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Plastid genome structure and loss of photosynthetic ability in the parasitic genus Cuscuta.

The genus Cuscuta (dodder) is composed of parasitic plants, some species of which appear to be losing the ability to photosynthesize. A molecular phylogeny was constructed using 15 species of Cuscuta in order to assess whether changes in photosynthetic ability and alterations in structure of the plastid genome relate to phylogenetic position within the genus. The molecular phylogeny provides evidence for four major clades within Cuscuta. Although DNA blot analysis showed that Cuscuta species have smaller plastid genomes than tobacco, and that plastome size varied significantly even within one Cuscuta clade, dot blot analysis indicated that the dodders possess homologous sequence to 101 genes from the tobacco plastome. Evidence is provided for significant rates of DNA transfer from plastid to nucleus in Cuscuta. Size and structure of Cuscuta plastid genomes, as well as photosynthetic ability, appear to vary independently of position within the phylogeny, thus supporting the hypothesis that within Cuscuta photosynthetic ability and organization of the plastid genome are changing in an unco-ordinated manner.

Cuscuta↗

[RAPD analysis of four species of Cuscuta in Shandong Province].

OBJECTIVE: To explore the genome difference of four species of Cuscuta in different hosts. METHOD: RAPD was used by 50 primers. RESULT: Four species of genus Cuscuta can be identified by 8 primers. Both Cuscuta chinensis and C. australis from Subg. Grammica had 3 bands whose molecular weights were 1.3 kb, 1.45 kb and 1.53 kb respectively. C. japonica and C. lupuliformis from Subg. Monogyna had a 1.48 kb specific band. Cuscuta of same subgenus had similar RAPD result and close genetic relationship. Same species of Cuscuta in different hosts showed DNA polymorphism. It indicated that hosts can affect genome of Cuscuta to some extent. CONCLUSION: RAPD can be used to identify the species of Cuscuta or same Cuscuta in different hosts.

Cuscuta↗

Heterochromatin and rDNA sites distribution in the holocentric chromosomes of Cuscuta approximata Bab. (Convolvulaceae).

Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.

Cell Division↗

Testing the phylogenetic position of a parasitic plant (Cuscuta, Convolvulaceae, asteridae): Bayesian inference and the parametric bootstrap on data drawn from three genomes.

Previous findings on structural rearrangements in the chloroplast genome of Cuscuta (dodder), the only parasitic genus in the morning-glory family, Convolvulaceae, were attributed to its parasitic life style, but without proper comparison to related nonparasitic members of the family. Before molecular evolutionary questions regarding genome evolution can be answered, the phylogenetic problems within the family need to be resolved. However, the phylogenetic position of parasitic angiosperms and their precise relationship to nonparasitic relatives are difficult to infer. Problems are encountered with both morphological and molecular evidence. Molecular data have been used in numerous studies to elucidate relationships of parasitic taxa, despite accelerated rates of sequence evolution. To address the question of the position of the genus Cuscuta within Convolvulaceae, we generated a new molecular data set consisting of mitochondrial (atpA) and nuclear (RPB2) genes, and analyzed these data together with an existing chloroplast data matrix (rbcL, atpB, trnL-F, and psbE-J), to which an additional chloroplast gene (rpl2) was added. This data set was analyzed with an array of phylogenetic methods, including Bayesian analysis, maximum likelihood, and maximum parsimony. Further exploration of data was done by using methods of phylogeny hypothesis testing. At least two nonparasitic lineages are shown to diverge within the Convolvulaceae before Cuscuta. However, the exact sister group of Cuscuta could not be ascertained, even though many alternatives were rejected with confidence. Caution is therefore warranted when interpreting the causes of molecular evolution in Cuscuta. Detailed comparisons with nonparasitic Convolvulaceae are necessary before firm conclusions can be reached regarding the effects of the parasitic mode of life on patterns of molecular evolution in Cuscuta.

Base Sequence↗

[Identification of semen Cuscutae by HPCE].

A method for identification of semen Cuscutae, a traditional Chinese herb, was developed. The proteins in semen Cuscutae were extracted under acid or basic conditions and separated by high performance capillary electrophoresis. The electrophoretograms of acidic or basic extractants from Cuscuta chinensis Lam., Cuscuta australis R. Br. and Cuscuta japonica Choisy showed significant differences, which can be used to identify the three different semen Cuscutae. The results of the identification for 13 pharmacognosical samples agreed well with those of scanning electronic microscopy and tissue microanalysis.

Drug Contamination↗

[Quantitative analysis of semen Cuscutae].

In this paper, the quantitative analysis of the total flavone in 7 species revealed the contents: Semen Cuscutae Chinensis (3.0%), Semen Cuscutae Australis (3.2%) as compared with Semen Cuscutae Europaea (4.5%). Semen Cuscutae Chinensis has the highest content of polysaccharide of about 5-6%. The experimental results provide a scientific basis the rational use of Semen Cuscutae.

Drugs, Chinese Herbal↗

Plastid transcription in the holoparasitic plant genus Cuscuta: parallel loss of the rrn16 PEP-promoter and of the rpoA and rpoB genes coding for the plastid-encoded RNA polymerase.

The holoparasitic plant genus Cuscuta comprises a range of species whose plastid genomes have different degrees of reductions in their coding capacity. In this study, four Cuscuta species, Cuscuta reflexa, C. gronovii, C. subinclusa and C. odorata, that possess substantial physiological differences, were analysed with respect to the sequence and promoter structure of the rrn16 gene coding for the ribosomal 16S rRNA. Whereas the coding region of this gene is highly conserved among all four Cuscuta species, significant differences were observed in the non-coding region 5' of rrn16 with respect to both the length of the intergenic region between rrn16 and trnV and the promoters used to initiate transcription of the rrn16 gene. In the green species C. reflexa, rrn16 transcription starts from a functional plastid-encoded RNA polymerase (PEP) promoter that is missing in the other three species, C. gronovii, C. odorata and C. subinclusa. Instead, a 15-nucleotide-long conserved sequence immediately upstream of the mapped 5' ends bearing the nuclear-encoded RNA polymerase (NEP) promoter motif could be identified in these three species. The lack of a PEP promoter in these species coincides with the loss of two genes that encode subunits of PEP (rpoA and rpoB).

5' Flanking Region↗

Plastids of three Cuscuta species differing in plastid coding capacity have a common parasite-specific RNA composition.

The chlorophyll containing holoparasitic species Cuscuta reflexa, the achlorophyllous species Cuscuta odorata and the intermediate species Cuscuta gronovii, which contains only traces of chlorophyll, were compared with respect to their plastid coding capacity and plastid gene expression at the level of RNA. While extensive deletions have taken place in the plastid DNA of the achlorophyllous species C. odorata, the green species C. reflexa has retained an almost complete plastid genome. Although the plastid genome of the intermediate species C. gronovii has suffered extensive deletions, in contrast to the plastid genome of C. odorata it has retained photosynthesis-related genes. Hybridization with radioactive 3'-labelled RNA revealed that in all three species only a small 'parasite-specific' portion of the plastid genome consisting of mainly rRNAs and tRNAs is represented at the level of steady-state RNA. Run-on transcription assays revealed that in plastids of C. reflexa the entire genome is transcribed. Hence, the subset of RNA species required for a parasitic lifestyle is preferentially stabilized in Cuscuta plastids.

3' Untranslated Regions↗

Characterization of two tomato aquaporins and expression during the incompatible interaction of tomato with the plant parasite Cuscuta reflexa.

A subtractive suppression hybridization technique was used to identify genes that were induced during early phases of the interaction between Cuscuta reflexa, a phanerogamic plant parasite and the incompatible host tomato (Lycopersicon esculentum Mill.). One of the identified genes encodes a new aquaporin (LeAqp2) from tomato. Its function was concluded from the swelling kinetics of LeAqp2-expressing Xenopus laevis oocytes under hypo-osmotic conditions. It was shown that, 6 h after attachment of the plant parasite, the corresponding mRNA accumulated in cells at and adjacent to the attachment site of Cuscuta, while artificial wounding did not modify steady-state LeAqp2- RNA levels. Expression of a close homologue named TRAMP (tomato-ripening-associated protein) was not affected by the plant-plant interaction. Levels of indole-3-acetic acid (IAA) in tomato tissue after infection by Cuscuta have been found to increase at a similar stage of infection. In contrast to the different behavior with respect to infection, IAA induced both LeAqp2 and TRAMP expression. The observed pattern of LeAqp2 expression during the interaction at a stage where cell elongation occurs together with the water-channel activity in the heterologous expression system suggest a function for LeAqp2 during the tomato-Cuscuta interaction.

Aquaporins↗

Determination of flavonoids in Semen Cuscutae by RP-HPLC.

Flavonoids contents in 40 samples of Semen Cuscutae collected from areas all around China were investigated. Five principal flavonoids, quercetin 3-O-beta-D-galactoside-7-O-beta-D-glucoside, quercetin 3-O-beta-D-apiofuranosyl-(1-->2)-beta-D-galactoside, hyperoside, quercetin and kaempferol were analyzed simultaneously by using a reversed phase liquid chromatograph system with 0.025 M phosphoric acid-methanol as mobile phase. The recovery of the method was 97.0-102.9%, and all the flavonoids showed good linearity (r > or = 0.9990) in a relatively wide concentration range. The results indicated that contents of flavonoids in S. Cuscutae varied significantly from species to species, locality to locality, and parasiting host to host. Cuscuta australis contained a much higher content of kaempferol than C. chinensis, and few flavonoids were detected in C. japonica. The quality of S. Cuscutae can be evaluated according to the contents of flavonoids.

China↗

Studies on Sequential Parasitism by Orobanche and Cuscuta on Petunia hybrida: Choline Kinase and Phospholipid.

Parasitism by Cuscuta and Orobanche on Petunia hybrida resulted in decreased choline kinase activity and phospholipids in the host shoots. The Cuscuta-infected host roots suffered a decline in phospholipid concentration with no appreciable change in enzyme activity, whereas the roots of the Orobanche-infected plants exhibited a substantial increase in phospholipid concentration despite a marked lowering in enzymic activity. Superimposition of infection by Cuscuta on Orobanche-infected plants resulted in an increase in both enzyme activity and phospholipid in host shoots; the host roots recorded a decline in phospholipid, although enzyme activity was increased. As compared to the filaments infecting singly, Cuscuta, in sequential infection, registered an increase in phospholipid concomitant with a fall in enzyme activity, whereas the root parasite revealed a lowered enzyme activity and a slight decrease in phospholipid. It is hypothesized that a physiological response to infection by root parasite was an accumulation of phospholipids at the region under infection, and to that by shoot parasite was an uptake of phospholipids by the parasite from the host; this was effected not by de novo synthesis but rather by mobilization from distal regions.

Journal Article↗

Macromolecular trafficking between Nicotiana tabacum and the holoparasite Cuscuta reflexa.

Transgenic tobacco plants expressing green fluorescent protein (GFP) under the control of the companion cell-specific promoter, AtSUC2, were parasitized by the holoparasite Cuscuta reflexa (dodder). GFP, moving in the translocation stream of the host, was transferred to the Cuscuta phloem via the absorbing hyphae of the parasite. An identical pattern of transfer was observed for the phloem-mobile probe, carboxyfluorescein. Following uptake by the parasite, GFP was translocated and unloaded from the Cuscuta phloem in meristematic sink tissues. Contrary to published data, these observations suggest the presence of a functional symplastic pathway between Cuscuta and its hosts, and demonstrate a considerable capacity for macromolecular exchange between plant species.

Arabidopsis↗

[Study on Enrichment process of total flavones from Semen cuscutae with macroporous resin].

OBJECTIVE: To study the optimal conditions and parameters for the enrichment process of total flavones in Semen cuscutae with macroporous resin. METHODS: With the enrichment degree of total flavones in Semen cuscutae as an index, the optimal conditions of the enrichment process were investigated. RESULTS: The aqueous extract from Semen cuscutae was taken into the macroporous resin column, then the resin was washed by distilled water to get rid of impurity and 30% and 70% ethannol to elute total flavones. The above conditions were optimal. CONCLUSIONS: The process is feasible to enrich effective ingredient flavones from Semen cuscutae.

Adsorption↗

[Studies of the herbal and botanical origins of seman Cuscutae].

Herbalogical study shows that Seman Cuscutae originated from Cuscuta chinensis is one of the most commonly used drugs in ancient times. Survey of botanical origins indicates that there are 9 species in Genus Cuscutae and 4 of them are commodities. The Cuscuta chinensis specified in current Chinese Pharmacopoeia (1985) is not the principal one.

China↗

Onset of puberty and ovarian steroidogenesis following adminstration of methanolic extract of Cuscuta reflexa Roxb. stem and Corchorus olitorius Linn. seed in mice.

The effect of methanolic extract (ME) of Cuscuta reflexa stem Roxb. and Corchorus olitorius Linn. seed on the onset of reproductive maturity and the ovarian steroidogenesis was studied by means of biochemical techniques. ME of Cuscuta reflexa stem and Corchorus olitorius seed treatment causes a remarkable delay in sexual maturation as evidenced by the age at vaginal opening and appearance of first estrus (cornified smear). The same treatment also results in a significant diminution of Delta(5)-3beta-hydroxysteroid dehydrogenase (HSD) and glucose-6-phosphate dehydrogenase (G-6-PD) activity along with a reduction in the weight of ovary, uterus and pituitary. On the basis of above data, it is assumed that the probable cause of delayed maturation in ME of Cuscuta reflexa stem and Corchorus olitorius seed treated mice is due to the suppressed ovarian steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗

The cell wall-modifying xyloglucan endotransglycosylase/hydrolase LeXTH1 is expressed during the defence reaction of tomato against the plant parasite Cuscuta reflexa.

A suppressive subtractive hybridization technique was used to identify genes, which were induced during the early phases of the interaction between dodder (Cuscuta reflexa), a phanerogamic parasite, and its incompatible host plant tomato. One of the identified genes encodes a tomato xyloglucan endotransglycosylase/hydrolase (XTH)--an enzyme involved in cell wall elongation and restructuring. The corresponding LeXTH1 mRNA accumulated 6 h after attachment of the parasite. In contrast, wounding did not influence the expression level. Subsequent to LeXTH1 mRNA accumulation, an increase in XTH activity at the infection sites as well as in adjacent tissues was observed. The effect of IAA on LeXTH1 expression was analyzed because the concentration of this phytohormone is known to increase in the tomato tissue during the interaction with the parasite. LeXTH1 mRNA accumulation was in fact induced by external application of auxin. However, in the auxin-insensitive tomato mutant diageotropica, Cuscuta induced LeXTH1-mRNA accumulated with a time course similar to wild type tomato. Thus, auxin appears not to be an essential signal for infection-induced LeXTH1 activation. Our data suggest a role for xyloglucan transglycosylation in defence reactions associated with the incompatible tomato- Cuscuta interaction.

Cell Wall↗

Transfer of phloem-mobile substances from the host plants to the holoparasite Cuscuta sp.

During the development of the haustorium, searching hyphae of the parasite and the host parenchyma cells are connected by plasmodesmata. Using transgenic tobacco plants expressing a GFP-labelled movement protein of the tobacco mosaic virus, it was demonstrated that the interspecific plasmodesmata are open. The transfer of substances in the phloem from host to the parasite is not selective. After simultaneous application of (3)H-sucrose and (14)C-labelled phloem-mobile amino acids, phytohormones, and xenobiotica to the host, corresponding percentages of the translocated compounds are found in the parasite. An open continuity between the host phloem and the Cuscuta phloem via the haustorium was demonstrated in CLSM pictures after application of the phloem-mobile fluorescent probes, carboxyfluorescein (CF) and hydroxypyrene trisulphonic acid (HPTS), to the host. Using a Cuscuta bridge (14)C-sucrose and the virus PVY(N) were transferred from one host plant to the another. The results of translocation experiments with labelled compounds, phloem-mobile dyes and the virus should be considered as unequivocal evidence for a symplastic transfer of phloem solutes between Cuscuta species and their compatible hosts.

Amino Acids↗

A large deletion in the plastid DNA of the holoparasitic flowering plant Cuscuta reflexa concerning two ribosomal proteins (rpl2, rpl23), one transfer RNA (trnI) and an ORF 2280 homologue.

We have determined the nucleotide sequence of a 5.3-kb region of the plastid DNA (ptDNA) from the heterotrophic holoparasitic plant Cuscuta reflexa. The cloned area contains genes for the D1-protein (32-kDa protein; psbA), tRNA(His) (trnH), ORF 740 (homologous to ORF 2280 from Nicotiana tabacum), ORF 77 (homologous to ORF 70), tRNA(Leu) (trnL) and a hypothetical ORF 55 which has no homology to any known gene among higher plants. This 5.3-kb area is colinear with a 12.4-kb region of tobacco ptDNA and has therefore undergone several deletions totalling 7.1 kb. Most of the missing nucleotides belong to one large deletion in the ptDNA of C. reflexa of approximately 6.5 kb. This deletion involves two ribosomal protein genes, rpl2 and rpl23, as well as the transfer RNA for Isoleucin (trnI) and a region encoding 1540 amino-acid residues of an ORF 2280 homologue, as compared to tobacco chloroplast DNA. This is remarkable since the remaining genes, especially the psbA gene, are highly conserved in C. reflexa. Furthermore, we found that the expression of the psbA gene is in the same range as in the autotrophic Ipomoea purpurea which belongs to the same family as Cuscuta (Convolvulaceae). Here we hypothesize a total loss of rpl2 and rpl23 in the entire genome of C. reflexa. The phylogenetic position of, and the evolutionary change of ptDNA from, Cuscuta are discussed.

Amino Acid Sequence↗