The eceriferum3 (cer3) locus encodes one of 21 gene products known to be involved in wax biosynthesis in Arabidopsis thaliana. Mutations at these loci are readily identified by their bright, dark-green stems when compared with the more glaucous wild-type plant. Clones of a gene which encodes a 795 amino acid open reading frame have been isolated by using plant DNA flanking the site of a T-DNA insertion in line BRL1. Molecular complementation of the cer3 mutant phenotype by clones of this gene establish that it corresponds to the CER3 gene. Although the 90 kDa predicted amino acid sequence of this gene shows no homology to any other known protein, the second exon of CER3 encodes a RRX12KK nuclear localization sequence (NLS). Southern blot and DNA sequence analysis revealed that the T-DNA is inserted 89 bp downstream of the translation termination codon of this gene. Northern blot hybridization of RNA isolated from the BRL1 mutant with the CER3 cDNA probe indicated that the transcript is absent in this mutant line. Unlike other CER genes that have been cloned to date, high levels of the CER3 transcript were found in all tissues from wild-type plants, that is, leaves, stems, roots, flowers, and apical meristems.

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We have characterized three mutant alleles of the CER2 gene of Arabidopsis thaliana by sequencing polymerase chain reaction products of this gene generated with template DNA isolated from lines MK1, BRL7, and BRL17. Sequence analysis of the amplification product from line BRL17 revealed a 17-bp deletion in the CER2 gene. The CER2 gene of BRL7 differs from the wild-type sequence by a 2-base substitution and 2-base insertion. As both of these lines were isolated from the transformant populations generated by Dr. K. Feldmann and his collaborators, we suggest that these small rearrangements may be caused by unsuccessful T-DNA insertions. Comparative sequence analysis of the sequence of line MK1 and the wild type revealed a single nucleotide substitution located 360 bp downstream from the intron-exon junction that changes a tryptophan triplet TGG to TGA; i.e., an opal non-sense mutation. In accordance with these observations, the cer phenotype of line MK1 was complemented by transformation with a fusion construct of the CaMV 35S promoter and the CER2 structural gene. Comparative analysis of the deduced amino acid sequences encoded by the CER2 gene from Arabidopsis and by the glossy2 gene from Zea mays revealed a significant similarity between them. This suggests that these gene products may have a similar biochemical function.

A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37-80 kb subpopulation, which was predominant in whole plants, to 18-34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5-10 kb significantly increased in suspension culture cells. In addition, a new peak (10-12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4-2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.

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