Enhanced formation of aromatic amino acids increases fragrance without affecting flower longevity or pigmentation in Petunia × hybrida

Purple Petunia × hybrida V26 plants accumulate fragrant benzenoid‐phenylpropanoid molecules and anthocyanin pigments in their petals. These specialized metabolites are synthesized mainly from the aromatic amino acids phenylalanine. Here, we studied the profile of secondary metabolites of petunia plants, expressing a feedback‐insensitive bacterial form of 3‐deoxy‐di‐arabino‐heptulosonate 7‐phosphate synthase enzyme (AroG*) of the shikimate pathway, as a tool to stimulate the conversion of primary to secondary metabolism via the aromatic amino acids. We focused on specialized metabolites contributing to flower showy traits. The presence of AroG* protein led to increased aromatic amino acid levels in the leaves and high phenylalanine levels in the petals. In addition, the AroG* petals accumulated significantly higher levels of fragrant benzenoid‐phenylpropanoid volatiles, without affecting the flowers' lifetime. In contrast, AroG* abundance had no effect on flavonoids and anthocyanins levels. The metabolic profile of all five AroG* lines was comparable, even though two lines produced the transgene in the leaves, but not in the petals. This implies that phenylalanine produced in leaves can be transported through the stem to the flowers and serve as a precursor for formation of fragrant metabolites. Dipping cut petunia stems in labelled phenylalanine solution resulted in production of labelled fragrant volatiles in the flowers. This study emphasizes further the potential of this metabolic engineering approach to stimulate the production of specialized metabolites and enhance the quality of various plant organs. Furthermore, transformation of vegetative tissues with AroG* is sufficient for induced production of specialized metabolites in organs such as the flowers.     

A peroxisomal thioesterase plays auxiliary roles in plant β‐oxidative benzoic acid metabolism

Peroxisomal β‐oxidative degradation of compounds is a common metabolic process in eukaryotes. Reported benzoyl‐coenzyme A (BA‐CoA) thioesterase activity in peroxisomes from petunia flowers suggests that, like mammals and fungi, plants contain auxiliary enzymes mediating β‐oxidation. Here we report the identification of Petunia hybrida thioesterase 1 (PhTE1), which catalyzes the hydrolysis of aromatic acyl‐CoAs to their corresponding acids in peroxisomes. PhTE1 expression is spatially, developmentally and temporally regulated and exhibits a similar pattern to known benzenoid metabolic genes. PhTE1 activity is inhibited by free coenzyme A (CoA), indicating that PhTE1 is regulated by the peroxisomal CoA pool. PhTE1 downregulation in petunia flowers led to accumulation of BA‐CoA with increased production of benzylbenzoate and phenylethylbenzoate, two compounds which rely on the presence of BA‐CoA precursor in the cytoplasm, suggesting that acyl‐CoAs can be exported from peroxisomes. Furthermore, PhTE1 downregulation resulted in increased pools of cytoplasmic phenylpropanoid pathway intermediates, volatile phenylpropenes, lignin and anthocyanins. These results indicate that PhTE1 influences (i) intraperoxisomal acyl‐CoA/CoA levels needed to carry out β‐oxidation, (ii) efflux of β‐oxidative products, acyl‐CoAs and free acids, from peroxisomes, and (iii) flux distribution within the benzenoid/phenylpropanoid metabolic network. Thus, this demonstrates that plant thioesterases play multiple auxiliary roles in peroxisomal β‐oxidative metabolism.     

Dynamic trajectories of volatile and non‐volatile specialised metabolites in ‘overnight’ fragrant flowers of Murraya paniculata

• Ephemeral flowers, especially nocturnal ones, usually emit characteristic scent profiles within their post‐anthesis lifespans of a few hours. Whether these flowers exhibit temporal variability in the composition and profile of volatile and non‐volatile specialised metabolites has received little attention.
• Flowers of Murraya paniculata bloom in the evenings during the summer and monsoon, and their sweet, intense fragrance enhances the plant's value as an ornamental. We aimed to investigate profiles of both volatile and non‐volatile endogenous specialised metabolites (ESM) in nocturnal ephemeral flowers of M. paniculata to examine whether any biochemically diverse groups of ESM follow distinct patterns of accumulation while maintaining synchrony with defensive physiological functions.
• Targeted ESM contents of M. paniculata flowers were profiled at ten time points at 2‐h intervals, starting from late bud stage (afternoon) up to the start of petal senescence (mid‐morning). Emitted volatiles were monitored continuously within the whole 20‐h period using headspace sampling. The ESM contents were mapped by time point to obtain a highly dynamic and biochemically diverse profile. Relative temporal patterns of ESM accumulation indicated that the active fragrance‐emitting period might be divided into ‘early bloom’, ‘mid‐bloom’ and ‘late bloom’ phases. Early and late bloom phases were characterised by high free radical generation, with immediate enhancement of antioxidant enzymes and phenolic compounds. The mid‐bloom phase was relatively stable and dedicated to maximum fragrance emission, with provision for strong terpenoid‐mediated defence against herbivores. The late bloom phase merged into senescence with the start of daylight; however, even the senescent petals continued to emit fragrance to attract diurnal pollinators.
• Our study suggests that dynamic relations between the different ESM groups regulate the short‐term requirements of floral advertisement and phytochemical defence in this ephemeral flower. This study also provided fundamental information on the temporal occurrence of emitted volatiles and internal pools of specialised metabolites in M. paniculata flowers, which could serve as an important model for pollination biology of Rutaceae, which includes many important fruit crops.

     

中间锦鸡儿CiCHIL克隆及其黄酮代谢功能研究

植物的次生代谢产物能够为植物的果实和花着色、有助于种子的形成、花粉的传播、适应外界逆境及防御害虫的攻击。苯丙烷代谢途径是植物中一个重要的次生代谢途径,苯丙烷代谢通路上有几条主要的分支,分支下游产生了上千种化合物。查耳酮异构酶（chalcone isomerase,CHI）是苯丙烷通路上的一个关键酶,催化查耳酮到黄烷酮的反应,主要作用是帮助底物进行正确的分子内环化反应,生成的黄烷酮类化合物成为苯丙烷代谢途径下游产物的底物。从中间锦鸡儿干旱胁迫抑制性削减杂交文库中克隆得到一个CHI基因家族成员,序列分析和系统进化分析表明,该基因属于CHIL基因亚家族成员,命名为CiCHIL。实时荧光定量PCR检测发现,该基因受UV-B诱导且过表达CiCHIL具有较强的抵御紫外胁迫的能力。对过表达株系进行RNA和蛋白质水平的检测,对挑选出来的过表达株系进行总黄酮含量检测,发现过表达株系总黄酮含量显著高于野生型。     

Scent engineering: toward the goal of controlling how flowers smell

Floral scent has an important role in the reproductive processes of many plants and a considerable economic value in guaranteeing yield and quality of many crops. It also enhances the aesthetic properties of ornamental plants and cut flowers. Many floral scent volatiles fall into the terpenoid or phenylpropanoid/benzenoid classes of compounds. Although the biochemistry of floral scent is still a relatively new field of investigation, in the past decade investigators have begun to identify 'scent genes'. Several of these genes, most of which, but not all, encode enzymes that directly catalyze the formation of volatile terpenoid or phenylpropanoid/benzenoid compounds, have now been used to manipulate, through genetic engineering techniques, the mix of volatiles emitted from the flowers of several plant species. The outcomes of these experiments, which are discussed here, have indicated that the genetic engineering approach to altering floral scents has potential; however, they have also revealed the limitations that result from our inadequate knowledge of the metabolic pathways responsible for scents and their regulation.     

Tomato phenylacetaldehyde reductases catalyze the last step in the synthesis of the aroma volatile 2-phenylethanol

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.     

Metabolic engineering of plant volatiles

Metabolic engineering of the volatile spectrum offers enormous potential for plant improvement because of the great contribution of volatile secondary metabolites to reproduction, defense and food quality. Recent advances in the identification of the genes and enzymes responsible for the biosynthesis of volatile compounds have made this metabolic engineering highly feasible. Notable successes have been reported in enhancing plant defenses and improving scent and aroma quality of flowers and fruits. These studies have also revealed challenges and limitations which will be likely surmounted as our understanding of plant volatile network improves.     

植物挥发物代谢工程在改良香气品质和植物防御中的应用

挥发物次生代谢在植物繁殖、植物防御和改良食物品质方面发挥着重要作用。近年来,随着参与挥发物生物合成的基因和酶类的鉴定以及代谢途径和调控机理等研究的不断发展和深入,挥发物代谢工程已经具备较高的可行性。应用代谢工程改良花、果实的香气品质以及提高植物防御能力的研究成效显著。主要介绍了这些方面的最新进展,同时也讨论了植物挥发物代谢工程应用存在的问题和挑战以及研究思路。     

Diverging pathways: Differential benzenoid and phenylpropanoid volatile production in Phlox subulata L. cultivars

Previous research into the genetic mechanisms of benzenoid and phenylpropanoid volatile biosynthesis has suggested the potential for metabolic flux, in which phenylalanine substrate dedicated to one pathway branch (i.e., benzenoid production) could alter volatile production in other pathways (i.e., phenylpropanoid production). However, little research has been conducted in planta to verify the validity of this hypothesis. We examined the emission rates of representative benzenoid and phenylpropanoid volatiles from seven cultivars of Phlox subulata L. to determine if cultivars had metabolic flux differences in terms of these two compound categories. Cultivars that produced large quantities of methyl benzoate and benzaldehyde were found to emit little or no phenylacetaldehyde and 2-phenylethanol, and vice versa. These results suggest that P. subulata cultivars experience phenylalanine substrate flux directed toward one pathway and away from the alternate branch. Such a pattern may be the result of differential selection pressures, in which gene expression has been altered to direct flux away from either benzenoid or phenylpropanoid production. Moreover, if these patterns hold true in wild populations, metabolic flux may lead to differential pollinator behavior and further phenotypic evolution. Future research using molecular tools could verify the role of metabolic flux in determining scent phenotypes and pinpoint the exact nature of the genetic mutations leading to phenotypic differences in odor.