Salinity is one of the environmental problems with greater impact on global food production,...
1
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. 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. [email protected] 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 4: Heterocygotic phenotype resistent to BASTA/Kanamycin at 7 days after sowing. K. Feldmann T-DNA INRA-Versailles T-DNA D. Weigel T-DNA W. Scheible and C. Somerville T- DNA M Figure 10: Germination percentage for tolerance to BASTA/Kanamycin in confirmed mutants. 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. Acq uir e Colle ction Prima r y Sc r eeni n g Growth in soil Sec ondary Scree ning Confir med Ph e notyp es Confi r med M utan t s 2.000 seeds/plate 500 seeds/plate 4.5-6 mM NE 4 mM NE BASTA/Kanamycin Figure 5: Homocygotic phenotype sensitive to BASTA/Kanamycin at 7 days after sowing. Figure 1: Schematic representation of the selection process of mutants tolerant to NE. • 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. 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 NE in INRA-Versailles mutants. 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 pGKB5 T-DNA • Feldmann T-DNA collection (N3115, N6500, N84442) • Loss of function lines generated by transformation of WS2 using p3850:1003 p3850:1003 T- DNA Co segre g ation Plasmid R escue popu lation S popu l at ion S 1 popu l at ion S 2 FUNDING: Spanish Government (ADYSARC – EUI2009-03985) and Valencian Government (PROMETEO/2010/038). Table 1. M utantC ollections,num beroflinesand seedsutilized in the screening and the confirm ed m utantstolerantto N E and BASTA/Kanam ycin obtained in M S + 4m M NE secondary screening.*Percentageofm utantslinesisolated com pared with thelinesused in theprim ary screening. M utants C ollections (population S) N ºoflines N ºof seeds Putative M utants (population S 1 ) Putative M utants (population S 2 ) Putative M utants (population S 2 ) C.Som ervilleand W .Scheible (~63.000) ~400.000 167 (0.26% )* 39 10 B+ D . W eigel (~22.672) ~100.000 183 (0.8% )* 69 7 B+ K. Feldm ann (~10.500) ~63.000 75 (0.71% )* 27 13 K+ IN RA-Versailles (~9.800) ~50.000 78 (0.79% )* 41 10 B+ 503 mutants 171 mutants 40 mutants WS4 is the control line.
-
Upload
beverly-hicks -
Category
Documents
-
view
218 -
download
1
Transcript of Salinity is one of the environmental problems with greater impact on global food production,...
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.
Identification of Arabidopsis mutants affected in K+ homeostasis
Martínez-Macías F, Muñoz-Bertomeu J, Serrano RInstituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de
Vera s/n 46022 Valencia, Spain. [email protected]
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 4: Heterocygotic phenotype resistent to BASTA/Kanamycin at 7 days after sowing.
K. Feldmann T-DNA
INRA-Versailles T-DNA
D. Weigel T-DNA W. Scheible and C. Somerville T-
DNA
M
Figure 10: Germination percentage for tolerance to BASTA/Kanamycin in confirmed mutants.
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.
Acq
uire
Col
lect
ion
Prim
ary
Scre
enin
g
Gro
wth
in s
oil
Seco
ndar
y Sc
reen
ing
Con
firm
ed P
heno
type
s
Con
firm
ed M
utan
ts
2.000 seeds/plate 500 seeds/plate
4.5-6 mM NE 4 mM NE
BASTA/Kanamycin
Figure 5: Homocygotic phenotype sensitive to BASTA/Kanamycin at 7 days after sowing.
Figure 1: Schematic representation of the selection process of mutants tolerant to NE.
• 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.
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 NE in INRA-Versailles mutants.
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
pGKB5 T-DNA
• Feldmann T-DNA collection (N3115, N6500, N84442)
• Loss of function lines generated by transformation of WS2 using p3850:1003
p3850:1003 T-DNA
Cos
egre
gatio
nPl
asm
id R
escu
e
popu
latio
n S
popu
latio
n S 1
popu
latio
n S 2
FUNDING: Spanish Government (ADYSARC – EUI2009-03985) and Valencian Government (PROMETEO/2010/038).
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.
Mutants Collections
(population S) Nº of lines
Nº of seeds
Putative Mutants
(population S1)
Putative Mutants
(population S2)
Putative Mutants
(population S2)
C.Somerville and W.Scheible
(~63.000) ~400.000 167 (0.26%)* 39 10 B+
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+
503 mutants 171 mutants 40 mutants
WS4 is the control line.
