Identification of Arabidopsis mutants affected in
+
K
homeostasis
Martínez-Macías F, Muñoz-Bertomeu J, Serrano R
Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n 46022
Valencia, Spain.
femarma@ibmcp.upv.es
Salinity is one of the environmental problems with greater impact on global food production, especially in semiarid areas such
as the Mediterranean region. Previous work has shown that sodium uptake is counteracted by increased potassium transport
or decreased H+ pumping and that mutants with the latter phenotypes can be obtained by screening for norspermidine (NE)
resistance (Alejandro et al, 2007, EMBO J 26: 3203). This polycation enters the cell by some unidentified system strongly
dependent on the electrical potential of the plasma membrane. As this biophysical parameter is determined by the relative
activities of H+-ATPase and K+ transport, mutants tolerant to NE would be more tolerant to Na+and would allow us to identify
regulators of H+ pumping and K+ influx. We have screened 100.000 mutant lines of Arabidopsis tagged with T-DNA (about
700.000 seeds) and obtained 40 mutants tolerant to this polycation, confirmed with germination percentages between 2 and 8
times greater than wild type.
2.000 seeds/plate
4.5-6 mM NE
503 mutants
500 seeds/plate
BASTA/Kanamycin
4 mM NE
171 mutants
40 mutants
Figure 1: Schematic representation of the selection process of mutants tolerant to NE.
Figure 2: Putative mutant isolated in the primary screening at 6 days
after sowing.
Figure 3: Homocygotic phenotype resistent to BASTA/Kanamycin
at 7 days after sowing.
Figure 5: Homocygotic phenotype sensitive to BASTA/Kanamycin
at 7 days after sowing.
K. Feldmann T-DNA
p3850:1003 T-DNA
Figure 4: Heterocygotic phenotype resistent to BASTA/Kanamycin
at 7 days after sowing.
INRA-Versailles TDNA
D. Weigel T-DNA
W. Scheible and
C. Somerville T-DNA
pGKB5 T-DNA
•Feldmann T-DNA collection
(N3115, N6500, N84442)
•Loss of function lines
generated by transformation of
WS2 using p3850:1003
Figure 6: Germination in MS medium + 4 mM
NE in Feldmann mutants. WS2 is the control
line.
•D. Weigel T-DNA
collection (N21995,
N21991, N23153)
•Activation tagging
lines
generated by
transformation of Col-7
using pSKI015
•INRA Versailles T-DNA collection (N5389, N5455,
N5600, N5866, N55047)
•Loss of function lines generated by
transformation of WS4 using pGKB5
Figure 8: Germination in MS medium + 4 mM
NE in Weigel mutants. Col-7 is the control line.
Figure 7: Germination in MS medium + 4 mM
WS4 is the control line.
NE in INRA-Versailles mutants.
•W. Scheible and
C.Somerville T-DNA
collection (N31100)
•Activation tagging
lines
generated by
transformation of Col-2
using pSKI015
Figure 9: Germination in MS medium + 4 mM
NE in Scheible and Somerville mutants. Col2 is
the control line.
M
Table 1. Mutant Collections, number of lines and seeds utilized in the screening and the
confirmed mutants tolerant to NE and BASTA/Kanamycin obtained in MS + 4mM NE
secondary screening.*Percentage of mutants lines isolated compared with the lines used in
the primary screening.
Putative
Putative
Putative
Mutants
Nº of
Mutants
Mutants
Mutants
Collections
Nº of lines
seeds
(population (population (population
(population S)
S1)
S2)
S2)
C.Somerville and
(~63.000) ~400.000 167 (0.26%)*
39
10 B+
W.Scheible
D. Weigel
(~22.672) ~100.000 183 (0.8%)*
69
7B+
K. Feldmann
(~10.500)
~63.000 75 (0.71%)*
27
13K+
INRA-Versailles
(~9.800)
~50.000 78 (0.79%)*
41
10B+
Figure 12: Southern blot analysis for Scheible and Somerville
mutants, isolated in the secondary screening. Genomic DNA
was cut with BamHI restriction enzyme, and membrane was
hybridaze with 35S probe.
Figure 10: Germination percentage for tolerance to BASTA/Kanamycin
in confirmed mutants.
FUNDING: Spanish Government
(PROMETEO/2010/038).
(ADYSARC
–
EUI2009-03985)
and
Valencian
Government