1. Guillermo E. Santa-María Instituto Tecnológico Chascomús (INTECH) 2009
Regulación de la adquisición de potasio en plantas y sus posibles implicancias tecnológicas
Guillermo E. Santa-María
Instituto Tecnológico Chascomús
(INTECH)
2009

2. Salt Resistance correlates with improved K+ retention in young leaves: K+/Na+ balance

3. K+-influx from diluted K+ solutions plays an important role in salt resistance

4. Simplified model for K+ influx from diluted K+ solutions
AKT1 K+ K+ H+ H+
ATP
ADP
HAK1
ATPase

5. The HAK1 mediated pathway
Hordeum vulgare HAK1 transporter (HvHAK1)
Canonic member of group I of KT-HAK-KUP
transporters

6. HvHAK1 contributes to inducible K+-influx - I
+K K E C VC A B
In-situ hybridization barley root
Fulgenzi et al. Plant Physiol 2008

7. HvHAK1 contributes to inducible K+-influx - II
0,5 1
1,5 2
2,5
718-J
718-T WT
Rb+-influx (umol g-1 h-1)
HvHAK1
Transgenic lines
Dashed: -K
Palid green: +K

8. Expression of HvHAK1 is transiently enhanced by salinization and parallels changes in the reduction of Na+-inhibitory effects on Rb+ uptake in barley roots
Fulgenzi, F. R., et al. Plant Physiol. 2008;147:
Copyright ©2008 American Society of Plant Biologists

9. Then, it could be argued that transgenic plants over- expressing HvHAK1 should display enhanced resistance to salinity

10. WT 718-J 718-T V366I-1 V366I-2 R591C-1 R591C-2 R591C-3
Peso Fresco (g)
WT J T V366I-1 V366I-2 R591C-1 R591C-2 R591C-3 d
bcd cd bc ab a
0,2
0,4
0,6
0,8 1
1,2
1,4
1,6 WT
718 (J)
718 (T)
J (1)
J (2)
K (1)
K (2)
K (3)
Expression of HvHAK1 in Arabidopsis does not improve plant resistance to salinity
Fresh Weight
WT J T
Mangano, Moriconi, Santa-María, umpublished

11. WHY ?

12. Arabidopsis vs Saccharomyces: complex and simple systems
Arabidopsis: 1- Strong background for K+ transport (athak5-atakt1 not available)
2- Accumulation of transcripts (i.e AtAMT1, SOS1) even under the control of the 35S promoter is enhanced in certain ionic environments (transcript stability)
Saccharomyces: 1- No background (trk1-trk2 available)
2- No evidence for a critical influence of the ionic enviroment on the accumulation of transcrips expressed under the control of a constitutive promoter

13. Rb+ uptake is enhanced by K+ starvation in HvHAK1-expressing yeast cells
Fulgenzi, F. R., et al. Plant Physiol. 2008;147:
Copyright ©2008 American Society of Plant Biologists

14. That increase should be linked to an increased Vmax

15. High sodium concentrations interfere with the increased contribution of HvHAK1 following K+ starvation in yeast cells
Fulgenzi, F. R., et al. Plant Physiol. 2008;147:
Copyright ©2008 American Society of Plant Biologists

16. Culture at moderate NaCl concentrations affects Rb+ transport mediated by HvHAK1 in yeast cells lacking Na+ exclusion systems
Fulgenzi, F. R., et al. Plant Physiol. 2008;147:
Copyright ©2008 American Society of Plant Biologists

17. K+ K+ HvHAK1 in S. cerevisiae Na+ HvHAK1 H+ Na+ H+ Na+ H+ ATPase ATP
ADP
ENA 1-4 K+
HvHAK1 H+ K+
Fulgenzi et al. (Plant Physiol. 2008)
TRK
Other systems
(NSC1), etc
Na+ H+
Na+
NHA1 H+
ATP
ADP
ATPase

18. Those results suggest that resistance to salinity could be improved by improving the Vmax of K+ transport mediated by HvHAK1 or by diminishing the relative inhibitory effect of Na+

19. Use of S. cerevisiae as a model system for screening HvHAK1 mutant versions

20. Generación de las mutantes
PCR, variando la concentración de nucleótidos.Primers sobre Porm y Term de Pypge15
HvHAK1 P T
+ Pypge15
Cotransformación en la cepa W3 (trk1-trk2-)de S.cerevisiae
Selección en medio P.A. bajo K+ alto Na+
Selección de transformantes en medio P.A. alto K+
Selección en medio P.A. bajo K+ (3000clones chequeados)
Se obtuvieron 4 mutantes

21. Mangano et al FEBS Letters, 2008

22. Mangano et al FEBS Letters, 2008

23. Mangano et al FEBS Letters, 2008

24. Then the improved resistance conferred by expressing the V366I or the R591 C versions must be related to improved K+ retention and to low Na+ accumulation

25. Improved Salt resiatnce correlates with improved Rb+-influx
Mangano et al FEBS Letters, 2008

26. Enhanced Vmax !!!!
Mangano et al FEBS Letters, 2008

27. The mutations do not change the regulatory effect exerted by the presence of high NaCl during culture
Mangano et al FEBS Letters, 2008

28. In the absence of ENA1-4 and NHA transporters, yeast cells expressing mutant versions display enhanced salt resistance and improved K+ nutrition but not improved Na+ exclusion
Mangano et al FEBS Letters, 2008

29. The V366I and R591 mutations confer resistance toi umltiple ionic stresses
Mangano et al FEBS Letters, 2008

30. An hypothesis linking improved K+-nutrition and improved resistance to ionic stresses
Glucosa
Aminoácidos
HvHAK1 K+ m
NH4+
Na+/ Li+ K+
ENA1-4 pH
ENA 1
Na+

31. 1- Regulatory effects on K+-influx must be taken in consideration
2- Reciprocal Interactions between K+ and Na+
3- Improved salt resistance can be eventually reached by improving the Vmax of K+ transport, proved that no differential inhibition by Na+ takes place

32. Laboratorio Fisiología de las Plantas
(INTECH)
Lic. Diana Lauff
Lic. Jorge Moriconi
Lic. Silvina Mangano
Dr. G. Santa María
Ing. Agr. Fabiana Fulgenzi
Lic. María Luisa Peralta
Dr. Cristian Danna
Dr. Augusto Vallejo
Gracias:
Alonso Rodríguez-Navarro, Francisco Rubio y Rosario Haro (Madrid)
Pere Puigdomenech (Barcelona)
Jorge Dubcovsky (Davis, CA)
Marcela Simontacchi (UBA)
Gabriela Tranquili
Antonio Díaz-Paleo (INTA)
Y muchos otros…