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Fluorescent proteins (FT) have become essential, biological research tools. Many novel genes have been cloned from a variety of species and modified for effective, stable, and strong expression in transgenic organisms. Although there are many applications, FT expression has been employed most commonly at the cellular level in plants. To investigate FT expression at the whole-plant level, particularly in flowers, petunia ‘Mitchell Diploid’ [MD (Petunia ×hybrida)] was genetically transformed with seven genes encoding FTs: DsRed2, E2Crimson, TurboRFP, ZsGreen1, ZsYellow1, rpulFKz1, or aeCP597. Each gene was cloned into a pHK-DEST-OE vector harboring constitutive figwort mosaic virus 35S promoter and NOS-terminator. These plasmids were individually introduced into the genome of MD by Agrobacterium tumefaciens–mediated transformation. Shoot regeneration efficiency from the cocultured explants ranged from 8.3% to 20.3%. Various intensities of red, green, and yellow fluorescence were detected from TurboRFP, ZsGreen1, and ZsYellow1-transgenic flowers, respectively, under ultraviolet light for specific excitation and emission filters. More than 70% of plants established from the regenerated shoots were confirmed as transgenic plants. Transgenic ZsGreen1 petunia generated strong, green fluorescence in all flower organs of T0 plants including petals, stigmas, styles, anthers, and filaments. Most of the chromophores were localized to the cytoplasm but also went into the nuclei of petal cells. There was a positive linear relationship (R2 = 0.88) between the transgene expression levels and the relative fluorescent intensities of the ZsGreen1-transgenic flowers. No fluorescence was detected from the flowers of DsRed2-, E2Crimson-, rpulFKz1-, or aeCP597-transgenic petunias even though their gene transcripts were confirmed through semiquantitative reverse transcriptase-polymerase chain reaction. T1 generation ZsGreen1 plants showed green fluorescence emission from the cotyledons, hypocotyls, and radicles, which indicated stable FT expression was heritable. Four homozygous T2 inbred lines were finally selected. Throughout this study, we demonstrated that ZsGreen1 was most suitable for generating visible fluorescence in MD flowers among the seven genes tested. Thus, ZsGreen1 may have excellent potential for better utility as a sensitive selectable marker.

Petunia × hybrida cv ‘Mitchell Diploid’ floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis ultimately produces floral volatiles derived sequentially from phenylalanine, cinnamic acid, and p-coumaric acid. In an attempt to better understand biochemical steps after p-coumaric acid production, we cloned and characterized three petunia transcripts with high similarity to p-coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT), and caffeoyl shikimate esterase (CSE). Transcript accumulation of PhC3H and PhHCT was highest in flower limb tissue during open flower stages. PhCSE transcript accumulation was also highest in flower limb tissue, but it was detected earlier at initial flower opening with a bell-shaped distribution pattern. Down regulation of endogenous PhC3H transcript resulted in altered transcript accumulation of many other FVBP network transcripts, a reduction in floral volatiles, and the emission of a novel floral volatile. Down regulation of PhHCT transcript did not have as large of an effect on floral volatiles as was observed for PhC3H down regulation, but eugenol and isoeugenol emissions were significantly reduced on the downstream floral volatiles. Together these results indicate that PhC3H is involved in FVBP biosynthesis and the reduction of PhC3H transcript influences FVBP metabolism at the network level. Additional research is required to illustrate PhHCT and PhCSE functions of petunia.

Seed germination and seedling vigor can be affected by environmental cues experienced by the mother plant. However, information about how the maternal environment affects seed quality is scarce in ornamental plants. This study aimed to investigate the effects of two different maternal environments on the seed germination and seedling vigor of Petunia × hybrida under a variety of abiotic stresses. Petunia mother plants were grown in either a greenhouse during the summer months or an indoor controlled-temperature-and-light environment. Collected seeds were subjected to external stressors, including polyethylene glycol (PEG), sodium chloride (NaCl), high temperature, and abscisic acid (ABA), to determine seed germination percentage and seedling vigor. Results indicated that seeds harvested from the mother plants grown in a controlled environment germinated better than seeds harvested from the mother plants grown in the greenhouse when suboptimal germination conditions were applied. Additionally, the seedlings from the controlled maternal environment performed better in both ABA and salinity stress tests than the greenhouse seedlings. Interestingly, the greenhouse seedlings displayed less reactive oxygen species (ROS) damage and lower electrolyte leakage than the controlled environment seedlings under dehydration stress. The difference in germination and seedling vigor of seeds from the two different maternal environments might be due to the epigenetic memory inherited from the mother plants. This study highlighted the strong impact of the maternal environment on seed germination and seedling vigor in Petunia and may assist in high-quality seed production in ornamental plants.

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and – in hypoxic conditions – nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here, we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of dextran sulfate sodium (DSS), after colitis induced by transfer of CD4+CD45RBhi cells, and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with inflammatory bowel disease (IBD) were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T-cell velocity and induced S1P1 degradation and retention of Naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function.

Light intensity, duration, direction, and wavelength are informative to plants. The biochemical circuits that connect specific light wavelengths to expression of specific genes and the metabolic networks they govern have been well defined. However, little emphasis has been placed on how discrete wavelengths of light, alone or in combination, may be applied to manipulate postharvest qualities of high-value horticultural crops. Using narrow-bandwidth LED light we test the hypothesis that discrete light wavelengths can affect the accumulation of volatile compounds known to affect aroma or taste in select flower and fruit products. Volatile benzenoid/phenylpropanoid emission from petunia flowers could be altered with light application. Levels of a key floral volatile, 2-phenylethanol, increased with a red and far-red light treatment. Similar experiments demonstrated that fruit volatile profiles of tomato, strawberry, and blueberry can be manipulated with specific light treatments. These results suggest that compounds affecting sensory qualities of flowers and fruits can be modified by adjustment of ambient light conditions. These findings open new areas of inquiry about how the fragrance and flavor of flowers and fruits may be improved with simple changes in postharvest light conditions.

Previously, when selecting for flavor in the University of Florida southern highbush blueberry (SHB, Vaccinium corymbosum L. hybrids) breeding program, sugar/acid ratios and breeder preference were the only factors considered. A more precise method of evaluating flavor would include volatile compounds that may also contribute to the flavor experience. Therefore, volatile profiles of five SHB cultivars (Farthing, FL01-173, Scintilla, Star, and Sweetcrisp) were compared using gas chromatography–mass spectrometry. All cultivars were harvested on four separate dates within the harvest season, and fruit from each cultivar were also harvested at four developmental stages on the first harvest date. Among the cultivars, soluble solids content and volatile production tended to increase with fruit maturity, whereas titratable acidity decreased. All volatile components were more variable than measures of sugars and acids during the harvest season. Many of the volatiles present varied significantly between harvest dates, resulting in significant genotype × environment interactions during the harvest season. A closer examination of linalool, trans-2-hexenol, trans-2-hexenal, hexanal, and 1-penten-3-ol, five volatile compounds commonly associated with blueberry flavor, showed cultivar, developmental stage, and harvest date differences for each volatile. ‘Star’ experienced the least variation through the harvest period.

R2R3-MYB transcription factors (TFs) are involved in diverse aspects of plant biology. Recently an R2R3-MYB was identified in Petunia x hybrida line P720 to have a role in the transcriptional regulation of floral volatile production. We propose a more foundational role for the R2R3-MYB TF EMISSION OF BENZENOIDS II (EOBII). The homolog of EOBII was isolated and characterized from P. x hybrida ‘Mitchell Diploid’ (MD) and Nicotiana attenuata. For both MD and N. attenuata, EOBII transcript accumulates to high levels in floral tissue with maximum accumulation at flower opening. When EOBII transcript levels are severely reduced using a stable RNAi (ir) approach in MD and N. attenuata, ir-EOBII flowers fail to enter anthesis and prematurely senesce. Transcript accumulation analysis demonstrated core phenylpropanoid pathway transcripts and cell wall modifier transcript levels are altered in ir-EOBII flowers. These flowers can be partially complemented by feeding with a sucrose, t-cinnamic acid, and gibberellic acid solution; presumably restoring cellular aspects sufficient for flower opening. Additionally, if ethylene sensitivity is blocked in either MD or N. attenuata, ir-EOBII flowers enter anthesis. These experiments demonstrate one R2R3-MYB TF can control a highly dynamic process fundamental to sexual reproduction in angiosperms: the opening of flowers.

Pantothenate kinase-associated neurodegeneration is a form of neurodegeneration with brain iron accumulation, characterized by a progressive movement disorder and prominent iron deposition in the globus pallidus. Formerly referred to as Hallervorden–Spatz syndrome, the disorder was renamed pantothenate kinase-associated neurodegeneration after discovery of the causative gene, PANK2. Although the pathological features of clinically characterized Hallervorden–Spatz syndrome have been described, the literature is confounded by the historical use of this term for nearly all conditions with prominent basal ganglia iron accumulation and by the fact that this term encompasses a genetically heterogeneous group of disorders, now referred to as ‘neurodegeneration with brain iron accumulation’. As a result, interpreting reports that precede molecular characterization of specific forms of neurodegeneration with brain iron accumulation is problematic. In the present studies, we describe neuropathological findings in six cases of molecularly confirmed pantothenate kinase-associated neurodegeneration. We identify prominent ubiquinated deposits in pantothenate kinase-associated neurodegeneration. We also characterize two distinct origins of spheroid bodies and delineate histological features of iron deposition. In so doing, we characterize fundamental features of the disease and redefine its nosological relationship to other neurodegenerative disorders.

The relationship between fragrance and vase life and the role of ethylene on volatile emission in cut rose flowers was investigated. No relationship was observed between the amounts of volatile compounds emitted and vase life when fragrant and non-fragrant rose cultivars were compared. Neither ethylene production nor respiration rate of flowers was directly related with vase life. Volatile production during vase life was differential and independent among volatiles originating from different biosynthetic groups. Ethylene did not play a role in the regulation of volatile emission in rose flowers. Endogenous ethylene production was very low in most of the cultivars and did not show autocatalytic production trends. Volatile emission patterns during vase life did not parallel endogenous ethylene production. Exogenous ethylene exposure had differential effects among all cultivars, regardless of the fragrance of the flower. Fragrant cultivar ‘Osiana’ was highly sensitive to exogenous ethylene, with petals abscising within 24 h of ethylene (1 LL −1) exposure while other fragrant cultivars ‘Erin’ and ‘Lovely Dream’ had low ethylene sensitivity. Volatile production was unaffected by exogenous ethylene. The results of this study indicate that volatile emission in cut roses is not regulated by endogenous or exogenous ethylene and occurs independently of petal senescence and/or abscission. These results provide a better understanding of the complexity of volatile emission in rose flowers. © 2010 Elsevier B.V. All rights reserved.

BACKGROUND: Tomatoes contain high levels of several carotenoids including lycopene and β-carotene. Beyond their functions as colorants and nutrients, carotenoids are precursors for important volatile flavor compounds. In order to assess the importance of apocarotenoid volatiles in flavor perception and acceptability, we conducted sensory evaluations of near-isogenic carotenoid biosynthetic mutants and their parent, Ailsa Craig. RESULTS: The carotenoid contents of these tomatoes were extremely low in the r mutant, increased in lycopene in old gold, and higher in tetra-cis-lycopene and ζ-carotene in tangerine. The volatiles derived from these carotenoids (β-ionone, geranylacetone and 6-methyl-5-hepten-2-one) were proportionally altered relative to their precursors. Fruits were also analyzed for soluble solids, sugars, acids and flavor volatiles. Consumer panels rated the r mutant lowest for all sensory attributes, while Ailsa Craig was generally rated highest. Old gold and tangerine were rated intermediate in two of the three harvests. CONCLUSIONS: Several chemicals were negatively correlated with at least one of the hedonic scores while several others were positively correlated with tomato flavor acceptability. The results permitted identification of positive and negative interactions of volatiles with tomato flavor. Copyright © 2010 Society of Chemical Industry

Persons with amnestic mild cognitive impairment (MCI) have subtle impairments in medical decision-making capacity (MDC). We examined the relationship between proton magnetic resonance spectroscopy (MRS) and MDC in MCI. Twenty-nine MCI patients and 42 controls underwent MRS to obtain ratios of N-acetylaspartate (NAA)/Creatine (Cr), Choline (Cho)/Cr, and myo-Inositol (mI)/Cr of the posterior cingulate. They also completed the Capacity to Consent to Treatment Instrument (CCTI), a vignette-based instrument measuring decisional standards of expressing choice, appreciating consequences of choice, providing rational reasons for choice, and understanding treatment choices. Patients showed abnormal MRS ratios of mI/Cr and Cho/Cr compared to controls, and impairments on the CCTI understanding and reasoning Standards. Performance on the Reasoning Standard of the CCTI was correlated with NAA/Cr (r = 0.46, p < 0.05). The relationship of NAA/Cr with decision-making suggests a role for posterior cortical neuronal functioning in performance of complex IADLs in MCI.

Ethylene is a gaseous plant hormone involved in many physiological processes including senescence, fruit ripening, and defence. Here the effects of pollination and wound-induced ethylene signals on transcript accumulation of benzoyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (PhBPBT) are shown in Petuniaxhybrida cv. Mitchell 'Diploid' (MD). In petunia, PhBPBT is responsible for the biosynthesis of both benzyl benzoate and phenylethyl benzoate from benzyl alcohol and phenylethanol, respectively. RNAi-silenced lines, with reduced PhBPBT transcript, displayed reduced benzyl benzoate emission, and increased benzyl alcohol levels. Detailed expression analysis showed that PhBPBT is regulated by both light and an endogenous circadian rhythm, while it is also differentially regulated in response to ethylene in a tissue-specific manner. Twenty-four hours following pollination of MD flowers, expression of PhBPBT decreases in the corolla, while it increases in the ovary after 48 h. This is caused by ethylene that is emitted from the flower coinciding with fertilization as this is not observed in transgenic ethylene-insensitive plants (CaMV35S::etr1-1; 44568). Ethylene is also emitted from vegetative tissue of petunia following mechanical wounding, resulting in an increase in PhBPBT expression in the leaves where expression is normally below detection levels. Indicative of this pattern of expression, we hypothesize that PhBPBT and subsequent benzyl benzoate production is involved in defence-related processes in the corolla prior to pollination, in the ovary immediately following fertilization, and in vegetative tissue in response to wounding.

*Summary Petunia flower petals emit large amounts of isoeugenol, which has been shown to be synthesized by isoeugenol synthase (PhIGS1) from an ester of coniferyl alcohol, hypothesized to be coniferyl acetate. This paper describes the identification and characterization of a novel petunia gene encoding an enzyme belonging to the BAHD acyltransferase family whose expression correlates with isoeugenol biosynthesis. RNAi suppression of this gene results in inhibition of isoeugenol biosynthesis. Biochemical characterization of the protein encoded by this gene showed that it has acetyltransferase activity and is most efficient with coniferyl alcohol among the alcohol substrates tested. Overall, these data support the conclusion that coniferyl acetate is the substrate of isoeugenol synthase.

In the past decade researchers have reported increasingly comprehensive cDNA libraries of genes associated with floral senescence in floral crops. Analysis of the function of these genes in flower senescence has depended on the slow process of stable transformation and regeneration of the plants. We have adopted an alternative strategy, using virus-induced gene silencing (VIGS) to evaluate the effects on flower senescence of silencing senescence-associated gene. Using purple flowered petunia as our test organism, we infect with a construct where the RNA2 of tobacco rattle virus (TRV) is modified by insertion of chalcone synthase (CHS, a key enzyme in anthocyanin biosynthesis). When virus infection occurs, the plant’s silencing mechanism silences CHS, resulting in white sectors or flowers. Infection with TRV containing a tandem construct of phytoene desaturase (PDS) and CHS resulted in characteristic photobleaching of leaves (due to impaired carotene synthesis), and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO). Whether the flowers were treated with ACC or pollinated, the white (silenced) sectors produced less ethylene and senesced later than the purple sectors. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of genes isolated from senescing floral tissues.

In many flowering plants, such as petunia (Petunia × hybrida), ethylene produced in floral organs after pollination elicits a series of physiological and biochemical events, ultimately leading to senescence of petals and successful fertilization. Here, we demonstrate, using transgenic ethylene insensitive (44568) and Mitchell Diploid petunias, that multiple components of emission of volatile organic compounds (VOCs) are regulated by ethylene. Expression of benzoic acid/salicylic acid carboxyl methyltransferase (PhBSMT1 and 2) mRNA is temporally and spatially down-regulated in floral organs in a manner consistent with current models for postpollination ethylene synthesis in petunia corollas. Emission of methylbenzoate and other VOCs after pollination and exogenous ethylene treatment parallels a reduction in PhBSMT1 and 2 mRNA levels. Under cyclic light conditions (day/night), PhBSMT mRNA levels are rhythmic and precede emission of methylbenzoate by approximately 6 h. When shifted into constant dark or light conditions, PhBSMT mRNA levels and subsequent methylbenzoate emission correspondingly decrease or increase to minimum or maximum levels observed during normal conditions, thus suggesting that light may be a more critical influence on cyclic emission of methylbenzoate than a circadian clock. Transgenic PhBSMT RNAi flowers with reduced PhBSMT mRNA levels show a 75% to 99% decrease in methylbenzoate emission, with minimal changes in other petunia VOCs. These results implicate PhBSMT1 and 2 as genes responsible for synthesis of methylbenzoate in petunia.

The plant hormone ethylene regulates many aspects of growth and development. Loss-of-function mutations in ETHYLENE INSENSITIVE2 (EIN2) result in ethylene insensitivity in Arabidopsis, indicating an essential role of EIN2 in ethylene signaling. However, little is known about the role of EIN2 in species other than Arabidopsis. To gain a better understanding of EIN2, a petunia (Petunia × hybrida cv Mitchell Diploid [MD]) homolog of the Arabidopsis EIN2 gene (PhEIN2) was isolated, and the role of PhEIN2 was analyzed in a wide range of plant responses to ethylene, many that do not occur in Arabidopsis. PhEIN2 mRNA was present at varying levels in tissues examined, and the PhEIN2 expression decreased after ethylene treatment in petals. These results indicate that expression of PhEIN2 mRNA is spatially and temporally regulated in petunia during plant development. Transgenic petunia plants with reduced PhEIN2 expression were compared to wild-type MD and ethylene-insensitive petunia plants expressing the Arabidopsis etr1-1 gene for several physiological processes. Both PhEIN2 and etr1-1 transgenic plants exhibited significant delays in flower senescence and fruit ripening, inhibited adventitious root and seedling root hair formation, premature death, and increased hypocotyl length in seedling ethylene response assays compared to MD. Moderate or strong levels of reduction in ethylene sensitivity were achieved with expression of both etr1-1 and PhEIN2 transgenes, as measured by downstream expression of PhEIL1. These results demonstrate that PhEIN2 mediates ethylene signals in a wide range of physiological processes and also indicate the central role of EIN2 in ethylene signal transduction.

Pollen viability, in-vivo pollen tube growth, fruit ripening, seed germination, seed weight, whole plant vigor, and natural fl ower senescence were investigated in homozygous and heterozygous transgenic ethylene-insensitive CaMV35S::etr1-1 petunias (Petunia ×hybrida 'Mitchell Diploidʼ). Homozygous or heterozygous plants were used to determine any maternal and/or paternal effects of the CaMV35S::etr1-1 transgene. All experiments except for those used to determine natural fl ower senescence characteristics were conducted in both high and low temperature greenhouses to determine the effect of temperature stress on transgenic plants when compared to wild-type. Results indicated that ethylene-insensitive plants had a decrease in pollen viability, root dry mass, seed weight, and seed germination. Fruit ripening, seed germination, and seed weight were maternally regulated. In contrast, the CaMV35S::etr1-1 transgene is completely dominant in its effect on natural fl ower senescence. The plant hormone ethylene is involved in many physiologi- cal processes in plants including fruit ripening, petal senescence, abscission, and seed germination (Abeles et al., 1992). The senes- cence of many fl owers is accompanied by a dramatic increase in ethylene production resulting in petal wilting, abscission or color fading. Treatment of ethylene-sensitive fl owers with chemical inhibitors of ethylene biosynthesis or perception reduces this burst of ethylene production and delays visual symptoms of corolla senescence (Jones and Woodson, 1997; Serek et al., 1995; White- head et al., 1984). Genetic modifi cation of ethylene biosynthetic and signal transduction pathways has provided further evidence of ethyleneʼs role in regulating fl ower senescence (Bovy et al., 1999; Savin et al., 1995; Wilkinson et al., 1997). Since many economically important fl oral crops are grown for their display, the control of ethylene perception is essential for increasing vase life and enhancing quality. Since one of the major concerns in the fl oriculture industry is fl ower longevity, research has been conducted to use genetic engineering on fl oriculture crops that are insensitive to ethylene. Wilkinson et al. (1997) transformed petunia with a dominant mu- tant Arabidopsis ethylene receptor, etr1-1, under the control of a constitutive Caulifl ower Mosaic Virus 35S (CaMV35S) promoter to produce ethylene-insensitivity throughout the whole plant. These petunias (44568) had an increase in natural and pollina- tion-induced fl ower longevity compared to wild type 'Mitchell Diploidʼ (MD) plants, but had physiological side effects that limit their commercial use (Gubrium et al., 2000; Wilkinson et al., 1997). Ethylene-insensitive petunias and tomatoes both showed a signifi cant reduction in adventitious root formation, and even exogenous treatments with auxin did not increase adventitious root formation to the level of untreated wild-type controls (Clark

Cytokinins have been shown to delay the onset of leaf senescence. The focus of this project was to produce transgenic petunia (Petunia ×hybrida) plants that over-produced endogenous cytokinins in a senescence specifi c manner. This was achieved by transforming plants with the IPT (isopentenyl transferase) gene driven by the senescence-associated transcriptional promoter, PSAG12. Two independent transgenic events produced T1 and T2 generation seedling lines that demonstrated the desired nonsenescent phenotype in progeny trials. These lines were used to evaluate the horticultural performance of PSAG12-IPT petunia plants in terms of delayed senescence, rooting of vegetative cuttings, lateral branch growth, fl ower number, fl oral timing, and fruit set. Although both lines displayed a delayed senescence phenotype the two PSAG12-IPT transgenic lines differed from each other in regard to other horticultural traits. In addition to delayed leaf senescence, line I-1-7 also demonstrated a decrease in adventitious rooting and an increased number of branches during plant production. Line I-3-18 also demonstrated a delayed leaf senescence phenotype; however, plants of this line were not greatly altered in any other horticultural performance traits in comparison to wild-type 'V26ʼ. IPT transcript was detected in young fully expanded leaves of both lines, although mRNA levels were higher in I-1-7 plants. A greater than 50-fold increase in IPT transcript abundance was detected in leaves of transgenic plants following drought stress. These results demonstrate that it is possible to use PSAG12-IPT to produce transgenic plants with delayed leaf senescence but differences in plant morphology between transgenic lines exist, which may alter horticultural performance characteristics. Senescence is the fi nal developmental process in the lifecycle of a leaf. It is a process through which the nutrients contained within the macromolecules (e.g., chlorophyll, proteins, nucleic acids) of leaf cells are metabolized to basic components and trans- ported to the growing shoot and reproductive organs of the plant. This strategy imparts a survival advantage for plants that grow under adverse conditions where nutrients are limiting (Nooden, 1988). Natural leaf senescence in many plants is characterized by yellowing or chlorosis of the lower leaves as nutrients and other components of the cells are degraded (esp. chlorophyll). In horticultural terms, chlorosis can decrease the aesthetic appear- ance and thereby decrease the salability of that plant. With so much at risk, it is no wonder that growers and researchers alike have tried to understand the components of leaf senescence in order to prevent it. One way to prevent leaf senescence is through the manipulation of cytokinins. Cytokinins are an important class of plant hormones that infl uence numerous aspects of plant growth and development. They have been shown to delay and, in some cases, reverse the leaf senescence process (Gan and Amasino, 1996). There is an inverse correlation between the concentration of cytokinins in leaves and the magnitude of leaf senescence. This can be dramati- cally seen as the formation of green islands on leaves infected by cytokinin-producing pathogens, such as Helminthosporium teres (Angra and Mandahar, 1991). The use of applied cytokinins to prevent senescence is limited because it is diffi cult to supply them

The molecular mechanisms responsible for postpollination changes in floral scent emission were investigated in snapdragon cv Maryland True Pink and petunia cv Mitchell flowers using a volatile ester, methylbenzoate, one of the major scent compounds emitted by these flowers, as an example. In both species, a 70 to 75% pollination-induced decrease in methylbenzoate emission begins only after pollen tubes reach the ovary, a process that takes between 35 and 40 h in snapdragon and approximately 32 h in petunia. This postpollination decrease in emission is not triggered by pollen deposition on the stigma. Petunia and snapdragon both synthesize methylbenzoate from benzoic acid and S-adenosyl-l-methionine (SAM); however, they use different mechanisms to downregulate its production after pollination. In petunia, expression of the gene responsible for methylbenzoate synthesis is suppressed by ethylene. In snapdragon, the decrease in methylbenzoate emission is the result of a decrease in both S-adenosyl-l-methionine:benzoic acid carboxyl methyltransferase (BAMT) activity and the ratio of SAM to S-adenosyl-l-homocysteine ("methylation index") after pollination, although the BAMT gene also is sensitive to ethylene.

Uniconazole was applied as a spray to the surface of container media prior to planting bedding plant plugs. This medium spray was compared to a standard whole-plant spray applied 2 weeks after planting. For petunia (Petunia ×hybrida Vilm.) and coleus (Solenostemon scutellarioides L.) the efficacy of the medium spray was similar to the whole-plant spray. However, for impatiens (Impatiens wallerana Hook. f.) and vinca [Catharanthus roseus (L.) G. Don.] the medium spray had greater efficacy than the whole-plant spray. Increased concentrations of uniconazole in the medium spray decreased plant height; however, the effect of higher concentrations was greater in a medium with out pine bark compared to a medium with pine bark as a component. In the above experiments, uniconazole was applied in a volume of 200 mL·m-2. In a test where spray volume varied, there was a negative linear relationship between plant height and spray volume. Chemical name used: (E)-(+)-(S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-pent-1-ane-3-ol (uniconazole).

Chlorophyll (Chl) and total soluble protein decreased and proteolytic activity increased over a 12-day period during dark-induced senescence in detached leaves of Tara, a yellowing cultivar (Y) of Dendranthema grandiflora. In Boaldi, a non-yellowing cultivar (NY), Chl and soluble protein remained near initial levels and little change in proteolytic activity was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins showed no major differences in banding patterns between the two cultivars at day 0; however, all of the resolved proteins were diminished in Tara by day 12. On the other hand, in NY Boaldi, the intensity of the protein bands did not change over the 12-day period. Attached and detached leaves exhibited similar senescence patterns for each cultivar. Ethylene (100 μl l -1 ) accelerated the rate of Chl loss in detached leaves of Tara, but had no effect on Boaldi. These observations suggest that Boaldi is a stay-green genotype, possibly a functional type. The results are discussed in relation to the role of ethylene in chrysanthemum leaf senescence.

ADDITIONAL INDEX WORDS. ACC (1aminocyclopropane-1-carboxylic acid), ethylene biosynthesis, flower petal abscission, pollination, seedling triple response SUMMARY. Experiments were conducted to determine if the seedling hypocotyl elongation and petal abscission assays could be used to identify differences in ethylene sensitivity among seedling geranium (Pelargonium ×hortorum) cultivars. When seedlings of six geranium cultivars were germinated and grown in the dark in the presence of the ethylene biosynthetic precursor 1-aminocyclopropane1-carboxylic acid (ACC) at various concentrations, they exhibited the triple response (measured as reduced hypocotyl length). While seedlings from all six cultivars were sensitive to ACC, ‘Scarlet Elite’ seedlings were most sensitive, and ‘Multibloom Lavender’, ‘Elite White’ and ‘Ringo 2000 Salmon’ seedlings were the least sensitive when germinated and grown on 20 mM [2022 mg·L–1 (ppm)] ACC. Florets representing three developmental stages of each of the six cultivars were exposed to 1 μL·L–1 of exogenous ethylene for 0, 30, or 60 min to determine if differences in cultivar sensitivity could be determined for petal abscission. Of the six cultivars tested, ‘Ringo 2000 Salmon’, ‘Multibloom Lavender’ and ‘Elite White’ were the least ethylene sensitive. Florets were also self-pollinated to test for cultivar differences in ethylene synthesis and subsequent petal abscission. Ethylene production and petal abscission were both promoted in selfpollinated florets compared to nonpollinated florets. ‘Ringo 2000 Salmon’, ‘Multibloom Lavender’ and ‘Elite White’ florets produced similar amounts of ethylene as all other cultivars, but abscised fewer petals after pollination. Our results indicate that the seedling hypocotyls elongation assay may be used to identify geranium cultivars with reduced sensitivity to ethylene. The data also suggest that genetic variability exists among geraniums for both ethylene sensitivity and biosynthesis. Ethylene induces premature floral abscission in numerous plant species (Abeles et al., 1992). Diploid zonal geraniums are among the most sensitive species with regard to ethylene-induced petal abscission; exposure of florets to 1 μL·L–1 ethylene can result in complete abscission in as little as 1 h (Evensen et al., 1993; Clark et al., 1997). Exposure to trace amounts of ethylene during postharvest transit and marketing can cause petal drop and significantly reduce the visual quality of geraniums (Armitage et al., 1980) and other important floriculture crops. Additionally, ethylene plays a role in the rapid abscission of the corolla following pollination (Abeles et al., 1992). A rapid increase in ethylene production by floral tissues after pollination, followed by accelerated corolla senescence and abscission, was reported for carnation (Dianthus caryophyllus) (Nichols, 1977), digitalis (Digitalis purpurea) (Stead and Moore, 1979, 1983), petunia (Petunia hybrida) (Pech et al., 1987; Whitehead et al., 1984), and geranium (Clark et al., 1997). Petal abscission by pollinated geranium florets is completely but reversibly blocked by 2,5 norbornadiene, an ethylene action inhibitor, indicating that endogenous ethylene perception is essential for the abscission response (Clark et al., 1997). Ethylene also has a regulatory effect on the growth of seedlings following germination. Dark-grown etiolated pea (Pisum sativum) seedlings exposed to exogenous ethylene exhibited: 1) decreased elongation of the hypocotyl (Arshad and Frankenburger, 1988; Koch and Moore, 1990); 2) increased hypocotyl diameter; and 3) exaggerated plumular hooks (Goeschl et al., 1966). This triple response of seedlings was used to identify ethyleneinsensitive mutants as a means to elucidate the mechanism of ethylene perception and action at the molecular level in arabidopsis (Arabidopsis thaliana) (Guzman and Ecker, 1990; Harpam et al., 1991) and tomato (Lycopersicon esculentum) (Lanahan et al., 1994). The effect of ethylene on hypocotyl elongation appears to be concentration-dependent; as exogenous ethylene concentration is increased, hypocotyl length is decreased up to a point of saturation (Goeschl and Kays, 1975; Harpham et al., 1991). Zonal geranium cultivars differ Environmental Horticulture Dept., University of Florida, Gainesville, FL 32611-0670.

A broccoli (Brassica oleracea var. botrytis L.) seedling bioassay was used to measure paclobutrazol activity and distribution in two growing media following drench or subirrigation applications. The bioassay exhibited a saturation-type response curve for paclobutrazol concentrations up to 1000 μg·L-1 in solution and 100 μg·L-1 in the media. The concentration of paclobutrazol required to achieve one-half of the maximum observed bioassay activity was 3-fold as high in bark-based commercial potting medium as in a peat-based medium. Less than 2% of applied paclobutrazol leached out during the drench application despite the collection of up to 50 mL of leachate per 120 mL of the solution (1000 μg·L-1) that was applied per 15-cm pot. Immediately following drench application, paclobutrazol concentrations in both media were highest in the uppermost 2.5 cm and decreased downward. By 3 weeks after treatment, drench-applied paclobutrazol had moved into lower depths. Distribution of paclobutrazol was limited to the bottom 2.5 cm of media when applied as a subirrigation soak. Chemical name used: (±)-(R*,R*)-β-[(4-chlorophenyl)methyl]-α-(1,1-dimethyl)-1H-1,2,4-triazole-1-ethanol (paclobutrazol).

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia ×hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more belowground root mass but fewer aboveground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated taproots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.

Three experiments were conducted to evaluate media component effects on paclobutrazol activity. In Expts. 1 and 2, a broccoli (Brassica oleracea var. botrytis L.) seedling bioassay was used to compare the activity of paclobutrazol at six concentrations (0-0.32 mg·L-1). Results from Expt. 1 indicated that an average of 4-, 5-, and 10-fold higher concentrations were required in old composted pine bark, fresh pine bark, and composted pine bark samples, respectively, to achieve the same activity observed in sphagnum peatmoss (peat) samples. Activity in coir was similar to that in peat while activity in vermiculite and perlite was greater than that in peat. Activity in a fibrous peat sample was greater than in two less-fibrous peat samples. Results from Expt. 2 indicated that paclobutrazol activity was reduced more in the fine (4 mm) fractions. In Expt. 3, petunia {Petunia hybrida Vilm. `Madness Red') was grown in a mixture of either 60% composted pine bark: 0% peat or 0% composted bark: 60% peat. The paclobutrazol concentration required to achieve the same size control was 14 times higher in the former mixture than in the latter. Thus, media components differ greatly in their influence on paclobutrazol activity and the bioassay procedure may serve as a useful tool for predicting media-paclobutrazol interactions. Chemical name used: (±)-(R*,R*)-β-[(4-chlorophenyl)methyl]-α-(l,l-dimethyl)-lH-l,2,4-triazole-l-ethanol (paclobutrazol).

Self-pollination of diploid zonal geranium (Pelargonium × hortorum L.H. Bailey) florets leads to a dramatic rise in ethylene production, followed by abscission within 4 h. Neither wounding of the stigma, pollination with tetraploid pollen, nor heat-killed self pollen could elicit as much ethylene production and petal abscission as self-pollination. A cDNA sharing sequence identity with ACC synthase (GACS2) and three different cDNAs sharing sequence identity with ACC oxidase (GACO1, GACO2, GACO3) were isolated from geranium pistils. Transcripts hybridizing with these probes increased slightly in response to self-pollination, but the degree of accumulation in response to various treatments did not correlate with ethylene production. When calculated on a per-plant-part basis, transcripts hybridizing with GACS2 were equally distributed among the stigma+style, sterile ovary, and ovary tissues, but transcripts hybridizing with the three ACC oxidase clones were differentially distributed. All transcripts were differentially expressed among the other tissues of the plant, with GACO1 being the most widely distributed. Ethylene production in geranium pistils was not autocatalytic. Propylene failed to induce ethylene production and ethylene did not induce the accumulation of ACC synthase or ACC oxidase transcripts. ACC accumulated in the stigma and style, and to a smaller extent in the sterile ovary, after pollination. These data support a model of pollination-induced ethylene production by post-transcriptional regulation of ethylene biosynthetic gene expression.

Rare mutations in the gene encoding for tau (MAPT, microtubule-associated protein tau) cause frontotemporal dementia-spectrum (FTD-s) disorders, including FTD, progressive supranuclear palsy (PSP) and corticobasal syndrome, and a common extended haplotype spanning across the MAPT locus is associated with increased risk of PSP and Parkinson's disease. We identified a rare tau variant (p.A152T) in a patient with a clinical diagnosis of PSP and assessed its frequency in multiple independent series of patients with neurodegenerative conditions and controls, in a total of 15 369 subjects. Tau p.A152T significantly increases the risk for both FTD-s (n = 2139, OR = 3.0, CI: 1.6-5.6, P = 0.0005) and Alzheimer's disease (AD) (n = 3345, OR = 2.3, CI: 1.3-4.2, P = 0.004) compared with 9047 controls. Functionally, p.A152T (i) decreases the binding of tau to microtubules and therefore promotes microtubule assembly less efficiently; and (ii) reduces the tendency to form abnormal fibers. However, there is a pronounced increase in the formation of tau oligomers. Importantly, these findings suggest that other regions of the tau protein may be crucial in regulating normal function, as the p.A152 residue is distal to the domains considered responsible for microtubule interactions or aggregation. These data provide both the first genetic evidence and functional studies supporting the role of MAPT p.A152T as a rare risk factor for both FTD-s and AD and the concept that rare variants can increase the risk for relatively common, complex neurodegenerative diseases, but since no clear significance threshold for rare genetic variation has been established, some caution is warranted until the findings are further replicated.

Floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is a complex and coordinate cellular process executed by petal limb cells of a Petuniaxhybrida cv. 'Mitchell Diploid' (MD) plant. In MD flowers, the majority of benzenoid volatile compounds are derived from a core phenylpropanoid pathway intermediate by a coenzyme A (CoA) dependent, beta-oxidative scheme. Metabolic flux analysis, reverse genetics, and biochemical characterizations of key enzymes in this pathway have supported this putative concept. However, the theoretical first enzymatic reaction, which leads to the production of cinnamoyl-CoA, has only been physically demonstrated in a select number of bacteria like Streptomyces maritimus through mutagenesis and recombinant protein production. A transcript hasbeen cloned and characterized from MD flowers that shares high homology with an Arabidopsis thaliana transcript ACYL-ACTIVATING ENZYME11 (AtAAE11) and the S. maritimus ACYL-COA:LIGASE (SmEncH). In MD, the PhAAE transcript accumulates in a very similar manner as bona fide FVBP network genes, i.e. high levels in an open flower petal and ethylene regulated. In planta, PhAAE is localized to the peroxisome. Upon reduction of PhAAE transcript through a stable RNAi approach, transgenic flowers emitted a reduced level of all benzenoid volatile compounds. Together, the data suggest that PhAAE may be responsible for the activation of t-cinnamic acid, which would be required for floral volatile benzenoid production in MD.

The effect of CO2 enrichment on carbohydrate status and postharvest leaf chlorosis of potted ‘Meijikatar’ miniature roses was investigated. Plants were grown in 350, 700 or 1050 μl l−1 CO2 until they reached flower bud coloration and placed into dark storage at 4°C or 16°C for 5 days to simulate shipping before placing in an interior environment with a photon flux of 30 μmol m−2 s−1 from cool white fluorescent light source for postharvest evaluation. Stomatal resistance was increased (∼25%) and aperture was decreased (∼36%) by 700 or 1050 compared to 350 μ1 1−1 CO2. The higher CO2 levels slightly (10%) increased leaf sucrose concentration, had no effect on stem sucrose concentration, and had little effect on whole plant reducing sugars. Leaf and stem starch concentration increased over three-fold as CO2 increased from 350 to 700 μ1 1−1. Regardless of storage temperature, carbon dioxide enrichment did not affect the overall visual quality or percentage leaf chlorosis after 5 days in the interior environment. Regardless of CO2 level, plants stored at 16°C showed three-fold increase in leaf chlorosis compared to plants stored at 4°C. Although CO2 enrichment increased the carbohydrate pools of the plants, our results do not support the hypothesis that increased carbohydrate supply plays a role in reduction of postharvest leaf chlorosis of potted miniature roses.

The biological effects of ethylene on plant growth and development have been documented for over a century. Ethylene profoundly influences many aspects of development, both pre- and postharvest (Abeles et al., 1992). Spoilage losses ascribed to ethylene effects are significant for both food and ornamental crops. The major agricultural losses associated with ethylene have spurred research on methodologies for control of its synthesis and action. We have been particularly interested in two physiological processes associated with high economic value: fruit ripening and flower senescence. This article addresses biotechnological approaches to control these two processes. There are multiple strategies for controlling ethylene synthesis and response. We will limit our discussion to those with which we have personal experience. Although we will not cover all of the potential target genes, the strategies outlined here apply to the other gene targets. Fruits in which ripening is controlled by ethylene are generally referred to as climacteric. Examples include tomato, melon, mango, papaya, and banana. Our efforts have focused on tomato as a model for several reasons. The tomato is relatively easy to genetically modify via Agrobacterium tumefaciens‐mediated transformation. It has a broad base of physiological, biochemical, and genetic research on which to build. It is also a highly important commercial crop, being the number one valued vegetable produced in Florida (Lucier, 2000). Partly because of its economic importance, much research has been focused on the molecular biology of tomato ethylene synthesis and perception. Like climacteric fruits, a subset of flowers produce and senesce in response to ethylene. One of the best understood of these species is petunia. A petunia flower will produce large amounts of ethylene within hours of pollination. Exposure of flowers to ethylene induces rapid loss of turgor that leads to corolla wilting within 1 to 2 d. Like tomato, petunia is readily transformable and has an excellent foundation of genetic, physiological, and biochemical research. All of these factors make it an excellent model system for genetic manipulation.

Experiments were conducted with four kinds of flowering plants to compare one-time vs. continuous application of paclobutrazol in subirrigation water. When a crop reached the stage at which it required growth regulator treatment, four concentrations of paclobutrazol were applied via subirrigation either one-time or continuously until the crop was terminated. Based upon regression equations, concentrations resulting in 30% size reduction for one-time applications of paclobutrazol were 0.01 mg·L-1 for Begonia ×semperflorens-cultorum `Cocktail Gin', 0.09 mg·L-1 for Impatiens wallerana Hook. `Super Elfin White', 0.2 mg·L-1 for Dendranthema ×grandiflorum (Ramat.) Kitamura `Tara', and 2.4 mg·L-1 for Petunia ×hybrida Vilm.-Andr. `Plum Crazy'. Respective optimal values for continuous application were 0.005, 0.02, 0.06, and 0.4 mg·L-1. Increasing the concentration for continuous application had a greater effect on paclobutrazol efficacy than did increasing the concentration for a single application. In a trial with impatiens `Super Elfin Salmon Blush', the paclobutrazol concentration was reduced 0%, 25%, 50%, 75%, or 100% (single application) for each successive subirrigation event following an initial application of 0.1 mg·L-1 of paclobutrazol. The 50%, 75%, and 100% reduction treatments provided similar levels of size control. Dilution was more important when the reduction rate was less than 50%. Chemical name used: (±)-(R*,R*)-β-[(4-chlorophenyl)methyl]-α-(1,1-dimethyl)-1H-1,2,4-triazole-1-ethanol (paclobutrazol).

Foliar anthocyanin production is frequently induced by phosphorus deficiency, but the adaptive significance of increased anthocyanin production under P stress, if any, remains unknown. In this study we hypothesised that if anthocyanin expression is an adaptive response to mitigate the stress effects of P deficiency, genotypes with constitutive anthocyanin expression would have greater tolerance to P stress than low anthocyanin-producing genotypes. Four studies were conducted in greenhouse, outdoor chamber and field conditions to compare genetically similar maize and coleus plants with contrasting anthocyanin accumulation (i.e. ‘red-leafed’ vs ‘green-leafed’). In low-P treatments, anthocyanin production did not consistently result in greater photosynthesis or biomass. In coleus, red-leafed phenotypes showed lower chlorophyll a/b ratios suggesting photoprotection by anthocyanins against degradation of light harvesting complex proteins. However, the opposite trend was observed in maize, where red-leafed phenotypes showed greater chlorophyll a/b ratios and lower qP (oxidation state of PSII). Based on results from the various treatments and growth conditions of this study, it could not be concluded that high foliar anthocyanin production confers a general functional advantage under low-P stress. More research comparing inducible vs constitutive production may help elucidate the role of anthocyanin biosynthesis in P deficiency responses.

The effects of carbon dioxide enrichment on growth, photosynthesis, and postharvest characteristics of `Meijikatar' potted roses were determined. Plants were grown in 350, 700, or 1050 μl CO2/liter until they reached 50% flower bud coloration and then were placed into dark storage for 5 days at 4 or 16C. Plants grown in 700 or 1050 μl CO2/liter reached the harvest stage earlier and were taller at harvest than plants produced in 350 μl CO2/liter, but there were no differences in the number of flowers and flower buds per plant among CO2 treatments. Plants grown in early spring were taller and had more flowers and flower buds than plants grown in late winter. Shoot and root growth of plants grown in 700 or 1050 μl CO2/liter were higher than in plants produced in 350 μl CO2/liter, with plants grown in early spring showing greater increases than plants grown in late winter. Immediately after storage, plants grown in 350 μl CO2/liter and stored at 4C had the fewest etiolated shoots, while plants grown in 1050 μl CO2/liter and stored at 16C had the most. Five days after removal from storage, chlorophyll concentration of upper and lower leaves had been reduced by ≈50% from the day of harvest. Carbon dioxide enrichment had no effect on postharvest leaf chlorosis, but plants grown in early spring and stored at 16C had the most leaf chlorosis while plants grown in late winter and stored at 4C had the least leaf chlorosis.

Summary In Petunia × hybrida cv. ‘Mitchell Diploid’ floral fragrance is comprised of 13 volatile benzenoids/phenylpropanoids derived from the aromatic amino acid phenylalanine. Several genes involved in the direct synthesis of individual floral volatile benzenoid/phenylpropanoid (FVBP) compounds, i.e. at the end of the pathway, have been isolated and characterized in petunia through reverse genetic and biochemical approaches. In an effort to understand the regulation of ‘upstream’ components in the FVBP system, we have cloned and characterized two CHORISMATE MUTASE (PhCM1 and PhCM2) cDNAs from petunia. PhCM1 has a transcript accumulation profile consistent with known FVBP genes, while PhCM2 showed a constitutive transcript accumulation profile. The plastid-localized PhCM1 is allosterically regulated by tryptophan but not phenylalanine or tyrosine. The total FVBP emission in PhCM1 RNAi knockdown petunias is reduced by approximately 60–70%, and total chorismate mutase activity in corolla tissue is reduced by 80–85% compared to control plants. These results show that PhCM1 is the principal CHORISMATE MUTASE responsible for the coupling of metabolites from the shikimate pathway to the synthesis of FVBPs in the corolla of Petunia × hybrida cv. ‘Mitchell Diploid’.

Senescence represents the last stage of floral development and is an active process that requires gene transcription and protein translation. A genetically controlled senescence program allows for the ordered degradation of organelles and macromolecules and the remobilization of essential nutrients from the petals. Petunia provides an excellent model system for studies of flower senescence because the plants flower profusely and have large floral organs amenable to molecular and biochemical analysis. While Petunia flowers have a finite lifespan that is under tight developmental control, petal senescence can be accelerated and synchronized by means of exogenous ethylene or by pollination. Petal senescence in Petunia is accompanied by decreased nucleic acid and protein content, DNA and nuclear fragmentation, and structural and compositional changes in the plasma membrane. These changes are correlated with increased mRNA abundance and enzyme activity of proteases, nucleases, and phospholipases. Major macronutrient levels in Petunia petals (collectively called the corolla) also decrease during senescence. These studies support cellular degradation and remobilization as the central functions of petal senescence. Ethylene is clearly involved in modulating the process, but the transcription factors and other components of the senescence signal transduction pathway(s) remain to be elucidated. Further studies focusing on early transcriptome changes during petal senescence will help to identify these early regulators, and subsequent studies of protein changes and the post-translational modification of senescence-related proteins will further our understanding of the pathways executing the senescence program.

Contamination of recirculated subirrigation water with growth retardants poses a potential problem for growers. Eight concentrations of ancymidol or paclobutrazol ranging from 0 to 100 μg·L-1 (0 to 1000 μg·L-1 for petunia) were supplied constantly in subirrigation water to potted plants to identify critical levels at which plant growth is affected. Concentrations of ancymidol resulting in 20% reduction in plant size relative to untreated controls were 3, 10, 98, 80, and 58 μg·L-1 for Begonia ×semperflorens-cultorum Hort. `Gin', chrysanthemum (Dendranthema ×grandiflora Kitam.) `Nob Hill', Impatiens walleriana Hook f. `Super Elfin Coral', Petunia ×hybrida Hort. Vilm.-Andr. `Madness Pink', and Salvia splendens Sell ex Roem. & Schult. `Red Hot Sally', respectively. Respective values for paclobutrazol were 5, 24, 17, 390, and >100 μg·L-1. The results provide useful information for managing potential growth retardant contamination problems or for applying growth retardants in subirrigation water. Chemical names used: α-cyclopropyl-α-(4-methoxyphenyl)-5-pyrimidinemethanol (ancymidol); (±)-(R*,R*)-β-[(4-chlorophenyl)methyl]-α-(1,1-dimethyl)-1H-1,2,4-triazole-1-ethanol (paclobutrazol).

Biotechnology has the potential to modify commercially important traits of crops, such as fruit size and stress tolerance. To date, the floricultural industry has not profited significantly from these possibilities to manipulate, for example, flower size. Cytokinins are known to be involved in many aspects of plant development, including cell division. Increasing the amount of cytokinins has the potential to increase the size of an organ, such as the flower or the fruit. The Agrobacterium tumefaciens cytokinin biosynthesis gene isopentenyltransferase (ipt) has been shown to increase cytokinin levels when introduced into plants. Moreover, it has a dramatic effect on the vegetative development of plants. The expression of the ipt gene under the control of the flower-specific Arabidopsis APETALA3 promoter in petunia (Petunia hybrida) increases the flower size dramatically, but with no effect on vegetative development. The resulting transgenic plants produced flowers with larger corolla diameter and greater total floral fresh weight. This strategy has the potential for use in the production of ornamental crops with large flowers and crop species with larger fruit.

The volatile compounds, 2-phenylacetaldehyde and 2-phenylethanol, are important for the aroma and flavor of many foods, such as ripe tomato fruits, and are also major constituents of scent of many flowers, most notably roses. While much work has gone into elucidating the pathway for 2-phenylethanol synthesis in bacteria and yeast, the pathways for synthesis in plants are not well characterized. We have identified two tomato enzymes (LePAR1 and LePAR2) that catalyze the conversion of 2-phenylacetaldehyde to 2-phenylethanol: LePAR1, a member of the large and diverse short-chain dehydrogenase/reductase family, strongly prefers 2-phenylacetaldehyde to its shorter and longer homologues (benzaldehyde and cinnamaldehyde, respectively) and does not catalyze the reverse reaction at a measurable rate; LePAR2, however, has similar affinity for 2-phenylacetaldehyde, benzaldehyde and cinnamaldehyde. To confirm the activity of these enzymes in vivo, LePAR1 and LePAR2 cDNAs were individually expressed constitutively in petunia. While wild type petunia flowers emit relatively high levels of 2-phenylacetaldehyde and lower levels of 2-phenylethanol, flowers from the transgenic plants expressing LePAR1 or LePAR2 had significantly higher levels of 2-phenylethanol and lower levels of 2-phenylacetaldehyde. The in vivo alteration of volatile emissions is an important step toward altering aroma volatiles in plants.

The petunia flower has served as a model for the study of several physiological processes including floral development, self-incompatibility, anthocyanin biosynthesis and ethylene signalling during senescence. More recently, Petunia hybrida ‘Mitchell' has been used to understand the complex regulation of volatile benzenoid biosynthesis, which occurs predominantly in flower petal tissues. Benzenoid biosynthesis is temporally and circadian controlled and is tightly down-regulated by ethylene during floral senescence. Using targeted transcriptomics and gene knockouts, both biosynthetic genes and a transcription factor regulating benzenoid synthesis have been recently discovered and characterized. It appears that benzenoid production is regulated predominantly by transcriptional control of the shikimate pathway, benzenoid biosynthesis genes and S -adenosyl-methionine cycle genes.

Plant senescence is regulated by a coordinated genetic program mediated in part by changes in ethylene, abscisic acid (ABA), and cytokinin content. Transgenic plants with delayed senescence are useful for studying interactions between these signaling mechanisms. Expression of ipt, a cytokinin biosynthetic gene from Agrobacterium tumefaciens, under the control of the promoter from a senescence-associated gene (SAG12) has been one approach used to delay senescence. We transformed petunia (Petunia x hybrida cv V26) with PSAG12-IPT. Two independently transformed lines with extended flower longevity (I-1-7-22 and I-3-18-34) were used to study the effects of elevated cytokinin content on ethylene synthesis and sensitivity and ABA accumulation in petunia corollas. Floral senescence in these lines was delayed 6 to 10 d relative to wild-type (WT) flowers. Ipt transcripts increased in abundance after pollination and were accompanied by increased cytokinin accumulation. Endogenous ethylene production was induced by pollination in both WT and IPT corollas, but this increase was delayed in IPT flowers. Flowers from IPT plants were less sensitive to exogenous ethylene and required longer treatment times to induce endogenous ethylene production, corolla senescence, and up-regulation of the senescence-related Cys protease phcp1. Accumulation of ABA, another hormone regulating flower senescence, was significantly greater in WT corollas, confirming that floral senescence was delayed in IPT plants. These results extend our understanding of the hormone interactions that regulate flower senescence and provide a means of increasing flower longevity.

Six cultivars of potted rose (Rosa ×hybrida L.) plants were evaluated for shipping stress-induced leaf chlorosis during holding at 8, 16, or 28C for 2, 4, or 6 days. `Meijikatar' showed more leaf chlorosis than the similar `Meirutral' at the higher simulated shipping temperatures and longer durations. Plants of `Meijikatar' were treated before simulated shipping with BA, TZ, or Promalin at 0, 25, 50, or 100 mg cytokinin/liter each, then paper-sleeved and stored in the dark in fiberboard boxes at 16C for 5 days. Plant quality 5 days after removal from storage was better with BA at 50 or 100 than at 0 mg·liter–1. All cytokinin-treated plants showed less leaf chlorosis than controls. Benzyladenine at 50 or 100 mg·liter–1 reduced leaf chlorosis when compared to all TZ treatments. There were no differences among treatments in the number of etiolated shoots per plant. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (benzyladenine, BA); trans-zeatin (TZ); gibberellic acid (GA4+7) + BA (Promalin).

To determine the potential for use of biotechnology to produce ethylene insensitive floriculture crops, we designed several experiments to measure horticultural performance of transgenic CaMV35S-etr1-1 petunias. Delayed floral senescence is observed at varying degrees in transgenic plants depending on production environment. Flowers grown in cooler greenhouse environments show greater delays in pollination-induced and natural flower senescence than those grown in warmer environments. Fruit set of self pollinated CaMV35S-etr1-plants was reduced compared to wild type plants, and was also dependent on production environment. Transgenic plants grown in warmer environments showed reduced fruit set after self pollination compared to those grown in cooler environments. Fruit ripening is delayed, and adventitious root formation is significantly reduced in transgenic CaMV35S-etr1 plants compared to wild type plants. These observations lead us to suggest that tissue specific ethylene insensitivity will be required to produce ethylene insensitive floriculture crops with extended flower life.

Dendranthema×grandiflorum (Ramat.) were grown in either a peat-based or pine bark—based medium and drenched with growth retardants at a range of concentrations to generate dose : response curves. The effect of ancymidol, paclobutrazol, and uniconazole on stem elongation was less in the pine bark—based than in the peat-based medium. Generally, the concentrations required to achieve the same response were 3- to 4-fold as high in the pine bark—based medium as in the peat-based medium. However, chlormequat was slightly more active in the pine bark—based medium than in the peat-based medium. Chemical names used: α-cyclopropyl-α—(4-methoxyphenyl)-5-pyrimidinemethanol (ancymidol); (±)-(R*,R*)-β-[(4-chlorophenyl)methyl]-α-(1,1-di methyl)-1H-1,2,4-triazole-1-ethanol (paclobutrazol); (E)-(RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pent -l-en-3-ol (uniconazole); 2-chloroethyltrimethylammonium chloride (chlormequat).

In this study, the temporal and spatial regulation of putative ethylene receptor genes was examined during ethylene and pollination-induced flower petal abscission of zonal geranium (Pelargonium × hortorum L.H. Bailey). We used the Arabidopsis thaliana ETR1 gene as a heterologous probe to isolate two full-length cDNA clones, GER1 and GER2, from an ethylene-treated geranium pistil cDNA library. Both cDNAs share a high degree of DNA sequence similarity to ETR1, and examinations of deduced amino acid sequences indicate that the proteins encoded by each gene have the conserved ethylene binding and response regulator domains found in ETR1. Experiments focused on determining the temporal regulation of these genes revealed that both genes are expressed in geranium florets much earlier than when the florets become responsive to ethylene treatment, which is sufficient to cause petal abscission in 1 hr. Both genes are expressed in pistils throughout floret development. Experiments focused on determining the spatial regulation of these genes revealed that both genes are expressed at moderate levels in leaves, pistils, anthers, and petals, and are expressed at very low levels in roots. Preliminary evidence suggests that GER2 is transcriptionally regulated by ethylene in pistils after exogenous ethylene treatment. Currently, the transcriptional regulation of these genes in pistils after pollination is unknown.