Ethylene-regulated floral volatile synthesis in petunia corollas

In many flowering plants, such as petunia (Petunia x 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 post-pollination 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 multiple phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two distinct protein lineages

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.     

Functional characterization of SlscADH1, a fruit-ripening-associated short-chain alcohol dehydrogenase of tomato

A tomato short-chain dehydrogenase-reductase (SlscADH1) is preferentially expressed in fruit with a maximum expression at the breaker stage while expression in roots, stems, leaves and flowers is very weak. It represents a potential candidate for the formation of aroma volatiles by interconverting alcohols and aldehydes. The SlscADH1 recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several volatile compounds present in tomato flavour with a strong preference for the NAD/NADH co-factors. The strongest activity was observed for the reduction of hexanal (K(m)=0.175mM) and phenylacetaldehyde (K(m)=0.375mM) in the presence of NADH. The oxidation process of hexanol and 1-phenylethanol was much less efficient (K(m)s of 2.9 and 23.0mM, respectively), indicating that the enzyme preferentially acts as a reductase. However activity was observed only for hexanal, phenylacetaldehyde, (E)-2-hexenal and acetaldehyde and the corresponding alcohols. No activity could be detected for other aroma volatiles important for tomato flavour, such as methyl-butanol/methyl-butanal, 5-methyl-6-hepten-2-one/5-methyl-6-hepten-2-ol, citronellal/citronellol, neral/nerol, geraniol. In order to assess the function of the SlscADH1 gene, transgenic plants have been generated using the technique of RNA interference (RNAi). Constitutive down-regulation using the 35S promoter resulted in the generation of dwarf plants, indicating that the SlscADH1 gene, although weakly expressed in vegetative tissues, had a function in regulating plant development. Fruit-specific down-regulation using the 2A11 promoter had no morphogenetic effect and did not alter the aldehyde/alcohol balance of the volatiles compounds produced by the fruit. Nevertheless, SlscADH1-inhibited fruit unexpectedly accumulated higher concentrations of C5 and C6 volatile compounds of the lipoxygenase pathway, possibly as an indirect effect of the suppression of SlscADH1 on the catabolism of phospholipids and/or integrity of membranes.     

Enhanced benzaldehyde formation by a monokaryotic strain of Pycnoporus cinnabarinus using a selective solid adsorbent in the culture medium.

A monokaryotic strain of the white-rot fungus Pycnoporus cinnabarinus was shown to produce, in a 2-L bioreactor culture, 100 mg.L-1 benzaldehyde (bitter almond aroma) from L-phenylalanine with a productivity of 33 mg.L-1.day-1. The addition of HP20 resin, a styrene divinylbenzene copolymer highly selective for benzaldehyde, enabled an eightfold increase in the production of benzaldehyde and a twofold increase in productivity. In the presence of HP20 resin, the production of 790 mg.L-1 benzaldehyde was concomitant with the synthesis of cinnamic acid derivatives of high organoleptic notes such as cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate.     

Generation of Phenylpropanoid Pathway-Derived Volatiles in Transgenic Plants: Rose Alcohol Acetyltransferase Produces Phenylethyl Acetate and Benzyl Acetate in Petunia Flowers

Esters are important contributors to the aroma of numerous flowers and fruits. Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs). Numerous homologous AATs from various plants have been characterized using in-vitro assays. To study the function of rose alcohol acetyltransferase (RhAAT) in planta, we generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter. Although the preferred substrate of RhAAT in vitro is geraniol, in transgenic petunia flowers, it used phenylethyl alcohol and benzyl alcohol to produce the corresponding acetate esters, not generated by control flowers. The level of benzyl alcohol emitted by the flowers of different transgenic lines was ca. three times higher than that of phenylethyl alcohol, which corresponded to the ratio between the respective products, i.e. ca. three times more benzyl acetate than phenylethyl acetate. Feeding of transgenic petunia tissues with geraniol or octanol led to the production of their respective acetates, suggesting the dependence of volatile production on substrate availability.     

茶树苯丙素类/苯环型挥发性物质的生物合成和胁迫响应的研究进展

茶(Camellia sinensis)的挥发性物质不仅会影响茶香的品质,也会在茶树中发挥重要的生态学功能。除了萜类和脂肪酸衍生类挥发性物质外,苯丙素类/苯环型挥发性物质(volatile phenylpropanoids/benzenoids, VPBs)也同样具有这些方面的功能。正因为具有这些功能,茶树中的VPBs也受到了人们越来越多的重视和研究。对近年来茶中一些具代表性VPBs的研究进展进行了综述,包括2-苯乙醇、苯乙醛等VPBs的生物合成及其对光胁迫的响应;1-苯乙醇和苯乙酮的生物合成途径及其对昆虫胁迫的响应;吲哚的生物合成及其在温度和机械损伤胁迫下的响应机制。此外,也探讨了VPBs中一些非传统的生物合成途径,以及这些途径在某些胁迫条件下的响应机制。     

A Solanum lycopersicum catechol-O-methyltransferase involved in synthesis of the flavor molecule guaiacol

O‐methyltransferases (OMT) are important enzymes that are responsible for the synthesis of many small molecules, which include lignin monomers, flavonoids, alkaloids, and aroma compounds. One such compound is guaiacol, a small volatile molecule with a smoky aroma that contributes to tomato flavor. Little information is known about the pathway and regulation of synthesis of guaiacol. One possible route for synthesis is via catechol methylation. We identified a tomato O‐methyltransferase (CTOMT1) with homology to a Nicotiana tabacum catechol OMT. CTOMT1 was cloned from Solanum lycopersicum cv. M82 and expressed in Escherichia coli. Recombinant CTOMT1 enzyme preferentially methylated catechol, producing guaiacol. To validate the in vivo function of CTOMT1, gene expression was either decreased or increased in transgenic S. lycopersicum plants. Knockdown of CTOMT1 resulted in significantly reduced fruit guaiacol emissions. CTOMT1 overexpression resulted in slightly increased fruit guaiacol emission, which suggested that catechol availability might limit guaiacol production. To test this hypothesis, wild type (WT) and CTOMT1 that overexpress tomato pericarp discs were supplied with exogenously applied catechol. Guaiacol production increased in both WT and transgenic fruit discs, although to a much greater extent in CTOMT1 overexpressing discs. Finally, we identified S. pennellii introgression lines with increased guaiacol content and higher expression of CTOMT1. These lines also showed a trend toward lower catechol levels. Taken together, we concluded that CTOMT1 is a catechol‐O‐methyltransferase that produces guaiacol in tomato fruit.     

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of <Emphasis Type="Italic">tomato spotted wilt virus</Emphasis> resistance

Background

Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance.

Results

The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV.

Conclusion

In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

     

Understanding in vivo benzenoid metabolism in petunia petal tissue

In vivo stable isotope labeling and computer-assisted metabolic flux analysis were used to investigate the metabolic pathways in petunia (Petunia hybrida) cv Mitchell leading from Phe to benzenoid compounds, a process that requires the shortening of the side chain by a C(2) unit. Deuterium-labeled Phe ((2)H(5)-Phe) was supplied to excised petunia petals. The intracellular pools of benzenoid/phenylpropanoid-related compounds (intermediates and end products) as well as volatile end products within the floral bouquet were analyzed for pool sizes and labeling kinetics by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Modeling of the benzenoid network revealed that both the CoA-dependent, beta-oxidative and CoA-independent, non-beta-oxidative pathways contribute to the formation of benzenoid compounds in petunia flowers. The flux through the CoA-independent, non-beta-oxidative pathway with benzaldehyde as a key intermediate was estimated to be about 2 times higher than the flux through the CoA-dependent, beta-oxidative pathway. Modeling of (2)H(5)-Phe labeling data predicted that in addition to benzaldehyde, benzylbenzoate is an intermediate between l-Phe and benzoic acid. Benzylbenzoate is the result of benzoylation of benzyl alcohol, for which activity was detected in petunia petals. A cDNA encoding a benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase was isolated from petunia cv Mitchell using a functional genomic approach. Biochemical characterization of a purified recombinant benzoyl-CoA:benzyl alcohol/phenylethanol benzoyltransferase protein showed that it can produce benzylbenzoate and phenylethyl benzoate, both present in petunia corollas, with similar catalytic efficiencies.     

Manipulation of flavour and aroma compound sequestration and release using a glycosyltransferase with specificity for terpene alcohols

Glycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP‐glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)‐hex‐3‐enol, contribute strongly to the ‘grassy‐green’ aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over‐expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan‐3‐ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over‐expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over‐expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3‐methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening.     

Constitutive activation of the jasmonate signaling pathway enhances the production of secondary metabolites in tomato

The phytohormone jasmonic acid (JA) regulates the synthesis of secondary metabolites in a wide range of plant species. Here, we show that exogenous methyl-JA (MeJA) elicits massive accumulation of caffeoylputrescine (CP) in tomato leaves. A mutant (jai1) that is defective in jasmonate perception failed to accumulate CP in flowers and MeJA-treated leaves. Conversely, a transgenic tomato line (called 35S::PS) that exhibits constitutive JA signaling accumulated high levels of leaf CP in the absence of jasmonate treatment. RNA blot analysis showed that genes encoding enzymes in the phenylpropanoid and polyamine pathways for CP biosynthesis are upregulated in MeJA-treated wild-type plants and in untreated 35S::PS plants. These results indicate that CP accumulation in tomato is tightly controlled by the jasmonate signaling pathway, and provide proof-of-concept that the production of some plant secondary metabolites can be enhanced by transgenic manipulation of endogenous JA levels.     

Characterization of L-phenylalanine metabolism to acetophenone and 1-phenylethanol in the flowers of Camellia sinensis using stable isotope labeling

Acetophenone (AP) and 1-phenylethanol (1PE) are the two major endogenous volatile compounds in flowers of Camellia sinensis var. Yabukita. Until now no information has been available on the biosynthesis of AP and 1PE in plants. Here we propose that AP and 1PE are derived from L-phenylalanine (L-Phe), based on feeding experiments using stable isotope-labeled precursors L-[(2)H(8)]Phe and L-[(13)C(9)]Phe. The subacid conditions in the flowers result in more hydrogenation than dehydrogenation in the transformation between AP and 1PE. Due to the action of some enzyme(s) responsible for the formation of (R)-1PE from AP in the flowers, (R)-1PE is the dominant endogenous steroisomer of 1PE. The modification of 1PE into nonvolatile glycosidic forms is one of the reasons for why only a little 1PE is released from the flowers. The levels of AP, 1PE, and glycosides of 1PE increase during floral development, whereas the level of L-Phe decreases. These metabolites occur mostly in the anthers.