In Situ and in Vitro Binding of Natriuretic Peptide Hormones in Tradescantia multiflora

Abstract: An increasing body of evidence suggests that in plants, as in vertebrates, biologically active natriuretic peptide (NP) hormones play an important role in the regulation of the osmotic and ionic balance. The evidence includes isolation and immunoaffinity purification of biologically active natriuretic peptide analogues (irPNP) from ivy that promoted stomatal opening and specifically, rapidly and transiently increased cGMP levels in root conductive tissue. In this study we demonstrate that I¹²⁵-rat atrial natriuretic peptide (rANP) binds to plasma membranes from leaf and stem tissue of Tradescantia multiflora and importantly, both unlabelled rANP and irPNP can competitively displace that binding. In addition, tissue section autoradiography reveals specific in situ binding of I¹²⁵-rANP to leaf and stem tissue. The findings are consistent with the presence of a biologically active NP system in plants and suggest that NPs signal through a dedicated receptor system.     

Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum ofTradescantia virginiana L.

Summary During an earlier investigation, microtubules were observed at the periphery of invasion processes in the developing syncytial tapetum ofTradescantia virginiana L. They were also associated with membranous sacs that accumulate adjacent to tetrads, with putative fusion sites where the tapetal plasmodium is initiated, and, in postmeiotic stages, with the perispore membrane that encloses the developing spore cells. Colchicine was administered to developing flower buds to investigate the roles of these microtubules. The results indicate that microtubules neither initiate nor guide the tapetal invasion of the loculus. The treatments, however, resulted in absence of cell coat from invasion processes and prevention of cell fusion. They also inhibited polarized migration of membrane sacs and removed the associated microtubules. The development of an organized secretory apparatus at the perispore membrane was disrupted, with subsequent disordered deposition of sporopollenin in the extracellular spaces of the partially-fused plasmodium. The results suggest that microtubules participate in the formation and internal spatial organization of the tapetal plasmodium, and establishment of a secretory surface that normally produces sporopollenin at the tapetum-microspore interface.     

Results and recommendations

In the first phase of a collaborative study by the International Programme on Chemical Safety (PRCS), four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), methyl nitrosurea (MNU), sodium azide (NaN₃) and maleic hydrazide (MH), and ethyl methanesulfonate (EMS) as a positive control were tested in four plant bioassays, namely the Arabidopsis embryo and chlorophyll mutation assay, the Tradescantia stamen hair assay (Trad-SH assay), the Tradescantia micronucleus assay (Trade-MCN), and the Vicia faba root tip assay. Seventeen laboratories from diverse regions of the world participated with four to six laboratories each using one plant assay. For the Arabidopsis assay, laboratories were in agreement with MNU and AG giving positive responses and NaN₃ giving a negative response. With the exception of one laboratory which reported MH as weakly mutagenic, no mutagenic response was reported for MH by the other laboratories. For the Vicia faba assay, all laboratories reported a positive response for MNU, AG, and MH, whereas two of the six laboratories reported a negative response for NaN₃. For the Trad-SH assay, MH was reported as giving a positive response and a positive response was also observed for MNU with the exception of one laboratory. NaN₃, which exhibited a relatively high degree of toxicity, elicited a positive response in three of the five laboratories. AG was found positive in only one of the two laboratories which tested this chemical. For the Trad-MCN assay, MNU and MH were reported as positive by all laboratories, while four out of five laboratories reported NaN₃ to be positive. Only one of three laboratories reported AG to be positive. The major sources of variability were identified and considered to be in the same range as found in similar studies on other test systems. Recommendations were made for minor changes in methodology and for initiating the second phase of this study.     

Light quality during growth of Tradescantia albiflora regulates photosystem stoichiometry, photosynthetic function and susceptibility to photoinhibition

Tradescantia albiflora (Kunth) was grown under two different light quality regimes of comparable light quantity: in red + far-red light absorbed mainly by photosystem I (PSI light) and yellow light absorbed mainly by photosystem II (PSII light). The composition, function and ultrastructure of chloroplasts, and photoinhibition of photosynthesis in the two types of leaves were compared. In contrast to regulation by light quantity (Chow et al. 1991. Physiol. Plant. 81: 175–182), light quality exerted an effect on the composition of pigment complexes, function and structure of chloroplasts in Tradescantia: PSII light-grown leaves had higher Chl a/b ratios, higher PSI concentrations, lower PSII/PSI reaction centre ratios and less extensive thylakoid stacking than PSI light-grown leaves. Light quality triggered modulations of chloroplast components, leading to a variation of photosynthetic characteristics. A larger proportion of primary quinone acceptor (Q_A) in PSI light-grown leaves was chemically reduced at any given irradiance. It was also observed that the quantum yield of PSII photochemistry was lower in PSI light-grown leaves. PSI light-grown leaves were more sensitive to photoinihibition and recovery was slower compared to PSII light-grown leaves, showing that the PSII reaction centre in PSI light-grown leaves was more easily impaired by photoinhibition. The increase in susceptibility of leaves to photoinhibition following blockage of chloroplast-encoded protein synthesis was greater in PSII light-grown leaves, showing that these leaves normally have a greater capacity for PSII repair. Inhibition of zeaxanthin formation by dithiothreitol slightly increased sensitivity to photoinhibition in both PSI and PSII light-grown leaves.     

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

The use of DNA markers to estimate the extent and nature of genetic variability in Solatium tuberosum cultivars

High levels of DNA polymorphism were detected in 27 Solanum tuberosum cultivars examined. Combinations of at least two c-DNA clones have been identified which in conjunction with EcoRI allow important UK potato cultivars to be characterised by their molecular profiles. The widely grown North American cultivar Russett Burbank was also successfully fingerprinted. Estimates of genetic diversity based on restriction fragment length polymorphism (RFLP) data indicate the important role that wild potato species and exotic germplasm have played in the development of the cultivars studied. A graphical method for simultaneously highlighting similarities and differences between genotypes for individual hybridising fragments is presented. This approach is particularly useful in identifying and recording fragments which are unique to certain genotypes. Two potato cultivars: Fiona and Morag produce unique RFLP profiles when digested with EcoRI and EcoRV and probed with a flax ribosomal DNA sequence. Both Fiona and Morag possess incomplete or partial (quantitative) type resistance to G. pallida which was transferred from S. vernei. The preferential transmission of the r-DN A fragments from S. vernei may indicate that this locus is associated with genetic factors controlling resistance to G. pallida.     

Fraying around the edges: negative effects of the invasive Tradescantia zebrina Hort. ex Bosse (Commelinaceae) on tree regeneration in the Atlantic Forest under different competitive and environmental conditions

Aims Invasive plants modify the structure and functioning of natural environments and threat native plant communities. Invasive species are often favored by human interference such as the creation of artificial forest edges. Field removal experiments may clarify if invasive plants are detrimental to native plant regeneration and how this is related to other local factors. We assessed the joint effect of environment and competition with the invasiveTradescantia zebrinaon tree species recruitment in an Atlantic Forest fragment.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis ofTradescantia virginiana leaf epidermal cells

Summary Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions — cortical actin patch — and in the establishment and maintenance of the division site —preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 g of cytochalasin B per ml but inhibited by 10 M oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.Abbreviations ADZ actin-depleted zone - DIC differential interference contrast - GMC guard mother cell - MT microtubule - PPB preprophase band - SMC subsidiary mother cell Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday