Juvenile-Specific Localization and Accumulation of a Rhamnosyltransferase and Its Bitter Flavonoid in Foliage,Flowers, and Young Citrus Fruits

1-2-Rhamnosyltransferase catalyzes the production of disaccharide-flavonoids that accumulate to 75% of dry weight. Vast energy is expended in a short time span to produce these flavonoids. The highest rhamnosyltransferase activities and immunodetected concentrations were observed in early development of Citrus grandis (pummelo), coinciding with up to 13% of fresh weight as naringin. The concentration of naringin in leaves, petals, receptacles, filaments, albedo, and flavedo drops drastically during development and correlates directly with a decrease in the activity and amounts of 1-2-rhamnosyltransferase. Anthers had minute rhamnosyltransferase activities and low concentrations of naringin. Conversely, high 1-2-rhamnosyltransferase activity and naringin concentrations appeared in both young and mature ovaries, as well as in young fruits. The total amounts of naringin in mature leaves decreased without detectable in vitro degradation of naringin in leaves. There was still a net accumulation of naringin in the albedo and flavedo of older fruit even though these tissues had only traces of 1-2-rhamnosyltransferase. Traces of enzyme synthesis in fruits, or import of the product from leaves, may explain the net accumulation of naringin in growing fruits. Unlike the late-expressed genes for glycosyltransferases in anthocyanin biosynthesis, the rhamnosyltransferases from Citrus are active only in juvenile stages of development.     

Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.     

Glyphosate Suppression of an Elicited Defense Response : Increased Susceptibility of Cassia obtusifolia to a Mycoherbicide

The major effort in developing pathogenic fungi into potential mycoherbicides is aimed at increasing fungal virulence to weeds without affecting crop selectivity. Specific suppression of biosynthesis of a phytoalexin derived from the shikimate pathway in Cassia obtusifolia L. by a sublethal dose (50 micromolar) of glyphosate increased susceptibility to the mycoherbicide Alternaria cassiae Jurair & Khan. Glyphosate applied with conidia suppressed phytoalexin synthesis beginning at 12 hours, but not an earlier period 8 to 10 hours after inoculation. The phytoalexin synthesis elicited by fungal inoculation was also suppressed by darkness. The magnitudes of virulence of the mycoherbicide in the dark or with glyphosate in the light were both higher than after inoculation in the light with the same concentration of conidia in the absence of glyphosate. Five times less inoculum was needed to cause disease symptoms when applied with glyphosate than without. Glyphosate did not render A. cassiae virulent on soybean (Glycine max), a crop related to the host. These results suggest that a specific inhibition of a weed's elicited defense response can be a safe way to enhance virulence and improve the efficacy of the mycoherbicide.     

The effect of chlorophyll-bleaching herbicides on growth,carotenoid and diosgenin levels in cell suspension cultures of Dioscorea deltoidea

Bleaching herbicides of the phenylpyridazinone group (norflurazon, metflurazon, SAN 9774 and SAN 9785) and difunon and amitrole were added to cell suspension cultures of Dioscorea deltoidea at 1μM. Difunon inhibited growth and carotenoid biosynthesis and metflurazon inhibited growth. Norflurazon was the only herbicide tested that influenced diosgenin production. Norflurazon increased the rate of diosgenin biosynthesis so that 180mg/l were obtained after 14 days of incubation as compared to 30 days for the control to reach the same level.     

Cell cultures vs. whole plants for measuring phytotoxicity I. The establishment and growth of callus and suspension cultures; definition of factors affecting toxicity on calli

Callus tissue has been isolated from 11 species, of 8 whichare ‘weeds’ from which callus isolation has notbeen previously described. Continuous suspension cultures offour species have been started. Growth conditions were optimizedfor high yield. Logarithmic phase and stationary phase cellsreact differently to some inhibitors. The larger the inoculumsize, the greater the concentration of inhibitor that must beused to achieve equal inhibition. Hormonal constitution of themedium affects inhibition by ‘auxin’ type herbicides.Growth kinetics, delayed response and recovery must be consideredin assessing phytotoxicity. Highly uniform and controlled proceduresare required for interspecific comparisons of many compounds. (Received January 7, 1977; )     

Infection of tubercles of the parasitic weed Orobanche aegyptiaca by mycoherbicidal Fusarium species

Progression of the infection by host-specific strains of Fusarium oxysporum and Fusarium arthrosporioides of Orobanche aegyptiaca (Egyptian broomrape) tubercles attached to tomato roots was tracked using light, confocal and electron microscopy. Mycelia transformed with the gene for green fluorescent protein were viewed using a confocal microscope. Fungal penetration was preceded by a rapid loss of starch, with approx. 10 % remaining at 9 h and no measurable starch at 24 h. Penetration into the Orobanche tubercles began by 12 h after inoculation. Hyphae penetrated the outer six cell layers by 24 h, reaching the centre of the tubercles by 48 h and infecting nearly all cells by 72 h. Most of the infected tubercles were dead by 96 h. Breakdown of cell walls and the disintegration of cytoplasm in and around the infected cells occurred between 48 and 96 h. Lignin-like material increased in tubercle cells of infected tissues over time, but did not appear to be effective in limiting fungal penetration or spread. Callose, suberin, constitutive toxins and phytoalexins were not detected in infected tubercles, suggesting that there are no obvious defence mechanisms to overcome. Both Fusarium spp. pathogenic on Orobanche produced fumonisin-like ceramide synthase inhibitors, while fusaric acid was produced only by F. oxysporum in liquid culture. The organisms do not have sufficient virulence for field use (based on glasshouse testing), suggesting that virulence should be transgenically enhanced or additional isolates sought.     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phytoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low β-carotene and in the norflurazon-treated phytoene-rich cells. These observations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil protects the cells against injury by excessive irradiation.     

Tandem constructs to mitigate transgene persistence: tobacco as a model

Some transgenic crops can introgress genes into other varieties of the crop, to related weeds or themselves remain as 'volunteer' weeds, potentially enhancing the invasiveness or weediness of the resulting offspring. The presently suggested mechanisms for transgene containment allow low frequency of gene release (leakage), requiring the mitigation of continued spread. Transgenic mitigation (TM), where a desired primary gene is tandemly coupled with mitigating genes that are positive or neutral to the crop but deleterious to hybrids and their progeny, was tested as a mechanism to mitigate transgene introgression. Dwarfism, which typically increases crop yield while decreasing the ability to compete, was used as a mitigator. A construct of a dominant ahasR (acetohydroxy acid synthase) gene conferring herbicide resistance in tandem with the semidominant mitigator dwarfing Delta gai (gibberellic acid-insensitive) gene was transformed into tobacco (Nicotiana tabacum). The integration and the phenotypic stability of the tandemly linked ahasR and Delta gai genomic inserts in later generations were confirmed by polymerase chain reaction. The hemizygous semidwarf imazapyr-resistant TM T1 (= BC1) transgenic plants were weak competitors when cocultivated with wild type segregants under greenhouse conditions and without using the herbicide. The competition was most intense at close spacings typical of weed offspring. Most dwarf plants interspersed with wild type died at 1-cm, > 70% at 2.5-cm and 45% at 5-cm spacing, and the dwarf survivors formed no flowers. At 10-cm spacing, where few TM plants died, only those TM plants growing at the periphery of the large cultivation containers formed flowers, after the wild type plants terminated growth. The highest reproductive TM fitness relative to the wild type was 17%. The results demonstrate the suppression of crop-weed hybrids when competing with wild type weeds, or such crops as volunteer weeds, in seasons when the selector (herbicide) is not used. The linked unfitness would be continuously manifested in future generations, keeping the transgene at a low frequency.     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phqtoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low & carotene and in the nor-urazon-treated phytoene-rich cells. These obseruations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil portects the cells against injury by excessive irradiation.     

Molecular biology of weed control

The vast commercial effort to utilize chemical and molecular tools to solve weed control problems has had a major impact on the basic biological sciences as well as benefits to agriculture, and the first generation of transgenic products has been successful, while somewhat crude. More sophisticated products are envisaged and expected. Biotechnologically-derived herbicide-resistant crops have been a considerable benefit, yet in some cases there is a risk that the same useful transgenes may introgress into related weeds, specifically the weeds that are hardest to control without such transgenic crops. Biotechnology can also be used to mitigate the risks. Molecular tools should be considered for weed control without the use of, or with less chemicals, whether by enhancing crop competitiveness with weeds for light, nutrients and water, or via allelochemicals. Biocontrol agents may become more effective as well as more safe when rendered hypervirulent yet non-spreading by biotechnology. There might be ways to disperse deleterious transposons throughout weed populations, obviating the need to modify the crops. This revised version was published online in August 2006 with corrections to the Cover Date.