Phalaris Coerulescens Research Articles

Identification of Changes in Wheat (Triticum aestivum L.) Seeds Proteome in Response to Anti–trx s Gene

BackgroundThioredoxin h (trx h) is closely related to germination of cereal seeds. The cDNA sequences of the thioredoxin s (trx s) gene from Phalaris coerulescens and the thioredoxin h (trx h) gene from wheat are highly homologous, and their expression products have similar biological functions. Transgenic wheat had been formed after the antisense trx s was transferred into wheat, and it had been certified that the expression of trx h decreased in transgenic wheat, and transgenic wheat has high resistance to pre-harvest sprouting.Methodology/Principal FindingsThrough analyzing the differential proteome of wheat seeds between transgenic wheat and wild type wheat, the mechanism of transgenic wheat seeds having high resistance to pre-harvest sprouting was studied in the present work. There were 36 differential proteins which had been identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). All these differential proteins are involved in regulation of carbohydrates, esters, nucleic acid, proteins and energy metabolism, and biological stress. The quantitative real time PCR results of some differential proteins, such as trx h, heat shock protein 70, α-amylase, β-amylase, glucose-6-phosphate isomerase, 14-3-3 protein, S3-RNase, glyceraldehyde-3-phosphate dehydrogenase, and WRKY transcription factor 6, represented good correlation between transcripts and proteins. The biological functions of many differential proteins are consistent with the proposed role of trx h in wheat seeds.Conclusions/SignificanceA possible model for the role of trx h in wheat seeds germination was proposed in this paper. These results will not only play an important role in clarifying the mechanism that transgenic wheat has high resistance to pre-harvest sprouting, but also provide further evidence for the role of trx h in germination of wheat seeds.

Transgenic barley with overexpressed PTrx increases aluminum resistance in roots during germination

A transgenic barley line (LSY-11-1-1) with overexpressed Phalaris coerulescens thioredoxin gene (PTrx) was employed to measure the growth, protein oxidation, cell viability, and antioxidase activity in barley roots during germination on the presence of 2 mmol/L AlCl(3) on filter paper. The results show that (1) compared with the non-transgenic barley, LSY-11-1-1 had enhanced root growth, although both were seriously inhibited after AlCl(3) treatment; (2) the degree of protein oxidation and loss of cell viability in roots of LSY-11-1-1 were much less than those in roots of non-transgenic barley, as reflected by lower contents of protein carbonyl and Evans blue uptakes in LSY-11-1-1; (3) activities of catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR) in LSY-11-1-1 root tips were generally higher than those in non-transgenic barley root tips, although these antioxidase activities gave a rise to different degrees in both LSY-11-1-1 and non-transgenic barley under aluminum stress. These results indicate that overexpressing PTrx could efficiently protect barley roots from oxidative injury by increasing antioxidase activity, thereby quenching ROS caused by AlCl(3) during germination. These properties raise the possibility that transgenic barley with overexpressed PTrx may be used to reduce the aluminum toxicity in acid soils.

Current situation of the Gaudinio fragilis—Hordeion bulbosi alliance in the Iberian Peninsula

This study reveals the occurrence of Phalaris coerulescens communities both in Portugal and the south of Andalusia (Spain). These grasslands present a variable cover ratio and have an average dominant vegetation size of about 100 cm. They tend to occur in river beds and in places which are sometimes water-logged, with a certain amount of organic matter inducing the occurrence of nitrophilous companion species. The soils are always siliceous, very poor in carbonates, but with a high proportion of sand. Consequently, flooding never lasts for long. The grasslands of Portugal are located in the Marianic—Monchiquensean unit, within the series of Pyro bourgaeanae—Queceto rotundifo-liae s. and Poterio agrimonioidis—Querceto suberis s. The new Senecio foliosi—Phalaridetum coerulescentis is characterized by Senecio jacobaea var foliosus, meanwhile, in the the south of Andalusia, the Hedysaro coronarii—Phalaridetum coerulescentis is described for the Aljibic and Gaditan—Onubensean Sectors, and, in the Rondean and Bermejensean areas, the Elymo repentis—Phalaridetum coerulescentis. These associations are included in the Gaudinio fragilis—Hordeion bulbosi alliance.

High-resolution mapping of theSandZloci ofPhalaris coerulescens

Self incompatibility (SI) in Phalaris coerulescens is gametophytically determined by two unlinked multi allelic loci (S and Z). Neither the S nor Z genes have yet been cloned. As part of a map-based cloning strategy, high-resolution maps of the S and Z regions were generated from distorted segregating populations using RFLP probes from wheat, barley, oat, and Phalaris. The S locus was delimited to 0.26 cM with two boundary markers (Xwg811 and Xpsr168) and cosegregated with Xbm2 and Xbcd762. Xbcd266 was the closest marker linked to Z (0.9 cM). A high level of colinearity in the S and Z regions was found in both self-incompatible and -compatible species. The S locus was localized to the subcentromere region of chromosome 1 and the Z locus to the long arm end of chromosome 2. Several rice BAC clones orthologous to the S and Z locus regions were identified. This opens the possibility of using the rice genome sequence data to generate more closely linked markers and identify SI candidate genes. These results add further support to the conservation of gene order in the S and Z regions of the grass genomes.

The development of sequence-tagged sites (STSs) in Lolium perenne L.: the application of primer sets derived from other genera

Genetic analysis, particularly the development of genetic linkage maps in forage grass species, lags well behind other members of the Poaceae. Comparative mapping within this family has revealed extensive conservation in gene and marker synteny among chromosomes of diverse genera. Recently, the ability to transfer mapped STS markers between barley and wheat has been demonstrated. The transfer of mapped STS markers between cereals and forage grasses could provide PCR-based markers for comparative mapping in these species providing they amplify homologous sequences. In this study, primers derived from three barley genes of defined function and a gene from Phalaris coerulescens were used to amplify homologous fragments in Lolium perenne. Primers derived from two barley and two oat cDNA clones were also tested along with eight barley and two Triticum tauchii STS markers. Twenty one primer pairs derived from 18 loci were tested. Eleven primer pairs (52%) amplified homologous sequences in L. perenne from ten (55%) of the loci targetted. Thirteen new STS markers were generated in L. perenne, of which ten have been mapped in barley or rye and amplify homologous sequences in L. perenne.

Cloning and expression of a distinct subclass of plant thioredoxins.

mRNAs encoding a novel thioredoxin were isolated from pollen RNA of Lolium perenne (LpTrx), Hordeum bulbosum (HbTrx), Phalaris coerulescens (PTrx) and Secale cereale (ScTrx). The cDNAs contain a single ORF of 393 bp encoding a protein of 131 amino acids. The predicted proteins showed highest homology to plant thioredoxins of the h class yet form a distinct subgroup that is characterized by a high level of sequence conservation (95.4-97.7% identity). GenBank searches revealed additional members of this subclass in tomato, soybean, rice and pine. LpTrx and PTrx were expressed as recombinant proteins in Escherichia coli and tested for thioredoxin activity. Both proteins displayed typical thioredoxin activity in the nonspecific insulin reduction assay, however, were not reduced by E. coli NADPH-dependant thioredoxin reductase.

Revisiting and Revising the Self-Incompatibility Genetics of Phalaris coerulescens

In the grasses, self-incompatibility is controlled by two unlinked loci, S and Z. Previously, Li et al. ([1994][1]) presented a differential screening technique to identify a putative S gene clone from Phalaris coerulescens. In this approach, a cDNA library made from mature pollen of P. coerulescens

Revisiting and Revising the Self-Incompatibility Genetics of Phalaris coerulescens

Revisiting and Revising the Self-Incompatibility Genetics of Phalaris coerulescens

(−)-Phalarine, a furanobisindole alkaloid from Phalariscoerulescens

Abstract Chemical examination of Phalaris coerulescens revealed a new alkaloid, phalarine.Extensive mass spectrometric and NMR spectroscopic examination of phalarine and itsmonoacetyl and monomethyl derivatives indicated a novel furano bis indole structure.

Oxindoles from Phalaris coerulescens

A phytochemical examination of various accessions of Phalaris coerulescens for the presence of toxic phenylethylamines, indoloamines and tetrahydro-β-carbolines revealed the presence of oxindoles previously not reported to occur in Phalaris species. One of the oxindoles, the structure of which was elucidated by spectroscopic methods, has not hitherto been reported to occur naturally and has been given the trivial name coerulescine.

A pollen-specific cDNA of Phalaris coerulescens shows homology to Zm13

Genes expressed by the male gametophyte have been divided into two classes, the early genes, which are expressed shortly after meiosis, and the late genes, which accumulate after microspore mitosis and are thought to play a role in germination, pollen-stigma interactions and early pollen tube growth (Mascarenhas 1989). Among the best-studied examples of late genes are Zm13 of maize (Hamilton et al. 1989: Hanson et al. 1989) and its homologs PS1 from rice (Zou et al. 1994) and LAT52 of tomato (Twell et al. 1989). Here we report the cDNA sequence of a Zm13 homologue from Phalaris coerulescens, a mediterranean grass which is used as a model system for the study of self-incompatibility in the Poaceae (Li et al. 1994). A cDNA library was constructed from pollen of Phalaris coerulescens in bacteriophage vector Lambda gt10. In order to isolate pollen-specific clones, this library was differentially screened with cDNA probes from leaves, roots, seeds, 7-day-old seedlings and young anthers around meiosis. AWPC-C7c was selected on the basis that it did not show hybridization with cDNA from any of the non-pollen tissues. A developmental RNA blot confirmed that AWPC-C7c expression is restricted to Nucleotide sequence data will appear in the EMBL, GenBank and DDBJ databases under the accession number U85499.

Self-incompatibility in the grasses: evolutionary relationship of the S gene from Phalaris coerulescens to homologous sequences in other grasses.

Self-incompatibility is widespread in the grasses and it is proposed that the grasses share a common incompatibility mechanism that is distinct from those operating in the dicotyledonous species studied in great detail. Where good genetic data are available, all grass species appear to have an incompatibility mechanism controlled by two unlinked loci, S and Z. A putative S gene has been cloned from Phalaris coerulescens. This gene is characterized by two major domains: an allele specificity domain and a thioredoxin catalytic domain. A family of sequences with varying degrees of homology to this gene has been identified among 15 grass species covering all subfamilies of the Poaceae. These S-related sequences appear to be present in the grass family regardless of self-compatibility. Evidence is presented to show that at least one of the sequences is transcribed, suggesting a functional gene. In contrast to the high expression of the S gene in Phalaris pollen, expression of the related gene in the pollen (or anthers) of the grass species examined was so low that RNA gel blot analysis failed to display a significant signal. However, reverse transcription-based polymerase chain reaction (RT-PCR) successfully amplified the region corresponding to the S thioredoxin domain from 10 of the grass species. With grasses other than Phalaris, RT-PCR showed limited success in amplifying the region corresponding to the S variable portion at the 5' end of the Phalaris S gene. Sequencing of the PCR-amplified S thioredoxin region from wheat, barley, rye and Dactylis revealed that this is a highly conserved gene with 94-97% sequence similarity with the corresponding Phalaris S gene. The conservation of sequence and ubiquitous expression of the gene across the grass family strongly suggest that the S-related gene is carrying out a significant biological function in the Poaceae. On the basis of these findings, a model for the evolution of the S self-incompatibility gene in the grasses is proposed.

A self‐fertile mutant of Phalaris produces an S protein with reduced thioredoxin activity

Gametophytic self-incompatibility in the Phalaris coerulescens is controlled by two unlinked genes, S and Z. Isolation of the S gene from the pollen of this grass species indicated that the C terminus has significant homology with thioredoxin H proteins. The protein from the C terminus, expressed in Escherichia coli, exhibits thioredoxin-life activity. This paper demonstrates that the C terminus of the S protein from an S complete mutant shows significant reduction in thioredoxin activity when compared with the wild-type form. Both pollen and stigma have lost self-compatibility in this mutant. Close examination of the lesions, which were found only in the C terminus of the mutant gene suggests that the substitution of a serine by an arginine is responsible for the reduced enzymatic activity. The association between reduced activity and the loss of the self-incompatibility provides evidence for a role of thioredoxin activity in the self-incompatibility reaction of this species.

Molecular mechanisms of self-incompatibility

Recent work has furthered our understanding of the molecules involved, and the diversity of mechanisms displayed, in self-incompatibility. S-RNases have been identified in two more plant families and their structure/function relationships are currently being dissected. Novel S genes have been cloned and expressed in Papaver rhoeas and Phalaris coerulescens, and the complexity of the S locus in Brassica is being unraveled. Present data implicate the involvement of protein kinase activity in all systems where pollen inhibition occurs at the stigma surface and RNase activity in those systems displaying stylar inhibition.

Thioredoxin activity in the C terminus of Phalaris S protein.

Self-incompatibility in the grass Phalaris coerulescens is controlled by two genes S and Z. Isolation and sequencing of two S alleles showed that they encode proteins that are highly conserved at the C terminus with significant homology to thioredoxin H proteins. In particular, the residues in and around the active site of thioredoxin, Trp-Cys-Gly-Pro-Cys, are perfectly conserved. The C terminus of the S protein has been expressed in Eschericia coli and purified to homogeneity on Ni-NTA resin. Functional assays showed that the protein has thioredoxin activity; it can act as a substrate for E. coli thioredoxin reductase and also catalyse the reduction of insulin by dithiothreitol. The possible role of thioredoxin-like activity of the S protein in mediating the incompatibility reaction in Phalaris is discussed.

Cloning a putative self-incompatibility gene from the pollen of the grass Phalaris coerulescens.

In Phalaris coerulescens, gametophytic self-incompatibility is controlled by two unlinked genes: S and Z. A probable S gene has now been isolated and sequenced. This represents a novel self-incompatibility gene isolated from pollen in the multilocus system of a monocotyledonous plant. The gene is approximately 3 kb long, split by five introns, and exclusively expressed in the mature pollen. The deduced amino acid sequences from the S1, S2, and part of the S4 alleles showed that the protein has a variable N terminus and a conserved C terminus. The sequence of a complete mutant at the S locus indicated that mutations in the conserved C terminus, a thioredoxin-like region, led to loss of function. We propose that the gene has two distinct sections, a variable N terminus determining allele specificity and a conserved C terminus with the catalytic function. The gene structure and its deduced protein sequences strongly suggest that this monocotyledon has developed a self-incompatibility system entirely different from those operating in the dicotyledons. The possible interactions between S and Z genes in both pollen and stigma are discussed.

Cloning a Putative Self-Incompatibility Gene from the Pollen of the Grass Phalaris coerulescens

Cloning a Putative Self-Incompatibility Gene from the Pollen of the Grass Phalaris coerulescens

Mutations affecting self-incompatibility in Phalaris coerulescens Desf. (Poaceae)

A total of 508 possible mutants affecting self-incompatibility in Phalaris coerulescens were obtained from a mutant trap experiment. None of these ‘mutants’, included mutations to a new functional incompatibility allele and 356 behaved as revertants. One hundred and fifty-two of the mutants were self-compatible and the methods of analysis of these mutants are described and the results from studying 37 of them are reported. Sixteen are mutants of the S locus including pollen-only mutants and one complete mutant; six are pollen-only mutants at the Z locus and 16 are mutants at a further locus T. This locus does not act to provide genetic diversity in specifying the incompatibility status of the pollen or style. Possible roles for this locus are suggested.

Stigma proteins of the two loci self-incompatible grass Phalaris coerulescens

Protein extracts from four self-incompatible genotypes of Phalaris coerulescens were subjected to analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultra-thin isoelectric focusing. A comparison between stigma, root and leaf extracts showed that there was no significant difference in electrophoretic or isoelectric focusing patterns between the genotypes for both root and leaf proteins. However, stigma protein patterns did vary between genotypes especially within the molecular weight region of 43 000–97 000 and within the pI range 5–7. The stigma-specific changes strongly suggest a link between the self-incompatible genotype and these stigma proteins. However, because there are two loci involved, it is not yet possible to precisely assign particular proteins to each S- or Z-allele.

The incompatibility loci as indicators of conserved linkage groups in the Poaceae

Linkage between an incompatibility locus and the locus of the gene encoding the enzyme glucose phosphoisomerase has been demonstrated for the grasses Alopecurus myosuroides, Phalaris coerulescens, Festuca pratensis, Holcus lanatus and Secale cereale suggesting the conservation of a common chromosomal element in the evolution of the Poaceae.