Knowing them by knocking them out : initial studies to characterize Arabidopsis annexin gene family function(s)

Annexins are a multigene family in most plant species and are suggested to play a role in wide variety of essential cellular processes. Analysis of paralogous blocks within the genome of Arabidopsis showed that AnnAt6 was a duplicated gene from AnnAt1 and AnnAt2. The motif analysis of the promoters of these three annexins indicated some putative functional redundancy but also agreed with the model that duplicated genes functionally diverge over time and undergo functional specialization that can principally be observed in their regulatory regions. This in silico analysis contributes important information which can be used to guide phenotypic analysis of mutants. The use of quantitative real time reverse transcription PCR (quantitative real time RT-PCR) to assess differential expression of annexins in different tissues from 5.5 day-old etiolated seedlings showed a trend of major expression of annexins in root and hypocotyl tissues compared to cotyledon tissue. This result supports previous data about the participation of annexins during growth and development. The light-dependent regulation of annexin gene expression was also examined using etiolated seedlings under red and far-red light conditions by quantitative real time RT-PCR. Red light treatment significantly altered transcript levels in hypocotyls and cotyledons for AnnAt5 and AnnAt6, respectively, and this early up-regulation was reversible by far-red light, implicating phytochrome as the receptor mediating this response. In order to begin testing the hypothesis that plant annexins have individual functions, over thirty annexin putative T-DNA insertional lines were obtained and screened. After a screen of these mutants, further work was focused on fourteen lines for which annexin insertion sites were confirmed. For AnnAt1 through AnnAt5 at least two allelic lines and for AnnAt6 and AnnAt7 one allelic line has been confirmed by PCR. For some of them the absence of mRNA was determined by RT-PCR which confirms that they are true knockouts. These annexin T-DNA insertion lines will allow for future phenotypic characterization and likely provide valuable information regarding individual functions of Arabidopsis annexins.Cellular and Molecular Biolog