Hiroko Hayama

Identification of a new expansin gene closely associated with peach fruit softening

Abstract Expansins are proteins that have been shown to contribute to fruit softening in tomato. However, expansins that have been correlated with loss of fruit firmness have not yet been identified in peach (Prunus persica (L.) Batsch). Along with the previously isolated PpExp1, two new expansin cDNAs, termed PpExp2 and PpExp3, were isolated from ripe peach fruit, and their mRNA expression patterns were characterized during fruit development and in other tissues, including the flower bud, leaf, and stem. All three expansins were detected in the fruit and not in the other tissues, but each showed differential patterns of mRNA accumulation during fruit development. The PpExp2 mRNA was constitutively expressed throughout fruit development but was abundant in Stage III, when the fruit expands exponentially and then matures. The PpExp1 and PpExp3 mRNAs were up-regulated at the onset of ripening, but PpExp1 was induced at an earlier stage. In order to identify the expansins whose expression correlates with the loss of peach fruit firmness, the mRNA expression levels of the three expansins were compared in the ripe fruit of the ‘Akatsuki’ and ‘Manami’ cultivars during postharvest storage. During storage, the ripe fruit of ‘Akatsuki’ rapidly softened as the level of ethylene increased significantly, while ‘Manami’ fruit remained firm and exhibited very low levels of ethylene production. The PpExp1 and PpExp2 mRNAs were constitutively detectable during the 8-day storage of both cultivars, whereas PpExp3 mRNA was detectable in ‘Akatsuki’ but hardly detectable in ‘Manami,’ suggesting that PpExp3 expression may be related to the changes in fruit firmness. To address the detailed role of PpExp3 in the loss of fruit firmness, the fruit of ‘Manami’ was treated by ethylene to artificially induce softening. The PpExp3 mRNA accumulation in the ethylene-treated ‘Manami’ was detectable and similar to that observed in ‘Akatsuki.’ These results show that, while several expansins show a general increase in expression levels during the later stages of fruit development, some isoforms show a greater association with softening than others. In this regard, PpExp3 is more likely to play a role in peach fruit softening than PpExp1 or PpExp2.

Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch)

The fruit of melting-flesh peach (Prunus persica L. Batsch) cultivars produce high levels of ethylene caused by high expression of PpACS1 (an isogene of 1-aminocyclopropane-1-carboxylic acid synthase), resulting in rapid fruit softening at the late-ripening stage. In contrast, the fruit of stony hard peach cultivars do not soften and produce little ethylene due to low expression of PpACS1. To elucidate the mechanism for suppressing PpACS1 expression in stony hard peaches, a microarray analysis was performed. Several genes that displayed similar expression patterns as PpACS1 were identified and shown to be indole-3-acetic acid (IAA)-inducible genes (Aux/IAA, SAUR). That is, expression of IAA-inducible genes increased at the late-ripening stage in melting flesh peaches; however, these transcripts were low in mature fruit of stony hard peaches. The IAA concentration increased suddenly just before harvest time in melting flesh peaches exactly coinciding with system 2 ethylene production. In contrast, the IAA concentration did not increase in stony hard peaches. Application of 1-naphthalene acetic acid, a synthetic auxin, to stony hard peaches induced a high level of PpACS1 expression, a large amount of ethylene production and softening. Application of an anti-auxin, α-(phenylethyl-2-one)-IAA, to melting flesh peaches reduced levels of PpACS1 expression and ethylene production. These observations indicate that suppression of PpACS1 expression at the late-ripening stage of stony hard peach may result from a low level of IAA and that a high concentration of IAA is required to generate a large amount of system 2 ethylene in peaches.

Molecular cloning of a ripening-related expansin cDNA in peach: evidence for no relationship between expansin accumulation and change in fruit firmness during storage

Summary A cDNA ( PchExp 1 ) encoding a ripening-regulated expansin was isolated from ripe peach fruit. PchExp 1 encodes a predicted protein of 252 amino acids including the predicted signal sequence. The phylogenetic tree based on the deduced amino acid sequences indicated that PchExp 1 was most closely related to apricot PaExp2 and strawberry ripening-regulated FaExp2 , and distantly to tomato ripening-regulated LeExp 1 . The accumulation of PchExp 1 mRNA was fruit-specific and ripening-regulated. Furthermore, it was enhanced abundantly in 6 h by ethylene treatment. However, the patterns of PchExp 1 mRNA abundance in the ripe fruit of Akatsuki, which lost its firmness rapidly after harvest, and Yumyeong, which retained its firmness after harvest, were very similar during storage for eight days, irrespective of their different traits regarding changes in fruit firmness. The accumulation of protein recognized by the antibodies for LeExp 1 is also detected in the ripe fruit of Yumyeong. The present study suggests that PchExp 1 may play an important role in peach fruit ripening, but the accumulation of PchExp 1 may not regulate the changes in fruit firmness during storage of the peach.

Changes in the levels of mRNAs for putative cell wall-related genes during peach fruit development☆

Cell wall changes are important factors for understanding fruit development. Four cDNA clones putatively encoding xyloglucan endotransglycosylase (pfPpXet1), expansin (pfPpExp2), sucrose synthase (pfPpSS1), and cellulose synthase (pfPpCesA1), which are all assumed to relate to cell wall modification, were isolated from developing fruit of peach (Prunus persica cv. Akatsuki), and the relationships between their mRNA accumulations and fruit development were investigated. Each of these four clones showed a different and characteristic pattern of mRNA expression. For example, the accumulation pattern of pfPpExp2 mRNA was in accordance with peach fruit development; i.e., it is abundant in the fruit when the fruit is growing and hardly detectable in the fruit when the fruit growth slows. The pfPpXet1 mRNA was detected in fruit in the earlier stages of development only. The result suggests that cell wall-related genes are differentially regulated during fruit development in peach.