Reduction of Benzenoid Synthesis in Petunia Flowers Reveals Multiple Pathways to Benzoic Acid and Enhancement in Auxin Transport

Abstract

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a β-oxidative or non-β-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between l-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.

Biosynthesis of plant‐derived flavor compounds

“…Transgenic petunia plants were generated in which expression of benzoyl-CoA: phenylethanol/benzyl alcohol benzoyltransferase (BPBT), the gene encoding the enzyme that uses benzoyl CoA and benzyl alcohol to make benzyl benzoate, was reduced or eliminated. Elimination of benzyl benzoate formation decreased the endogenous pool of benzyl acid and methyl benzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzyl benzoate to benzoic acid formation (Orlova et al, 2006). Labeling experiments with 2 H 5 -phenylalanine revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than phenylalanine, possibly phenylpyruvate.…”

A comparative analysis of characteristic floral scent compounds in Prunus mume and related species

“…Although a large amount of benzaldehyde exists in the endogenous extracts of various Prunus species (Fig. 3), as in the case of Petunia hybrid 35) , the non-aromatic species appear to lack the molecular apparatus for reducing benzaldehyde to benzyl alcohol or part of the benzyl alcohol is converted to other products due to the existence of competitive pathways. Moreover, the increase of benzaldehyde content after treatment in P. persica indicated that the conversion of benzaldehyde to benzyl alcohol was reversible.…”

An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase

“…Petunia hybrida cv: Mitchell wild-type (Ball Seed Co., West Chicago, IL, USA) and transgenic PhADT1 RNAi, PhPPA-AT RNAi and crossed PhADT1xPhPPA-AT RNAi plants were grown under standard greenhouse conditions 41 with a light period from 6:00 h to 21:00 h. Crossed lines were screened for the presence of transgenes by PCR on genomic DNA using gene-specific primers: forward 5 0 -GCTAAGCTGGAGT GCACATT-3 0 , and reverse 5 0 -ACCTGGTATGACCTTCACGA-3 0 for PhPPA-AT and forward, 5 0 -ATTTCGAACAAGCGTACACC-3 0 and reverse 5 0 AATTGT GAGCGCCTTAGGTA-3 0 for PhADT1. Scent collection and targeted metabolic profiling of petunia petals were performed as described previously 16 , with the exception of scent collection time, which was from 18:00 h to 22:00 h.…”

“…For the PhPPY-AT RNAi construct a synthetic DNA, containing two spliced PhPPY-AT cDNA fragments of the coding region corresponding to nucleotides 363-891 and 363-687, the latter in antisence orientation to form a hairpin structure, was synthesized (Genscript, Piscataway, NJ, USA). EcoRI and BamHI sites were introduced at the 5 0 and 3 0 ends during DNA synthesis and the resulting synthetic DNA fragment was subcloned into the EcoRI/BamHI site of pRNA69 vector containing LIS promoter 41 . Next, the part of constructed plasmid containing the LIS promoter and the two PPY-AT fragments in opposite orientation separated by the intron was cut out using SacI/NotI and subcloned into the pART27 simpler binary vector 45 .…”