Effects of bisphenol A on the microtubule arrays in root meristematic cells of Pisum sativum L.

Bisphenol A (BPA), a widely used chemical in the plastics industry that displays weak oestrogenic properties, is an emerging environmental pollutant, potentially harmful to living organisms. The presumed cytotoxicity of BPA to plant cells has been poorly studied. To understand how BPA might influence plant cell division and affect the underlying cytoskeleton, the effects of BPA on the microtubule (MT) arrays of meristematic root-tip cells of Pisum sativum L. were investigated. Root tips of young seedlings were exposed to 20, 50 and 100 mg/L BPA for 1, 3, 6, 12 and 24 h. The effects of each treatment were determined by means of confocal laser scanning microscopy after immunolabelling of tubulin and counterstaining of DNA, and by use of light and transmission electron microscopy. It was found that BPA affected normal chromosome segregation, hampered the completion of cytokinesis and deranged interphase and mitotic MT arrays. BPA effects were dependent on the stage of each cell at the time of BPA entrance. Moreover, BPA induced the formation of macrotubules with a mean diameter of 32 ± 0.14 nm, compared with 23 ± 0.70 nm for the MT arrays in untreated cells. Finally, all MT arrays and macrotubules were depolymerised upon longer treatment. Taken together, the data suggest that BPA exerts acute anti-mitotic effects on meristematic root-tip cells of P. sativum, MT arrays constitute a primary sub-cellular target of BPA toxicity, and the manifested chromosomal abnormalities could be attributed to the disruption of the MT cytoskeleton.     

Mitotic index of meristematic cells and root growth of Pisum sativum is affected by inositol cycle modulators

Relationships between cell division and inositol cycle modulation caused by different effectors in roots of Pisum sativum were studied. Stimulation of the inositol cycle by myoinositol increased the mitotic index of meristematic cells and root length, while the inhibition of the cycle with Li+ and a heavy metal Gd3+ considerably decreased mitotic activity and growth. Exposure of roots to 10 mM CaCl2 and 15 mM myoinositol resulted in the accumulation of chromosome aberrations. Changes in the activity of inositol cycle are assumed to be involved in the root growth control.     

Proteolytic activity in relationship to senescence and cotyledonary development in Pisum sativum L.

Changes in the weight and in the chlorophyll, free amino-acid and protein content of developing and senescing, vegetative and reproductive organs of Pisum sativum L. (cv. Burpeeana) were measured, and the proteolytic activity in extracts from the senescing leaf and the subtended pod was followed in relation to these changes. Protein content decreased in the ageing leaf and pod while it increased in the developing cotyledon. The proteolytic activity of the leaf did not increase as the leaf protein content decreased. In contrast, proteolytic activity in the subtended pod increased while the protein level decreased. The proteolytic activity in the extracts from the ageing organs was greater than the rates of protein loss. The proteolytic activity of leaf and pod extracts was greater on protein prepared from the respective organ than on non-physiological substrates. Proteolysis was increased by 2-mercaptoethanol and ethylenediaminetetraacetate but was not influenced by addition of ATP to the reaction mixture. The pH optimum was at 5.0. Free amino acids did not accumulate in the senescing leaf or pod when protein was degraded in each organ. It is suggested that these amino acids were quickly metabolized in situ or translocated to sink areas in the plant, especially to the developing seeds.     

Rapid multiple shoot production from cotyledonary node explants of pea (Pisum sativum L.)

Summary Multiple shoots were produced, from cotyledonary node explants of pea (Pisum sativum L.) cultured on MS medium containing 1 mg 1⁻¹ BAP. The number of buds formed could be increased by scraping the nodes before culture or by increasing the cytokinin concentation. However, cytokinin levels over 5 mg 1⁻¹ increasingly produced shoots that were vitrified and dificult to root. With all the genotypes tested, developing buds were visibile as soon as 5 days after culture and elongated shoots could be removed after 21 days., Histological studies indicated, that the buds and shoots were formed from superficial layers of tissue. The efficiency of this regeneration system compared favorably with previosly published methods. The rapid, genotype-independent, high frequency system described here may be of use in the production of transgenic pea plants.     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

Biosynthesis of nucleic acids in pea (Pisum sativum L.) buds released of dominance

Biosynthesis of nucleic acids in the pea buds released from apical dominance by decapitation has been studied. The rate of biosynthesis of all nucleic acid classes separated by means of methylated albuminkieselgur (MAK) column chromatography was found to be increased in released pea buds as judged by the extent of orotic-6-¹⁴C acid incorporation. This increase has been observed in all experimental points examined (15, 24, 36 and 48 h after decapitation), being the most pronounced 15 h after decapitation. After that a certain decrease of the specific radioactivities of nucleic acids has been registered, but even 48 h after decapitation specific activities of the main nucleic acid classes were higher in comparison with those found in dominant buds. Variations of the free pyrimidine nucleotide pools were about 14% suggesting that this parameter did not influence the extent of the uptake of labelled orotic acid.     

Lipoxygenase heterogeneity in Pisum sativum

Antibodies raised against two pea (Pisum sativum L. cv. Birte) seed lipoxygenases have been used to analyze lipoxygenase heterogeneity in seeds and in other organs. At least seven different polypeptides were identified in vivo; five of these were identified as precursors synthesized in vitro. The developmental appearance of the seed polypeptides has been analyzed and early and late forms were identified. Limited N-terminal sequence data indicated further heterogeneity when compared with sequences predicted from cDNAs.Abbreviations cDNA complementary DNA - DAF days after flowering - HPLC high-performance liquid chromatography - Ig immunoglobulin - kb kilobase - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SSC 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0 This work was supported by the Agricultural and Food Research Council via a grant-in-aid to the John Innes Institute. We acknowledge financial support from the Commission of the European Communities Biotechnology Action Programme; grant No. 0063-UK.     

Underreplication of repetitive DNA in polyploid cells of Pisum sativum.

Earlier reported results of a difference in the amount of DNA between 2C meristematic plumula cells and 2C cortex cells of the epicotyls are confirmed by fluorometric Feulgen-DNA determinations. Renaturation experiments of isolated DNA suggest that this difference in DNA is probably correlated with an underreplication of some highly repetitive DNA sequences in cells preparing endomitosis.     

Plasmatubules in transfer cells of pea (Pisum sativum L.)

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461–465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.     

Ultrastructure of meristematic cells of dormant and released buds in Tradescantia paludosa

The lateral bud meristems of Tradescantia paludosa show a characteristic cytohistological zonation during dormancy. The cells comprising this so called ‘zone of inhibition’, which is located at the extreme tip of the bud apex, rarely synthesize nuclear DNA or undergo mitotic division. These nuclei are as large as prophase nuclei, yet contain only telophase (2C) amounts of DNA and significantly lower amounts of histone as compared to the 2C nuclei of the actively dividing cells.Ultrastructural observations of the nuclei in the ‘zone of inhibition’ show that a large proportion of the chromatin is organized as less condensed, diffuse, euchromatin fibrils; however, the chromatin of the actively dividing nuclei of the cells outside the ‘zone of inhibition’ or in the released bud meristems is organized to a greater extent as condensed clumps of heterochromatin. When the dormancy is released, the nuclei in the ‘zone of inhibition’ synthesize DNA and histone and undergo cell division in approx. 4 days. Striking changes in the organization of chromatin fibrils take place during this transition period. The diffuse chromatin fibrils of the nuclei in the ‘zone of inhibition’ progressively become more and more condensed as the cell prepares to undergo the first mitotic division after the release of dormancy. This change which is coupled with the synthesis of histones in the nuclei of the ‘zone of inhibition’ suggests a prominent structural role of these basic proteins in the organization of the chromatin. The large volume of 2C nuclei of the ‘zone of inhibition’ seems, therefore, to result not from a great nuclear mass, but probably from a relatively small degree of condensation of chromatin.     

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 #-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.     

Matromorphy in Pisum sativum L.

Summary The possibility of obtaining instant pure breeding lines by matromorph seed development in Pisum sativum L. has been investigated. Two types of maternal parents, namely, homozygous for the recessive marker genes and heterozygous for the dominant marker genes were pollinated with Lathyrus odoratus and the P174 variety of Pisum sativum L. carrying dominant markers. For both pollinators, induction of matromorphy by prickle pollination, irradiated pollen and IAA treatment was examined. Promising matromorphs were identified in the M₁ generation which were studied in the M₂ generation for assessing their genetic status with respect to homozygosis. The success of pod set varied from zero to 28% with a varying number of matromorphic seeds following different treatments. The possible mechanisms for matromorphic origin have been discussed. The evidence presented herein favours induction of matromorphy in peas for the production of homozygous stocks. In addition, the recovery of double recessive seed markers of the maternal parents along with plant markers from the paternals has prospective implications in plant breeding as an alternative tool to recurrent back crossing.     

Phytochrome-mediated bud development in Pisum sativum

Summary Light-grown dwarf peas were disbudded except for a single lateral bud, then transferred to darkness at 24° C. During the dark period the seedlings were irradiated daily for 5 or 7 min with R or FR. The buds exposed to R developed into shoots faster than those irradiated with FR. The R effect was FR reversible, and the FR effect was R reversible. The P_fr form of phytochrome thus promoted shoot growth including cell division, DNA and RNA synthesis.Abbreviations R red - FR far-red - P phytochrome     

Auxin Physiology of Dwarfism in Pisum sativum

Similar levels of diffusible auxin are measured for the apices of both Little Marvel (dwarf) and Alaska (normal) cultivars of the pea when grown in sunlight and darkness. In sunlight, however, diffusible auxin disappears in the subtending internode of the Little Marvel plant but remains at 50 per cent of the level of the apex in the subtending internode of the Alaska plant. The enzyme preparation from the apex of the dwarf plant converts tryptophan and tryptamine to IAA more readily than that from the normal plant. Indoleacetyl aspartate synthetase activity is also higher in the dwarf plant than in the normal plant and the dwarf plant contains four times as much conjugate as the normal plant with or without treatment with gibberellic acid. Gibberellic acid (GA) does not affect the induction of the synthetase enzyme nor the enzymatic formation of indoleacetyl aspartate. The growth induced by GA is the result of an increased synthesis of auxin.     

Peculiarities in PMC Meiosis of Pisum sativum

Summary Nemesia strumosa plants were discovered which had styles capable of discriminating among incompatible pollen tubes from different pollinators, allowing growth of some but not others. All but 3 of 26 families tested had at least some members with discriminating styles (DS). Presence and level of DS was independent of S genotype. Plants with pseudo-self-compatiblity (PSC) levels greater than 10% had the trait, though many plants with strong DS had PSC levels less than 10%. Self pollination of highly DS plants produced mostly DS offspring, but of differing sensitivities. Some progenies from crosses between a family of highly DS plants and unrelated, probably low DS plants segregated half DS and half non-DS, while others consisted of mostly DS or mostly non-DS. The DS phenomenon is probably caused by PSC genes.Scientific Journal Series Paper No. 11,677 of the Minnesota Agricultural Experiment Station