Endo-β-1,4-glucanases are involved in peach fruit growth and ripening, and regulated by ethylene

During peach (Prunus persica [L.] Batsch) fruit development and ripening the cell wall undergoes several structural and biochemical changes driven by several hydrolases. Among these, the endo-beta-1.4-D-glucanase (EGase, EC 3.2.1.4), or cellulase, may play a crucial role. Involvement of EGase throughout development and ripening of the fruit of cv. Redhaven was assessed by monitoring enzyme activity, specific polypeptide accumulation and gene transcription. During the four stages of growth EGase activity was high during S1 and in the early S2, declined during S3, and increased with the onset of ripening (S4). Two isoforms with isolectric points of 6.5 and 9.5 were identified. The pI 6.5 EGase was the only form present during the early stages of growth, whereas the pi 9.5 EGase was most abundant during ripening. The same isoforms were present in leaf and fruit abscission zones. The antibody raised against the pi 9.5 EGase, purified from lear abscission zones, cross-reacted with a protein of 54 kDa. A cDNA clone of 753 bp encoding peach EGase was obtained by RT-PCR. EGase transcripts, detectable only after amplification of total RNA by RT-PCR, were observed during S1, and at the preclimacteric and climacteric stages. However, the strongest hybridisation occurred at ripening, in correspondence with the maximal enzyme activity and polypeptide accumulation, which took place before the ethylene climacteric and in the early stage of flesh softening. Propylene treatments reduced EGase activity during the early stage of fruit growth but dramatically enhanced enzyme activity and the related transcript accumulation at ripening, and accelerated the loss of firmness. In fruit treated with 2,5-norbornadiene the softening process was strongly inhibited and the rise in EGase transcripts and activity did not take place. The results point to the EGases being involved in early fruit growth and the initial phases of softening. The presence of two isoforms and the dual effect of propylene on enzyme activity suggest that different EGase genes operate during the early and late developmental stages in peach fruit.