A Protein Kinase, Interacting with Two

A Protein Kinase,Interacting with Two Calcineurin B-like Proteins,Regulates K+Transporter AKT1in Arabidopsis

Jiang Xu,1,2Hao-Dong Li,1,2Li-Qing Chen,1,2Yi Wang,1,2Li-Li Liu,1,2Liu He,1and Wei-Hua Wu1,*

1State Key Laboratory of Plant Physiology and Biochemistry,College of Biological Sciences,China Agricultural University, National Plant Gene Research Centre(Beijing),Beijing100094,China

2These authors contributed equally to this work.

*Contact:wuwh@https://www.wendangku.net/doc/b51412620.html,

DOI10.1016/j.cell.2006.06.011

SUMMARY

Potassium is an essential mineral element for plant growth and development.Although it is known that plants absorb and transport K+ through membrane transporters,it remains un-clear how these transporters are regulated. Here we show that the protein kinase CIPK23, encoded by the LKS1gene,regulates K+uptake under low-K+conditions.Lesion of LKS1sig-ni?cantly reduced K+uptake and caused leaf chlorosis and growth inhibition,whereas over-expression of LKS1signi?cantly enhanced K+ uptake and tolerance to low K+.We demon-strate that CIPK23directly phosphorylates the K+transporter AKT1and further?nd that CIPK23is activated by the binding of two calci-neurin B-like proteins,CBL1and CBL9.We pro-pose a model in which the CBL1/9–CIPK23 pathway ensures activation of AKT1and en-hanced K+uptake under low-K+conditions. INTRODUCTION

Potassium is an essential element for plant growth and de-velopment,and it plays crucial roles in many fundamental processes in living plant cells,such as osmoregulation, electrical neutralization,regulation of membrane potential, cotransport of sugars,and so on(Clarkson and Hanson, 1980).Normal plant growth and development require milli-molar K+in the soil or growth medium.However,typical K+ concentration at the interface of roots and soils is within micromolar range(Schroeder et al.,1994).Under low-K+ stress,most plants show K+-de?cient symptoms,typically leaf chlorosis and subsequent inhibition of plant growth and development(Mengel and Kirkby,2001).It is known that different genotypes of a plant species show varied K+utilization ef?ciency(Epstein,1978),which suggests

that K+uptake and translocation in plants is genetically controlled.

Plants absorb and transport K+ions through a number of transporters that vary in kinetics,energy coupling,and reg-ulation(Schroeder et al.,1994;Ve′ry and Sentenac,2003). Although many plant K+transporters have been identi?ed (see the review by Ve′ry and Sentenac,2003and references therein)and some of them have been functionally charac-terized,such as AKT1in K+nutrition(Lagarde et al.,1996; Hirsch et al.,1998;Spalding et al.,1999;Ivashikina et al., 2001)and KAT1in stomatal movements(Ichida et al., 1997),little is known about the regulatory mechanism of these K+transporters.It is well known that some K+chan-nels in animal cells are regulated by speci?c kinases(Jan and Jan,1997).However,it remains unknown whether K+transporters in plant cells are functionally regulated by protein phosphorylation,although circumstantial evidence exists for interaction between protein kinases and K+ transporters(Li et al.,1998;Mori et al.,2000).Che′rel et al.(2002)reported the effect of AtPP2CA on AKT2activ-ity,suggesting possible regulation of plant K+channels by protein phosphorylation and dephosphorylation.Despite some pharmacological studies investigating regulation of plant K+transport systems by protein kinases,these data do not necessarily infer a direct effect of the regulatory proteins on K+transporters(Che′rel,2004).

Here we report the identi?cation and characterization of a regulatory pathway for K+uptake in Arabidopsis,in which a Ser/Thr protein kinase(CIPK23)phosphorylates K+transporter AKT1and enhances K+uptake,particu-larly under low-K+stress.Furthermore,two calcineurin B-like(CBL)proteins were identi?ed as the upstream reg-ulators of CIPK23.In addition,activation of AKT1by CIPK23and CBL1or CBL9was further con?rmed by voltage-clamp recording using a Xenopus oocyte expres-sion system and by patch-clamp recording from root-cell protoplasts.All evidence supports the conclusion that there exists an AKT1-mediated and CBL/CIPK-regulated K+uptake pathway in higher plants that plays crucial roles in plant K+uptake,particularly under K+-de?cient conditions.

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RESULTS

Isolation of Arabidopsis Low-K +-Sensitive Mutant lks1

Arabidopsis low-K +-sensitive (lks )mutants were isolated from EMS-mutagenized M 2seedlings (see Experimental Procedures ).Among a number of putative M 3mutants,lks1showed signi?cant lks phenotype.On low-K +(LK,100m M)medium,the lks1mutants (lks1-1and lks1-2)stopped growing after 4days,and their cotyledons and leaves showed chlorotic phenotype after 10days (Figure 1A).The growth of wild-type seedlings also slowed down under LK conditions,but their leaves remained healthy (Figure 1A).As leaf chlorosis is a typical symptom under K +-de?cient conditions (Mengel and Kirkby,2001),we took leaf chlorosis as the standard lks phenotype for further analysis in this study.We noted that root growth of the lks1mutants maintained a faster pace of elongation than the wild-type during the ?rst 4days after transfer to LK medium (Figure 1A).It should also be noted that the lks phenotype of the mutants was high ammonium dependent (see Figure S2in the Supplemental Data available with this article online),which is consistent with the previous report (Spalding et al.,1999).

Genetic analyses showed that lks1-1and lks1-2harbor monogenic recessive mutations in a nuclear gene and that the two mutations in two independent lines,lks1-1(in Landsberg erecta background)and lks1-2(in Columbia background),are allelic to each other (Figure 1A and Table S1).The analyses of the F 2generations from a cross be-tween either lks1-1and wild-type (Ler)or lks1-2and wild-type (Col)revealed a 3:1segregation ratio of wild-type over lks1(Table S1).

LKS1Encodes CBL-Interacting Protein Kinase 23,which Positively Regulates K +Uptake

Map-based cloning was carried out using an F 2popula-tion,and LKS1was located within BAC clone F12P21of chromosome I (Figure 1B)and ?nally identi?ed to en-code CIPK23(At1g30270;Figure 1B),a member of the CBL-interacting protein kinase family (Luan et al.,2002).CIPKs form a plant-speci?c family of Ser/Thr kinases structurally similar to the SNF1kinase from yeast and AMPK from mammalian systems (Shi et al.,1999).

The

Figure 1.Isolation and Phenotype Test of the lks1(Low-K +-Sensitive)Mutants and Map-Based Cloning of the LKS1Gene

(A)Phenotype comparison between wild-type Arabidopsis (Ler and Col)and lks1mutants (lks1-1,lks1-2,and F 1progenies of \lks1-13_lks1-2and \lks1-23_lks1-1)grown in MS medium and LK (100m M K +)medium for 10days.

(B)Positional cloning of LKS1.The LKS1gene was identi?ed as encod-ing CIPK23(At1g30270).In the LKS1structure,the ?lled boxes repre-sent exons and the lines represent introns.The sites of mutation and T-DNA insertion are shown in red.

(C)RT-PCR veri?cation of LKS1expression in lks1-3(SALK_036154).Elongation factor 1a (EF )was used as loading control.

(D)Phenotype comparison between wild-type Arabidopsis (Col)and lks1-2,lks1-3,and F 1progenies of the mutants after growth in LK me-dium for 10days.

(E–H)Expression of LKS1as determined by LKS1::GUS .(E)Seven-day-old seedling.(F)Adult leaf.

(G and H)Cross-and vertical sections of roots from a 15-day-old seed-ling,respectively.Scale bars in (G)and (H)equal 50m m.

(I)GUS activity in various materials under control (MS)or LK condi-tions.A2-4,A3-6,and C1-5are three independent LKS1::GUS trans-genic lines.Data are shown as means ±SEM (n =4).(J)Northern analysis of LKS1in roots (R)and shoots (S).

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single-nucleotide mutation C1317T in the lks1-1allele (Figure1B)caused a change of Ala199to Val in the amino acid sequence located at the activation loop,a highly con-served domain in CIPKs(Guo et al.,2001).The mutation C3039T in the lks1-2allele led to Leu447-to-Phe substitu-tion in the domain near the C terminus of CIPK23(Fig-ure1B).A T-DNA insertion line(SALK_036154)containing a T-DNA insertion in the seventh intron of LKS1(Figure1B) was named lks1-3in this study.Figure1C shows that the T-DNA insertion in lks1-3disrupted the transcription of the LKS1gene.The lks1-3mutants,like lks1-1and lks1-2,dis-played the lks phenotype(Figure1D),indicating that these mutations are allelic.The F1generations of the lks1-33 lks1-1and lks1-33lks1-2crosses exhibited the same lks phenotype as their parents(Figure1D;Table S1),fur-ther con?rming that the phenotype of lks1alleles resulted from loss of function of the LKS1gene.

Tissue expression pro?les of the LKS1gene were ana-lyzed by creating transgenic plants carrying the LKS1pro-moter-b-glucuronidase(GUS)gene fusion.Histochemical GUS activity analysis indicated that CIPK23was broadly expressed in whole seedlings(Figure1E).In particular, GUS activity was high in vascular bundles(Figures1E–1H).In root tissues,LKS1::GUS activity was strong in cor-tex and endodermis cells(Figures1G and1H)that are in-volved in K+uptake and translocation(Pilot et al.,2003). The results suggest that CIPK23may play an important role in K+uptake and translocation.The quantitative re-sults of GUS enzymatic activity assays showed that CIPK23expression was induced when plants were grown on LK medium(Figure1I).Northern blot analysis further con?rmed that the low-K+treatment strongly induced the expression of LKS1in roots(Figure1J).

To gain more information on the function of CIPK23,we constructed LKS1-overexpressing lines and analyzed the phenotype of these plants.The elevated expression of LKS1mRNA in transgenic lines was veri?ed by Northern blot(Figure2A).All LKS1-overexpressing lines showed very signi?cant low-K+-tolerant phenotype compared to the control lines(Figure2B).The phenotype tests showed

that,in LK medium,the seedlings of the LKS1-overex-pressing lines kept growing even after1month,whereas the lks1mutants died completely after3weeks(data not shown).These results indicate that overexpression of LKS1enhances plant tolerance to low-K+stress.One of the potential mechanisms for this phenotype may be asso-ciated with K+uptake.To test this possibility,we deter-mined the K+uptake rates of the lks1mutants,LKS1-over-expressing lines,and wild-type plants.Two independent LKS1-overexpressing lines showed the highest K+uptake rates among all tested materials(Figure2C),whereas the K+uptake rates of the lks1mutants were the lowest (Figure2C).As shown in Table S2,the LKS1-overexpress-ing lines had the highest V max and the lowest K m and C min for K+uptake among the plants tested,while the lks1mu-tants had much lower V max and increased K m and C min compared to wild-type plants(Table S2).Compared with the lks1-3mutants,the V max for K+uptake of two

LKS1-overexpressing lines increased by about40%,while the K m for K+uptake of these two transgenic lines de-creased to89m M and81m M,respectively,compared to 105m M for wild-type plants and135m M for lks1-3mutants. The results demonstrate that overexpression of LKS1re-sults in signi?cant increases in the maximum K+uptake rate and K+uptake af?nity.The C min for K+uptake of the LKS1-overexpressing lines Col+LKS1-2and Col+LKS1-4 was decreased to33m M and35m M,respectively,com-pared to61m M for the wild-type plants(Table S2),indi-cating that the LKS1-overexpressing plants may initiate K+uptake at much lower[K+]compared to wild-type plants.

Identi?cation of Potassium Channel AKT1as the Downstream Target of CIPK23

Some studies in animal cells have shown that K+trans-porters,such as Kv channels,are regulated by

protein Figure2.Overexpression of LKS1Enhances Arabidopsis Tol-erance to Low-K+Stress by Increasing K+Uptake

(A)Northern analysis of LKS1expression in six independent

SUPER::LKS1transgenic lines.

(B)Phenotype tests of LKS1-overexpression lines in MS(left)or LK me-

dium(right)for10days.

(C)Comparison of K+uptake by K+-depletion methods between vari-

ous materials.Data are presented as means±SEM(n=3).

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kinases (see the review by Jan and Jan,1997).The ?nding that CIPK23participates in the regulation of K +uptake in Arabidopsis led us to hypothesize that CIPK23may regu-late K +uptake by phosphorylation of a K +transporter (or transporters).To test the hypothesis,we ?rst screened for potential downstream targets of CIPK23by examining phenotypes of the various Arabidopsis T-DNA insertion lines for K +transporter genes.The candidates for the tar-gets were selected based on their possible functions in

K +uptake and/or their expression patterns (Ve

′ry and Sentenac,2003).The selected T-DNA insertion mutants included akt1(SALK_071803),atkc1(SALK_140579),kup1(SALK_148762),kup3(SALK_002622),hak5(SALK_005604),etc.The ?anking sequences of the T-DNA insertion sites were veri?ed,and RT-PCR analyses con?rmed that the expression of the selected genes was disrupted in these mutants (Figure 3A;Figure S8).Among these mutants,akt1showed the same lks phenotype as lks1(Figure 3B;Figures S1and S2).The atkc1mutants ex-hibited a phenotype similar to lks1and akt1mutants in their root growth during the ?rst 4days under low-K +stress;however,the leaves of atkc1mutants did not show chlo-rotic phenotype like lks1mutants did (Figure 3B).It has been reported that both AKT1and AtKC1are preferentially expressed in the cortex and endodermis of roots (Lagarde et al.,1996;Pilot et al.,2003)where LKS1promoter activity is strong as well (Figures 1G and 1H).The same or similar phenotypes of the lks1,akt1,and atkc1mutants and similar expression patterns of LKS1,AKT1,and AtKC1genes sug-gested that CIPK23may regulate AKT1and/or AtKC1.To test whether CIPK23would interact with AKT1and AtKC1,we used the yeast two-hybrid method to test the potential interaction of the cytosolic region of AKT1(cAKT1)or AtKC1(cAtKC1)(Pilot et al.,2003)with CIPK23.cAKT1includes 564amino acids from His294to Ser857,and cAtKC1includes 333amino acids from His330to Phe662.As shown in Figure 3C,CIPK23speci?cally inter-acted with cAKT1,but not with cAtKC1,consistent with the observation that the akt1and lks1mutants displayed the same phenotype (Figure 3B).However,CIPK9,which is closely related to CIPK23,did not interact with cAKT1in the yeast two-hybrid assays (Figure S3),which demon-strates the speci?c interaction of CIPK23with

AKT1.

Figure 3.CIPK23Interacts with AKT1

(A)RT-PCR analysis to verify expression of various K +transporter genes in their T-DNA insertion lines.

(B)akt1,an AKT1T-DNA insertion line,showed the same lks pheno-type as lks1mutants after growth in LK medium for 10days.

(C)CIPK23interacted with the cytosolic region of AKT1in yeast two-hybrid assays.The blank pACT2vector was used as control.

(D)BiFC assays of CIPK23interaction with AKT1in vivo.The expres-sion of AKT1alone (AKT1-YN/pUC-SPYCE)was used as control.(E)In vitro phosphorylation of AKT1by CIPK23.AtKC1served as neg-ative control.GST-CIPK23(CA):a constitutively active form of CIPK23fused with GST (77kDa);GST-cAKT1:the cytosolic region of AKT1fused with GST (89kDa);GST-cAtKC1:the cytosolic region of AtKC1fused with GST (64kDa).

(F)Northern analysis of LKS1expression in two independent SUPER ::LKS1transgenic lines in akt1background.

(G)Phenotype tests of the various materials as indicated.Plants were grown in LK medium for 10days.

(H)Decreased K +uptake in akt1mutants.Data are shown as means ±SEM (n =3).

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The in vivo interaction between these two proteins was con?rmed using bimolecular ?uorescence complementa-tion (BiFC)assays (Walter et al.,2004).The BiFC results shown in Figure 3D not only demonstrate the in vivo inter-action between two tested proteins but also show the spe-ci?c localization of the interacting proteins at the plasma membranes (PM).In vitro protein phosphorylation assays further demonstrated the interaction of these two proteins.A constitutively active form of CIPK23(CIPK23(CA))is generated by deleting the binding site of CBLs (FISL/NAF motif)(Guo et al.,2001;Albrecht et al.,2001)and changing Thr190to Asp in the activation loop (Guo et al.,2001).CIPK23(CA)was produced as a GST fusion and was used to phosphorylate the cytosolic region of AKT1and AtKC1.The results showed that CIPK23phos-phorylated cAKT1,but not cAtKC1(Figure 3E).The ge-netic evidence further proved this https://www.wendangku.net/doc/b51412620.html,pared with the transgenic lines of SUPER ::LKS1in a wild-type or lks1background (Figure 2B),the transgenic lines of SUPER ::LKS1in the akt1background (veri?ed by North-ern blot;Figure 3F)did not rescue the lks phenotype (Figure 3G).All of these data are consistent with the hy-pothesis that AKT1functions downstream of CIPK23.Fur-ther evidence demonstrating this conclusion came from the similar kinetic parameters of K +uptake for lks1and akt1mutants (Figure 3H;Table S2).Compared with the V max for K +uptake of wild-type plants,the V max for K +up-take of the lks1-2,lks1-3,and akt1mutants similarly decreased by 23%,23%,and 17%,respectively (Table S2).The K m and C min for K +uptake of these three mutants were also changed to a similar extent (Table S2).

The results of K +content measurements showed that,among the tested materials,the root K +content of wild-type plants (Col)and the LKS1-overexpressing line (Col+LKS1-4)was much higher than that of the lks1and akt1mutants after cultured in MS medium for 4,7,or 10days (Figure 4A),while wild-type plants and the LKS1-overexpressing line had shoot K +content similar to that of the lks1and akt1mutants (Figure 4B).The LKS1-over-expressing line had the highest K +content in both roots (Figure 4C)and shoots (Figure 4D)after growth in LK me-dium for 4,7,or 10days.In addition,the K +content in shoots of lks1and akt1mutants was not signi?cantly dif-ferent from that in shoots of wild-type plants under the normal conditions (Figure 4B),whereas the K +content in shoots of lks1and akt1mutants was signi?cantly different from that in shoots of wild-type plants under low-K +con-ditions (Figure 4D).The results suggest that CIPK23-regu-lated AKT1may play important roles in translocating K +from roots to shoots at the root endodermis and vascular tissues when plants are subjected to low-K +stress.This may explain the chlorotic phenotype in the lks1and the akt1mutants under the low-K +stress.

AKT1-overexpressing lines were also constructed and tested,but no signi?cant low-K +-responsive phenotype was observed (data not shown).It has been

reported

Figure https://www.wendangku.net/doc/b51412620.html,parison of K +Content in Various Plant Materials

(A and B)K +content of roots (A)and shoots (B)after growth in MS medium for the indicated times.

(C and D)K +content of roots (C)and shoots (D)after treatment under low-K +conditions for the indicated times.Data are shown as means ±SEM (n =4).

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that AKT1expression level is high in roots (Lagarde et al.,1996;Reintanz et al.,2002)and that this expression is not induced by K +starvation (Lagarde et al.,1996;Pilot et al.,2003).This,together with our results,suggests that the ex-pression level of AKT1is suf?cient to meet the need for K +uptake under most conditions and that the limitation for AKT1function may lie in its phosphorylation status regu-lated by CIPK23.

CBL1and CBL9Are Involved in Upstream Regulation of CIPK23

Activity of a CIPK is regulated by one or more CBLs (Shi et al.,1999;Albrecht et al.,2001;Luan et al.,2002),a unique family of calcium binding proteins in plants (Kudla et al.,1999;Ishitani et al.,2000).Yeast two-hybrid assays were conducted to identify the particular CBL (or CBLs)that in-teracts with CIPK23.As shown in Figure 5A,CIPK23inter-acted with six members of the CBL family,including CBL1,CBL2,CBL3,CBL5,CBL8,and CBL9.Quantitative b -ga-lactosidase activity assays showed that,among these six CBLs,CBL1and CBL9displayed the strongest interaction with CIPK23(Figure 5B).To test the genetic link between these CBL s and LKS1,the T-DNA insertion lines for these two CBL genes were tested for their potential low-K +sen-sitivity.Expression of CBL1and CBL9was disrupted in

cbl1(SALK_110426)and cbl9(SALK_142774)mutants,re-spectively,and in cbl1cbl9double mutants (Figure 5C;Figure S8).Although the cbl1and cbl9single mutants did not show the typical lks phenotype under LK conditions,the cbl1cbl9double mutants showed the same lks pheno-type as the lks1or akt1mutants (Figure 5D),indicating that CBL1and CBL9have overlapping functions.The low-K +sensitivity of the cbl1cbl9double mutants was comple-mented after the mutants were transformed by CBL1(Fig-ures 5E and 5F)driven by its own promoter.

Furthermore,wild-type plants transformed with SUPER ::CBL1or SUPER ::CBL9(Figure 6A)showed en-hanced low-K +-tolerant phenotype (Figure 6B;Figure S5)similar to the LKS1-overexpressing lines (Figure 2B).How-ever,overexpression of CBL1or CBL9in the lks1mutants did not complement the low-K +sensitivity of lks1mutants (Figure 6B),and overexpression of LKS1in the cbl1cbl9double mutants did not complement the low-K +sensitivity of the cbl1cbl9double mutants (Figures 6C and 6D).This genetic evidence demonstrates that CBL1and CBL9are the upstream positive regulators of CIPK23.

Although CBL5,like CBL1and CBL9,showed strong interaction with CIPK23in our yeast two-hybrid assays (Figures 5A and 5B),the CBL5-overexpressing lines in wild-type background did not show any

signi?cant

Figure 5.Interaction of CIPK23with CBL1and CBL9

(A)Comparative yeast two-hybrid interaction analysis of CIPK23with all members of the Arabidopsis CBL family.

(B)Quantitative results of the b -galactosidase assays of positive clones containing CIPK23/CBL complex.Data are presented as means ±SEM (n =3).(C)RT-PCR analysis of CBL1and CBL9expression in cbl1and cbl9mutants and cbl1cbl9double mutants.(D)Phenotype tests of the various materials as indicated.Plants were grown in MS and LK medium for 10days.(E)RT-PCR analysis of CBL1expression in the complementation line (cbl1cbl9/CBL1)of cbl1cbl9double mutants.

(F)Phenotype tests of the various materials as indicated.Photographs were taken after 10days of growth in MS or LK medium.

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enhancement of low-K +tolerance under low-K +stress (Figure S5).Thus,CBL5may not be involved in regulation of the AKT1-mediated K +uptake pathway.

As mentioned above,the CBLs speci?cally interact with the FISL/NAF domain of the CIPKs (Guo et al.,2001;Al-brecht et al.,2001).As shown in Figure 6E,the FISL-dele-

tion form of CIPK23(CIPK23-FISL)lost its interaction with CBL1or CBL9.The in vivo interaction between CIPK23and CBL1or CBL9was con?rmed using BiFC assays as shown in Figure 6F.In addition,BiFC results also demon-strated that the two pairs of interacting proteins were spe-ci?cally localized at the PM (compare to Figure S6),

which

Figure 6.Functional Con?rmation of Inter-action between CIPK23and CBL1or CBL9

(A)Northern analysis of CBL1or CBL9expression in the various transgenic lines as indicated.

(B)Phenotype tests of the various materials as in-dicated.Photographs were taken after 10days of growth in MS or LK medium.

(C)Northern blot analysis of LKS1expression in the various materials as indicated.

(D)Phenotype tests of the various materials as in-dicated.Photographs were taken after 10days of growth in MS or LK medium,respectively.

(E)Comparative yeast two-hybrid analysis be-tween CBL1or CBL9and CIPK23or CIPK23-FISL (FISL-deletion form of CIPK23).

(F)BiFC assays of CIPK23interaction with CBL1(left panel)and CBL9(right panel)in vivo.The co-expression of CIPK23-FISL with CBL1or CBL9was used as control (bottom images in each panel).

(G)Northern blot analysis of LKS1(CA)expression in the various materials as indicated.

(H)Phenotype tests of the various materials as in-dicated.

(I and J)K +content in roots (I)and shoots (J)of the tested materials as indicated.K +content was measured after low-K +treatment for the indicated times.Data are presented as means ±SEM (n =4).

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Figure7.AKT1-Mediated Inward Currents Are Activated by CIPK23and CBL1in https://www.wendangku.net/doc/b51412620.html,evis Oocytes and Are Regulated by CIPK23in Arabidopsis Root Cells

(A)AKT1current recordings in https://www.wendangku.net/doc/b51412620.html,evis oocytes.Whole-cell currents were recorded in oocytes with injection of cRNA of,from top to bottom,AKT1, AKT1+CIPK23,AKT1+CBL1,AKT1+CIPK23(CA),AKT1+CIPK23+CBL1,AKT1+CIPK9+CBL1,and AKT1+CIPK23+CBL5.

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is consistent with the notion that the CIPK23-CBL1(or CIPK23-CBL9)complex regulates the activity of PM-local-ized AKT1.Furthermore,deletion of the FISL/NAF domain resulted in nonfunctional CIPK23in vivo.Overexpression of LKS1(CA)in wild-type plants or in the cbl1cbl9double mutants did not increase low-K+tolerance of the trans-genic plants(Figures6G and6H).

In addition,overexpression of CBL1or CBL9signi?-cantly increased K+content in both roots and shoots un-der low-K+conditions(Figures6I and6J),while the disrup-tion of CBL1and CBL9expression in the cbl1cbl9double mutants decreased K+content,particularly in shoots(Fig-ures6I and6J).

Transgenic plants harboring CBL1::GUS and CBL9:: GUS constructs were produced to analyze the promoter activity of these two genes in different plant tissues.The GUS histochemical assays showed that expression pat-terns of CBL1and CBL9were similar to CIPK23and were particularly abundant in root tips and vascular bun-dles in the stem and the leaf(data not shown).Given that both CIPK23and AKT1are abundantly expressed in root tips and vascular bundles,these results may further sup-port the notion that CBL1and CBL9regulate CIPK23/ AKT1-mediated K+uptake.

Activation of AKT1-Mediated Inward K+Currents

by CIPK23and CBL1(or CBL9)in Xenopus Oocytes Arabidopsis KAT1-mediated K+currents have been suc-cessfully and repeatedly recorded in Xenopus oocytes (see the review by Che′rel,2004and references therein). However,observation of AKT1-mediated K+currents has not been successful in Xenopus oocytes so far,although Arabidopsis AKT1(Gaymard et al.,1996),and its ortholog in potato,SKT1(Zimmermann et al.,1998),-mediated K+ currents have been recorded in insect cells.Considering that the activity of AKT1is regulated by CIPK23and CBL1(or CBL9),we hypothesized that coexpression of AKT1with CIPK23and CBL1(or CBL9)may activate AKT1-mediated K+currents in oocytes.Not surprisingly, as shown in Figure7A,AKT1-mediated K+inward currents were activated under hyperpolarization conditions after the cRNAs of AKT1,CIPK23,and CBL1were coinjected into the oocytes.Substitution of CBL1cRNA with CBL9 cRNA showed the same results(data not shown).The ab-sence of either CIPK23or CBL1(or CBL9)resulted in com-plete inactivation of AKT1activity(Figure7A).Furthermore, substitution of either CBL1cRNA with CBL5cRNA or CIPK23cRNA with CIPK9cRNA also resulted in complete inactivation of the AKT1currents(Figure7A).In addition, substitution of CIPK23cRNA with CIPK23(CA)cRNA also resulted in inactivation of the AKT1currents,which is con-sistent with our BiFC assay results?nding that FISL/NAF-deleted CIPK23did not interact with CBL1or CBL9 (Figure6F).

The AKT1-mediated inward currents were external K+ dependent,and the AKT1channels were highly selective for K+over Na+(Figures7B and7C).The measured rever-sal potentials(E rev)of AKT1currents at various external [K+]correlated well with the theoretical K+equilibrium po-tentials(Figure7D)under the indicated conditions. Figure7E show the AKT1-mediated inward currents un-der the low-K+conditions.Similar to the previous observa-tions reported by Cao et al.(1992)and Ve′ry et al.(1995),the time-dependent endogenous inward currents were devel-oped when the control oocytes(injected with water)were hyperpolarized to membrane potentials more negative thanà160mV.This hyperpolarization-activated endoge-nous inward current was K+independent(compare the three upper recordings at different external[K+]shown in Figure7E).However,after the coexpression of AKT1to-gether with CBL1and CIPK23,the time-activated and ex-ternal-K+-dependent inward currents were observed at 100m M external K+when the membrane voltages were clamped to more negative than aboutà150mV.The esti-mated E rev for K+currents at100m M external K+was about à150mV.Given that the estimated[K+]in oocytes is be-tween80mM and120mM(Descal,1987),the K+equilib-rium potentials(E K)are betweenà169mV andà179mV at20oC.The shift of E rev to more positive voltages might partially result from hyperpolarization-activated endo-genous membrane permeability to other ions(Descal, 1987),and it might be also partially induced by a possible slight permeability of AKT1to Na+,particularly at low-K+ (100m M)and high-external-Na+(95.9mM)conditions,

(B)CIPK23/CBL1-activated AKT1currents in oocytes were external K+dependent.Currents were recorded from oocytes coinjected with the mixed cRNAs of AKT1,CIPK23,and CBL1.The changes of[K+]in the bath solutions were made by substitution of KCl with NaCl,and the total concentration of K+and Na+in the bath was maintained at96mM.

(C)I-V relationship of AKT1steady-state currents in oocytes versus applied voltages under the various external[K+].The data in(C)and(D)are pre-sented as means±SEM(n=5).

(D)Dependence of the measured E rev(closed circles)of AKT1currents in oocytes on the external K+.The solid line shows the calculated K+equilibrium potentials(assuming the[K+]in oocytes to be about100mM).

(E)External K+-dependent AKT1inward currents in oocytes under low-K+conditions.The three upper recordings show background inward currents in the oocytes injected with water(Control),and the three lower recordings show external-K+-dependent inward currents in oocytes injected with mixed cRNAs of AKT1,CIPK23,and CBL1under various external[K+]as indicated.The changes of[K+]in the bath solutions were made by substitution of KCl with NaCl,and the total concentration of K+and Na+in the bath was maintained at96mM.

(F)Patch-clamp whole-cell recordings in Arabidopsis root-cell protoplasts.The plant materials used for root-cell protoplast isolation are indicated above each recording.The voltage protocols as well as time and current scale bars for the recordings are shown.The methods used for the whole-cell recordings are described in Experimental Procedures.

(G)I-V relationship of the steady-state whole-cell currents in root-cell protoplasts.The data are derived from the recordings as shown in(F)and are presented as means±SEM.

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even though AKT1channels are highly selective for K+ over Na+.

Regulation of AKT1-Mediated Inward K+Currents by CIPK23and CBL1(or CBL9)in Root-Cell Protoplasts To further con?rm the regulation of AKT1-mediated inward K+currents by CIPK23and CBL1(or CBL9)in vivo,patch-clamp whole-cell recordings were conducted using root-cell protoplasts.As shown in Figures7F and7G,inward K+currents were activated in the presence of CIPK23as well as CBL1and CBL9(wild-type)and inactivated in the absence of CIPK23(lks1mutant)or in the double mutant of CBL1and CBL9(cbl1cbl9mutant).The inward K+cur-rents in the root cells of AKT1overexpressors were not sig-ni?cantly different from those in the root cells of wild-type plants(Figures7F and7G),which is consistent with our ob-servation that the AKT1-overexpressing lines did not show enhanced low-K+-tolerant phenotype(data not shown).

DISCUSSION

A Novel K+Uptake Regulatory Pathway

Here we report that CIPK23,a member of CIPK family, serves as a positive regulator of K+transporter AKT1,par-ticularly under low-K+stress,and that two Ca2+sensors of the CBL family,CBL1and CBL9,act as positive upstream regulators of CIPK23.In Figure8,we propose a working model to illustrate this regulatory pathway.Low-K+stress signals may trigger the cytosolic Ca2+signal and lead to activation of calcium sensors CBL1and CBL9,which localize to the PM.The CBL proteins interact with CIPK23 and may recruit CIPK23to the PM,where AKT1is phosphorylated by CIPK23.As the result,AKT1is acti-vated for K+uptake under low-K+conditions.Our results demonstrate that this pathway,at least including CBL1/ CBL9,CIPK23,and AKT1,plays important roles in plant K+acquisition,particularly under low-K+conditions.It is noteworthy that the shoot K+content in the lks1mutants was lower than that in the akt1mutants(Figure4D),which suggests that CIPK23might also regulate other K+trans-porters in addition to AKT1.

The Role of AKT1in K+Uptake and Its Regulation under Low-K+Stress

It is known that AKT1is one of the most important K+trans-porters in Arabidopsis(Lagarde et al.,1996;Hirsch et al., 1998),mediating growth-sustaining uptake of K+into roots over a wide range of external K+concentrations and mainly contributing to high-af?nity K+uptake within low-K+con-centration ranges(Hirsch et al.,1998;Spalding et al., 1999).These?ndings are consistent with our results show-ing that the loss of function of CIPK23or AKT1in the lks1or the akt1mutants,respectively,resulted in decrease of K+ uptake and accumulation and that the leaves of the lks1 and the akt1mutants exhibited chlorotic phenotype under low-K+conditions.AtKC1,as a silent K+channel(Reintanz et al.,2002),interacts with AKT1and modulates K+uptake by forming AtKC1/AKT1heterotetramers(Dreyer et al., 1997;Reintanz et al.,2002;Pilot et al.,2003).Our results demonstrate that AtKC1is not the direct target of CIPK23. In addition to the leaf chlorosis that has been considered as a low-K+-sensitive phenotype throughout this study, the lks1and akt1mutants as well as cbl1cbl9double mu-tants consistently showed faster paces of root elongation than wild-type plants during the?rst4days after the seed-lings were transferred to LK medium.One possible expla-nation for the coappearance of these two phenotypes is that not only does the K+acquisition mediated by AKT1 play critical roles in overall K+uptake,the AKT1-mediated pathway also may somehow contribute to K+translocation from roots to shoots.The decrease of K+content in the lks1and akt1mutant shoots(Figure4D),probably caused by inhibition of K+translocation from roots to shoots,may result in leaf chlorosis.On the other hand,inhibition of K+ translocation from roots to shoots may cause more K+ac-cumulation in mutant roots than in wild-type roots,which may consequently result in faster growth of mutant roots in LK medium.However,understanding the detailed mechanism of this interesting phenotype of the lks1and akt1mutants will require further study.

CBL1and CBL9:Positive Upstream Regulators

of CIPK23

Both CBL1and CBL9localize to the PM when expressed as GFP fusion proteins(Figure S6).Thus,CBL1and CBL9may not only play a role in activation of CIPK23, they may also?rst recruit CIPK23to the PM so that acti-vated CIPK23can be more effective for activation of AKT1.CBL1and CBL9are known to be involved in plant responses to water stress(see the review by Batistic and Kudla,2004).While CBL1is ABA independent,CBL9is in-volved in ABA-mediated signaling.It was

hypothesized 1356Cell125,1347–1360,June30,2006a2006Elsevier Inc.

that the?nal signal output of CBL1/CIPK1versus CBL9/ CIPK1would be determined by the ratio of the two com-plexes in speci?c cells(Batistic and Kudla,2004).For reg-ulation of CIPK23in our study,both CBL1and CBL9are positive upstream regulators,and they have overlapping functions.Disruption of CBL1might lead to more effective CBL9due to removal of cooperation with CBL1and vice versa.On the other hand,overexpression of either CBL1 or CBL9can increase its positive regulation to CIPK23,en-hance K+uptake,and consequently result in a low-K+tol-erant phenotype.

Observation of AKT1Currents in Xenopus Oocytes The functional expression of AKT1and its homologs from different plant species has been investigated in different heterozygous systems,including yeast(Bertl et al., 1997),Sf9insect cells(Gaymard et al.,1996),HEK cells (Fuchs et al.,2005),and Xenopus oocytes(Philippar et al.,1999;Hartje et al.,2000).For the case of AKT1and its homolog expression in oocytes,divergent phenomena have been reported.AKT1was electrically silent when ex-pressed alone in Xenopus oocytes(Dreyer et al.,1997), while it was functionally expressed in yeast(Bertl et al., 1997)and insect cells(Gaymard et al.,1996),and its rice homolog OsAKT1was functionally expressed in HEK cells (Fuchs et al.,2005).The AKT1homologs LKT1(Hartje et al., 2000),SKT1(Hartje et al.,2000),and ZMK1(Philippar et al., 1999)were functionally expressed in oocytes.Before we conduct a thorough investigation to understand these complex and divergent phenomena,we propose several possible explanations based on the available information. First,functional expression of a plant channel protein in a heterozygous system may rely on the presence of an ap-propriate regulator(or regulators)in this expression sys-tem.Different heterozygous cells may contain different functional regulatory components;for instance,yeast cells contain CIPK-like regulatory factor SNF1(Celenza and Carlson,1986),and Xenopus oocytes might contain SNF1-like AMPK(NCBI accession number AAL73336). Second,the regulation mechanisms of homologous chan-nels from different species may vary.This possibly results from differences in their amino acid sequences and conse-quent protein structures,as suggested by the reported cases of the members of AKT1subfamily expressed in oo-cytes(Dreyer et al.,1997;Philippar et al.,1999;Hartje et al., 2000).Third,channel-protein assembly may be important for its functional expression as indicated by Dreyer et al. (1997)and Reintanz et al.(2002).In addition,functional ex-pression of a plant channel protein in a heterozygous sys-tem may depend on its transcription,translation,and post-translational events.

The genetic and biochemical evidence presented in this study demonstrates that activation of AKT1requires coex-istence of CIPK23and CBL1or CBL9.Obviously,AKT1 currents cannot be observed in the absence of CIPK23 and CBL1or CBL9in oocytes.Our success in functionally characterizing AKT1currents for the?rst time using Xenopus oocytes as a heterozygous expression system

provides the direct evidence supporting our conclusion presented here.

Phosphorylation:A Universal Regulatory Mechanism for Plant Ion Transporters?

The previously reported SOS pathway,including SOS3 (CBL4),SOS2(CIPK24),and SOS1(a Na+/H+antiporter), is involved in plant responses to salt stress(Xiong et al., 2002).The SOS pathway is directly related to Na+trans-port and is also associated with K+acquisition(Xiong et al.,2002),probably because of competitive transport between Na+and K+ions.Considering the previous re-ports together with our results here,it is plausible to further hypothesize that regulation of various plant ion trans-porters via their phosphorylation status might be a univer-sal mechanism.

EXPERIMENTAL PROCEDURES

Mutant Isolation,Plant Materials,and Growth Conditions

For the low-K+-sensitive(lks)Arabidopsis mutant isolation,EMS-muta-genized M2seeds were germinated on MS medium containing0.8% (w/v)agar and3%(w/v)sucrose at22oC under constant illumination at60m mol mà2sà1for4days.Then,M2seedlings were transferred to LK medium to screen for the putative lks mutants,whose cotyledons showed chlorotic phenotype after7–10days.The[K+]for the LK me-dium was determined by testing phenotypes of wild-type(wt)Arabi-dopsis seedlings in medium containing different[K+],and the?nal [K+]in the LK medium was100m M throughout this study.The LK me-dium was made by modi?cation of MS medium.KNO3and KH2PO4in MS medium were replaced by NH4NO3and NH4H2PO4,respectively;

0.8%(w/v)K+-depleted agar(see Supplemental Experimental Proce-

dures)was added;and the?nal[K+]was100m M,adjusted by adding KNO3and determined by atomic absorption spectrophotometry.

The putative lks mutants were grown under normal conditions to har-vest M3seeds,and the lks phenotype was further con?rmed with M3 seedlings in LK medium.The selected M3mutants were backcrossed with the wt plants,and phenotypes of their F1and F2generations were tested in LK medium for genetic analyses.The T-DNA insertion lines, including lks1-3(SALK_036154),akt1(SALK_071803),atkc1 (SALK_140579),kup1(SALK_148762),kup3(SALK_002622),hak5 (SALK_005604),cbl1(SALK_110426),and cbl9(SALK_142774)were ordered from the Arabidopsis Biological Resource Center(http:// https://www.wendangku.net/doc/b51412620.html,/abrc/).The information for the molecular con?r-mation of the T-DNA insertion lines is summarized in Figure S8.

For seed harvest and hybridization,Arabidopsis plants were grown in potting soil mixture(rich soil:vermiculite=2:1,v/v)and kept in growth chambers at22oC with illumination at120m mol mà2sà1for a14hr daily light period.The relative humidity was approximate 70%(±5%).

Map-Based Cloning of LKS1

The lks1-1(Landsberg erecta background)mutants were crossed with Columbia wild-type plants,and the lks1-2(Columbia background)mu-tants were crossed with Landsberg erecta wild-type plants to create mapping populations.A total of3700individual F2plants were selected for chromosomal mapping of LKS1.

Vector Constructions and Arabidopsis Transformation

The LKS1::GUS construct was generated by fusing the LKS1promoter fragment(2.57kb)in front of the b-glucuronidase(GUS)coding se-quence in pCAMBIA1381vector.The SUPER::LKS1construct was generated by cloning the coding sequence of LKS1into pBIB vector un-der control of the SUPER promoter(Li et al.,2001).The constructs of

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CBL1::GUS,CBL9::GUS,SUPER::CBL1,SUPER::CBL5,SUPER:: CBL9,and SUPER::LKS1(CA)were generated.For the generation of complementation lines of the cbl1cbl9double mutants,CBL1was cloned into pCAMBIA1300vector.To introduce the Thr190-to-Asp mu-tation and create the FISL/NAF deletion construction for constitutively active CIPK23,primer-based site-directed mutation(50-GGTGTTCCA CAGGTGTCGTGAAGTAACCCATCC-30,50-GGATGGGTTACTTCACG ACACCTGTGGAACACC-30)and the deletion(50-GAAGAAGGACTCA AAACACCAGTAAAACAAATGGGGCTGG-30,50-AGTTTTTCGTTTCAC CAGCCCCATTTGTTTTACTGGTGTTTTGAGT-30)were generated,re-spectively.Arabidopsis transformation with Agrobacterium(strain GV3101)was carried out by the?oral dip method(Clough and Bent, 1998).

GUS Assays

The T3homozygous transgenic plants of LKS1::GUS,CBL1::GUS,and CBL9::GUS were stained by submersion in staining solution(Lagarde et al.,1996).GUS-stained plants and tissues were?xed with2%(w/v) paraformaldehyde in0.1M PBS and,after dehydration,embedded in hydroxyethyl methacrylate(LR White)and sliced into3m m sections. Extractable GUS enzymatic activity was quantitatively determined.

Northern Blot Analysis

For low-K+inducement analysis,7-day-old seedlings were treated in 1/2MS or low-K+-containing medium for12hr.Total RNA of roots or shoots was isolated separately.In assays of mRNA expression in the transgenic lines,total RNA of whole seedlings was isolated.Probes were labeled with32P using random primer labeling reagents(Pharma-cia)and hybridized to RNA blotted onto nylon membrane.Hybridiza-tion signals were imaged using a phosphorimager.

Kinetic Analysis of K+Uptake

For K+-depletion experiments(Drew et al.,1984),7-day-old seedlings were pretreated in starvation solution(0.2mM CaSO4,5mM MES [pH5.7]adjusted with Tris)at22oC for48hr.The tests began5min after transfer of seedlings to the depletion solution(0.25mM KNO3,0.2mM CaSO4,5mM MES[pH5.7]adjusted with Tris).The experiments were conducted at22oC in the light(60m mol mà2sà1).The solution samples were collected at different time points as indicated.For K+content measurements in plant tissues,the4-day-old Arabidopsis seedlings were transferred from MS medium to LK medium and treated for differ-ent times as indicated.The plant tissues were treated in a muf?e fur-nace at575oC for5hr and then dissolved in0.1N HCl.[K+]of the sam-ples was measured by atomic absorption spectrophotometry.

Yeast Two-Hybrid Assays

The coding sequences of CIPK23,CIPK23-FISL,and CIPK9were cloned into pAS2vector.The coding sequences for cytosolic regions of AKT1(from H294)and AtKC1(from H330)(Pilot et al.,2003)as well as the coding sequences for CBLs were cloned into the pACT2 vector.The vectors were transformed into yeast strain AH109for yeast two-hybrid tests.The positive clones of CIPK23/AKT1and CIPK23/ CBL complexes were selected and assayed for b-galactosidase activity.

BiFC Assays

BiFC assays were performed as described previously(Walter et al., 2004).For generation of the BiFC vectors,the coding regions of LKS1 and LKS1-FISL were cloned via XbaI-XhoI into pUC-SPYCE,resulting in CIPK23-YC and CIPK23-FISL-YC;the coding regions of CBL1and CBL9were cloned via BamHI-XhoI into pUC-SPYNE,resulting in CBL1-YN and CBL9-YN;and the coding region of AKT1was cloned via XbaI-XhoI into pUC-SPYNE,resulting in AKT1-YN.Mesophyll pro-toplasts were isolated from5-week-old wild-type plants(ecotype Co-lumbia)and transformed with the tested pairs of constructs as de-scribed(https://www.wendangku.net/doc/b51412620.html,/sheenweb/protocols_reg. html).Fluorescence of YFP in the transformed protoplasts was imag-ined using a confocal laser scanning microscope(LSM510,Carl Zeiss) after the protoplasts were incubated at23oC for12–18hr.

Expression Vectors,Puri?cation of GST Fusion Proteins,

and In Vitro Kinase Assays

The respective coding sequences were cloned into pGEX-4T-2(Phar-macia)to generate GST-CIPK23(CA,constitutively active),GST-cAKT1(the cytosolic region),and GST-cAtKC1(the cytosolic region) (Pilot et al.,2003)constructs.These GST fusion proteins were ex-pressed in E.coli strain BL21and were puri?ed using glutathione Se-pharose4B(Pharmacia).The in vitro kinase assays were performed in buffer(containing,in mM:20Tris-HCl[pH7.2],2.5MnCl2,2.5MgCl2, 0.5CaCl2,1DTT;1m Ci[g-32P]ATP was added in a?nal volume of50m l) at25oC for30min.Reaction aliquots(25m l)were resolved by SDS-PAGE and exposed to the phosphorimager.

In Vitro Transcription and Expression in Xenopus Oocytes

The coding sequences of AKT1,LKS1,CIPK9,CBL1,CBL5,CBL9,and LKS1(CA)were cloned into pGEMHE vector.The cRNAs were tran-scribed in vitro using the T7RiboMAX Large Scale RNA Production Sys-tem(Promega).Oocytes were isolated from https://www.wendangku.net/doc/b51412620.html,evis and injected with cRNAs.The oocytes were injected with distilled water(50nl as control), AKT1cRNA(28ng in50nl),AKT1and CIPK23cRNA mixture(14:14ng in50nl),AKT1and CBL1cRNA mixture(14:14ng in50nl),AKT1and CIPK23(CA)cRNA mixture(14:14ng in50nl),the cRNA mixture of AKT1,CIPK23,and CBL1(12:8:8ng in50nl),the cRNA mixture of AKT1, CIPK9,and CBL1(12:8:8ng in50nl),or the cRNA mixture of AKT1, CIPK23,and CBL5(12:8:8ng in50nl).Before used in voltage-clamp re-cordings,the injected oocytes were incubated at17oC in modi?ed Barth’s solution containing(in mM)88NaCl,1.0KCl,0.91CaCl2, 0.33Ca(NO3)2,0.82MgSO4,2.4NaHCO3,and10HEPES-NaOH(pH 7.5)supplemented with gentamycin(0.1mg/ml)and streptomycin (0.1mg/ml).

Two-Electrode Voltage-Clamp Recording from Xenopus Oocytes

Whole-cell recordings were performed2days after cRNA injection.A two-electrode voltage-clamp technique was applied using a Gene-Clamp500B ampli?er(Axon Instruments)at room temperature ($20oC).The microelectrodes were?lled with3M KCl.The bath solu-tion contained(in mM)96KCl,1.8MgCl2,1.8CaCl2,and10HEPES-NaOH(pH7.2).To analyze the external K+dependence of AKT1 currents,the K+concentration in the bath solution was changed by sub-stitution of KCl with an equivalent amount(in mol)of NaCl.Whole-cell currents were?ltered at1kHz and digitized through a Digidata1322A AC/DC converter using Clampex9.0software(Axon Instruments).

Patch-Clamp Whole-Cell Recording from Root-Cell Protoplasts Root-cell protoplasts were isolated from5-day-old primary roots of Arabidopsis seedlings.The enzyme solution was prepared by dissolv-ing1.5%(w/v)cellulysin(Calbiochem),1.5%(w/v)cellulase RS(Yakult Honsha Co.),0.1%(w/v)pectolyase Y-23(Seishin Pharmaceutical Co.),and0.1%(w/v)BSA in standard solution containing(in mM)10 K+glutamate,2MgC12,1CaC12,350sorbitol,and5MES(pH5.8) adjusted with Tris.The primary roots were cut into small pieces and in-cubated in the enzyme solution at23oC for40min to release root-cell protoplasts.The protoplasts were?ltered through80m m nylon mesh and washed with standard solution.The mixture was centrifuged at 1603g for5min,and the pellet was resuspended with standard so-lution and kept on ice before use.Standard whole-cell recording tech-niques were applied(Hamill et al.,1981).The contents of the bath and pipette solutions for the whole-cell recordings were the same as de-scribed(Ivashikina et al.,2001).The patch-clamp recordings were conducted at about20oC in dim light.Whole-cell currents were re-corded using an Axopatch200A ampli?er(Axon Instruments)con-nected to a computer via an interface(TL-1DMA Interface,Axon Instruments).

1358Cell125,1347–1360,June30,2006a2006Elsevier Inc.

Supplemental Data

Supplemental Data include Supplementary Experimental Procedures, two tables,and eight?gures and can be found with this article online at https://www.wendangku.net/doc/b51412620.html,/cgi/content/full/125/7/1347/DC1/.

ACKNOWLEDGMENTS

We thank Dr.Emily Limam(University of Southern California,USA)for providing us with pGEMHE vector for the experiments using oocytes and Dr.Jo¨rg Kudla(Institut fu¨r Botanik und Botanischer Garten,Uni-versita¨t Mu¨nster,Germany)for providing us with the vectors pUC-SPYCE and pUC-SPYNE for BiFC assays.We also thank Dr.Jiayang Li(Institute of Genetics and Developmental Biology,Chinese Academy of Sciences),Dr.Zhizhong Gong(China Agricultural University),and Drs.Li-Geng Ma and Yan Guo(National Institute of Biological Sci-ences,Beijing)for helpful discussions and suggestions for the experi-mental protocols as well as technical assistance.We are grateful to Dr.Sarah Assmann(Penn State University,USA)and Dr.Sheng Luan (University of California,Berkeley,USA)for critical reading of the man-uscript.This work was supported by competitive National Science Foundation of China Research Grants(#30421002and#30370124), the Chinese National Key Basic Research Project(#2006CB100100), and the Chinese National High Technology Research and Develop-ment Program(#2003AA222010)(W.-H.W.).

Received:January27,2006

Revised:April10,2006

Accepted:June13,2006

Published:June29,2006

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