Pea Gene Research Articles

Identification of RAPD markers linked to the rust (Uromyces fabae) resistance gene in pea (Pisum sativum)

Two RAPD markers linked to gene for resistance (assayed as pustule number cm−2 leaf area) to rust [Uromyces fabae (Pers.) de Bary] in pea (Pisum sativum L.) were identified using a mapping population of 31 BC1F1 [HUVP 1 (HUVP 1 × FC 1] plants, FC 1 being the resistant parent. The analysis of genetics of rust resistance was based on the parents, F1, F2, BC1F1 and BC1F2 generations. Rust resistance in pea is of non-hypersensitive type; it appeared to be governed by a single partially dominant gene for which symbol Ruf is proposed. Further, this trait seems to be affected by some polygenes in addition to the proposed oligogene Ruf. A total of 614 decamer primers were used to survey the parental polymorphism with regard to DNA amplification by polymerase chain reaction. The primers that amplified polymorphic bands present in the resistant parent (FC 1) were used for bulked segregant analysis. Those markers that amplified consistently and differentially in the resistant and susceptible bulks were separately tested with the 31 BC1F1 individuals. Two RAPD makers, viz., SC10-82360 (primer, GCCGTGAAGT), and SCRI-711000 (primer, GTGGCGTAGT), flanking the rust resistance gene (Ruf) with a distance of 10.8 cM (0.097 rF and LOD of 5.05) and 24.5 cM (0.194 rF and a LOD of 2.72), respectively, were identified. These RAPD markers were not close enough to Ruf to allow a dependable maker-assisted selection for rust resistance. However, if the two makers flanking Ruf were used together, the effectiveness of MAS would be improved considerably.

Shoot and inflorescence branching

A major aspect of postembryonic plant development is the formation of secondary axes of growth: vegetative branches, inflorescence branches, or flowers. The first step in side-shoot development is the establishment of lateral meristems in the axils of leaves. GRAS-, MYB-, and bHLH-type transcription factors act as key regulators of early steps in this process. The REVOLUTA subfamily of HD-ZIP transcription factors controls the organization of lateral meristems. Whereas the development of lateral meristems into lateral buds is only poorly understood, recent studies have provided new insights into the regulation of lateral bud outgrowth. The MORE AXILLARY GROWTH (MAX) genes of Arabidopsis and the RAMOSUS (RMS) genes of pea are involved in the production, perception, and transduction of a signal that inhibits lateral bud outgrowth. Synthesis of this not-yet-identified hormone is positively regulated by the main shoot tip through auxin signalling.

The Last Reaction Producing Brassinolide Is Catalyzed by Cytochrome P-450s, CYP85A3 in Tomato and CYP85A2 in Arabidopsis

Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.

Expression of the Symbiotic nod4 Pea Gene against Various Genotypic Backgrounds

Experiments on the effect of genotypic environment on the expression of the nod4 gene, responsible for supernodulation in pea, were performed. The genotypic background was found to affect the manifestation of both major symbiosis-related traits (number of nitrogen-fixing bacterial nodules and nitrogenase activity) and productivity-related traits (stem height, seed number, and seed weight), which form the pleiotropic complex of the mutant gene. Using recurrent selection, we developed supernodulating lines significantly exceeding the original mutant line and studied them up to generations F5-F6. Of special importance is the fact that these lines showed high levels of nodulation and nitrogen fixation. The results are discussed from the viewpoint of breeding.

Identification and mapping of polymorphic RAPD markers of pea (Pisum sativum L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.

Brassinosteroid deficiency due to truncated steroid 5alpha-reductase causes dwarfism in the lk mutant of pea.

The endogenous brassinosteroids in the dwarf mutant lk of pea (Pisum sativum) were quantified by gas chromatography-selected ion monitoring. The levels of castasterone, 6-deoxocastasterone, and 6-deoxotyphasterol in lk shoots were reduced 4-, 70-, and 6-fold, respectively, compared with those of the wild type. The fact that the application of brassinolide restored the growth of the mutant indicated that the dwarf mutant lk is brassinosteroid deficient. Gas chromatography-selected ion monitoring analysis of the endogenous sterols in lk shoots revealed that the levels of campestanol and sitostanol were reduced 160- and 10-fold, respectively, compared with those of wild-type plants. These data, along with metabolic studies, showed that the lk mutant has a defect in the conversion of campest-4-en-3-one to 5alpha-campestan-3-one, which is a key hydrogenation step in the synthesis of campestanol from campesterol. This defect is the same as that found in the Arabidopsis det2 mutant and the Ipomoea nil kbt mutant. The pea gene homologous to the DET2 gene, PsDET2, was cloned, and it was found that the lk mutation would result in a putative truncated PsDET2 protein. Thus it was concluded that the short stature of the lk mutant is due to a defect in the steroidal 5alpha-reductase gene. This defect was also observed in the callus induced from the lk mutant. Biosynthetic pathways involved in the conversion of campesterol to campestanol are discussed in detail.

A Promoter from Pea Gene DRR206 Is Suitable to Regulate an Elicitor-Coding Gene and Develop Disease Resistance

ABSTRACT Plant nonhost disease resistance is characterized by the induction of multiple defense genes. The pea DRR206 gene is induced following inoculation with pathogens and treatment with abiotic agents, and moderately induced by wounding. A deletion series of DRR206 promoter segments was fused with the beta-glucuronidase (GUS) reporter gene and transiently transferred to tobacco, potato, and pea. GUS activity revealed that two upstream regions of the DRR206 promoter were particularly important for activation in the three plant species. Putative cis regulatory elements within the DRR206 promoter included a wound/pathogen- inducible box (W/P-box) and a WRKY box (W-box). Gel shift assays with nuclear extracts from treated and untreated tissue with the W/P-box revealed both similar and unique protein-DNA complexes from pea, potato, and tobacco. Tobacco was stably transformed with gene constructs of the DRR206 promoter fused with a DNase elicitor gene from Fusarium solani f. sp. phaseoli, FsphDNase. Pathogenicity tests indicated that the FsphDNase elicitor conferred resistance against Pseudomonas syringae pv. tabaci and Alternaria alternata in tobacco. Transgenic potatoes showed some sensitivity to the FsphDNase gene providing less protection against Phytophthora infestans. Thus, the elicitor-coding gene, FsphDNase, is capable of generating resistance in a heterologous plant system (tobacco) when fused with defined regions of the pea DRR206 promoter.

Structure and expression of 12-oxophytodienoate reductase (subgroup I) genes in pea, and characterization of the oxidoreductase activities of their recombinant products.

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.

Analysis of pea HMG‐I/Y expression suggests a role in defence gene regulation

SUMMARY HMG-I/Y proteins are characterized by the presence of AT-hook motifs, DNA binding domains that recognize AT-rich tracts of DNA. By facilitating protein:protein and protein:DNA interactions in the vicinity of these AT-rich binding sites, HMG-I/Y positively or negatively regulates gene expression. Several pea defence gene promoters have AT-rich tracts of DNA that are potential targets for modulation via HMG-I/Y. In this study, a comparison of the expression of a pea defence gene (DRR206) mRNA relative to the expression of HMG-I/Y mRNA was monitored by Northern analysis following the inoculation of a fungal pathogen, Fusarium solani or treatment with chitosan and a F. solani DNase (Fsph DNase). In pea pod endocarp tissue, HMG-I/Y expression was observed at high levels in untreated tissue and at lower levels 6 h following inoculation or wounding of the tissue. Western blots with an antipea HMG-I/Y polyclonal antibody also revealed that pea HMG-I/Y is expressed at decreased levels 6 h following inoculation or elicitor treatment. HMG-I/Y extracted from pea caused alterations in the gel migration of radio-labelled AT-rich sequences from the pea DRR206 promoter, suggesting that similar interactions could exist in vivo. Agroinfiltration was utilized to express the pea HMG-I/Y gene in tobacco containing a chimeric gene fusion of a promoter from the PR gene, DRR206, and the beta-glucuronidase (GUS) reporter gene. Transient expression of pea HMG-I/Y led to a decrease in GUS reporter gene activity in the heterologous tobacco system. These data implicate pea HMG-I/Y abundance in the down-regulation of DRR206 gene expression, and possibly HMG-I/Y depletion in the expression of defence genes in pea.

MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea.

Shoot branching is inhibited by auxin transported down the stem from the shoot apex. Auxin does not accumulate in inhibited buds and so must act indirectly. We show that mutations in the MAX4 gene of Arabidopsis result in increased and auxin-resistant bud growth. Increased branching in max4 shoots is restored to wild type by grafting to wild-type rootstocks, suggesting that MAX4 is required to produce a mobile branch-inhibiting signal, acting downstream of auxin. A similar role has been proposed for the pea gene, RMS1. Accordingly, MAX4 and RMS1 were found to encode orthologous, auxin-inducible members of the polyene dioxygenase family.

A supaeromaculata mutation affects heterochrony in pea

Mutations in Aero1 promote the silver flecking (aeromaculata) commonly seen on wild‐type pea (Pisum sativum L.) leaves, resulting in a phenotype known as supaeromaculata. We show here the MIII/122 line carries a supaeromaculata mutation allelic to the type aero allele (aero1‐1). MIII/122 (aero1‐10) and aero1‐1 were also found to attain an adult leaf form, flower and undergo apical arrest earlier than their wild‐type progenitor, cv. Virtus and cv. Torsdag, respectively. Principal component analyses indicated that in both cases these characteristics co‐segregated with the supaeromaculata phenotype. These results suggest that Aero1 is associated with the timing of plant development. Plants carrying an aero1 mutation become fully adult and reproductively competent sooner than their wild‐type counterparts, suggesting Aero1 is required to delay these aspects of plant development. Thus, Aero1 represents a previously unrecognized heterochronic gene in the garden pea.

Multiple plastid signals regulate the expression of the pea plastocyanin gene in pea and transgenic tobacco plants.

The expression of nuclear genes encoding photosynthesis-related proteins is regulated by signals from plastids. To investigate how the pea PetE gene encoding plastocyanin is regulated by plastid signals, the effects of norflurazon, lincomycin and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), specific inhibitors of plastid-located processes generating plastid signals, have been examined. RNA-gel blot analysis of 7-day-old pea and tobacco seedlings containing the pea PetE gene showed that treatment with norflurazon and lincomycin, but not DCMU, decreased the accumulation of transcripts of pea PetE and endogenous Lhcb1 genes. Analysis of chimeric PetE gene constructs in tobacco seedlings showed that an intact PetE mRNA 5' terminus and elements within the PetE coding region were required to confer sensitivity to norflurazon and lincomycin, suggesting post-transcriptional regulation. Analysis of 4-week-old tobacco plants containing chimeric PetE constructs showed that DCMU treatment decreased the accumulation of pea PetE and Lhcb1 transcripts, but had opposite effects on the transcription of the genes in nuclear run-on assays. DCMU upregulated transcription from the pea PetE promoter whereas transcription of tobacco Lhcb1 genes was decreased. These experiments provide evidence for multiple plastid signals operating at different developmental stages and affecting transcriptional and post-transcriptional processes regulating expression of the pea PetE gene.

Effect of mutations in the pea genes Sym33 and Sym40. II. Dynamics of arbuscule development and turnover.

Two symbiotic pea (Pisum sativum L.) mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread formation in symbiotic root nodules were characterised with respect to dynamics of arbuscule development at 15 degrees C and 24 degrees C. Mutation of sym33 decreased mycorrhiza colonisation at both temperatures and delayed arbuscule development at 15 degrees C, whereas mutation of sym40 accelerated mycorrhiza colonisation and arbuscule senescence at 24 degrees C. The differences between the mutants and the wild-type were more pronounced at 24 degrees C, a temperature close to the optimum for pea growth. The results demonstrate that both pea genes are important in the control of arbuscular mycorrhiza development and can be considered necessary for the tripartite symbiosis in pea.

Effect of mutations in the pea genes Sym33 and Sym40. I. Arbuscular mycorrhiza formation and function.

Two pea (Pisum sativum L.) symbiotic mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread development and function in symbiotic root nodules have been characterised in terms of mycorrhizal colonisation of roots, shoot and root biomass accumulation and shoot and root phosphorus (P) content. The mutation in gene sym33 decreased mycorrhizal colonisation of roots (except arbuscule abundance in mycorrhizal root fragments, which increased) but did not change the effectiveness of mycorrhiza function. The mutation in sym40 did not affect either of these processes. Both mutants showed differences in plant development compared with the wild-type line SGE. The mutants had delayed flowering and pod ripening, and shoot/root biomass ratios and P accumulation also differed from those of SGE. These observations suggest that the gene mutations cause systemic changes in plant development.

STS Markers for Comparative Mapping in Legumes

DNA primers for 37 genes have been developed in pea (Pisum sativum L.). Two-thirds of these primers also amplify orthologous sequences in lentil (Lens culinaris). The primers were designed to be complementary to highly conserved sequences in exons of known genes. In addition, most of the priming sequences were selected to be 1000 to 3000 bp distant on the genomic DNA and to amplify a fragment that contained at least one intron. Segregating sequence polymorphism in mapping populations of recombinant inbred lines (RILs) derived from wide crosses in Pisum was observed by restriction of the amplified fragment with endonucleases recognizing four-base restriction sites. Successful mapping of 36 of these genes in pea demonstrated the utility of these primers for mapping, and it appears likely that the primers should have general utility for comparative mapping in legumes.

Identification of ultraviolet-B responsive genes in the pea, Pisum sativum L.

In the present work, UV-B-repressible and UV-B-inducible genes were identified in the pea, Pisum sativum L., by rapid amplification of 3′ cDNA ends through use of the polymerase chain reaction. Of the UV-B-repressible clones, psUVRub and psUVDeh represent genes encoding Rubisco activase and dehydrin, respectively. A third clone, psUVZinc, did not correspond closely in overall nucleotide sequence to any gene registered in GenBank; however, a short deduced peptide shared similarity with the photosystem-II reaction center X protein of the chlorophyll a+c-containing alga, Odontella sinensis. The UV-B-inducible clones, psUVGluc, psUVAux and psUVRib, were related to genes encoding β-1, 3-glucanase, auxin-repressed protein, and a 40S ribosomal protein, respectively. The modulation of these pea genes indicates how UV-B, through its actions as a physical stressor, affects several important physiological processes in plants.

Characterization of a tissue-specific and developmentally regulated beta-1,3-glucanase gene in pea (Pisum sativum).

As part of a search for seed coat-specific expressed genes in Pisum sativum cv. Finale by PCR-based methods, we identified and isolated a cDNA encoding a beta- 1,3-glucanase, designated PsGNS2. The deduced peptide sequence of PsGNS2 is similar to a subfamily of beta-1,3-glucanases, which is characterized by the presence of a long amino acid extension at the C-terminal end compared to the other beta-1,3-glucanases. PsGNS2 is expressed in young flowers and in the seed coat and is weakly expressed in vegetative tissues (roots and stems) during seedling development. It is not inducible by environmental stress or in response to fungal infection. In developing pea flowers the transcript is detectable in all four whirls. In the seed coat the expression is temporally and spatially regulated. High abundance of the transcript became visible in the seed coat when the embryo reached the late heart stage and remained until the mid seed-filling stage. In situ hybridization data demonstrated that the expression of PsGNS2 is restricted to a strip of the inner parenchyma tissue of the seed coat, which is involved in temporary starch accumulation and embryo nutrition. This tissue showed also less callose deposits than the other ones. The 5' genomic region of PsGNS2 was isolated and promoter activity studies in transgenic Medicago truncatula showed a seed-specific expression. Highest activity of the promoter was found in the seed coat and in the endosperm part of the seed.

IDENTIFICATION OF MOLECULAR MARKERS LINKED TO POWDERY MILDEW (Erysiphe pisi Syd.) RESISTANCE MUTATED GENES IN PISUM SATIVUM L.

The aim of this work was to find DNA markers linked to powdery mildew resistance genes in pea (Pisum sativum L.). Two resistant mutant lines selected after ethylnitrosurea chemical mutagenesis of the commercial varieties Frilene and Solara and the respective two original susceptible varieties were included in this study. The screening of 360 decamer primers enabled the identification of two RAPD markers linked to one of the mutant resistance genes (Frilene mutant), which need further confirmation in a segregant F2 progeny. The same set of primers did not enable the identification of any molecular marker linked to the other resistance gene from the Solara mutant. For the identification of AFLP markers, 20 EcoRI/MseI primer combinations have already been tested, but none have revealed any polymorphism. Other primer combinations are currently being used in order to find additional molecular markers.

New Gene Crt(Curly Roots) Controlling Pea (Pisum sativum L.) Root Development

Using ethyl methane sulfonate (EMS) treatment of the seeds of line SGE, a new mutant of pea (Pisum sativum L.) with alterations in root development was obtained. The mutant phenotype depends on the density of the growth substrate: on sand (a high density substrate) the mutant forms a small compact curly root system whereas on vermiculite (a low density substrate) differences between the root systems of the mutant and wild type plants are less pronounced. Genetic analysis revealed that the mutant carries a mutation in a new pea gene designedcrt (curly roots). Gene crt has been localized in pea linkage group V. The mutant line named SGEcrt showed increased sensitivity to exogenous auxin and an increased concentration of endogenous indole-3-acetic acid (IAA) in comparison with the wild type line SGE.

Photosynthetic electron flow regulates transcription of the psaB gene in pea (Pisum sativum L.) chloroplasts through the redox state of the plastoquinone pool.

Plants respond to changing light conditions by altering the stoichiometry between components of the photosynthetic electron transport chain of chloroplast thylakoids. We measured specific run-on transcription of the chloroplast genes psaB, psbA and rbcL in pea (Pisum sativum L.) seedlings grown under three different conditions of illumination: light selective for photosystem I (PSI-light); light selective for photosystem II (PSII-light); and a combination of PSI- and PSII-light (mixed light, ML). The transcriptional rate of the psaB gene increased under PSII-light and decreased under PSI-light, while the transcriptional rates of the psbA and rbcL genes were affected only in a non-specific way. Similar effects also occurred in plants grown under ML and switched to either PSI- or PSII-light for 4 h. Addition of the inhibitors of photosynthetic electron transport 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) influenced psaB transcription in isolated, illuminated chloroplasts: DCMU addition resulted in oxidation of the plastoquinone pool and decreased transcription of psaB; DBMIB addition resulted in reduction of the plastoquinone pool and increased transcription of psaB. The experimental results obtained in vivo and in vitro provide evidence for coupling between the redox state of plastoquinone and the rate of transcription of the psaB gene in pea.