Wild-type Pea Research Articles

Flower development in pisum sativum: from the war of the whorls to the battle of the common primordia

The ontogeny of pea (Pisum sativum L.) flowers, as in many legume and nonlegume plant species, proceeds through a very different sequence of events from the same process in Antirrhinum majus and Arabidopsis thaliana. Using scanning electron microscopic analysis, we have characterized the early development of wild-type pea flowers and selected morphological characters or markers to break it down into different developmental stages. We used these markers as tools to characterize early alterations in flower development of several pea floral homeotic mutants. These mutants display phenotypes resembling those of: (1) floral meristem identity mutations, frondosus (brac); (2) class A mutations, calix carpellaris (cc); (3) class B mutations, stamina pistilloida (stp-1 and stp-2); and (4) class C mutations, petalosus (pe). According to the homeotic transformations observed in the pea floral mutants, it would appear feasible that the identity and developmental pattern of the four organ types in pea flowers are governed by at least the same three developmental functions, A, B, and C, proposed for the two model systems. However, our results suggest that, in pea, although these functions do have a similar role in the specification of organ identity shown by their counterparts in Arabidopsis or Antirrhinum, they may differ in the control of other processes, such as floral determinacy, organ number, or leaf development. The more remarkable features of pea flower ontogeny were the existence of four common primordia to petals and stamens, the early carpel primordium initiation, and the abaxial-adaxial unidirectional initiation of organ primordia within each different floral whorl, in contrast to the centripetal and sequential floral ontogeny in other plants. Organ differentiation within each of these common primordia appears to be a complex process that plays a central role in the ontogeny of pea flowers. Analysis of flower developmental pea homeotic mutants suggests that A, B, and C functions are necessary for the correct differentiation of organs from common primordia and that, in addition to its role in the specification of petals and stamens, B function, would be involved in conferring common primordia identity. Copyright 1999 Wiley-Liss, Inc.

Cellular localization of a plant protein PSAM 1 in arbuscular mycorrhizas of Pisum sativum.

Psam 1 is a single-copy gene which is activated during early plant-fungal interaction in wild-type pea inoculated with Glomus mosseae and which codes for PSAM 1, a putative protein of 108 amino acids. A synthetic peptide was designed of 108 amino acids. A synthetic peptide was designed in an antigenic region of this protein to produce a polyclonal antibody against PSAM 1 and to investigate its cellular localization. Western blot analysis revealed that a polypeptide of about 14.5 kDa accumulated more in mycorrhizal than non-mycorrhizal pea roots. The PSAM 1 antigen was immunolocated in planta in arbuscule-containing cells of mycorrhizal roots and especially in the cytoplasm surrounding young arbuscules in cortical cells, which suggests that its accumulation is somehow related to the symbiotic state of these cells.

Differential display analysis of RNA accumulation in arbuscular mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene.

Differential RNA display was used to analyze gene expression during the early steps of mycorrhiza development on Pisum sativum following inoculation with Glomus mosseae. Seven out of 118 differentially displayed cDNA fragments were subcloned and sequenced. One fragment corresponded to part of the fungal 25S ribosomal RNA gene and a second one showed similarity to a human Alu element. The others were derived from plant genes of unknown function. One of the fragments was used for the isolation of a full-length cDNA clone. It corresponded to a single-copy gene (psam1) which is induced during early symbiotic interactions, and codes for a putative transmembrane protein. Northern and RNA dot blot analyses revealed enhanced accumulation of psam1 RNA after inoculation with G. mosseae of wild-type pea and an isogenic mutant deficient for nodule development (Nod-, Myc+).

Immunocytological evidence for abnormal symbiosome development in nodules of the pea mutant line Sprint2Fix− (sym31)

Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix− (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often enclosed within one peribacteroid membrane, or were found within a vacuole-like compartment. In wild-type nodules, the monoclonal antibody JIM18, which recognizes a plasmalemma glycolipid antigen, bound to the juvenile peribacteroid membrane, and did not recognize the mature peribacteroid membrane. However, in the mutant, the antibody bound to all peribacteroid membranes within the nodule, suggesting that differentiation of the peribacteroid membrane was arrested. Another antibody, MAC266, recognized plant glycoproteins which normally accumulate in symbiosomes at a late stage of nodule development. Binding of this antibody was much reduced within mutant nodules, labelling only a few mature cells. Similarly, MAC301, which normally recognizes a lipopolysaccharide epitope expressed on differentiated bacteroids prior to the induction of nitrogenase, failed to react with rhizobial cell extracts isolated from nodules of thesym31 mutant. On the basis of these developmental markers, the symbiosomes ofsym31 nodules appeared to be blocked at an early stage of development. The distribution of infection structures was also found to be abnormal in the mutant nodules. Models of symbiosome development are presented and discussed in relation to the morphological and developmental lesions observed in thesym31 mutant.

Developmental changes in the gibberellin-induced growth response in stem segments of light-grown pea genotypes

The effects of GA on stem elongation were studied using segments from one tall and three dwarf light-grown pea genotypes varying in endogenous hormone content. Stem segments were cut at two distinct ages: when the fourth internode was at about 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Light microscopy and flow cytometry were used to demonstrate that GA does not induce cell division in excised pea stem segments. The growth studied here was strictly elongation. Measurement of final segment length after 48 hours and high resolution measurement of growth kinetics over 20 hours using an angular position transducer were done on segments treated with hormone solutions. Our data indicate that the action of GA on stem elongation can be classified into two distinct modes. The first, apparent in early-expansion stem segments, shows distinct growth kinetics and is independent of the endogenous IAA concentration of the segments. Quantitation of IAA by GC/MS in early-expansion segments of wild type pea incubated with gibberellin shows that an increase in IAA concentration is part of the GA response in such segments. The second mode of GA action is evinced in mid-expansion segments. Whereas there is no short term (<20 h) response to GA alone (as determined by growth kinetics), there is a long term (48 h) response whose magnitude decreases across the genotypes with decreasing endogenous hormone content. Growth responses indicate that in mid-expansion segments exogenous GA acts by enhancing IAA action but appears to be unable to augment endogenous IAA content. Contradictory reports of the response of excised stem segments to GA can be reconciled when tissue genotype and developmental stage are considered.

The Control of Pinna Morphology in Wildtype and Mutant Leaves of the Garden Pea (Pisum sativum L.)

The compound leaf of the wildtype pea exhibits basal stipules, proximal leaflets, and distal tendrils, which are profoundly modified by several recessive genes thought to act as homeotic mutations. The acacia (tl) gene appears to replace simple tendrils with leaflets in the distal region, while the afila (af) gene appears to replace the leaflets with branched tendrils in the proximal region. Morphological analyses of all homozygous and heterozygous combinations of this genetic system indicate that these genes affect many other features of leaf morphology, including leaf length, petiole length, pinna number, and branching order. Moreover, these genes are apparently expressed within the context of preexisting proximal and distal regions in the developing leaf. Pinna identity on the leaves from different genotypes is therefore regulated by the interaction among relative pinna position, morphological region, and genetic background. Current models of pea leaf development fail to account for several characteristics of pea leaves, such as (1) the leaf phenotype of the double recessive mutant called pleiofila, which consists of novel compound pinnae with very small terminal leaflets, (2) mixed pairs of proximal and distal pinnae, and (3) intermediate narrow leaflets/broad tendrils in certain heterozygotes. Starting with the assumption that the pleiofila leaf represents the ground state of the pea leaf, the available evidence indicates that the dominant Af and Tl genes are acting as heterochronic mutations that determine the timing of lamina formation and axis termination, with the result that these genes cause the observed morphological changes in pinna identity.

Flowering in Pisum: Identification of a new ppd allele and its physiological action as revealed by grafting

The late flowering, quantitative long day habit of wild type pea (Pisum sativum L.) is conferred by the joint presence of dominant genes Sn, Dne and Ppd. Grafting studies have shown that flowering in wild type plants is delayed under short days by formation of a graft‐transmissible inhibitor and that the early flowering, day neutral mutants sn and dne are deficient in this inhibitor. However, the physiological action of the Ppd gene has not been examined by grafting and the possibility exists that the ppd mutation causes early flowering and a day neutral habit by blocking response to, rather than synthesis of, the inhibitor. We here identify a second, more severe (probably null) mutant allele (ppd‐2) at the Ppd locus and show that flowering was delayed by 4 nodes in a ppd‐2 shoot grafted to a wild type stock, and promoted by 13 nodes in a wild type shoot grafted to a ppd‐2 stock. Thus a ppd‐2 shoot can respond to inhibitor donated by a wild type stock but a ppd‐2 stock is unable to provide sufficient inhibitor to prevent early flower initiation in a wild type shoot. We conclude genes Sn, Dne and Ppd each control steps in the synthesis of the flower inhibitor. Grafts among the sn, dne and ppd mutants gave an indication that the three genes may act in the sequence Sn, Ppd, Dne, but possible cases of physiological complementation need to be tested using null mutants in the same genetic background.

Functional organization of thylakoid membranes in viable pea mutants with low chlorophyll content

The functional organizations of thylakoid membranes from wild type pea (Pisum sativum L. cv. Kapital) and two viable mutants with low chlorophyll (Chl) contents were compared. Nuclear mutations in mutants 7 and 42 led to two‐ and three‐fold decrease in total chlorophyll content, respectively. In spite of low Chl content mutants showed 80% photosynthetic activity, biological productivity, and seed production. It has been shown that mutant membranes differed from that of wild type by Chl distribution between the pigment‐protein complexes and by stoichiometry of the main electrontransport complexes. The ratio photosystem I (PSI): photosystem II (PSII): cytochrome (Cyt) bjf complex: Chl was 1:1.1:1.2:650 in wild type chloroplasts, 1:1.8:1.7:600 in mutant 7, and 1:1.5:1.9:350 in mutant 42. PSI‐ and PSII‐dependent electron‐transport activities were enhanced in the mutants per mg Chl in proportion to number of reaction centers. The activity of the non‐cyclic electron‐transport chain increased in proportion to PSII and Cyt bjf complexes. The amount of ATP synthetase per unit of Chl as estimated by H−ATPase activity was much greater in mutant thylakoids, which is favorable for photosynthetic energy transduction. The low content of the light‐harvesting complexes (LHC) in mutants is compensated by an increase of the number of PSII and Cyt bjf complexes, which eliminates the bottleneck at the site of plastoquinone oxidation.

Chitinase andβ1,3-glucanase isoforms expressed in pea roots inoculated with arbuscular mycorrhizal or pathogenic fungi

Chitinases were studied in an endomycorrhiza-resistant mutant and wild type pea (Pisum sativum L. cv. Frisson) in order to characterize plant hydrolases specific to pathogenic (Aphanomyces euteiches andChalara elegans) or mycorrhizal (Glomus mosseae) root interactions. Stimulation of constitutive and induction of new chitinase activities was detected by native PAGE for acidic proteins in both pea genotypes inoculated with pathogenic fungi. In contrast, a different additional chitinase isoform was induced inG. mosseae-colonized roots. This isoform was also not elicited in chemically-stressed roots, confirming its mycorrhiza-specificity. Investigations of basic chitinase andβ-1,3-glucanase activities provided further evidence for differential pea responses during pathogenic and symbiotic interactions.

Fluorescence and photochemical characterization of phytochrome in de-etiolated pea mutant lip

The red-light-absorbing form of phytochrome, Pr, and its phototransformation into the first stable photoproduct, lumi-R, under red light ( λ r = 667 nm) and back under far-red light ( λ fr = 696 nm) at 85 K were investigated with the use of low temperature fluorescence spectroscopy in the stems and roots of lip mutant pea seedlings, which have many of the characteristics of light-grown plants even when grown in the dark (S. Frances et al., Plant Cell, 4 (1992) 1519–1530). The spectroscopic and photochemical parameters of Pr were found to be similar in wild-type and lip mutant etiolated seedlings. The two Pr forms with high and low extents of phototransformation into lumi-R, i.e. Pr′ and Pr″ respectively, detected at low temperatures (V. Sineshchekov, Photochem. Photobiol., 59 (1994) 77–86) were shown to be roughly equivalent in the wild-type and the lip mutant pea. Fluence time-response curves for the phototransformation of Pr′ into lumi-R and back in lip stems revealed three subpopulations of Pr similar to those obtained in the wild-type pea. The total phytochrome content [P tot] determined from the Pr fluorescence intensity in the stems of the mutant was approximately 30% of the wild type. However, mutant and wild-type roots contained similar amounts of Pr. Thus any contribution to the mutant phenotype made by phytochrome may be due to changes in the total concentration of phytochrome rather than variations in the relative content or modification of the various Pr species.

Chitinase isoforms in roots of various pea genotypes infected with arbuscular mycorrhizal fungi

Chitinase activity has been investigated in mycorrhiza-resistant (myc −), non-nodulating (nod −) isogenic pea ( Pisum sativum L.) mutants in an attempt to understand the reasons for such resistance to symbiotic fungi. Activities from control and Glomus mossease-inoculated roots of myc − mutants were compared to the corresponding mycorrhizal (myc +), nodulating (nod +) wildtype pea genotype cv. Frisson. A pea mutant only deficient for the nod − character [myc +, nod −] was also studied. Two acidic and two basic chitinase isoforms were detected in control roots of all peas tested after native polyacrylamide gel electrophoretic (PAGE) separation of proteins at pH 8.9 or pH 4.3. No difference in basic chitinase isoforms patterns occurred in the various pea genotypes in the presence of G. mosseae. However, as soon as 1 week after infection, an additional acidic chitinase isoform was observed in extracts of G. mosseae-colonized roots from the wildtype pea genotype and the [myc +, nod −] mutant. The isozyme had a molecular mass of approximately 27 kDa, estimated by sodium dodecyl sulfate (SDS)-PAGE under non-reducing conditions, and exhibited a faint lysozyme activity by lysis of Micrococcus luteus cells. It was not detected in the [myc −, nod −] pea mutant, unless plants were grown under conditions which increased the number of appressoria. The host origin of the 27 kDa chitinase isoform was indicated by its presence after infection with another Glomus species, and from the fact that it was not detected in a mixture of germinated spores and mycelia of G. mosseae.

Initial characterization of a pea mutant with light-independent photomorphogenesis.

We have identified a mutant of pea cultivar Alaska that has many of the characteristics normally associated with light-grown seedlings even when grown in complete darkness. We have designated this mutant lip1, for light independent photomorphogenesis. Etiolated wild-type pea seedlings are white to slightly yellow in color and have a distinct morphology characterized by elongated epicotyls and buds containing unexpanded leaves with small, undifferentiated cells. In contrast, mutant seedlings grown under the same conditions are yellow in color and have short epicotyls and expanded leaves showing clear cellular differentiation. Transmission electron microscopy revealed partially developed, agranal plastids in the dark-grown mutant, unlike wild-type seedlings that contain etioplasts with prolamellar bodies. The mutant also exhibits a much shorter lag period for chlorophyll accumulation when etiolated seedlings are transferred from darkness to white light. The dark-grown mutant has 10-fold less spectrally detectable phytochrome, which can be attributed to a 10-fold reduction in the level of the PHYA polypeptide. Cab, Fed1, and RbcS transcripts are present in dark-grown mutant seedlings at levels comparable to those produced in light-grown material. The levels of these transcripts show a normal decrease when green plants grown for 15 days in a light/dark cycle are transferred to continuous darkness. However, transcript levels remain high during dark treatment of seedlings grown for 9 days in continuous light, indicating that the dark adaptation response in this mutant is developmentally plastic. The lip1 mutant has several features in common with the deetiolated Arabidopsis mutants det1, det2, and cop1. However, there are also several important differences, including varying effects on phytochrome levels, organ-specific gene expression, plastid development, and response to dark adaptation.

Histological Evidence that a Defence Response of the Host Plant Interferes with Nodulin Gene Expression in Vicia sativa Root Nodules Induced by an Agrobacterium Transconjugant

Summary The Agrobacterium transconjugant LBA2712 harbours a complete R. legummosarum sym plasmid in an Agrobacterium chromosomal background. On Vicia sativa (common vetch) this strain induces nodules in which a number of plant cells is filled with bacteria. The leghaemoglobin (Lb) genes were not found to be expressed in these nodules, suggesting that the Rhizobium sym plasmid-borne genes are not sufficient for inducing Lb gene expression. Detailed structural analyses on the nodules induced by LBA2712 indicated that the bacteria are subject to degradation as soon as they are released from the infection thread. An immunocytological analysis of developing wild-type pea root nodules using a leghaemoglobin antiserum showed that leghaemoglobin is only detectable as from the third to fifth cell layer of the late symbiotic zone. Thus, the differentiation into infected and uninfected cells precedes leghaemoglobin accumulation. In combination with the structural analysis of the nodules formed by the Agrobacterium transconjugant, it is concluded that the Agrobacterium -induced nodules do not reach the developmental stage in which the leghaemoglobin genes normally are induced, despite the release of the bacteria from the infection thread. As the results presented indicate that apparently a host defence mechanism interferes with nodulin gene expression, it follows that an Agrobacterium transconjugant does not allow conclusions with respect to the involvement of sym plasmid-borne genes in the induction of the expression of late nodulin genes.

Synthesis and assembly of thylakoid membrane proteins in isolated pea chloroplasts: The chlorophyll [formula omitted] of the photosystem II reaction center

The apoproteins of two minor chlorophyll (Chl) a- complexes which are thought to be part of the photosystem II (PS II) reaction center have been identified among the polypeptides synthesized in isolated chloroplasts from a chlorophyll b- less pea mutant and wild-type pea. Peptide mapping of the products of proteolysis confirmed that the incorporated radioactivity was an integral part of the apoproteins. It is shown that the newly formed apoproteins had been integrated into the thylakoid membrane, based on their release with octyl-β- d-glucopyranoside. Labelling of the complexes in isolated chloroplasts was dependent upon light as an energy source, and displayed characteristic sensitivity towards inhibitors of photophosphorylation and organellar protein synthesis.

Dynamics of extractable carbohydrates in Pisum sativum. II. Carbohydrate content and photosynthesis of pea cuttings in relation to irradiance and stock plant temperature and genotype

Rooting ability was studied for cuttings derived from stock plants of wild type pea seedlings and seedings of two mutants deficient in photosystem II activity and chlorophyll. Stock plants were grown at 15, 20, 25 or 30°C at 38 W m‐2. Cuttings were rooted at 20°C and at an irradiance of 16 or 38 W m‐2. The rooting ability seemed to be correlated with the initial carbohydrate content only at 38 W m‐2. Based on the findings of the present study it may be concluded that for pea seedlings the growth temperature is more important than photosynthesis as regards accumulation of extractable carbohydrates. During the rooting period carbohydrates are necessary for root formation, but the effect of the iradiance on the number of roots formed is not mediated by the carbohydrate content. Under specific rooting conditions it is possible to correlate the initial carbohydrate content with the rooting capacity of the cuttings within a phenotype, but not always when different phenotypes are considered. The results indicate a connection between the metabolic activity of the cuttings and their ability to form adventitious roots.

Races ofPseudomonas pisiand sources of resistance in field and garden peas

Abstract Evidence is presented for the occurrence of two races of Pseudomonas pisi. Seven isolates from New Zealand and overseas, pathogenic to cv. Early Onward but non-pathogenic to cv. Partridge, were designated race 1. Similarly 16 isolates, pathogenic to cv. Partridge but non-pathogenic to cv. Early Onward, were designated race 2. Tests on eight pea cultivars showed that two were resistant to race 1 but not race 2, three were susceptible to both races, and three were susceptible to race 1 but resistant to race 2. In a search for resistance in other cultivars and wild-type peas two lines, 3080 and cv. Vinco, were apparently resistant to both races under glasshouse conditions. The significance of race 2 to the present disease problem in New Zealand is discussed.