Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence

Several cDNAs for mRNAs that change in abundance during tomato leaf senescence were isolated. In this paper we report molecular cloning and expression analysis of two cysteine proteases. SENU2 is identical to the cDNA C14 which encodes a cysteine protease previously shown to be expressed in response to extremes of temperature in tomato fruit [43]. SENU3 cDNA clone was 1.2 kb in length and hybridized to a transcript of 1.4 kb which suggested that the clone was not full-length. The missing 5' end was isolated using rapid amplification of cDNA ends (RACE). Southern blot analysis of tomato genomic DNA indicates that SENU3 is encoded by a single or low copy gene. SENU3 was also shown to have significant homology with known cysteine proteases. These two senescence-associated cysteine proteases are also expressed during other developmental processes, including seed germination, consistent with a role in protein turnover. SENU2 and SENU3 mRNAs were detectable in young fully expanded leaves and increased in abundance with leaf age, reaching a maximum during the later stages of visible leaf senescence. Such a pattern of expression suggests that the onset of leaf senescence is a gradual event. Analysis of senescence in transgenic plants deficient in ethylene biosynthesis, in which leaf senescence is delayed, indicated that enhanced accumulation of SENU2 and SENU3 mRNA was similarly delayed but not prevented.