Characterization of oncogene-silenced transgenic plants: implications for Agrobacterium biology and post-transcriptional gene silencing.

SUMMARY Agrobacterium tumefaciens tumorigenesis is initiated by the horizontal transfer of a suite of oncogenes that alter hormone synthesis and sensitivity in infected plant cells. Transgenic plants silenced for the iaaM and ipt oncogenes are highly recalcitrant to tumorigenesis, and present a unique resource to elucidate fundamental questions related to Agrobacterium biology and post-transcriptional gene silencing (PTGS). The oncogene-silenced transgenic tomato line 01/6 was used to characterize A. tumefaciens growth in planta and to screen for iaaM and ipt sequence variants. Even in the absence of macroscopic and microscopic indications of tumorigenesis, A. tumefaciens is capable of long-term survival in the hypocotyl tissues of the 01/6 line. A. tumefaciens growth, however, is significantly reduced in the 01/6 line, with populations decreased by 96% relative to wild-type at 52 days post-inoculation. In addition, the 01/6 line displayed suppression of tumorigenesis against all 35 tested strains of A. tumefaciens. High target homology is an absolute requirement of PTGS, therefore this result suggests that regions of the iaaM and ipt oncogenes are very highly conserved across most A. tumefaciens strains. Finally, graft transmissibility of oncogene silencing was assessed by grafting various non-silenced tomato genotypes on to the 01/6 line. Phenotypic and molecular evidence (tumorigenesis and absence of small interfering RNAs, respectively) suggest that oncogene silencing is not graft-transmissible, at least to wild-type and antisense iaaM-over-expressing genotypes.

Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis.

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in the dioecious species, Pistacia vera. Progenies from two female parents pollinated by a common male parent were studied. Two bulks of DNA were made in each cross, one from males and one from females, by pooling an equal weight of fresh leaves from each individual contributing to the bulk prior to DNA extraction. DNA was extracted from each bulked sample and from each of the contributing individuals. DNA was also extracted from 14 cultivars of P. vera and from 94 open-pollinated, fewweeks-old P. vera seedlings of unknown sex. Seven hundred different decamer oligonucleotide primers were used to perform DNA amplification, with 1 of these (OPO08) producing a 945 bp amplification band that was present only in the bulked female samples and absent in the bulked male samples of the two crosses. The relationship between band presence and female sex expression was conserved in every individual obtained from the two crosses and in the 14 cultivars unrelated to the crosses. We propose that this band is tightly linked to the gene(s) that control sex determination in pistachio. The OPO08945 RAPD marker could be used in a breeding program to screen the gender of pistachio plants long before they reach reproductive maturity, resulting in considerable savings of time and economic resources. In order to verify that assumption we screened 94 additional seedlings with the OPO08 primer and obtained results consistent with a 1∶1 male:female ratio.

Maintenance of turgor by rapid sealing of puncture wounds in leaf epidermal cells.

When leaf epidermal cells are puncture wounded with a glass microcapillary tip, a small droplet of fluid is discharged and then evaporates, leaving a solid residue on the cell surface. For puncture wounds of about 3.5 micrometers in diameter, this process is complete within 2 to 3 seconds. A second puncture wound also exhibits a similar discharge, indicating the persistence of some turgor pressure within the cell, despite damage to the cell wall. Direct measurement of turgor on the large epidermal cells of Tradescantia virginiana L. demonstrated that turgor was substantially maintained (91-96%) after puncture wounding. Anatomical and histochemical evidence suggests that the damaged portion of the cell wall was sealed with an amorphous plug of material comprised of pectinaceous polysaccharides. Rapid sealing of puncture wounds and the maintenance of turgor in epidermal cells may be an important functional component of plant adaptation to physical damage such as that caused by insect feeding.

The initiation and organization of microtubules in germinating pear (Pyrus communis L.) pollen.

To study microtubule organization in germinating pear (Pyrus communis L., cv., Bartlett) pollen, we removed the pollen wall by freeze-fracturing before treating the resultant pollen protoplasts by conventional immunofluorescence procedures. Results reveal that axial bundles of microtubules are present in the generative cell of both inactivated and activated pollen grains. Microtubules are not present in the vegetative cells of inactivated pollen, but they are present in the vegetative cells of activated pollen grains. Microtubule nucleation occurs in the vegetative cell cortex. Subsequently, the microtubules grow as branching arrays through most of the vegetative cell cortex except at the apertures where they form localized converging or criss-cross patterns. Eventually, in a germinated pollen grain, the microtubules form network-like arrays through most of the pollen grain and a collar of short arrays at the base of the pollen tube. It is suggested that the role of vegetative cell microtubules in pollen germination is indirect through their mediation of the conformational changes in actin organization that are essential for pollen germination.

Salinity stress increases cytoplasmic ca activity in maize root protoplasts.

High concentrations of NaCl immediately elevated cytoplasmic Ca activity in maize (Zea Mays L. cv Pioneer 3377) root protoplasts, as measured with the fluorescent probe Indo-1. The effect of salinity was inhibited by Li pretreatment but restored by inositol, suggesting that phosphoinositides mediate the stress response.

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation.

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.

Microfluorometry of pectic materials in the dehiscence zone of almond (Prunus dulcis [Mill.] DA Webb) fruits.

We examined the middle lamella and the primary and secondary walls in almond pericarp dehiscence zone cells using a fluorescent cytochemical method which permitted specific, quantitative detection of pectic cell wall materials. Glycol methacrylate-embedded sections were stained with coriphosphine and pectin-specific fluorescent emissions at 630 nm were quantified using green excitation (546 nm). Examination of sectioned material extracted with purified pecto-lytic enzyme preparations was used to demonstrate the relative specificity of the staining reaction for pectic substances.

Zymolyase removes callose from germinating pollen and pollen tube walls.

The impermeable callosic wall that surrounds germinating pollen grains and pollen tubes is removed by brief treatment with Zymolyase, an enzyme preparation that hydrolyzes linear glucose polymers with beta-1,3 linkages. Zymolyase treatment does not interfere with pollen tube growth or cylosis in the vegetative cell.

Displacement of ca by na from the plasmalemma of root cells : a primary response to salt stress?

A microfluorometric assay using chlorotetracycline (CTC) as a probe for membrane-associated Ca(2+) in intact cotton (Gossypium hirsutum L. cv Acala SJ-2) root hairs indicated displacement of Ca(2+) by Na(+) from membrane sites with increasing levels of NaCl (0 to 250 millimolar). K(+)((86)Rb) efflux increased dramatically at high salinity. An increase in external Ca(2+) concentration (10 millimolar) mitigated both responses. Other cations and mannitol, which did not affect Ca(2+)-CTC chelation properties, were found to have no effect on Ca(2+)-CTC fluorescence, indicating a Na(+)-specific effect. Reduction of Ca(2+)-CTC fluorescence by ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid, which does not cross membranes, provided an indication that reduction by Na(+) of Ca(2+)-CTC fluorescence may be occurring primarily at the plasmalemma. The findings support prior proposals that Ca(2+) protects membranes from adverse effects of Na(+) thereby maintaining membrane integrity and minimizing leakage of cytosolic K(+).