Abstract
Phenylalanine ammonia-lyase (PAL) is the crucial enzyme of the phenylpropanoid pathway, which plays an important role in plant disease resistance. To understand the function of PAL in Picea asperata, in this study, the full-length cDNA sequence of the PAL gene from this species was isolated and named PaPAL. The gene contains a 2160-bp open reading frame (ORF) encoding 720 amino acids with a calculated molecular weight of 78.7 kDa and a theoretical isoelectric point of 5.88. The deduced PaPAL protein possesses the specific signature motif (GTITASGDLVPLSYIA) of phenylalanine ammonia-lyases. Multiple alignment analysis revealed that PaPAL has high identity with other plant PALs. The tertiary structure of PaPAL was predicted using PcPAL from Petroselinum crispum as a template, and the results suggested that PaPAL may have a similar function to that of PcPAL. Furthermore, phylogenetic analysis indicated that PaPAL has a close relationship with other PALs from the Pinaceae species. The optimal expression condition of recombinant PaPAL in Escherichia coli BL21 (DE3) was 0.2 mM IPTG (isopropyl β-D-thiogalactoside) at 16 °C for 4 h, and the molecular weight of recombinant PaPAL was found to be approximately 82 kDa. Recombinant PaPAL was purified and exhibited high PAL activity at optimal conditions of pH 8.6 and 60 °C. Quantitative real-time PCR (qRT-PCR) showed the PaPAL gene to be expressed in all tissues of P. asperata tested, with the highest expression level in the needles. The PaPAL gene was induced by the pathogen (Lophodermium piceae), which caused needle cast disease, indicating that it might be involved in defense against needle cast disease. These results provide a basis for understanding the molecular mechanisms of the PAL gene in the process of P. asperata disease resistance.
Highlights
To adapt to changes in the ecological environment and related biotic and abiotic stresses, conifers have evolved a diverse defense system as one of their main strategies for survival [1]
Young needles were collected from healthy P. asperata and P. asperata infected by the pathogen (Lophodermium piceae, its information is shown in Figure S1 of Supplementary Material) causing needle cast disease in May, July, September and November
Phenylalanine ammonia-lyase (PAL)-mediated catalysis rate-limiting step of theis phenylpropanoid which levels of asperata were examined during infection by the pathogen (Lophodermium controls the biosynthesis of secondary metabolites in plants, such as lignins, flavonoidspiceae) and causing needleincast disease
Summary
To adapt to changes in the ecological environment and related biotic and abiotic stresses, conifers have evolved a diverse defense system as one of their main strategies for survival [1]. The conifer defense system synthesizes a variety of secondary metabolites when attacked by pathogens, mainly a variety of phenolic compounds, including highly polymerized physical barriers such as lignin, which prevent the invasion of pathogens [2]. These phenolic defense compounds are primarily synthesized by the phenylpropanoid pathway, an important secondary metabolic pathway that produces various key molecules required for the plant defense system and systemic resistance [3,4,5]. Rosmarinic acid is synthesized from the precursor molecules L -phenylalanine and L -tyrosine, which may play a role in plant defense against fungal and bacterial infections [8]. Salicylic acid is produced by a branch of the Forests 2019, 10, 613; doi:10.3390/f10080613 www.mdpi.com/journal/forests
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