Ca²⁺-signals. These results overcome furthermore the theory that Calcium signals are induced by pressure, which might also be a step during Cuscuta ssp. infection.Open in a separate windowFigure 1Cuscuta reflexa infection induces calcium-signals in aequorin-expressing tomato. Left: Bright field; middle: light emission representing Ca²⁺-signals at the infection site ∼30 h post onset of the parasite; signals were monitored with a ccd camera. Right: overlay.The fact that calcium fluxes act as a second messenger in several stress responses such as cold shock, wind, touch, osmotic stress,⁹ phytohormone signalling pathways,¹⁰ plant—symbiotic interactions^10–12 or also plant pathogen interaction^13–15 complicates the interpretation of the signals that are induced by Cuscuta reflexa. One possibility could be that visible Ca²⁺-signals are part of a signalling pathway where also SA (salicylic acid) or/and JA (jasmonic acid) play an important role. Recently, Runyon et al.¹⁶ could show that tomato plants infected with Cuscuta pentagona respond with a strong induction of JA and SA 24–36 h post infections. This time frame correlates with our described calcium signals and it has been previously described that calcium fluxes might be a part of the JA- and SA-signalling cascade.The tomato—dodder interaction, however, represents an exception among dicotyledonous plants because tomato generates a hypersensitive response which is part of a successful resistance reaction.^7,16 In this particular case characteristic components of C. reflexa must be sensed by its host plant. These factors indicate “non-self” for the host plant, probably following a model comparable to the MAMP concept where characteristic molecular patterns of a pathogen are recognized in host plants via pattern recognition receptors and subsequently trigger defence responses.^17,18 But sensing and signalling in host plants takes place not only in the case of an “incompatible” interaction. The developmental phenomenons of a dodder—plant interaction in a “compatible” interaction are nearly a miracle. In this case, the parasite is completely tolerated and achieves the attachment and the penetration of the host plant. It interferes in developmental processes and manipulates its host to develop vascular tissues, to build up chimerical cell walls and interspecific symplastic cell connections.^16,17 Finally, it is connected to the host plant and starts to withdraw nutrients and carbohydrates^19,20 by mimicking endogenous sinks. Such a tolerated interaction reminds of an interaction of plants with bacterial or fungal symbionts, where also Ca²⁺-signals have been described and well characterized.^11,12 In the case of Cuscuta ssp.—host interaction a lot of further studies have to be done to discover all important steps of signalling cascades.     

A new species of Cuscuta (Convolvulaceae) from Michoacán, Mexico

A new species, Cuscuta cotijana, is described and illustrated from the Trans-Mexican Volcanic Belt in northwestern Michoacán, Mexico. The species is most closely related to Cuscuta jalapensis, C. mitriformis, C. rugosiceps and C. lindsayi, from which it differs by the large spur-like projections on the outer calyx lobes and the ovoid to conical capsule with a small intrastylar aperture.     

Intracellular localisation of hexokinase in Cuscuta reflexa

In Cuscuta reflexa 16% of the hexokinase activity was associated with the particulate fraction and the rest in the 105 000 g, 1 hr supernatant. In a sucrose gradient, hexokinase activity banded with an organelle at a mean density of 1.20 g cm^?3, coinciding with the mitochondrial marker, cytochrome c oxidase. Fractionation of isolated mitochondria by digitonin showed the presence of the enzyme in the outer membrane along with its marker rotenone-insensitive NADH cytochrome c reductase. No latent form of hexokinase was detected.     

Sterols and fatty acids of some non-photosynthetic angiosperms

Sterols and fatty acids were extracted and identified from three parasitic angiosperms, Cuscuta campestris, Monotropa uniflora and M. hypopitys. Each plant contained the typical 16 and 18-carbon fatty acids of angiosperms, but the partially-photosynthetic Cuscuta contained much larger quantities of linolenic acid than the non-green Monotropa species which had smaller amounts of linolenic acid characteristic of non-photosynthetic tissue. Sterol quantity was three times higher in Cuscuta than in the Monotropa species. Sitosterol was the major sterol in all species with smaller amounts of campesterol and cholesterol.     

Taxonomic status of Cuscuta nevadensis and C. veatchii (Convolvulaceae) in North America

The taxonomy ofCuscuta nevadensis andC. veatchii is investigated.Cuscuta nevadensis is more closely related toC. veatchii andC. denticulata than toC. salina, and the former two taxa are accepted as species. A summary of relevant taxonomic and biological information is provided, including synonymy, distribution and ecology, keys, and a comparison of the morphology of flowers and seeds are examined. The morphological basis of vivipary inC. nevadensis is discussed. The status ofCuscuta vivipara, an invalid name in recent use, is clarified.     

Host-produced ethylene is required for marked cell expansion and endoreduplication in dodder search hyphae

The genus Cuscuta comprises stem holoparasitic plant species with wide geographic distribution. Cuscuta spp. obtain water, nutrients, proteins, and mRNA from their host plants via a parasitic organ called the haustorium. As the haustorium penetrates into the host tissue, search hyphae elongate within the host tissue and finally connect with the host’s vascular system. Invasion by Cuscuta spp. evokes various reactions within the host plant’s tissues. Here, we show that, when Arabidopsis (Arabidopsis thaliana) is invaded by Cuscuta campestris, ethylene biosynthesis by the host plant promotes elongation of the parasite’s search hyphae. The expression of genes encoding 1-aminocylclopropane-1-carboxylic acid (ACC) synthases, ACC SYNTHASE2 (AtACS2) and ACC SYNTHASE6 (AtACS6), was activated in the stem of Arabidopsis plants upon invasion by C. campestris. When the ethylene-deficient Arabidopsis acs octuple mutant was invaded by C. campestris, cell elongation and endoreduplication of the search hyphae were significantly reduced, and the inhibition of search hyphae growth was complemented by exogenous application of ACC. In contrast, in the C. campestris-infected Arabidopsis ethylene-insensitive mutant etr1-3, no growth inhibition of search hyphae was observed, indicating that ETHYLENE RESPONSE1-mediated ethylene signaling in the host plant is not essential for parasitism by C. campestris. Overall, our results suggest that C. campestris recognizes host-produced ethylene as a stimulatory signal for successful invasion.

Growth of Cuscuta campestris search hyphae is inhibited in ethylene-deficient Arabidopsis mutants, suggesting that host-derived ethylene acts as a stimulatory signal for parasitism by Cuscuta spp.     

Modelling of the flows and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa Roxb. and its host Lupinus albus L.: II. Flows between host and parasite and within the parasitized host

A recently developed empirically based modelling technique wasused to quantify uptake, flow and utilization of C and N inLupinus albus L., uninfected and parasitized by Cuscuta reflexaRoxb. plants over a 12 d period during flowering and early fruitsetting of the host. The modelling combined data on molar C:Nratios in host phloem and pressure-induced xylem sap, net incrementsof C and N in host and parasite plant parts and respiratorylosses of C. The modelling of the solute transfer between hostand Cuscuta was achieved by assuming non-specific intake fromthe xylem. The models predicted that Cuscuta derived 99.5% ofits carbon and 93.6% of its nitrogen demand from the host phloem.The overriding sink strength of the parasite diverted most ofthe basipetally translocated host assimilates and massivelycompeted with the host root and inhibited fruit setting. Carbonincorporation in Cuscuta consumed 56%, respiration 24% and secretionby extrafloral nectaries 1.8% of the current host photosynthate.Root respiration was inhibited by 59% and carbon was mobilizedfrom host root and leaves. Competition by the parasite for Nwas even more severe and Cuscuta incorporated nitrogen equalling223% of current fixation, but N₂ fixation of the host was severelyrestricted to 37%. Withdrawal of N from host phloem led to severelosses of N from leaves and the root and marked decreases inN concentration. It required massive xylem-to-phloem transferof N, because the xylem as the major supply route for N wasnot exploited substantially by Cuscuta. The results are discussedin relation to likely causes for parasite-induced pathogeniceffects, suggesting that Cuscuta affected the host adverselyby depriving it mainly of its nitrogen, but that causal to incipientnitrogen deficiency and restricted N2 fixation was the superiorsink potential of Cuscuta, which prevented adequate supply ofassimilates to the nodulated root. The dominating sink potentialof Cuscuta is compared with the similarly strong sink competitionexerted by fruits at the stage of seed filling in annual plants. Key words: Cuscuta reflexa, Lupinus albus, parasitism, carbon, nitrogen, phloem, xylem, transport     

Cuticle development and ultrastructure: evidence for a procuticle of high osmium affinity

Transmission electron microscopy was used to investigate the development and ultrastructure of the cuticles of the bladder primordium and other parts of Utricularia, the stem of Cuscuta gronovii, and the leaves of Athanasia parviflora. In all materials investigated, except the apical meristem of Cassytha pubescens, the first-formed cuticle, named the procuticle, was very electron dense and apparently amorphous in texture. Later, the procuticle changed its ultrastructural appearance: in all species having a procuticle it lost much of its electron density. Simultaneously, it developed into a lamellar structure in U. lateriflora and Cuscuta, and became part of a lamellar cuticle proper. In U. sandersonii and Athanasia the procuticle generally remained without visible structure. The velum of the pavement epithelium of Utricularia is considered to be a slightly modified procuticle which has become loosened from the epithelial cells and stretched.