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Comparative Study
. 1999 Apr;119(4):1261-70.
doi: 10.1104/pp.119.4.1261.

Molecular cloning and characterization of apricot fruit polyphenol oxidase

T Chevalier  1 , D de Rigal, D Mbéguié-A-Mbéguié, F Gauillard, F Richard-Forget, B R Fils-Lycaon
Affiliations
Comparative Study

Molecular cloning and characterization of apricot fruit polyphenol oxidase

T Chevalier et al. Plant Physiol. 1999 Apr.

Abstract

A reverse transcriptase-polymerase chain reaction experiment was done to synthesize a homologous polyphenol oxidase (PPO) probe from apricot (Prunus armeniaca var Bergeron) fruit. This probe was further used to isolate a full-length PPO cDNA, PA-PPO (accession no. AF020786), from an immature-green fruit cDNA library. PA-PPO is 2070 bp long and contains a single open reading frame encoding a PPO precursor peptide of 597 amino acids with a calculated molecular mass of 67.1 kD and an isoelectric point of 6.84. The mature protein has a predicted molecular mass of 56.2 kD and an isoelectric point of 5.84. PA-PPO belongs to a multigene family. The gene is highly expressed in young, immature-green fruit and is turned off early in the ripening process. The ratio of PPO protein to total proteins per fruit apparently remains stable regardless of the stage of development, whereas PPO specific activity peaks at the breaker stage. These results suggest that, in addition to a transcriptional control of PPO expression, other regulation factors such as translational and posttranslational controls also occur.

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Figures

Figure 1
Figure 1
SDS-PAGE of purified apricot PPO. MW, Molecular size markers; F IE, most active fraction eluted from the DEAE-Sepharose CL6B ionic exchange column.
Figure 2
Figure 2
IEF in liquid medium of apricot PPO. IEF was performed at 4°C for 3 d in a pH gradient (3.5–5.0). On each fraction of 1.5 mL collected, pH was measured and PPO activity was assayed.
Figure 3
Figure 3
Optimal alignment of PPOs from several plant species. Accession nos.: apricot, no. AF020786; apple, no. P43309 (Boss et al., 1995); grape, no. P43311 (Dry and Robinson, 1994); fava bean, no. 418754 (Cary et al., 1992); Virginian pokeweed, no. D45385 (Joy et al., 1995); tomato, no. Q08296 (Newmann et al., 1993). A dot refers to identity with apricot. A space denotes a gap introduced for improved alignment. Single underlined amino acid residues correspond to the transit peptide. Domain I of transit peptide is marked by •. Domain II of transit peptide is marked by ⋄. The “n-region” of domain II of transit peptide is shaded in blue. The thylakoid transfer domain of the domain II of transit peptide is shaded in green. Shown as bold letters are the hydrophobic amino acids of thylakoid transfer domain and the precleavage site. The first amino acid residue of the mature protein is shaded in black. Double underlined amino acid residues correspond to the sequence obtained from N-terminal sequencing of the purified protein. Copper domains A and B of the mature protein are shaded in yellow. His residues predicted to be copper-binding ligands are boxed.
Figure 4
Figure 4
Genomic DNA analysis of PA-PPO. Genomic DNA (20 μg per lane) was digested with EcoRI (lane 1), HindIII (lane 2), and EcoRI and HindIII (lane 3), hybridized with an RT-PCR PPO fragment from apricot and washed at low stringency. λDNA codigested with EcoRI and HindIII (Promega) was used as a molecular mass marker.
Figure 5
Figure 5
Expression of PA-PPO gene during ripening of apricot fruit. Fifteen micrograms of total RNA from apricot fruit was used at five stages of development: IM1 (immature green 1), BR (breaker), BR+ (breaker +), HR (half-ripe), and OR (overripe). The blot was hybridized to an RT-PCR PPO fragment from apricot and washed at high stringency.
Figure 6
Figure 6
Changes in PPO activity per fruit, protein per fruit, and PPO specific activity during ripening of var Bergeron apricot. Six stages of development were considered: IMG 1 (immature green 1), MG (mature green), BR (breaker), HR (half-ripe), RI (fully ripe), and OR (overripe).
Figure 7
Figure 7
Amounts of PPO protein during ripening of apricot fruit. Total proteins from apricot fruit at six stages of development: IM1 (immature green 1), MG (mature green), BR (Breaker), HR (half-ripe), RI (fully ripe), OR (overripe) were used. Proteins (10 μg per lane) of each stage were separated on SDS-PAGE, transferred and immunoblotted with anti-apple PPO crude serum. Molecular size markers are in lane 1.
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