The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O 2 , CO 2 , and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme⅐(substrate or cofactor)⅐NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O 2 complexes. It is shown here that the ternary Fe(II)ACCO⅐ACC⅐NO complex is readily formed, but no Fe(II)ACCO⅐ascorbate⅐NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15 N or 17 O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the ␣-amino and ␣-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O 2 bind to the iron as a step in O 2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O 2 , thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

A cDNA library was constructed from poly(A)(+)RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)(+)-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.

The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified.

We present in this paper the structural analysis of two members of a small cellulase gene family, designated cel1 and cel2, from avocado. These genes were isolated by screening a lambda EMBL3 genomic library with a ripening-induced cellulase cDNA. Restriction endonuclease and Southern blot analyses showed that the cel1 gene is highly homologous to the cellulase cDNA and thus represents a ripening-related cellulase gene. The other cellulase gene, cel2, is closely related to cel1, but is divergent at its 5' end. The nucleotide sequence of a 5 kb region encompassing the cel1 gene was determined. Four previously characterized cellulase cDNAs from ripe fruit are identical to the eight exons of the cel1 gene. RNase protection and primer extension analyses were used to define the transcription start site of cel1 and to quantitate cel1 transcripts in ripening fruit. The cel1 mRNA was present at a low level in unripe fruit and increased 37-fold during ripening. Partial DNA sequence analysis of cel2 and comparison to the cel1 sequence revealed a high degree of similarity both at the DNA and deduced amino acid sequence levels. No characterized cellulase cDNAs derived from ripe fruit represent cel2 transcripts. These data suggest that the cel1 gene is responsible for a major portion, if not all, of the cellulase transcripts in ripe fruit. The DNA sequence of 1.4 kb of 5' flanking DNA of the cel1 gene was compared to the upstream sequence of other ethylene-regulated genes. Several interesting upstream sequence motifs were identified and are discussed.