Characterization of CONSTANS, an Arabidopsis gene that promotes flowering.

Robson, Frances Clare (1999). Characterization of CONSTANS, an Arabidopsis gene that promotes flowering. PhD thesis The Open University.



Under long day conditions Arabidopsis thaliana plants grow vegetatively for approximately two weeks before they initiate flowering while under short day conditions the vegetative period lasts around six weeks. The constans (co) mutant flowers later than wild type under long days but at approximately the same time as wild type under short days. This suggests that the CO product promotes flowering in response to long days. The CO gene was cloned previously by chromosome walking and encodes a 373 amino acid protein containing two zinc fingers suggesting that it binds DNA and is involved in regulation of gene expression.

A series of mutant alleles of the CO gene was characterized and the effect of each mutation on flowering time examined. All seven co mutants are semi-dominant, with the heterozygotes having flowering times and leaf numbers intermediate between wild type and the homozygous co parent, and all differ quantitatively with respect to flowering time and leaf number.

The co alleles were characterized at the molecular level and the mutations were clustered into two regions of the gene that define important domains of the predicted CO protein. Five of the mutations affect the zinc finger region near the N-terminus of the protein and the remaining two mutations affect adjacent amino acids in a basic domain near the C-terminus of the protein. Both of these domains and the individual amino acids affected by the mutations are highly conserved. Homology searches revealed a family of at least 14 closely related genes in Arabidopsis and homologues in a number of other plant species.

The seven co mutations all cause in-frame changes and none were unambiguous null alleles. To investigate the effect of loss of CO function a putative null allele, co-8, was isolated after gamma-irradiation. This was characterized at the molecular level and found to contain a 1.3 kb deletion which removes approximately 1 kb of the promoter and 0.3 kb of the coding sequence at the 5’ end of the CO ORF that includes the zinc finger domain. The new co-8 mutant has an intermediate flowering time with respect to the seven previously isolated co mutants, and is also semi-dominant.

A number of constructs designed to express derivatives of the CO protein were introduced into Arabidopsis to investigate whether it was possible to repress flowering by the creation of dominant negative mutations. However, with the exception of one or two small effects these did not greatly affect flowering time.

A Brassica napus homologue of the CO gene was isolated. The predicted protein is 74% identical to the Arabidopsis CO protein, with the greatest conservation over the zinc fingers (91% identical) and C-terminal basic domain (94% identical). The homologue was able to complement the Arabidopsis co-2 mutation and restore wild type flowering time.

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