Cellulase activity was measured in soybean (Glycine max) leaf abscission zones, flower abscission zones, pod abscission zones, apical buds, and adventitious rooting hypocotyls. Immunoprecipitation data showed that a cellulase immunologically similar to the bean abscission cellulase (isoelectric point 9.5) is present in soybean leaf, flower, and pod abscission zones, but is not present in soybean apical buds or rooting hypocotyls. cDNA and genomic clones for two different soybean genes were identified and show sequence similarity with the bean abscission cellulase clone pBAC10. The cDNA clone pSAC1, isolated from a soybean abscission cDNA library, hybridized to transcripts in soybean leaf, flower, and pod abscission zones. Although ethylene has been shown to play a role in the increase in cellulase activity associated with both abscission and adventitious root initiation, no signal was seen for hybridization of the soybean abscission cellulase clone, pSAC1, to RNA from soybean adventitious rooting hypocotyls. In addition, no soybean abscission cellulase transcripts were detected in apical buds. Transcripts for a second soybean cellulase gene (SC2) were not detected in any of the tissues surveyed.

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

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We have isolated a cytochrome c gene from Arabidopsis thaliana (cv. Columbia), which is the first cytochrome c gene to be cloned from a higher plant. Genomic DNA blot analysis indicates that there is only one copy of cytochrome c in Arabidopsis. The gene consists of three exons separated by two introns. Gene features such as regulatory regions, codon usage, and conserved splicing-specific sequences are all present and typical of dicotyledonous plant nuclear genes. We have constructed phenograms and cladograms for cytochrome c amino acid sequences and histone H3, alcohol dehydrogenase, and actin DNA sequences. For both cytochrome c and histone H3, Arabidopsis clusters poorly with other higher plants. Instead, it clusters with Neurospora and/or the yeasts. We suggest that perhaps this observation should be considered when using Arabidopsis as a model system for higher plants.

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