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The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.

The involvement of abscisic acid (ABA) in the process of leaf abscission induced by 1-aminocyclopropane-1-carboxylic acid (ACC) transported from roots to shoots in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings grown under water stress was studied using norflurazon (NF). Water stress induced both ABA (24-fold) and ACC (16-fold) accumulation in roots and arrested xylem flow. Leaf bulk ABA also increased (8-fold), although leaf abscission did not occur. Shortly after rehydration, root ABA and ACC returned to their prestress levels, whereas sharp and transitory increases of ACC (17-fold) and ethylene (10-fold) in leaves and high percentages of abscission (up to 47%) were observed. NF suppressed the ABA and ACC accumulation induced by water stress in roots and the sharp increases of ACC and ethylene observed after rewatering in leaves. NF also reduced leaf abscission (7-10%). These results indicate that water stress induces root ABA accumulation and that this is required for the process of leaf abscission to occur. It was also shown that exogenous ABA increases ACC levels in roots but not in leaves. Collectively, the data suggest that ABA, the primary sensitive signal to water stress, modulates the levels of ethylene, which is the hormonal activator of leaf abscission. This assumption implies that root ACC levels are correlated with root ABA amounts in a dependent way, which eventually links water status to an adequate, protective response such as leaf abscission

The N-demethylation of the pyridazinone pro-herbicide metflurazon into norflurazon implies a toxification in photosynthetic organisms. This is confirmed by quantitative structure activity relationships determined for two unicellular green algae, Chlorella sorokiniana and Chlorella fusca; however, the latter is 25 to 80 times more sensitive to metflurazon. This sensitivity is linked to differences in the N-demethylase activity of both algae, as determined by an optimized in vivo biotransformation assay. Apparent Km values of the metflurazon-N-demethylase indicate a 10-fold higher affinity for this xenobiotic substrate for Chlorella fusca. Furthermore, algal metflurazon-N-demethylation is characterized by distinct variations in activity, depending on the stage of cell development within the cell cycle. Several well-established inhibitors of cytochrome P450-mediated reactions, including piperonylbutoxide, 1-aminobenzotriazole, 1-phenoxy-3-(1H-1,2,4-triol-1yl)4-hydroxy-5,5-di methylhexane, and tetcyclacis, as well as cinnamic acid, a potential endogenous substrate, inhibited the N-demethylation of metflurazon. The results suggest that the N-demethylation of metflurazon by both algae is mediated by a cytochrome P450 monooxygenase. The determination of antigenic cross-reactivity of algal proteins with heterologous polyclonal antibodies originally raised against plant P450s, anti-cinnamic acid 4-hydroxylase (CYP73A1), anti-ethoxycoumarin-O-dealkylase, anti-tulip allene oxidase (CYP74), and an avocado P450 (CYP71A1) or those of bacterial origin, CYP105A1 and CYP105B1, suggests the presence of distinct P450 isoforms in both algae

A comparison was made of the effects of abscisic acid (ABA) and the ABA biosynthesis inhibitor, norflurazon on the interaction between soybean leaves and Phytophthora sojae. Inoculation of leaves of cv. Harosoy resulted in a compatible interaction typified by the presence of spreading, water soaked lesions with ill-defined margins while inoculation of cv. Haro 1272 resulted in an incompatible interaction with lesions restricted to the inoculation site. Activity of phenylalanine ammonia lyase (PAL) slowly increased in the compatible interaction but in the incompatible interaction there was a rapid rise in activity within 8 h after inoculation. When Haro 1272 plants were treated with ABA the normally incompatible interaction with race 1 was changed to what resembled a compatible interaction and activity of PAL was reduced to control levels. There was no visible effect on the compatible combination. In contrast when plants of cv. Harosoy were treated with norflurazon the normally compatible interaction with race 1 was changed to that which resembled an incompatible interaction and PAL activity increased to high levels rapidly. There was no effect of norflurazon on the incompatible interaction of cv. Haro 1272 with race 1. Stomata on leaves of cv. Harosoy treated with norflurazon closed within 2 h of inoculation resembling the response of stomata in normal incompatible interactions but not compatible interactions where stomata remained open. On leaves of cv. Harosoy treated with norflurazon at sites 3 and 20 mm from the inoculation point stomata also closed. These results extend and confirm the idea that ABA is a molecule that may regulate the outcome of the interaction between soybeans and P. sojae.

Synthesis of carotenoids in higher plants occurs in the plastids, but all of the required enzymes are coded for in the nuclear genome and are post-transcriptionally imported into the plastid compartment. Regulation of the synthesis of the enzymes is poorly understood. The two-step desaturation of phytoene to zeta-carotene, carried out by the enzyme phytoene desaturase (PDS), is one of the earliest steps in the pathway and has been studied in several systems. Previous analyses of phytoene-accumulating tissue suggested that there may be feedback regulation of PDS gene transcription, with higher expression in white tissue. To investigate this regulation further, we examined phytoene-accumulating tissue in Arabidopsis thaliana (L.) Heynh. Two types of phytoene-accumulating tissue were studied: Norflurazon-bleached plants and white sectors from the immutans variegation mutant. Based on competitive RT-PCR measurements of PDS mRNA and immunochemical detection of PDS protein, we determined that there is no significant induction of PDS gene expression specific to white tissue, indicating that PDS expression is independent of the pigment status of the cells. Reasons why our results differ from those in other systems are discussed.

To study the mechanisms that integrate pigment and chlorophyll a/b-binding apoprotein biosynthesis during light-harvesting complex II assembly, we have examined beta-glucuronidase (GUS) enzyme activities, chlorophyll contents, and cell sizes in fluorescence-activated, cell-sorting-separated single cells from transgenic Arabidopsis thaliana wild-type and immutans variegation mutant plants that express an Lhcb (photosystem II chlorophyll a/b-binding polypeptide gene)/GUS promoter fusion. We found that GUS activities are positively correlated with chlorophyll content and cell size in green cells from the control and immutans plants, indicating that Lhcb gene transcription is coordinated with cell size in this species. Compared with the control plants, however, chlorophyll production is enhanced in the green cells of immutans; this may represent part of a strategy to maximize photosynthesis in the green sectors to compensate for a lack of photosynthesis in the white sectors of the mutant. Lhcb transcription is significantly higher in pure-white cells of the transgenic immutans plants than in pure-white cells from norflurazon-treated, photooxidized A. thaliana leaves. This suggests that immutans partially uncouples Lhcb transcription from its normal dependence on chlorophyll accumulation and chloroplast development. We conclude that immutans may play a role in regulating Lhcb transcription, and may be a key component in the signal transduction pathways that control chloroplast biogenesis

We have analyzed plastid and nuclear gene expression in tobacco seedlings using the carotenoid biosynthesis inhibitor norflurazon. mRNA levels for three nuclear-encoded chlorophyll-binding proteins of photosystem I and photosystem II (CAB I and II and the CP 24 apoprotein) are no longer detectable in photobleached seedlings, whereas those for other components of the thylakoid membrane (the 33- and 23-kD polypeptides and Rieske Fe/S polypeptide) accumulate to some extent. Transgenic tobacco seedlings with promoter fusions from genes for thylakoid membrane proteins exhibit a similar expression behavior: a CAB-beta-glucuronidase (GUS) gene fusion is not expressed in herbicide-treated seedlings, whereas PC-, FNR-, PSAF-, and ATPC-promoter fusions are expressed, although at reduced levels. All identified segments in nuclear promoters analyzed that have been shown to respond to light also respond to photodamage to the plastids. Thus, the regulatory signal pathways either merge prior to gene regulation or interact with closely neighboring cis elements. These results indicate that plastids control nuclear gene expression via different and gene-specific cis-regulatory elements and that CAB gene expression is different from the expression of the other genes tested. Finally, a plastid-directing import sequence from the maize Waxy gene is capable of directing the GUS protein into the photodamaged organelle. Therefore, plastid import seems to be functional in photobleached organelles

Broadleaf weed and yellow nutsedge control with herbicide programs containing pendimethalin and combinations of fomesafen, fluometuron, and norflurazon applied alone or with POST-directed applications of MSMA or fluometuron plus MSMA was evaluated. Soil-applied herbicide combinations containing fomesafen controlled yellow nutsedge better than combinations of norflurazon and fluometuron but did not provide better entireleaf, ivyleaf, pitted, and tall morningglory or sicklepod control. Fluometuron plus MSMA controlled morningglories and sicklepod more effectively than MSMA. Seed cotton yield was greater in one of two years when fomesafen was applied and was associated with better yellow nutsedge control.

Norflurazon (4-chloro-5-(methylamino)-2-(alpha,alpha, alpha-trifluoro-m-tolyl-3(2H)-pyridazinone))is a pre-emergent herbicide used in cranberry (Vaccinium macrocarpon Ait.) cultivation to control annual grasses, sedges, and broadleaf weeds. Cranberries are an economically important crop in New England, Wisconsin, and other parts of the northern USA. The biodegradation of norflurazon in the high organic matter, acidic soil characteristic of cranberry bogs has been shown to proceed slowly (Savin and Amador, 1998). The potential effects of cranberry cultivation practices--soil moisture control, fertilization, sand addition, and herbicide application rate--on mineralization of norflurazon in a bog soil were evaluated in a laboratory study. Optimal soil moisture for norflurazon mineralization was between 80 and 90% of waterholding capacity (WHC) in soil from the Oi and A horizons. Saturating the soil reduced the rate of norflurazon mineralization significantly. By contrast, soil respiration was maximal at 25% of WHC in both horizons. Addition of inorganic P increased soil respiration, but did not affect norflurazon mineralization significantly. Addition of inorganic N plus P increased soil respiration in the A, but not Oi, horizon and significantly decreased norflurazon mineralization in the Oi horizon. Sand addition had no significant effect on norflurazon mineralization. Mineralization was affected by herbicide application rate, with the rate of mineralization increasing proportionally with increasing concentration from 0.75 to 7.5 mg norflurazon kg-1 soil. The mineralization of 14C-norflurazon was slow for all of the agronomic practices evaluated, indicating that the potential for norflurazon to accumulate in cranberry bog soils may be high

We have used the carotenoid biosynthesis inhibitor norflurazon to study the relationship between chloroplast and nuclear gene expression and the mechanisms by which plastid mRNA accumulation is regulated in response to photooxidative stress. By treating 4-week-old hydroponic spinach plants (Spinacea oleracea), we were able to determine the response at two distinct stages of chloroplast development. For all parameters studied, differences were found between the norflurazon-treated young and mature leaves. Young leaves lost essentially all pigment content in the presence of norflurazon, whereas mature leaves retained more than 60% of their chlorophyll and carotenoids. The accumulation of plastid mRNA was determined for several genes, and we found a decrease in mRNA levels for all genes except psbA in herbicide-treated young leaves. For genes such as atpB, psbB, and psaA, there was a corresponding change in the relative level of transcription, but for psbA and rbcL, transcription and mRNA accumulation were uncoupled. In norflurazon-treated mature leaves, all plastid mRNAs except psaA accumulated to normal levels, and transcription levels were either normal or higher than corresponding controls. This led to the conclusion that plastid mRNA accumulation is regulated both transcriptionally and posttranscriptionally in response to photooxidative stress. Although direct photooxidative damage is confined to the plastid and peroxisome, there is a feedback of information controlling the transcription of nuclear-encoded plastid proteins. Considerable evidence has accumulated implicating a \"plastid factor\" in this control. Therefore, the expression of several nuclear-encoded plastid proteins and the corresponding mRNAs were determined. Although the levels of both the small subunit of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b-binding protein and corresponding mRNAs were reduced, a 28-kilodalton chloroplast RNA-binding protein and corresponding mRNA were at normal levels in norflurazon-treated plants. Changes in mRNA and protein levels were not the result of a general loss due to photooxidation but rather the result of selective stabilization of certain components. The response of both genomes to photooxidative stress is discussed in terms of the postulated plastid factor