Status of Arabidopsis annexin TDNA project

1
Molecular evolution of annexins in Arabidopsis
thaliana and Oriza sativa genomes
Cantero et al. 2006
2
Ortholog vs Paralog
Ortholog gene homology traces back to a
speciation event Paralog gene homology traces
back to a duplication event
3
Model for annexin functions in plants
4
Zucchini annexin binding to F-actin affinity
column
5
Localization of annexin at the tip of
emerging and growing Nicotiana pollen tubes
6
Status of Arabidopsis annexin T-DNA project

• Obtained and tested more than 40 TMRI/ABRC
  putative T-DNA insertion annexin lines have
  two putative independent lines for six of the
  eight Arabidopsis annexins
• Majority of the lines contained more than one
  T-DNA insertion, so have backcrossed the
  homozygotes to obtain lines with single T-DNA
  insertion have fifteen lines with single
  insertions
• Currently determining which lines with single
  T-DNA insertions are true knockouts using gene
  specific primers and RT-PCR
• Characterizing each knockout line, does
  individual annexin knockouts show phenotypes?
  Altered growth, stress and/or gravitropic
  responses, defective pollen germination and tube
  elongation, defective root hair elongation, other
  processes affected?
• Creating double annexin knockouts

7
RT-PCR of wild type and putative knock out plants
with gene specific primers
1 2 3 4 5 6 7 8 9 10
11 12 13
Lane 1 DNA marker 2 WT Apt1 3WT AnnAt1 4
KO(AnnAt1) Apt1 5 KO(AnnAt1)AnnAt1, 6 WT Apt1
7 WT AnnAt5 8 KO(Ann5) Apt1 9 KO(AnnAt5)
AnnAt5 10 WT Apt1 11 WT AnnAt7 12
KO(AnnAt7) Apt1 13 KO(AnnAt7) AnnAt7 APT1 size
300 bp Annexin size 1 Kb
8
Phenotype analysis of annAt5 mutants grown in
vessels
annAt5-1
Wild type Col
What triggered this phenotype?
9
T-DNA mutants for three annexins show impaired
orientation in the dark
Wild type
mutant
10
AnnAt2 localization in the endodermis site for
shoot gravity perception