Supplemental Material

Gene identification from the Populus trichocarpa genome

For this purpose we used sequences of already characterized proteins from Arabidopsis and the BLAST algorithm at the Doe Joint genome initiative web site ( Potassium: Potassium channels of the Shaker type play a major role in K+ nutrition, growth, development and movement (for review see Gambale and Uozumi 2006). Comparing conserved protein regions of Arabidopsis and Populustremula x P. tremuloides with predicted protein sequences of Pop. trichocarpa we identified three already characterized (Langer et al. 2002 and 2004) and seven new putative poplar K+ channel genes, which in analogy to the Arabidopsis channels are namedKPT1, PTKC1, PTKC2, PTK2, PKT1, PKT1b, PKT6, PTORK, PTORK2 and PTORK3 and grouped accordingly.

Potassium uptake channels (Fig.6): KPT1 and PTK2 have already been cloned and characterized (Langer et al. 2002 and 2004). KPT1 is like its Arabidopsis ortholog KAT1 expressed in guard cells, where it is like KAT1 the most important K+ uptake system (Anderson et al. 1992; Mäser et al. 2001). In contrast to its Arabidopsis counterpart, KPT1 is also expressed in buds, where it is correlated with bud break. KAT1, the first cloned plant K+ channel is expressed mainly in guard cells and other epidermal cells of the leaves. PKT1 and PKT1b homologous to AKT1 have been expected to be involved in K+ uptake by the roots, whereas PTKC1 and PTKC2 like ATKC1 we predicted to be expressed in leaves and roots (Sentenac et al. 1992; Lagarde et al. 1996; Hirsch et al. 1998; Ivashikina et al. 2001). PTK6 and PTKC transcripts could not be detected within the experiment samples. PTK6 is an ortholog of the pollen specific AKT6/SPIK (Mäser et al. 2001; Mouline et al. 2002). Expression of this K+ channel is expected to be restricted to flowers or pollen, organs which were not subjects of this study. Since all AKTC like channels investigated so far are expressed in root tissues, it, however, might not be excluded that the PTKC primers derived from the Populus trichcarpa genome did not fit with the P. canescens cDNA..

Potassium release channels (Fig. 6): In Arabidopsis, one of the two K+ release channels, GORK, is mainly expressed in guard cells, where it is important for stomatal closure. This channel also mediates K+ release into root vascular tissues in general (Ache et al. 2000; Ivashikina et al. 2001). The other one, SKOR is restricted to roots, where it mediates the release of K+ from the xylem parenchyma into the xylem vessels (Gaymard et al. 1998).

PTORK expression was mainly detected in poplar roots, rays and guard cells. In the latter PTORK is probably, like GORK, involved in the stomatal movement (Langer et al.2002, Arend et al. 2005). PTORK2 and PTORK3 share more homology with SKOR.

AKT2-type (Fig. 6): PTK2 from poplar is a phloem localized channel and, like the week voltage dependent ortholog AKT2 from Arabidopsis, able to transport potassium in both directions (Marten et al 1999, Ache et al. 2001, Ivashikina et al. 2001, Langer et al. 2002, Arend et al. 2005). This channel type is predominantly expressed in the phloem and involved in the regulation of the sugar loading into the phloem (Ache et al. 2001, Deeken et al. 2002, Carpaneto et al. 2005).

Sodium related proteins: We found genes discussed as key elements in salt stress response. PtSOS1 (P. tr. Salt overly sensitive) a putative sodium exporter and PtHKT1 a putative high affinity potassium transporter, are orthologs of AtSOS1 and AtHKT1, respectively, which both are located in the plasma membrane of Arabidopsis (Pardo et al. 2006 and references therein). NhaD was described as an Na+/H+ antiporter of halotolerant bacteria (Nozaki et al. 1998, Dzioba et al. 2002) and a gene with sequence similarity to this antiporter class was recently characterized in Populus euphratica (PeNhaD1, Ottow et al. 2005).

Constitutively expressed genes: Besides the actin 2 ortholog (Langer et al. 2002 and 2004) we used poplar ß-tubulin(EMBL AY353093) as housekeeping genes. The high affinity potassium transporter PtKUP did not react to any treatment in previous studies (Langer et al. 2002 and 2004) served also used as a control.

To follow changes in the content of the growth hormone auxin during salt stress we used orthologs of the auxin-amidohydrolase genes ILL3 and IAR3 (LeClere et al. 2002). No significant changes were found (Fig. S1d, e).

To monitor the effect of salt stress on the expression pattern of water channels in roots we used gene expression of PtaPIP1.1, PIP1.2, PIP2.2, PIP2.3, PIP2.4, and PIP2.5 as marker (Marjanovic et al. 2005). In contrast to drought stress (Marjanovic et al. 2005), no significant general alteration of water channel transcripts could be observed under salt stress. Only PIP2.3 expression was 3fold elevated under salt exposure (Fig. S1f).

References:

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Anderson JA, Huprikar SS, Kochian LV, Lucas WJ, Gaber RF (2002) Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. PNAS 89:3736-3740.

Arend M, Stinzing A, Wind C, Langer K, Latz A, Ache P, Fromm J, Hedrich R (2005) Polar localised poplar K+ channel capable of controlling electrical properties of wood forming cells. Planta 223:140-148.

Carpaneto A, Geiger D, Bamberg E, Sauer N, Fromm J, Hedrich R (2005) Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under control of sucrose gradient and pmf. J Biol. Chem. 280:21437-21443.

Deeken R, Geiger D, Fromm J, Koroleva O, Ache P, Langenfeld-Heyser R, SauerN,May ST, Hedrich R (2002) Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis. Planta 216:334-344.

Dzioba J, Ostroumov E, Winogrodzki A, Dibrov P (2002) Cloning, functional expression in Escherichia coli and primary characterization of a new Na+/H+ antiporter, NhaD, of Vibrio cholerae. Molecular and Cellular Biochemistry 229:119-124.

Gambale F, Uozumi N (2006) Properties of shaker-type potassium channels in higher plants. Journal of Membrane Biology210:1-19.

Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J, Michaux-Ferriere N, Thibaud JB, Sentenac H (1998) Identification and disruption of a plant Shaker-like outward channel involved in K+ release into the xylem sap. Cell 94:647-655.

Hirsch R, Lewis BD, Spalding EP, Sussmann MR (1998) A role for the AKT1 potassium channel in plant nutrition. Science 280:918-921.

Ivashikina N, Becker D, Ache P, Meyerhoff O, Felle HH, Hedrich R (2001) K+ channel profile and electrical properties of Arabidopsis root hairs. FEBS Letters 25503:1-7.

Lagarde D, Basset M, Lepetit M, Conejero G, Gaymard F, Astruc S, Grignon C (1996) Tissue-specific expression of ArabidopsisAKT1 gene is consistent with a role in K+ nutrition. Plant Journal 9:195-203.

Langer K, Ache P, Geiger D, Stinzing A, Arend M, Wind C, Regan S, Fromm J,Hedrich R (2002) Poplar potassium transporters capable of controlling K+ homeostasis and K+- dependent xylogenesis. Plant Journal 32:997-1009.

Langer K, Levchenko V, Fromm J, Geiger D, Steinmeyer R, Lautner S, Ache P,Hedrich R (2004) The poplar K+ channel KPT1 is associated with K+ uptake during stomatal opening and bud development. Plant Journal 37:828-838.

LeClere S, Tellez R, Rampey R, Seiichi PTM, Bartel B (2002) Characterization of a family of IAA-amino acid conjugate hydrolases from Arabidopsis. Journal of Biological Chemistry 277:20446-20452.

Marjanovic Z,Nehls U, Hamp R (2005) Mycorrhiza formation enhances adaptive response of hybrid poplar to drought. Annals of the New YorkAcademy of Sciences 1048:496–499.

Marten I, Hoth S, Deeken R, Ache P, Ketchum KA, Hoshi T., Hedrich R (1999) AKT3, a phloem-localized K+ channel, is blocked by protons. PNAS 96:7581-7586.

Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Amtmann A, Maathuis FJ, Sanders D, Harper JF, Tchieu J, Gribskov M, Persans MW, Salt DE, Kim SA, Guerinot ML (2001) Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiology 126:1646-1667.

Mouline K, Very AA, Gaymard F, Boucherez J, Pilot G, Devic M, Bouchez D, Thibaud JB, Sentenac H (2002) Pollen tube development and competitive ability are impaired by disruption of a Shaker K+ channel in Arabidopsis. Genes and Development 16:339-350.

Nozaki K, Kuroda T, Mizushima T, Tsuchiya T (1998) A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus. Biochimica et Biophysica Acta 1369:213-220.

Pardo JM, Cubero B, Leidi EO, Quintero FJ (2006) Alkali cation exchangers: roles in cellular homeostasis and stress tolerance. JEB57:1181-1199.

Supplement Figure S1a: Expression profiles upon salt stressed trees (black bars) and untreated controls (white bars) by real time PCR. Data represent the detailed origin of figure 7. (mean ± SD, n = 6)

Supplement Figure S1b:

Supplement Figure S1c:

Supplement Figure S1d:

Supplement Figure S1e:

Supplement Figure S1f: Water channel transcripts in roots

S-table 2 Primers used in real time PCR

PtACT2fwd
PtACT2rev / 5’-CCCAGAAGTCCTCTT-3’
5’-ACTGAGCACAATGTTAC-3’
TUBfwd
TUBrev / 5‘-gatttgtccctcgcgctgt-3‘
5‘-tcggtataatgacccttggcc-3‘
KPT1fwd
KPT1rev / 5’-TATCCACAGGCAGCTTCA-3’
5’-GCCATCTCGAATGACAC-3’
PKT1fwd
PKT1rev / 5‘-CCCAAAACAGTCATAAT-3‘
5‘-TCAGCGACAAACATAAT-3‘
PKT1bfwd
PKT1brev / 5‘-AACCAACTATTCGACCT-3‘
5‘-CGGGTGAGATGTCGAA-3‘
PTK2fwd
PTK2rev / 5‘-ATGCGATATACACCTG-3‘
5‘-TGCTCACCCTAATACA-3‘
PKT6fwd
PKT6rev / 5‘-AAATAGGATGCAGACGA-3‘
5‘-CAGAAGCGTTCTTACCA-3‘
PTKC1fwd
PTKC1rev / 5‘-TCTCATTGTCCATGCTG-3‘
5‘-CAATACCTGATCTGTTCC-3‘
PTKC2fwd
PTKC2rev / 5‘-CCTTGTTGTCCGTTCC-3‘
5‘-GGGTGGCATACCACTG-3‘
PTORKfwd
PTORKrev / 5‘-TGATGAAGCTCGTATTG-3‘
5‘-GTAACCACCTGAAGATT-3‘
PTORK2fwd
PTORK2rev / 5‘-CATGGGGTGCAAAAGAAC-3‘
5‘-AACTTCTGGCCATCATCG-3‘
PTORK3fwd
PTORK3rev / 5‘-ACACTCCACTTGACGAG-3‘
5‘-GCCACCATCAATCATGTT-3‘
PtKUPfwd
PtKUPrev / 5‘-CCCAAACTTTACAGGA-3‘
5‘-TCGCCTTAATATGAGAGT-3‘
PtKIN2fwd
PtKIN2rev / 5‘-CTGACAATACCCAGAAG-3‘
5‘-CGTAGACATCACCTGTT-3‘
PtHKT1fwd
PtHKT1rev / 5‘-TCTCATTTGCGTCTCAG-3‘
5‘-CCATGTTACTCCACCTT-3‘
PtSOS1fwd
PtSOS1rev / 5‘-ATGGTGGTCTTATGAGC-3‘
5‘-ACGATGCGGTTCTTAAA-3‘
PttDTfwd
PttDTrev / 5‘-TCCAAATTGCGAAGACG-3‘
5‘-CTCCTAGTGTAGGCATGA-3‘
PcILL3fwd
PcILL3rev / 5’-CAATTCACTCCCTGCACTCACC-3’
5’-CAATGCTGCCCCAATTGAAA-3’
PcIAR3fwd
PcIAR3rev / 5’-GTGTCTGTCACCACAATGGACG-3’
5’-CCGAGTACAACGGTTTCTGGAA-3’
PttPIP1.1fwd
PttPIP1.1rev / 5’-TCTTCAACAAGGACAGCG-3’
5’-GAATGGCTCTGATCACAAC-3’
PttPIP1.2fwd
PttPIP1.2rev / 5’-TCTACCACCAGATAGTCATC-3’
5’-AGTTGTTGAGAGATGTGATTC-3’
PttPIP2.2fwd
PttPIP2.2rev / 5’-TTGGCGCTGCTGTCATC-3’
5’-ATGCTGCGGCTGCTAATG-3’
PttPIP2.3fwd
PttPIP2.3rev / 5’-GTTCATCTTGAGAGCAGC-3’
5’-ATCTTTGCGTACGTTCC-3’
PttPIP2.4fwd
PttPIP2.4rev / 5’-TGTTATCTACAACCAAGACAAG-3’
5’-GATGAATTGGTGGTAGAAAG-3’
PttPIP2.5fwd
PttPIP2.5rev / 5’-CTTTATATCACCAATACGTCC-3’
5'-GATGGAAGTATTTCAGACC-3’