Aromatic amino acid decarboxylase is involved in volatile phenylacetaldehyde production in loquat (Eriobotrya japonica) flowers

Abstract

Post anthesis, loquat flowers emit volatile benzenoids, including phenylacetaldehyde, phenylethyl alcohol, and 2-phenethyl benzoate. Previous studies have shown that pyridoxal phosphate-dependent aromatic L-amino acid decarboxylase (AADC) produces phenylacetaldehyde from L-phenylalanine. Here, two AADC genes (EjAADC1 and EjAADC2) were isolated from loquat (Eriobotrya japonica) flowers. The EjAADC1 and EjAADC2 proteins showed approximately 72% and 55% identity, respectively, to a rose AADC homolog that has phenylacetaldehyde synthase activity. Transcript analyses indicated that EjAADC1 was specifically expressed in petals, with the highest level of expression in fully opened flowers; the petals showed high levels of volatile benzenoids, including phenylacetaldehyde. In contrast, EjAADC2 was expressed at a lower level than EjAADC1 in all tested tissues, including leaves and developing flowers. Functional characterization of a recombinant EjAADC1 protein expressed in Escherichia coli showed that it catalyzes the formation of phenylacetaldehyde from L-phenylalanine in a pyridoxal phosphate-dependent manner. Our results suggest that EjAADC1 is mainly responsible for the biosynthesis of volatile benzenoids in loquat flowers.

Keywords: aromatic amino acid decarboxylase; loquat; phenylacetaldehyde; volatile benzenoid.

Figure 1. Biosynthesis of volatile benzenoid phenylacetaldehyde in higher plants. Proposed biosynthetic pathway leading to phenylacetaldehyde and its derivatives found in loquat flowers. AADC, Aromatic amino acid decarboxylase; PAAS, Phenylacetaldehyde synthase; AAS, aromatic aldehyde synthase; AAAT, aromatic amino acid aminotransferase; PPDC, phenylpyruvic acid decarboxylase.

Figure 2. Sequence analysis of EjAADCs. (A) A phylogenetic tree of two loquat AADC genes and other related amino acid decarboxylase genes of different plant species. Phylogenetic analysis was performed in MEGA6 using the neighbor joining method. The scale bar represents 0.2 amino acid substitutions per site. At, Bn, Ca, Cr, Os, Op, Pc, Ph, Ps, Rh, Sl, Tf, Mt, and Car indicate Arabidopsis thaliana, Brassica napus, Camptotheca acuminata, Catharanthus roseus, Oryza sativa, Ophiorrhiza pumila, Petroselinum crispum, Petunia hybrida, Papaver somniferum, Rosa hybrida, Solanum lycopersicum, Thalictrum flavum, Medicago truncatula, and Cicer arietinum, respectively. AAS, GAD, SDC, TDC, PAAS, TYDC, AADC, and HDC indicate acetaldehyde synthase, glutamate decarboxylase, serine decarboxylase, tryptophan decarboxylase, phenylacetaldehyde synthase, tyrosine decarboxylase, aromatic amino acid decarboxylase, and histidine decarboxylase, respectively. The AADC family is indicated by grey shading. (B) Sequence alignment of EjAADC and known phenylacetaldehyde synthesizing proteins. The conserved pyridoxal 5′-phosphate-binding residue is indicated by an asterisk. The triangle indicates the important residue for aldehyde synthase activity.

Figure 3. Expression of EjAADC genes. (A) RPKM values of AADC genes from loquat flowers by RNA-Seq. (B) Tissue specific expression of EjAADC genes examined with semi-quantitative RT-PCR. (C) Tissue specific expression of EjAADC1 gene examined by quantitative RT-PCR.

Figure 4. Conversion of phenylalanine to phenylacetaldehyde by EjAADC1. Product analysis of the reaction catalyzed by EjAADC1 by GC-MS.