Petunia × hybrida floral scent production is negatively affected by high‐temperature growth conditions

Increasing temperatures due to changing global climate are interfering with plant–pollinator mutualism, an interaction facilitated mainly by floral colour and scent. Gas chromatography–mass spectroscopy analyses revealed that increasing ambient temperature leads to a decrease in phenylpropanoid‐based floral scent production in two Petunia × hybrida varieties, P720 and Blue Spark, acclimated at 22/16 or 28/22 °C (day/night). This decrease could be attributed to down‐regulation of scent‐related structural gene expression from both phenylpropanoid and shikimate pathways, and up‐regulation of a negative regulator of scent production, emission of benzenoids V (EOBV). To test whether the negative effect of increased temperature on scent production can be reduced in flowers with enhanced metabolic flow in the phenylpropanoid pathway, we analysed floral volatile production by transgenic ‘Blue Spark’ plants overexpressing CaMV 35S‐driven Arabidopsis thaliana production of anthocyanin pigments 1 (PAP1) under elevated versus standard temperature conditions. Flowers of 35S:PAP1 transgenic plants produced the same or even higher levels of volatiles when exposed to a long‐term high‐temperature regime. This phenotype was also evident when analysing relevant gene expression as inferred from sequencing the transcriptome of 35S:PAP1 transgenic flowers under the two temperature regimes. Thus, up‐regulation of transcription might negate the adverse effects of temperature on scent production.We demonstrate that petunia flowers produce less volatile phenylpropanoid compounds, in both scent bouquets and internal pools, in response to elevated temperatures. We reveal that the decrease in floral scent is correlated with reduced transcript levels of scent‐related genes, and that the adverse effect of high temperature can be negated by expressing transcriptional up‐regulators. We believe that the conclusions and implications drawn from the original data presented in our manuscript will be of particular interest to a broad spectrum of your readers, particularly in view of recent changes in global climate and the risk of environmental disruption of plant–pollinator mutualism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112003/1/pce12486-sup-0001-si.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/112003/2/pce12486.pd

At The Intersection of Phenylpropanoid Metabolism in Petunia

Plants are sessile organisms which in comparison with animals, had to develop a different set of mechanisms to adapt to environmental queues, such as abiotic and biotic stresses. An even more complex strategy must have developed through evolution to ensure reproductive success. Plant secondary metabolites, are represented by far more than the approximately 100,000 low molecular weight compounds described so far. Volatile organic compounds one group of secondary metabolites is important for several ecological functions. They act as attractants for pollinators and seed disperser to enhance reproduction and genetic variation, as well as representing a defense mechanism against herbivores as well as pathogens, and they allow for plant-plant communication. This work has focused on three critical steps in phenylpropanoids/benzenoid metabolism, which represent the second largest class of volatile organic compounds (Knudsen and Gershenzon, 2006). Arabidopsis thaliana predominantly produces terpenoid compounds, however, closer investigation of the Arabidopsis genome revealed the presence of two genes designated as aromatic L-amino acid decarboxylases (AADCs) with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in phenylacetaldehyde production. This work includes the biochemical characterization and functional analysis in planta of an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. Petunia (Petunia hybrida cv \u27Mitchell Diploid\u27) in contrast to Arabidopsis produces a unique blend of volatile phenylpropanoid/benzenoid compounds. Benzoic acid (BA) and its derivatives can be generated via two different routes the β-oxidative and non-β-oxidative pathway. The first reaction in the β-oxidative route is the activation of cinnamic acid (CA) to its CoA thioester, likely catalyzed by a 4-coumarate:CoA ligase (4CL) type enzyme. Investigation of this particular step lead to isolation of a cinnamoyl-CoA ligase (CNL) and 4CL enzyme, both of which were biochemically characterized and analyzed for their gene expression profiles. An RNAi strategy was applied to enable evaluation of CNL function in planta. 4CL was further analyzed for its impact on the production of the phenylpropenes eugenol and isoeugenol. Isoeugenol is one of the major volatile compounds released by Petunia hybrida cv \u27Mitchell Diploid\u27. These phenylpropenes are derived from phenylalanine (Phe) and share the initial biosynthetic steps with lignin formation. Important intermediates in this biochemical pathway are hydroxycinnamic CoA esters, formed by activation of hydroxycinnamic acid derivatives, likely catalyzed by 4CL. This study includes the in planta analysis of 4CL function

A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems

Abstract Background Metabolic fluxes represent the functional phenotypes of biochemical pathways and are essential to reveal the distribution of precursors among metabolic networks. Although analysis of metabolic fluxes, facilitated by stable isotope labeling and mass spectrometry detection, has been applied in the studies of plant metabolism, we lack experimental measurements for carbon flux towards lignin, one of the most abundant polymers in nature. Results We developed a feeding strategy of excised Arabidopsis stems with 13C labeled phenylalanine (Phe) for the analysis of lignin biosynthetic flux. We optimized the feeding methods and found the stems continued to grow and lignify. Consistent with lignification profiles along the stems, higher levels of phenylpropanoids and activities of lignin biosynthetic enzymes were detected in the base of the stem. In the feeding experiments, 13C labeled Phe was quickly accumulated and used for the synthesis of phenylpropanoid intermediates and lignin. The intermediates displayed two different patterns of labeling kinetics during the feeding period. Analysis of lignin showed rapid incorporation of label into all three subunits in the polymers. Conclusions Our feeding results demonstrate the effectiveness of the stem feeding system and suggest a potential application for the investigations of other aspects in plant metabolism. The supply of exogenous Phe leading to a higher lignin deposition rate indicates the availability of Phe is a determining factor for lignification rates

MOESM1 of A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems

Additional file 1. Figure S1. A simplified pathway illustrating the enzymes and metabolites involved in lignin biosynthesis. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate CoA ligase; HCT, hydroxycinnamoyl CoA:shikimatehydroxycinnamoyl transferase; C3′H, p-coumaroyl shikimate 3′-hydroxylase; CSE, caffeoyl shikimate esterase; CCoAOMT, caffeoyl CoA O-methyltransferase; F5H, ferulate5-hydroxylase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl CoA reductase; CAD, cinnamyl alcohol dehydrogenase. SALDH, sinapaldehydedehydrogenase. Figure S2. Excised stems incubated in tubes with MS medium in growth chamber. (A) An Arabidopsis stem was excised and placed into a 1.5 mL tube containing liquid MS medium. (B) Arabidopsis stems incubated in MS medium were placed in a rack to perform feeding experiment (picture taken from side). (C) Stems were sitting away from each other to mimic their growth in the soil (picture taken from top). Figure S3. Medium absorbed by the excised stems during the feeding process. The loss of medium from each tube with an excised stem was measured after feeding for 0, 40, 90, 180, and 240 min. Data represented mean ± SD (n = 45). Figure S4. Hierarchical clustering of labeling percentage profiles of soluble phenylpropanoids from the base of Arabidopsis stems supplied with 1 mM [13C6]-Phe over the feeding time course. The averaged labeling percentage data of each metabolite over the time course from Figure 5 were clustered based on squared Euclidian distance. Figure S5. Metabolic profiles of soluble phenylpropanoids from the base of Arabidopsis stems supplied with 1 mM [13C6]-Phe over the feeding time course. Sum of endogenous and 13C6 labeled compounds was quantified with LC/MS-MS and normalized to fresh weight of stem tissue. The plot of each metabolite measured was placed above its name on the pathway. Dashed lines mean multiple steps. Data represent mean ± SD (n = 3)

Adult plant resistance in maize to northern leaf spot is a feature of partial loss-of-function alleles of Hm1.

Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild-type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR