Ethylene-sensitivity regulates proteolytic activity and cysteine protease gene expression in petunia corollas*

Abstract

To investigate ethylene's role in petal senescence, a comparative analysis of age-related changes in total protein, protease activity, and the expression of nine cysteine protease genes in the corollas of ethylene-sensitive Petuniaxhybrida cv. Mitchell Diploid (MD) and ethylene-insensitive (35S:etr1-1; line 44568) transgenic petunias was conducted. The later stages of corolla senescence in MD flowers were associated with decreased fresh weight, decreased total protein, and increased proteolytic activity. Corolla senescence was delayed by approximately 8 d in etr-44568 transgenic petunias, and decreases in corolla fresh weight, protein content, and maximum proteolytic activity were similarly delayed. Protease inhibitor studies indicated that the majority of the protease activity in senescing petals was due to cysteine proteases. Nine cysteine proteases expressed in petals were subsequently identified. Northern blot analysis indicated that six of the nine cysteine proteases showed increased transcript abundance during petal senescence. One of these cysteine proteases, PhCP10, was detected only in senescing tissues. Expression of four of the senescence-associated cysteine proteases was delayed, but not prevented in etr-44568 flowers. The other two senescence associated cysteine proteases had high levels of transcript accumulation in etr-44568 corollas at 8 d after flower opening, when MD flowers were senescing. These patterns suggest that age-related factors, other than ethylene, were regulating the up-regulation of these genes during flower ageing. The delay in visible symptoms and biochemical and molecular indicators of senescence in ethylene-insensitive flowers is consistent with the concept that ethylene modulates the timing of senescence pathways in petals.