Peach (Prunus persica) Endopolygalacturonase cDNA Isolation and mRNA Analysis in Melting and Nonmelting Peach Cultivars

Abstract

Two distinct partial cDNAs, PRFl and PRF3, similar in sequence to previously described polygalacturonases, were amplified from ripe peach (Prunus persica 1. Batsch cv Flavorcrest) fruit cDNA by the polymerase chain reaction. PRF1-related RNA was present in fruit from early ripening at levels not detected by northern analysis. PRF3-related RNA was readily detectable in ripe fruit by northern analysis. PRF3 was used to isolate a cDNA with a complete open reading frame, PRFS, from a XZAP li cDNA library prepared from poly(A)+ RNA of ripe peach fruit. PRF5 coded for a predicted protein of 393 amino acids with a molecular mass of 41,500 D. The derived amino acid sequence of PRFS included a putative leader sequence of 23 amino acids, followed by a sequence that matched the N terminus of endopolygalacturonase protein purified from ripe peach fruit. By northern analysis, PRF3-related RNA was undetectable in firm, unripe Flavorcrest fruit. It appeared at low levels as a 1.7-kb transcript in fruit that had begun to ripen and soften and was very abundant in ripe fruit that had undergone the "melting" stage of softening. The marked increase in PRF3-related RNA levels took place over a period of less than 2 d at 20°C and coincided with the climacteric peak in ethylene evolution. Levels of 1 -aminocyclopropane-1 -carboxylate oxidase-related RNA increased during ripening at a much earlier stage than levels of PRF3-related RNA. Lower levels of 1.7-kb RNA transcript were detected by PRF3 in ripe fruit of the melting cultivar Fragar, which are firmer than Flavorcrest fruit. In ripe fruit of the nonmelting cultivar Carolyn, PRF3 detected a 1.45-kb RNA transcript that was present at low levels. Transcripts of a peach polygalacturonase-related genomic sequence were not detected in ripening fruit.

Three Different Polygalacturonases Are Expressed in Tomato Leaf and Flower Abscission, Each with a Different Temporal Expression Pattern

“…The TAPG gene products from tomato most likely encode endopolygalacturonases due to their high percent identity with the PFPG (62% identity), which was shown to encode an endopolygalacturonase (Lester et al, 1994). Moreover, in the dendrogram shown in Figure 3 the fruit and abscission PGs are grouped separately from the pollen PGs.…”

“…The TAPG2 cDNA includes an approximately 150-bp poly(A) tail at the 3' end. The first 18 amino acids of the deduced amino acid sequence includes a hydrophobic core of amino acids and a cleavage site indicative of a signal peptide (von Heijne, 1983 (Delia Penna et al, 1986), and PFPC (Lester et al, 1994) Percent identities for the alignments of peptide sequences are shown in boldface. signal peptide) has a predicted molecular mass of 40.1 kD, identical to that of the TAPG1 peptide, and a pi of 8.08.…”

“…flower buds (el 84), X72291; and cedar pollen, D29772. Table I shows the relative percent identities for the nucleotide and peptide sequences for the three TAPGs (TAPG1, TAPG2, and TAPG4), TFPG (Delia Penna et al, 1986), and PFPG (Lester et al, 1994). The TAPGs are clearly different from the TFPGs (38-41% amino acid sequence identities), putting them into a separate subfamily of tomato PG genes.…”

Single nucleotide polymorphism analysis of the endopolygalacturonase gene in peach and its potential use in crossbreeding programs

“…In particular, endopolygalacturonase (endo-PG, EC 3.2.1.15) is an enzyme involved in the depolymerization of pectin, a major component of cell walls that is extensively disassembled during the late stages of fruit softening (Hayama et al, 2003(Hayama et al, , 2006aTatsuki et al, 2013). On this same note, the lack of a melting phase in nonmelting flesh (NMF) genotypes has been shown to be caused by a deletion of the endo-PG genes or a truncation of their mRNAs, which, in turn, causes an absence of the immunodetectable endo-PG protein (Lester et al, 1994;Brummell et al, 2004). These data indicate that endo-PG-mediated pectin modification may play an important role in the later stages of softening and textural changes during the ripening of the peach.…”

“…At present, published studies have mainly focused on analyzing the activities and gene expression of endo-PG (Lester et al, 1994;Ma et al, 1999;Hayama et al, 2003;Brummell et al, 2004;Tatsuki et al, 2013); only a few studies have yet to analyze SNPs (Hayama et al, 2006a). In one of these studies, the selected fragments of an endo-PG gene (1172 bp) from the genomic DNA isolated from 2 peach cultivars were subsequently amplified, cloned, and sequenced, showing 31 SNPs, 2 main deletions of 17 bp each, a 2-bp deletion, and a 2-bp insertion (Morgutti et al, 2006).…”