Cellular and subcellular localization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methylbenzoate in snapdragon flowers.

The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed by S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators.     

Effects of various chemical agents and early ethylene production on floral senescence of Hibiscus syriacus L.

To understand the factors that induce floral senescence in Hibiscus syriacus L., we have investigated the effects of various chemical agents on flower senescence at two different flowering stages, before and after full bloom, as well as the relationship between flower longevity and endogenous ethylene production before full bloom. Treatments with ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ethephon enhanced floral senescence, while aminoethoxyvinylglycine (AVG) promoted flower longevity regardless of treatment timing. Although ethanol slightly extended flower longevity, abscisic acid (ABA), nitric oxide, boric acid and sucrose, which have been reported to affect flower longevity or senescence, had no effect on H. syriacus floral senescence. The polyamine spermine (SPM), methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SPM biosynthesis, and cycloheximide (CHI) accelerated flower senescence when applied before full bloom, but had no effect when applied after full bloom. SPM, MGBG and CHI treatments resulted in enhanced ethylene production during flower opening, and the promotion of flower senescence is mediated by ethylene production prior to full bloom. Furthermore, endogenous ethylene, spontaneously produced before blooming, was closely associated with floral senescence. These results suggest that ethylene production during flower opening plays a key role in determining the timing of Hibiscus flower senescence.     

A Role for Auxin in Flower Development

Auxin has long been implicated in many aspects of plant growth and development including flower development. However, the exact roles of auxin in flower development have not been well defined until the recent identification of auxin biosynthesis mutants. Auxin is necessary for the inltiation of floral primordia, and the disruption of auxin biosynthesis, polar auxin transport or auxin signaling leads to the failure of flower formation. Auxin also plays an essential role in specifying the number and Identity of floral organs. Further analysis of the relationship between the auxin pathways and the known flower development genes will provide critical information regarding mechanisms of organogenesis and pattern formation in plants.     

Effects of pollination on floral attraction and longevity

The end of a flower's attraction to pollinators may be due toa range of visible cues such as permanent flower closure, acolour change, and withering or abscission of the petals. Floralattraction may be reduced by pollination. Pollination-inducedconclusion of floral attraction is often due to a colour changeor to flower closure. This may or may not be followed by a reductionin floral longevity, defined as the time to petal withering,wilting or shattering. In a few species floral longevity isincreased following pollination-induced flower closure or apollination-induced change in colour. Floral attraction, therefore,has to be disting uished from floral longevity. A literature survey shows that pollination rapidly reduces floralattraction in numerous orchids, but among other plant familiesonly about 60 genera have been found to show pollination-inducedshortening of floral attraction. Although only a few specieshave been investigated, it was invariably established that theeffect of pollination is blocked by inhibitors of ethylene synthesisor ethylene perception, hence is mediated by ethylene. The flowersthat cease to be attractive to pollinators, shortly followingpollination, tend to be from families that are known mainlyto comprise species in which flower longevity, petal colour,or flower closure, is sensitive to exogenous ethylene. Thisindicates that the effect of pollination on floral attractionis generally mediated by endogenous ethylene. Numerous species reportedly show a decrease in the period offloral attraction after exposure to ethylene, whereas only fora small number of species a decrease in the period of floralattraction induced by pollination has been observed. This discrepancymay be due to the greater attention that has been paid to theeffects of ethylene. Nonetheless, the possibility remains thatendogenous ethylene has a role in changing perianth form andcolour in addition to signalling the occurrence of pollination. Key words: Ethylene sensitivity, flower closure, flower longevity, pollination, petal colour, petal wilting, petal withering, petal abscission     

Molecular cloning and characterization of senescence-related genes from carnation flower petals

The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased production of ethylene which plays an important role in regulating this developmental event. Three senescence-related cDNA clones were isolated from a cDNA library prepared from mRNA isolated from senescing petals. These cDNAs are representative of two classes of mRNAs which increase in abundance in senescing petal tissue. The mRNA for one class is present at low levels during the early stages of development and begins to accumulate in mature petals prior to the increase in ethylene production. The accumulation of this mRNA is reduced, but not eliminated, in petals treated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, or silver thiosulfate, an ethylene action inhibitor. In contrast, expression of the second class of mRNAs appears to be highly regulated by ethylene. These mRNAs are not detectable prior to the rise in ethylene production and increase in abundance in parallel with the ethylene climacteric. Furthermore, expression of these mRNAs is significantly inhibited by both aminooxyacetic acid and silver thiosulfate. Expression of these mRNAs in vegetative and floral organs was limited to floral tissue, and predominantly to senescing petals.     

Expression of ethylene biosynthetic and receptor genes in rose floral tissues during ethylene-enhanced flower opening

Ethylene production, as well as the expression of ethylene biosynthetic (Rh-ACS1-4 and Rh-ACO1) and receptor (Rh-ETR1-5) genes, was determined in five different floral tissues (sepals, petals, stamens, gynoecia, and receptacles) of cut rose (Rosa hybrida cv. Samantha upon treatment with ethylene or the ethylene inhibitor 1-methylcyclopropene (1-MCP). Ethylene-enhanced ethylene production occurred only in gynoecia, petals, and receptacles, with gynoecia showing the greatest enhancement in the early stage of ethylene treatment. However, 1-MCP did not suppress ethylene production in these three tissues. In sepals, ethylene production was highly decreased by ethylene treatment, and increased dramatically by 1-MCP. Ethylene production in stamens remained unchanged after ethylene or 1-MCP treatment. Induction of certain ethylene biosynthetic genes by ethylene in different floral tissues was positively correlated with the ethylene production, and this induction was also not suppressed by 1-MCP. The expression of Rh-ACS2 and Rh-ACS3 was quickly induced by ethylene in gynoecia, but neither Rh-ACS1 nor Rh-ACS4 was induced by ethylene in any of the five tissues. In addition, Rh-ACO1 was induced by ethylene in all floral tissues except sepals. The induced expression of ethylene receptor genes by ethylene was much faster in gynoecia than in petals, and the expression of Rh-ETR3 was strongly suppressed by 1-MCP in all floral tissues. These results indicate that ethylene biosynthesis in gynoecia is regulated developmentally, rather than autocatalytically. The response of rose flowers to ethylene occurs initially in gynoecia, and ethylene may regulate flower opening mainly through the Rh-ETR3 gene in gynoecia.     

Involvement of Ethylene Biosynthesis and Signalling in the Transition from Male to Female Flowering in the Monoecious Cucurbita pepo

It is well established that ethylene is the main hormonal regulator of sexual expression in the Cucurbitaceae family, controlling not only the sexual fate of individual floral buds, but also the female flower transition, that is, the time at which the first female flower appears and therefore the number of female flowers per plant. Although sex determination of individual flower buds is known to be controlled by specific ethylene biosynthesis ACS genes in melon and cucumber, the role of ethylene genes in the control of the transition to female flowering is still unknown. We have identified two contrasting monoecious inbred lines of Cucurbita pepo, Bolognese (Bog) and Vegetable spaghetti (Veg), which differ in female flower transition but not in flower development. In Bog, which is very sensitive to ethylene, the transition to female flowering is very early, whereas in Veg, which is much less sensitive to ethylene, the transition occurs much later. In this article we compare the production of ethylene and the expression profiles of seven genes involved in the biosynthesis, perception, and signalling of ethylene in the two contrasting lines. Bog, with earlier female flower transition, showed higher ethylene production and CpACO1 expression in the apex at an earlier stage of plant development, when Bog is already producing female flowers, but Veg has not transitioned to female flowering yet. Moreover, the expression of the ethylene receptor and CTR-like genes in the apex of Veg and Bog plants indicates that these genes negatively regulate female flower transition during the earlier stages of plant development. The earlier transition to female flowering in Bog is not only associated with a higher production of ethylene in the apex but also with a premature decline of ethylene negative regulators (receptors and CTR-like) in the apex of the plant. These results provide the basis for a model that explains the regulation of female flowering transition in monoecious cucurbits.     

Genetic aspects of floral fragrance in plants

It is generally assumed that compounds are emitted from flowers in order to attract and guide pollinators. Due to the invisibility and the highly variable nature of floral scent, no efficient and reliable methods to screen for genetic variation have been developed. Moreover, no convenient plant model systems are available for flower scent studies. In the past decade, several floral fragrance-related genes have been cloned; the biosynthesis and metabolic engineering of floral volatiles have been studied with the development of biotechnology. This review summarizes the reported floral fragrance-related genes and the biosynthesis of floral scent compounds, introduces the origin of new modification enzymes for flower scent, compares different methods for floral fragrance-related gene cloning, and discusses the metabolic engineering of floral scent. Finally, the perspectives and prospects of research on floral fragrance are presented. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 4, pp. 437–446.     

Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.)

A number of Cucurbita pepo genotypes showing instable monoecy or partial andromonoecy, i.e. an incomplete conversion of female into bisexual flowers, have been detected. Given that in melon and cucumber andromonoecy is the result of reduction of ethylene production in female floral buds, caused by mutations in the ethylene biosynthesis genes CmACS7 and CsACS2; we have cloned and characterized two related C. pepo genes, CpACS27A and CpACS27B. The molecular structure of CpACS27A and its specific expression in the carpels of female flowers during earlier stages of flower development suggests that this gene is the Cucurbita ortholog of CmACS7 and CsACS2. CpACS27B is likely to be a paralogous pseudogene since it has not been found to be expressed in any of the analyzed tissues. CpACS27A was sequenced in Bolognese (Bog) and Vegetable Spaghetti (Veg), two monoecious inbred lines whose F2 was segregating for partial andromonoecy. The Bog allele of CpACS27A carried a missense mutation that resulted in a substitution of the conserved serine residue in position 176 by an alanine. Segregation analysis indicated that this mutant variant is necessary but not sufficient to confer the andromonoecious phenotype in squash. In concordance with its involvement in stamen arrest, a reduction in CpACS27A expression has been found in bisexual flower buds at earlier stages of development. This reduction in CpACS27A expression was concomitant with a downregulation of other ethylene biosynthesis and signaling genes during earlier and later stages of ovary development. The role of CpACS27A is discussed regarding the regulation of ethylene biosynthesis and signaling genes in the control of andromonoecy-associated traits, such as the delayed maturation of corolla and stigma as well as the parthenocarpic development of the fruit.     

Conversion of leaves into petals in Arabidopsis

More than 200 years ago, Goethe proposed that each of the distinct flower organs represents a modified leaf [1]. Support for this hypothesis has come from genetic studies, which have identified genes required for flower organ identity. These genes have been incorporated into the widely accepted ABC model of flower organ identity, a model that appears generally applicable to distantly related eudicots as well as monocot plants. Strikingly, triple mutants lacking the ABC activities produce leaves in place of flower organs, and this finding demonstrates that these genes are required for floral organ identity [2]. However, the ABC genes are not sufficient for floral organ identity since ectopic expression of these genes failed to convert vegetative leaves into flower organs. This finding suggests that one or more additional factors are required [3, 4]. We have recently shown that SEPALLATA (SEP) represents a new class of floral organ identity genes since the loss of SEP activity results in all flower organs developing as sepals [5]. Here we show that the combined action of the SEP genes, together with the A and B genes, is sufficient to convert leaves into petals.     

Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence

Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC 4.4.1.14) genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity.Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.