Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms

Abstract

Background: Phenylalanine ammonia-lyase (PAL; E.C.4.3.1.5) is a key enzyme of the phenylpropanoid pathway in plant development, and it catalyses the deamination of phenylalanine to trans-cinnamic acid, leading to the production of secondary metabolites. This enzyme has been identified in many organisms, ranging from prokaryotes to higher plants. Because Nelumbo nucifera is a basal dicot rich in many secondary metabolites, it is a suitable candidate for research on the phenylpropanoid pathway.

Results: Three PAL members, NnPAL1, NnPAL2 and NnPAL3, have been identified in N. nucifera using genome-wide analysis. NnPAL1 contains two introns; however, both NnPAL2 and NnPAL3 have only one intron. Molecular and evolutionary analysis of NnPAL1 confirms that it is an ancient PAL member of the angiosperms and may have a different origin. However, PAL clusters, except NnPAL1, are monophyletic after the split between dicots and monocots. These observations suggest that duplication events remain an important occurrence in the evolution of the PAL gene family. Molecular assays demonstrate that the mRNA of the NnPAL1 gene is 2343 bp in size and encodes a 717 amino acid polypeptide. The optimal pH and temperature of the recombinant NnPAL1 protein are 9.0 and 55°C, respectively. The NnPAL1 protein retains both PAL and weak TAL catalytic activities with Km values of 1.07 mM for L-phenylalanine and 3.43 mM for L-tyrosine, respectively. Cis-elements response to environmental stress are identified and confirmed using real-time PCR for treatments with abscisic acid (ABA), indoleacetic acid (IAA), ultraviolet light, Neurospora crassa (fungi) and drought.

Conclusions: We conclude that the angiosperm PAL genes are not derived from a single gene in an ancestral angiosperm genome; therefore, there may be another ancestral duplication and vertical inheritance from the gymnosperms. The different evolutionary histories for PAL genes in angiosperms suggest different mechanisms of functional regulation. The expression patterns of NnPAL1 in response to stress may be necessary for the survival of N. nucifera since the Cretaceous Period. The discovery and characterisation of the ancient NnPAL1 help to elucidate PAL evolution in angiosperms.

Figure 1

Gene structure of the PAL family, NnPAL1, NnPAL2 and NnPAL3, in Nelumbo nucifera. The green bars represent exons, and the red bars represent the conserved nucleotide sequences encoding the phenylalanine and histidine ammonia-lyase signature (GTITASGDLVPLSYIA). The black lines represent introns. The numbers 0, 1 and 2 represent the intron phase.

Figure 2

Phylogenetic tree of the phenylalanine ammonia lyase gene family. The amino acid sequences are aligned and the maximum likelihood tree as constructed using the program PhyML 3.0. The numbers at the nodes are the bootstrap values (>50%) from the maximum likelihood (ML). The other BI and NJ trees are shown in Additional file 5, Figure 5(A) and Figure 5(B). The numbers associated with the branches are the ML bootstrap support values and posterior probabilities. NnPAL1 is marked with a red dot, and the dicotyledon and monocotyledon clades are marked with carmine and green dots, respectively. Three clades, gymnosperm I, gymnosperm II and gymnosperm III, of Pinus taeda are marked with light green, pink and blue dots, respectively.

Figure 3

Prediction of NnPAL1 secondary structure and tertiary structure. (A) Prediction of the NnPAL1 secondary structure. The blue, pink, red, and green regions represent the alpha helix, random coil, extended strand, and beta turn, respectively. (B) The three domains of the predicted tertiary structure of NnPAL1 established by homology-based modelling (9 strictly conserved residues are marked).

Figure 4

Expression (A) and purification (B) of recombinant NnPAL1. A: The total proteins from E. coli BL21 are harvested at 4 h, 8 h and 12 h after post-induction, and 1 and 2 represent the total proteins of E .coli BL21 harbouring the pET28a(+) vector and recombinant pET28a(+)-NnPAL1 vector, respectively. B: A series of imidazole buffer concentration gradients (10 mM, 50 mM, 100 mM, 200 mM); lane1: the supernatant of the E. coli BL21 lysate harbouring the pET28a(+) vector; lane2: (native control) the supernatant of the E. coli BL21 lysate harbouring the pET28a(+)-NnPAL1 vector; lane3: the supernatant of the flow through of the Ni-IDA column for three replicates; lane 4, lane 5, lane 6, lane 7 and lane 8: the products washed with 10 mM, 20 mM, 50 mM, 100 mM, and 200 mM imidazole buffer, respectively.

Figure 5

Transcription of NnPAL1 under different treatments. A 250 μM ABA, B 100 ng/ml IAA, C ultraviolet light treatment, DNeurospora crassa (fungi) treatment, E drought treatment. The leaves obtained from the treated seedlings of N. nucifera are used as samples. β-actin is used as an internal control for all samples. The vertical bars represent the means ± SE (n = 3 replicates, SE < 0.5).

Figure 6

Phylogenetic tree of the phenylalanine ammonia lyase from prokaryote and animal HAL, fungal PAL and plant PAL.