Diversity and evolution of pollen-ovule production in <Emphasis Type="Italic">Cuscuta</Emphasis> (dodders,Convolvulaceae) in relation to floral morphology

Cuscuta (dodder, Convolvulaceae) is a genus of about 200 species of obligate stem parasites with subcosmopolitan distribution. The diversity of pollen and ovule production was surveyed in 128 species and ten varieties. Taxa were assigned to Cruden’s mating system categories based on their pollen-ovule ratios. Variation and correlations among floral characters were analyzed using regression and ANOVA, while the mating system categories were subjected to a linear discriminant and canonical variates analysis to assess their cohesiveness. Our data strongly suggest that most Cuscuta species possess a wide range of mixed-mating systems. Whereas four ovules develop in each flower, pollen production varies over three orders of magnitude. Several Cuscuta taxa are highly outcrossing, but no species could be identified that are exclusively selfing. The transition from the one-style flowers of subg. Monogynella to the two-style flowers of subgenera Cuscuta and Grammica, and from simultaneous to sequential maturation of the two stigmas in the latter subgenus, has decreased the role of herkogamy as a facilitator of outcrossing. These evolutionary changes are associated with an increase of species richness in subgenus Cuscuta, and especially in subgenus Grammica. Morphological features were not individually found to have a strong correlation to the mating system, but in general, larger corollas and stigmas were associated with greater pollen-ovule ratios. Cuscuta presents some puzzling results when considered in light of the sex allocation theory, as only some infrageneric lineages demonstrate the predicted pollen size-number tradeoff, while Cuscuta gracillima complex (in subgenus Grammica) displays an unexpected negative relationship between pollen size and style length. The relationship between host range and mating system is discussed, prompting further research into the co-evolution of pollination systems and life history traits between parasites and their host species.     

Diversity,evolution, and function of stomata bearing structures in Cuscuta (dodders,Convolvulaceae): From extrafloral nectar secretion to transpiration in arid conditions

Cuscuta includes ca. 200 species of functionally holoparasitic plants grouped in four subgenera: Monogynella, Cuscuta, Pachystigma, and Grammica. Multicellular structures with stomata in Cuscuta are represented by extrafloral nectaries (ENs), reported from the stems of one Monogynella species, and stomatiferous protuberances (SPs), which are non-secretory. These latter structures had been noted on the stems of three Grammica species more than a century ago but entirely forgotten until recently when similar, non-secretory SPs were reported on the flowers of several new Grammica species. Here we study for the first time: (1) the extent of occurrence, diversity and evolution of secretory (ENs) and non-secretory (SPs) multicellular structures in Cuscuta, and (2) the function of SPs. We undertook a character evolution study of ENs and SPs on the stems and flowers of 136 Cuscuta taxa, and examined the structure/ultrastructure of SPs. ENs are inferred as primitive and characterize subg. Monogynella. SPs are derived in the remaining subgenera; they are ubiquitous on the flowers of Cuscuta and Pachystigma, but absent on their stems. Subgenus Grammica species develop two functional types of stems during their life cycle: vegetative, exploratory stems with very low stomatal densities (and no SPs), and reproductive, haustorial stems with numerous SPs. Moreover, 24 species from nine clades of subg. Grammica have evolved morphologically diverse floral SPs with systematic significance. To preliminarily ascertain SP function, we determined in the field the water uptake of Tithonia tubiformis plants parasitized or not by Cuscuta costaricensis, a species with both stem and floral SPs, and the stomatal conductance of dodder stems and flowers, as well as host leaves. Water uptake of parasitized hosts was significantly higher compared to non-parasitized plants, even after host leaves were removed, both during the day and night. The increased water uptake of parasitized hosts and stomatal conductance values suggest a transpiration role for the SPs, which is also confirmed by their lacunar structure. Grammica species with floral SPs grow in arid areas or characterized by a pronounced dry season during flowering/fruiting, which suggests that SPs may have evolved to stimulate the host water uptake during these phenophases.     

Modulation of key nitrogen assimilating enzymes by NAA and in vitro culture in Cuscuta reflexa

Nitrogen assimilation in the callus of an angiosperm holoparasitic plant, Cuscuta reflexa, has been investigated by studying the level of key enzymes of the nitrogen assimilation pathway, namely nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), during its growth in the absence and presence of NAA. The activity of all these enzymes in culture exhibited a developmental profile of an initial increase followed by a decrease. The presence of NAA increased the activity of all the enzymes throughout the culture period without altering their developmental profiles. Isozyme profiles of GS and GDH in the callus of C. reflexa were analyzed by PAGE and direct in gel activity staining. In the absence of NAA, the callus exhibited one isozyme of GS and two isozymes of GDH. NAA treatment led to the development of one additional isozyme of GS. Further stimulating effect of NAA on the activity of each of these enzymes was also evident by in gel activity staining of the isozymes. A comparison of the levels of NR, GS, GOGAT and GDH in field vines of C. reflexa, leaves of its host plant, Catheranthus with those of Cuscuta callus, led to the observation that all the nitrogen assimilating enzymes except GDH, were absent in the field vines of C. reflexa. Callus and field vines revealed a preponderance of GDH as compared to GS activity, while a reverse trend was observed in the host plant. The data are suggestive of ammonia assimilation through GDH pathway in this parasite.     

Gypsy moth herbivory induced volatiles and reduced parasite attachment to cranberry hosts

Interactions between species can have cascading effects that shape subsequent interactions. For example, herbivory can induce plant defenses that affect subsequent interactions with herbivores, pathogens, mycorrhizae, and pollinators. Parasitic plants are present in most ecosystems, and play important roles in structuring communities. However, the effects of host herbivory on parasitic plants, and the potential mechanisms underlying such effects, are not well known. We conducted a greenhouse study to ask whether gypsy moth (Lymantria dispar) damage, host cultivar, and their interaction affected preference of the stem parasite dodder (Cuscuta spp.) on cranberry hosts (Vaccinium macrocarpum). We then assessed the mechanisms that could underlie such effects by measuring induced changes in phytohormones and secondary compounds. We found that damage by gypsy moths delayed dodder attachment by approximately 0.3 days when dodder stems were added 2 days after damage, and reduced attachment by more than 50% when dodder stems were added 1 week after host plant damage. Gypsy moth damage significantly increased jasmonic acid (JA) levels, total volatile emissions, and the flavonol, quercetin aglycone, suggesting possible mechanisms underlying variation in dodder ability to locate or attach to hosts. Dodder preference also differed between cranberry cultivars, with the highest attachment on the cultivar that had significantly lower levels of total volatile emissions and total phenolic acids, suggesting that volatile composition and phenolics may mediate dodder preference. Our results indicate that herbivory can reduce subsequent attachment by a highly damaging parasitic plant, demonstrating the potential importance of early damage for shaping subsequent species interactions.     

Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, <Emphasis Type="Italic">Cuscuta reflexa</Emphasis> and <Emphasis Type="Italic">Cuscuta gronovii</Emphasis>

Background  

The holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana.     

Sink-stimulated photosynthesis and sink-dependent increase in nitrate uptake: nitrogen and carbon relations of the parasitic association Cuscuta reflexa–Ricinus communis

Ricinus communis L. was grown under limiting N supply in quartz sand culture, fed with 0.2, 1 or 5 mol m^?3 NO₃^?, or in liquid culture with 0.022, 0.05 or 0.5 mol m^?3 NO₃^?. Some of the plants were infected with Cuscuta reflexa Roxb. As occurred for the host, dry matter production and growth of C. reflexa were severely depressed with decreasing N supply to the host. When parasitized by C. reflexa, the shoot and root dry weight of Ricinus was diminished at all levels of N nutrition, but the total dry weight of host plus parasite was almost the same as that of uninfected Ricinus. In contrast to the situation in Lupinus albus (Jeschke et al. 1994b), infection by Cuscuta resulted in increased tissue N levels in the host and the N content of the system Ricinus plus C. reflexa was the same or even somewhat larger than that of uninfected plants. This indicated a sink-dependent stimulation of nitrate uptake. As a result of decreased root weights, nitrate uptake g^?1 FW was stimulated by 80, 60 or only 40% at 0.2, 1 or 5 mol m^?3 nitrate supply. Increased nitrate uptake was reflected, particularly at low N supply, in xylem transport; xylem sap nitrate concentrations were substantially elevated, while those of amino acids were decreased in parasitized plants. This indicated an inhibition of nitrate assimilation in roots of parasitized plants under limiting N supply. Besides these effects on N relations, C. reflexa induced a substantial sink-dependent stimulation of net photosynthesis in host leaves and a concomitant increase in stomatal opening and transpiration. This stimulation depended on the relative sink size induced by Cuscuta, on nitrogen nutrition and on leaf age, indicating that delayed senescence of leaves contributes to the overall effects of Cuscuta on its host. The Cuscuta-induced inhibition of nitrate assimilation in the roots and the increase in nitrate uptake suggest that nitrate reduction was shifted towards the leaves in the presence of C. reflexa. The stimulating effects of C. reflexa in the Ricinus-Cuscuta association are compared with the strongly inhibitory effects occurring in the tripartite association L. albus–Rhizobium–Cuscuta reflexa.     

Sink-stimulated photosynthesis, increased transpiration and increased demand-dependent stimulation of nitrate uptake: nitrogen and carbon relations in the parasitic association Cuscuta reflexa-Coleus blumei

When parasitizing Coleus blumei Benth., grown in quartz sandculture and fed with 0.2, 1 or 5 mM nitrate, the biomass productionof Cuscuta reflexa Roxb. was inhibited to a similar extent asthat of the host supplied limiting concentrations of nitrate.In the presence of Cuscuta the growth and dry matter increaseof the host plant was severely inhibited. However, dry matterproduction of host plus parasite was only slightly less thanor at 0.2 mM nitrate almost the same as that of uninfected Coleusplants. Under all conditions of nitrate nutrition, parasitismby Cuscuta led to a substantial increase in photosynthesis inhost leaves under light-saturating conditions and in transpiration.Particularly with 0.2 mM and mM     

Phytochelatins govern zinc/copper homeostasis and cadmium detoxification in Cuscuta campestris parasitizing Daucus carota

Cuscuta sp., known with the common name of “dodder”, is an obligate parasite capable of invading stems and leaves of a wide range of host plants. Dodder stem usually coils counterclockwise around the host and, within a few days, develops haustorial structures at each point of contact. As soon as dodder haustoria reach host vascular bundles, they start tapping water, photosynthates and minerals. Metal ions such as zinc (Zn) and copper (Cu) are essential for dodder growth and metabolism, although an exceedingly high (over-homeostatic) supply of these micronutrients can result in growth inhibition and cellular toxicity. Even more so, non-essential metals such as cadmium (Cd), if transferred from the host to the parasite, need to be neutralized by timely detoxification mechanisms. In this work, we showed that Cuscuta campestris Yuncker establishes effective haustorial connections with leaf petioles and blades of Daucus carota L. (carrot), with the consequent transfer of Cd and essential metals (such as Zn and Cu) from the host vascular bundles to the parasite. Following up to this point, we detected the presence in the parasite of significant amounts of glutathione and phytochelatins, even in the absence of Cd exposure. This suggests that thiol peptides in dodder might be particularly important for Zn and Cu homeostasis as well as for Cd detoxification. Finally, we demonstrated that dodder is capable of synthesizing phytochelatins on its own, rather than massively importing them from the host, and also provided evidence for the existence of an endogenous, constitutively expressed, dodder's phytochelatin synthase.     

Diet mixing in a parasitic plant: adaptation or constraint?

Some herbivores deliberately consume a mixed diet, either to obtain a superior mix of nutrients or to avoid consuming too much of any one toxin. Few studies have examined diet mixing in parasitic plants, which typically have very broad host ranges. We offered the parasitic plant Cuscuta indecora (dodder), a range of mixtures of two hosts (Iva frutescens and Borrichia frutescens) in the greenhouse, and observed correlations between the host community and Cuscuta infection in the field. In the greenhouse, Cuscuta performed better on mixtures with a higher relative abundance of Iva. Cuscuta selectively foraged on whichever host was more abundant (diet switching), the exact opposite of the behavior that would be expected if diet mixing was advantageous. In the field, the intensity of Cuscuta infections was decreased by the presence of non-hosts (grasses), not strongly affected by the presence of intermediate hosts, and increased by the presence of Borrichia. We conclude that Cuscuta does not obtain nutritional benefits from a broad diet, but instead is constrained by its relative lack of mobility to attack hosts of intermediate value. In general, the lack of mobility of parasitic plants compared to herbivores probably selects for broad host ranges in parasitic plants.     

Cuscuta reflexa invasion induces Ca2+ release in its host

Cuscuta reflexa induces a variety of reaction in its hosts. Some of these are visual reactions, and it is clear that these morphological changes are preceded by events at the molecular level, where signal transduction is one of the early processes. Calcium (Ca²⁺) release is the major second messenger during signal transduction, and we therefore studied Ca²⁺ spiking in tomato during infection with C. reflexa. Bioluminescence in aequorin‐expressing tomato was monitored for 48 h after the onset of Cuscuta infestation. Signals at the attachment sites were observed from 30 to 48 h. Treatment of aequorin‐expressing tomato leaf disks with Cuscuta plant extracts suggested that the substance that induced Ca²⁺ release from the host was closely linked to parasite haustoria.     

Significance of Cuscutain,a cysteine protease from <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>, in host-parasite interactions

Background  

Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified.     

Trehalose Toxicity in Cuscuta reflexa: SUCROSE CONTENT DECREASES IN SHOOT TIPS UPON TREHALOSE FEEDING

Trehalose, an α,α-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [¹⁴C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding.     

Development of Cuscuta species on a partially incompatible host: induction of xylem transfer cells

Summary.  The growth of dodders, Cuscuta reflexa and Cuscuta japonica, on the partially incompatible host poinsettia (Euphorbia pulcherrima) is studied. Poinsettia responds by bark growths to the formation of the dodder haustoria and prevents dodder from obtaining normal growth. The growth instead becomes extremely branched, coral-like, and dodder lacks the ability to form haustoria. After a period of coral-like growth, long shoots sprout, resembling the normal growth. These long shoots mark an ending phase for dodder, which dies shortly after without having flowered. During the coral-like growth phase, dodder develops transfer cells in the parenchyma cells bordering the vessels of the xylem in the shoot. The transfer cells have not been observed when dodder is grown on the compatible host Pelargonium zonale. A coral-like growth phase has also been observed at the establishing phase when dodder is grown in vitro on agar; later a more normal growth form takes over. In this coral phase, xylem transfer cells are also developed. The fluorochromes carboxyfluorescein and Texas Red were loaded into the host in the phloem and xylem, respectively, and detection of these fluorochromes in the dodder stem indicated that a functional haustorial contact developed for both vascular systems. The results show that Cuscuta spp. have the genetic ability to develop xylem transfer cells and use this in response to developmental stress. Received June 12, 2002; accepted August 26, 2002; published online March 11, 2003     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a susceptible host plant for the holoparasite <Emphasis Type="Italic">Cuscuta </Emphasis>spec

Arabidopsis thaliana and Cuscuta spec. represent a compatible host–parasite combination. Cuscuta produces a haustorium that penetrates the host tissue. In early stages of development the searching hyphae on the tip of the haustorial cone are connected to the host tissue by interspecific plasmodesmata. Ten days after infection, translocation of the fluorescent dyes, Texas Red (TR) and 5,6-carboxyfluorescein (CF), demonstrates the existence of a continuous connection between xylem and phloem of the host and parasite. Cuscuta becomes the dominant sink in this host–parasite system. Transgenic Arabidopsis plants expressing genes encoding the green fluorescent protein (GFP; 27 kDa) or a GFP–ubiquitin fusion (36 kDa), respectively, under the companion cell (CC)-specific AtSUC2 promoter were used to monitor the transfer of these proteins from the host sieve elements to those of Cuscuta. Although GFP is transferred unimpedly to the parasite, the GFP–ubiquitin fusion could not be detected in Cuscuta. A translocation of the GFP–ubiquitin fusion protein was found to be restricted to the phloem of the host, although a functional symplastic pathway exists between the host and parasite, as demonstrated by the transport of CF. These results indicate a peripheral size exclusion limit (SEL) between 27 and 36 kDa for the symplastic connections between host and Cuscuta sieve elements. Forty-six accessions of A. thaliana covering the entire range of its genetic diversity, as well as Arabidopsis halleri, were found to be susceptible towards Cuscuta reflexa.     

Metabolite changes with induction of Cuscuta haustorium and translocation from host plants

Cuscuta is a stem holoparasitic plant without leaves or roots, parasitizing various types of host plants and causing major problems for certain crops. Cuscuta is known as a generalist and, thus, must have unique parasite strategies to cope with different host plants. For elucidating metabolic responses and mechanisms of parasitization, metabolomic approaches using GC/MS were applied. We compared five stages of Cuscuta japonica: early stage seedlings, with far red light (FR) cue, with contact signal, haustorium induced seedlings by both signals and adult plant parasites on host plants. Sugars, amino acids, organic acids, nucleic acids, and polyols were identified from the polar phase fraction. The apical part contained metabolite profiles different from the haustorium induced part or the basal part. Amino acid and some organic acids were up-regulated for haustorium induction but decreased after parasitization. After attachment to different host plants, metabolite profiles of Cuscuta japonica changed dramatically due to the absorption of specific host plant metabolites such as pinitol. Cuscuta seedlings attached to pinitol rich host plants contained more pinitol and showed different profiles from those attached to plants having less or lacking pinitol.     

Trehalose Toxicity in Cuscuta reflexa: CORRELATION WITH LOW TREHALASE ACTIVITY

A toxic effect of α,α-trehalose in an angiospermic plant, Cuscuta reflexa (dodder), is described. This disaccharide and its analogs, 2-aminotrehalose and 4-aminotrehalose, induced a rapid blackening of the terminal region of the vine which is involved in elongation growth. From the results of in vitro growth of several angiospermic plants and determination of trehalase activity in them, it is concluded that the toxic effect of trehalose in Cuscuta is because of the very low trehalase activity in the vine. As a result, trehalose accumulates in the vine and interferes with some process closely associated with growth. The growth potential of Lemna (a duckweed) in a medium containing trehalose as the carbon source was irreversibly lost upon addition of trehalosamine, an inhibitor of trehalase activity. It is concluded that, if allowed to accumulate within the tissue, trehalose may be potentially toxic or inhibitory to higher plants in general. The presence of trehalase activity in plants, where its substrate has not been found to occur, is envisaged to relieve the plant from the toxic effects of trehalose which it may encounter in soil or during association with fungi or insects.     

Modelling of quinolizidine alkaloid net flows in Lupinus albus and between L. albus and the parasite Cuscuta reflexa: new insights into the site of quinolizidine alkaloid synthesis

The present work reveals new and completely different conclusionsabout the alkaloid economy of symbiotically fed Lupinus albusand L. albus parasitized by Cuscuta reflexa in the study periodof 43–55 d after sowing of lupin. Net flows of alkaloidswithin lupin and between host and parasite were calculated usingthe molar ratio of alkaloid nitrogen: total nitrogen combinedwith known net flows of nitrogen in the transport fluids andanalysing alkaloid accumulation in plant organs by HRGC. Incontrast to previous studies, quinolizidine alkaloids were predictedto be synthesized mainly in the root of L. albus and to be predominantlytransported via xylem to the apical plant shoot organs. Parasitismby C. reflexa for 12 d induced a decline of alkaloid contentin the host L. albus up to 53% compared to control plants andalkaloid synthesis was halved—apparently due to a shortageof the precursor lysine. In spite of an additional decreasein nitrogen levels at the second harvest, the host-parasitesystem showed a1.3-fold higher alkaloid content than the controlplants, 63% of the total alkaloids being attracted by Cuscuta.This indicates (a) restriction of catabolic processes withininfected lupins, (b) a massive shift of nitrogen metabolismin the direction of alkaloids and (c) an enormous sink potentialof Cuscuta for nitrogenous compounds. Although xylem was foundto be the main translocation system for alkaloids, the modellingof alkaloid flows predicts Cuscuta to derive only 4.5% of itstotal alkaloid supply from the xylem and 95.5% from the phloem.By analogy with nitrogen flows, this finding requires xylemphloemtransfers which were assumed to occur within the stem axis oflupin. A similar proportion regarding the contribution of xylemand phloem to the supply of Cuscuta was obtained for the netflows of two selected alkaloids, lupanine and 13