David W. M. Leung

Chelated lead transport in Pinus radiata: an ultrastructural study

Abstract Pinus radiata plants, approximately 12 months old, were exposed to Pb(NO3)2 in hydroponic culture, with and without the addition of the chelating agents H-EDTA and EDTA, for 1 week. Subsequently lead uptake was quantified in treated materials by flame atomic absorption spectrometry. While lead supplied in unchelated form accumulated predominantly in root tissue, lead chelated with either H-EDTA or EDTA was transported principally to the needles. With transmission electron microscopy, ultrastructural observations were carried out on ultra-thin sections derived from lead-treated P. radiata tissues. In root tissues, lead supplied in unchelated form was found exclusively in cell walls. In needles, lead accumulation due to treatment incorporating chelators was found in sites adjacent to cell walls and in intercellular spaces. The morphology of cellular components, such as mitochondria, dictyosomes, ribosomes and endoplasmic reticula, does not appear to have been adversely affected by the presence of lead.

Chelated lead transport in Chamaecytisus proliferus (L.f.) link ssp. proliferus var. palmensis (H. Christ): an ultrastructural study

Abstract Chamaecytisus palmensis plants growing in hydroponic culture were exposed to Pb(NO3)2, with and without the addition of the chelating agents H-EDTA and EDTA, for 1 week. Subsequently lead uptake was quantified in treated materials by flame atomic absorption spectrometry. Unchelated lead accumulated predominantly in root tissue, while lead chelated with either H-EDTA or EDTA was taken up principally by the shoots. With transmission electron microscopy, ultrastructural observations were carried out on ultra-thin sections derived from lead-treated C. palmensis tissues. Unchelated lead was found in cell walls, bacteroids and mitochondria in root nodule tissue, and in middle lamellae and intercellular spaces in root tissues. In roots, chelated lead was found in mitochondria, and in shoot tissues it was found in chloroplasts, pit membranes, and plasmodesmata.

Onion is a monocotyledonous host for Agrobacterium

Abstract Onion (Allium cepa cvs Pukekohe Longkeeper and Early Longkeeper) bulbs and leaves were inoculated with 25 virulent strains of Agrobacterium. Eleven strains of Agrobacterium tumefaciens, one of A. rubi and six of A. rhizogenes induced tumorous growths. One A. rhizogenes strain, HRI produced a root-like structure arising from a tumour at the base of onion bulbs. The majority of these tumours produced nopaline, suggesting that transformation has occurred. In contrast, neither nopaline nor octopine was ever detected in extracts of tumour-free inoculation sites which had been inoculated with virulent or avirulent strains. Tumours appeared earlier on bulbs inoculated with Agrobacterium cultured in the presence of acetosyringone.

Involvement of plant chitinase in sexual reproduction of higher plants

Abstract Higher plants do not contain chitin but chitin-hydrolytic enzyme or chitinase activity is present in extracts of seeds, roots or leaves. Large increases of the enzyme activity have been found in the latter two organs following attack by chitin-containing phytopathogens or pests. This has led to the widespread notion that plant chitinase is a defence protein. However, the present study reports that active chitinase enzyme is present in the extracts of healthy and non-senescent petunia flower tissues. Furthermore, almost all the flower chitinase activity is localized in the stigma and increases about five-fold therein following anther dehiscence. This strongly suggests the plant chitinase has a heretofore unrecognized specific function in the sexual reproduction of higher plants.

Lysophosphatidic acid acyltransferase-β: a novel target for induction of tumour cell apoptosis

Phosphatidic acid (PA) is a component of cellular membranes that is also a mediator of certain cell signalling functions associated with oncogenesis. These include ras/raf/Erk and Akt/mTor [1-3]. The authors have investigated whether it would be possible to interrupt these known oncogenic pathways through the inhibition of lysophosphatidic acid acyltransferase (LPAAT), an enzyme that catalyses the biosynthesis of PA. The expression and activity of the LPAAT-β isoform are elevated in human tumours, and the respective gene displays transforming capacity when overexpressed in vitro. Inhibition by either genetic means or by isoform-specific small molecules results in a block to cell signalling pathways and apoptosis. Furthermore, the small-molecule inhibitors of LPAAT-β are not cytotoxic to a number of normal cell types, including primary bone marrow progenitors, indicating a differential dependence of tumour cells on LPAAT-β function. These discoveries indicate that LPAAT-β represents a potential novel cancer therapy target.

Starch Accumulation Is Associated with Adventitious Root Formation in Hypocotyl Cuttings of Pinus radiata

This study of the internal physiology of adventitious root formation in Pinus radiata was performed without the potential complications from microbial contamination and nutritional stress. Hypocotyl cuttings of radiata pine were cultured in half strength MS nutrient medium supplemented with IBA (indole-3-butyric acid), IBA + kinetin, kinetin, or without phytohormones (control). Averages of 8.35 and 0.08 roots per cutting were formed in IBA and in growth regulator-free treatments, respectively. No roots were formed in IBA + kinetin, kinetin, or sucrose-free treatments at day 30 after excision of hypocotyls. Changes in fresh weight, sugar, and starch content were measured at established developmental stages associated with adventitious root formation. Sucrose-supplemented medium was required for higher levels of sugar, starch, and root formation in rooting region of IBA-treated hypocotyls. Starch accumulation, in particular, seems to have potential as a biochemical marker before root primordium emergence in light of the following observations in the IBA treatment. Starch began to build up preferentially in cells involved in or in close proximity to potential sites of new root primordium formation (that is, the cells on the inside of the cortex and the pith) before any visible organized root primordia and then began to disappear during root primordium formation. The substantial starch accumulation associated with IBA treatment was not observed in the IBA + kinetin, kinetin alone, growth regulator-free, sucrose-free treatments (nonrooting treatments) or in the nonrooting region of hypocotyls treated with IBA.

Factors influencing the growth of micropropagated shoots and in vitro flowering of gentian

About 70% of the shoots developed from nodal explants ofGentiana triflora flowered in vitroondouble strength WPM medium containing 3% (w/v) sucrose, 0.5mg/l BA after 12 weeks of culture in a growth room at 22°Cwith continuous illumination (PPFD = 60μmolm−2 s−1). The influences oninvitro shoot development and flowering of several factors includingthe position of the explant, requirements for sucrose, cytokinin orGA3, variations of pH and photosynthetic photon flux density (PPFD)were investigated. In vitro flowering but not shootdevelopment of G. triflora decreased notably withincreaseddistance from the apex of the shoot, indicating the presence of a “floralgradient” in the micropropagated shoots. Conversely, as little as 0.01mg l−1 GA3 in the medium promotedshootdevelopment but even up to 0.2 mg l−1GA3 did not induce in vitro flowering.Even though BA could substitute GA3 for a high level of shootdevelopment, it also promoted a high level of in vitroflowering at the PPFD of 60 μmolm−2 s−1. Sucrose was required for shootdevelopment and flowering in vitro and higher levels ofPPFD could not compensate effectively for the omission of the sugar from themedium. In general, the effects of different concentrations of BA in the mediumor variations of pH on shoot development and flowering invitro were found to be influenced by PPFD. A novel observation isthat precocious flowering of micropropagated gentian shoots did not occur ifthey were first cultured for 5 weeks in the dark before transfer to the lightcondition.

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis, cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), α-galactosidase and β-mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo-β-mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.

Elevation of extracellular β-1,3-glucanase and chitinase activities in rose in response to treatment with acibenzolar-S-methyl and infection by D. rosae

Summary Changes in activities of β-1,3-glucanase and chitinase were determined in intercellular fluids of leaves of in vitro rose shoots at various times after treatment with acibenzolar-S-methyl (BTH, a benzothiadiazole derivative; trade name Bion 50WG) or inoculation with Diplocarpon rosae . The results indicate that BTH treatment led to enhanced activities of β-1,3-glucanase and chitinase in the intercellular spaces of rose leaves. An increase in extracellular β-1,3-glucanase and chitinase activities was also found in D. rosae -infected leaves. However, the increase in enzyme activities occurred much more rapidly and was more strongly enhanced in D. rosae -infected leaves that were previously treated with BTH, suggesting that the increased β-1,3-glucanase and chitinase may play a role in restricting the development of disease symptoms on the rose leaves infected with D. rosae . The expression patterns of rose β-1,3-glucanase and chitinase isoforms were investigated on native PAGE with specific staining techniques. A similar induction pattern of these enzymes was observed in both treatments. The increased β-1,3-glucanase and chitinase activities are mainly due to the enhanced expression of β-1,3-glucanase isoform G2 and chitinase isoforms C1 and C2. These isoforms are likely to be a part of rose defense responses to pathogen attack.

A comparison of in vitro with in vivo flowering in gentian.

Young nodal explants of Gentiana triflora Pall. var. axillariflora were cultured in a woody plant medium (WPM) supplemented with B5 vitamins, sucrose (3%) and kinetin (2.0 μM). A novel observation was made in that in vitro flowering occurred following development of the axillary bud of the cultures. A comparison was made between in vitro and in vivo flowers. Although smaller in size, the in vitro flowers were morphologically comparable to the in vivo ones. Flowers from both sources were semi-opened or not opened. The colour of the in vitro flowers was paler than those in vivo. Stigma development was generally poor in both in vitro and in vivo flowers. Pollen viability was over 90% in both types of flowers. About 11% and 34% of pollen from in vitro and in vivo flowers, respectively, germinated on WPM containing 100 g l−1 sucrose solidified with 10 g l−1 agar. Hand pollination of stigma could raise viable seed production in in vivo-flowering plants from 0.3 o/o (i.e. without aided pollination) to 3% but none in in vitro-flowering plants where only seed-like structures, probably unfertilised ovules, were found.