Identification of the laccase-like multicopper oxidase gene family of sweet cherry (Prunus avium L.) and expression analysis in six ancient Tuscan varieties

Abstract

Laccase-like multicopper oxidases (LMCOs) are versatile enzymes used as biocatalysts performing the oxidation of different substrates of industrial relevance, with or without the intervention of a mediator. They have attracted a lot of interest for biotechnological applications in light of their eco-friendliness: they indeed oxidize the substrate(s) by coupling the four electron reduction of the final acceptor, molecular oxygen (O 2 ), to water. Plant LMCOs represent a still poorly studied, important class of oxidoreductases controlling e.g. the post-harvest quality of fruits and enabling the tailoring of designer energy crops. We here sought to identify the LMCOs in Prunus avium L., whose fruits are rich in bioactive molecules, but are also highly perishable. The goal was to analyze them using bioinformatics (phylogenetic and in silico structural analyses) and to perform a targeted expression study on a subset of genes in six ancient varieties from Tuscany, all threatened by genetic erosion. These sweet cherry varieties contain higher amount of bioactive molecules, as compared to commercial counterparts. The results shown demonstrate strikingly different gene expression patterns in the six ancient varieties (‘Benedetta’, ‘Carlotta’, ‘Crognola’, ‘Maggiola’, ‘Morellona’, ‘Moscatella’) belonging to the Tuscan Regional Bank of Germplasm, as compared to a widely used commercial one (‘Durone’). The motivation of this study is the economic importance of P. avium and the involvement of LMCOs in post-harvest fruit parameters, like color. The results presented pave the way to follow-up researches on LMCOs of sweet cherry exploring post-harvest fruit parameters (e.g. anthocyanin stability responsible for pericarp browning and the preservation of the appealing red color), as well as developmental processes, like stony pit formation.

Tuscan Varieties of Sweet Cherry Are Rich Sources of Ursolic and Oleanolic Acid: Protein Modeling Coupled to Targeted Gene Expression and Metabolite Analyses

“…The expression analysis highlights different values among the Tuscan varieties (Figure 11). ‘Crognola’ and ‘Carlotta’ are among the varieties showing the highest expression levels of OSC s and CYP85 s. This finding is very interesting if one considers the recently reported higher content of phenolic compounds in these ancient Italian sweet cherries [33] and the higher expression levels of genes encoding laccase-like multicopper oxidases [29]. ‘Benedetta’ shows higher levels than all the other varieties only for XP_021819928.1.…”

“…Previously, we successfully used the Mcule tool to analyze the docking of various substrates in laccases from Tuscan sweet cherry varieties [29]. However, compared to the accessible active site of laccases, OSCs have a more hydrophobic and closed catalytic pocket (Figure 4).…”

“…The sweet cherry fruits used in this study were sampled in 2017, as previously described [29], from eighteen-year-old cherry trees (ancient local varieties of P. avium on P-HL-B rootstocks) grown in the experimental field of the CNR in Ivalsa Follonica (GR, Italy, coordinates 42° 55′ 59″ N, 10° 45′ 57″ E). Sampling took place in May 2017.…”

“…The RNA extraction procedure, quality control and quantification, as well as reverse transcription and real-time PCR conditions have been previously detailed [29]. The primers used for the reference genes have been previously published [29], those for eTIF4E , GAPDH , OSC s and CYP8 5s are indicated in Table 2. They were designed with Primer3Plus () and checked with OligoAnalyzer 3.1 ().…”

Identification of Jasmonic Acid Biosynthetic Genes in Sweet Cherry and Expression Analysis in Four Ancient Varieties from Tuscany

“…The 18-year-old sweet cherry trees were grown in an experimental field of the National Research Council (CNR) in Follonica (Tuscany, Italy). The coordinates of the experimental field were previously reported [31]. The fruits were harvested by picking them all over the tree canopy at an average height of 1.70–1.90 m from the ground.…”