Download Isolation of AtSUC2 promoter-GFP

The Plant Journal (2003) 36, 931±945
doi: 10.1046/j.1365-313X.2003.01931.x
TECHNICAL ADVANCE
Isolation of AtSUC2 promoter-GFP-marked companion cells
for patch-clamp studies and expression pro®ling
Natalya Ivashikina1,y, Rosalia Deeken1,y, Peter Ache1, Erhard Kranz2, Benjamin Pommerrenig3, Norbert Sauer3 and
Rainer Hedrich1,
1
Lehrstuhl fuÈr Molekulare P¯anzenphysiologie und Biophysik, Julius-von-Sachs-Institut fuÈr Biowissenschaften,
UniversitaÈt WuÈrzburg, Julius-von-Sachs-Platz 2, 97082 WuÈrzburg, Germany,
2
Institut fuÈr Allgemeine Botanik, Zentrum fuÈr Angewandte Molekularbiologie der P¯anzen, UniversitaÈt Hamburg,
Ohnhorststr. 18, 22609 Hamburg, Germany, and
3
Lehrstuhl Botanik II, Molekulare P¯anzenphysiologie, UniversitaÈt Erlangen-NuÈrnberg, Staudtstrasse 5, 91058 Erlangen,
Germany
Received 30 July 2003; accepted 16 September 2003.
For correspondence (fax ‡49 931 8886157; e-mail [email protected]).
y
These authors contributed equally to this work.
Summary
K‡ channels control K‡ homeostasis and the membrane potential in the sieve element/companion cell
complexes. K‡ channels from Arabidopsis phloem cells expressing green ¯uorescent protein (GFP) under
the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RTPCR. Single green ¯uorescent protoplasts were selected after being isolated enzymatically from vascular
strands of rosette leaves. Companion cell protoplasts, which could be recognized by their nucleus, vacuole
and chloroplasts, and by their expression of the phloem-speci®c marker genes SUC2 and AHA3, formed the
basis for a cell-speci®c cDNA library and expressed sequence tag (EST) collection. Although we used
primers for all members of the Shaker K‡ channel family, we identi®ed only AKT2, KAT1 and KCO6 transcripts. In addition, we also detected transcripts for AtPP2CA, a protein phosphatase, that interacts with
AKT2/3. In line with the presence of the K‡ channel transcripts, patch-clamp experiments identi®ed distinct
K‡ channel types. Time-dependent inward rectifying K‡ currents were activated upon hyperpolarization
and were characterized by a pronounced Ca2‡-sensitivity and inhibition by protons. Whole-cell inward
currents were carried by single K‡-selective channels with a unitary conductance of approximately 4 pS.
Outward rectifying K‡ channels (approximately 19 pS), with sigmoidal activation kinetics, were elicited
upon depolarization. These two dominant phloem K‡ channel types provide a versatile mechanism to
mediate K‡ ¯uxes required for phloem action and potassium cycling.
Keywords: Arabidopsis thaliana, phloem, companion cells, EST library, potassium channels, laser microdissection.
Introduction
In higher plants, photosynthates formed in source leaves
are translocated to sink tissues via the sieve element/
companion cell (SE/CC) complexes. Functional SEs are
interconnected by plasmodesmata, which provide a lowresistance pathway for solute transport. Functioning of the
enucleate SE is sustained by metabolically active CCs connected to SE by branched plasmodesmata (van Bel and
Kempers, 1997; Oparka and Turgeon, 1999). Symplasts of
ß 2003 Blackwell Publishing Ltd
SE/CC complexes in Arabidopsis are isolated from adjacent
cells (van Bel and van Rijen, 1994) so that loading and unloading of the phloem require an apoplastic step controlled
by the plasma membrane transporters of the phloem cells.
Because of high K‡ concentration in the phloem sap and
the stimulation of sugar translocation by K‡ (reviewed by
Marschner et al., 1997; Pate and Jeschke, 1995), it has been
proposed that cycling of K‡ and other mineral nutrients
931
932 Natalya Ivashikina et al.
between source and sink tissues is required to maintain both
the membrane potential of SE/CC and the osmotic potential
of the phloem (van Bel and Kempers, 1997; Marschner et al.,
1996, 1997, and references therein). From changes in the
phloem electrical potential in response to different apoplastic K‡ concentrations, potassium channels were supposed to
provide a major pathway for K‡ entry and release in the
phloem (Ache et al., 2001). Among the ®ve plant Shaker K‡
channel subfamilies, members of the AKT2 subfamily were
found to be expressed in phloem tissues of Arabidopsis
thaliana (Lacombe et al., 2000; Marten et al., 1999), Zea
mays (Bauer et al., 2000; Philippar et al., 1999), Vicia faba
(Ache et al., 2001) and Populus (Langer et al., 2002). The
AKT2 gene is under the control of light and sugars in line
with a role in regulating the entry of photosynthates into
the phloem (Ache et al., 2001; Deeken et al., 2000, 2002).
Deeken et al. (2002) recently demonstrated that the control
of the SE/CC membrane potential and sugar loading was impaired in AKT2-de®cient plants and that the sucrose content
of their sieve tubes was only 50% of that in wild-type plants.
Heterologously expressed AKT2/3 channels are blocked
by protons and Ca2‡ (Lacombe et al., 2000; Marten et al.,
1999) and display two distinct gating modes characterized
by time-dependent and instantaneous current components
(Dreyer et al., 2001). In a yeast two-hybrid screen, the
protein phosphatase AtPP2CA was identi®ed as an interacting partner to AKT2/3 (Vranova et al., 2001). Co-expression of AtPP2CA with AKT2 in animal cells increased inward
recti®cation of the channel (Cherel et al., 2002). In addition
to AKT2/3, KAT2 channels have also been found in the
phloem of A. thaliana Columbia ecotype (Pilot et al.,
2001). When expressed in Xenopus oocytes, KAT2 mediates inward rectifying currents with a single channel conductance of approximately 7 pS. In contrast to AKT2/3,
KAT2 homomers are proton-activated and Ca2‡-insensitive
(Pilot et al., 2001; Lacombe, personal communication).
Previous studies showed that Arabidopsis lines expressing GFP can be used to study the electrical properties of
certain cell types of the root (Kiegle et al., 2000; Maathuis
et al., 1998). In our investigation into the nature and properties of phloem-expressed K‡ channels, we applied RT-PCR
and patch-clamp techniques to phloem protoplasts that had
been isolated from transgenic Arabidopsis plants expressing GFP under control of the companion cell-speci®c
AtSUC2 promoter (Imlau et al., 1999). Individual GFPexpressing protoplasts were selected to analyse their
K‡ channel transcript composition and K‡-dependent electrical properties after being isolated enzymatically from
vascular strands. We identi®ed members of the A. thaliana
K‡ channel superfamily capable of controlling phloem
potassium uptake, release and cycling. In addition to ion
channel genes, we also discuss the unique companion cell
expression pro®le (EST collection) with respect to SE/CC
autonomy, stress tolerance and hormone action.
Results
Isolation of phloem- and mesophyll-specific cDNAs
The successful application of the laser capture microdissection technique to plant cells has been reported recently
(Asano et al., 2002; Kerk et al., 2003; Nakazono et al., 2003).
Differentially expressed genes were identi®ed in vascular
tissues from maize and rice phloem tissues composed of
functionally different cell types. These microarray studies
and EST collections, however, lacked expression pro®les
for the companion cell-speci®c proton ATPase and sucrose/
proton symporter, as well as for the phloem-localized
potassium channels. To bridge this gap, we applied the
laser microdissection and pressure catapulting (LMPC)
technique to the vascular-rich ¯ower stalk of Arabidopsis.
Following the excision of about 150 individual phloem
sectors (Figure 1a), mRNA was isolated and probed for
the presence of transcripts for a phloem K‡ channel,
sucrose carrier and H‡ pump. Using RT-PCR with primers
speci®c for the well-known phloem transporters, we identi®ed three phloem-speci®c expressed genes SUC2
(sucrose carrier), AKT2 (K‡ channel) and AHA3 (H‡ pump;
Figure 1b; Imlau et al., 1999; Marten et al., 1999; Truernit
and Sauer, 1995). These results were in line with the ones of
Doering-Saad et al. (2002), who reported on mRNA in barley phloem sap. In addition to these transcripts, we also
detected expression of SUC3, a gene encoding another
sucrose transporter (Figure 1b), which has been previously
found in the phloem periphery (Meyer et al., 2000). The
presence of SUC3 suggested that the mRNA originated
from different phloem cell types such as sieve elements,
companion cells and parenchyma cells. Because companion cell transcripts could not be isolated by LMPC without
contamination by mRNA from other cell types, we then
enzymatically isolated companion cells expressing GFP
under the control of the AtSUC2 promoter. Vascular strands
were excised from major veins of fully developed rosette
leaves from transgenic A. thaliana AtSUC2 promoter-GFP
plants (Figure 2a). Release of protoplasts from phloem tissue was observed 1 h after enzyme application (Figure 2b).
With blue light excitation, 20±30% of the protoplast population showed green ¯uorescence (Figure 2c,d). One hundred
and forty-®ve individual ¯uorescent protoplasts containing
chloroplasts and/or vacuole(s) were collected under an
epi¯uorescence microscope and, after washing, were transferred to PCR lysis buffer using glass micropipettes. As a
control, we collected 150 non-¯uorescing mesophyll protoplasts. Real-time RT-PCR was used to quantify marker
gene transcripts in the individual cDNAs in order to both
characterize the protoplast type and to test for contamination of phloem protoplasts by mesophyll cells (and
vice versa). Transcripts of the sucrose transporter SUC2
(Truernit and Sauer, 1995) and the proton ATPase AHA3
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
Isolation of companion cells for patch-clamp and expression profiling
933
Figure 1. RT-PCR analysis of phloem samples obtained by LMPC technique.
(a) Arabidopsis phloem (left) was excised from in¯orescence stalk cross-sections by a laser beam (middle) and catapulted into a reaction cap (right) for further
analysis.
(b) RT-PCR products ampli®ed from RNA of LMPC-excised phloem segments. Identi®cation of SUC2, AKT2 and AHA3 transcripts in phloem samples only. M ˆ l
PstI marker, C ˆ reference transcript.
(De Witt and Sussman, 1995) were detected in the phloem
fraction only, whereas SUC3 transcripts were found in
both phloem and mesophyll protoplasts (cf. Figures 1b
and 3a). Quanti®cation, however, revealed that SUC3
expression was most prominent in the mesophyll cells,
while only background levels (7%) were found in the
phloem (Figure 3b, cf. Meyer et al., 2000). From the distribution of these marker gene transcripts, we concluded
that the preparations of phloem and mesophyll cells were
not cross-contaminated.
Companion cell cDNA library and EST collection
Figure 2. Isolation of phloem protoplasts.
(a) GFP ¯uorescence in the vascular tissue of a rosette leaf from AtSUC2
promoter-GFP plants.
(b) Release of ¯uorescent protoplasts from phloem tissue during enzymatic
digestion.
(c, d) Protoplasts derived from vascular strands. Bright ®eld image (c) and
epi¯uorescence image (d).
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
We used the mesophyll-free companion cell mRNA to
generate a cDNA library and partially sequenced 2000
individual clones. About 56% of the Arabidopsis gene
sequences were identi®ed and they formed the foundation
for a steadily increasing EST collection. Within this group,
33% encoded unknown proteins while others encoded
previously described phloem-expressed genes (Nakazono
et al., 2003), as well as a unique selection of genes most
likely required for sieve tube function and survival, hormone action and pathogen defence. Singlets as well as
contigs of up to 40 identical sequences were present within
the latter fraction (Table 1). Putative functions were
assigned to the cDNAs when predictions and scores were
identical in all three data bases used for analysis: BLASTX
against Swissprot plant proteins, BLASTN against Arabidopsis coding sequences ( introns, UTRs) and BLASTN
against Arabidopsis genes (‡introns, ‡UTRs). Finally, genes
were subgrouped into 10 functional clusters, which were
then related to the number of identi®ed cDNAs (Figure 4):
934 Natalya Ivashikina et al.
references therein), brassinosteroid insensitive1 (BRI1),
involved in the signal transfer of brassinosteroids (Wang
et al., 2001) and PIN3, a component of the lateral auxin
transport system regulating tropic growth (Friml et al.,
2002). In contrast, DIR1, involved in systemic acquired
resistance (Maldonado et al., 2002), and the polar auxin
transport-related PIN6 were detected only in companion
cells. The expression patterns of these two PIN genes were
in line with the differential expression of PIN3 and PIN6
deduced from the analysis of Arabidopsis mutants defective in interfascicular ®bre differentiation (Zhong and Ye,
2001).
K‡ channel transcripts in companion cells
Figure 3. Marker transcripts of phloem and mesophyll cells.
(a) RT-PCR products of mesophyll cell (MC) and companion cell (CC) protoplasts separated in an 1% tris-borate-ethylenediaminetetraacetic acid
(TBE)±agarose gel.
(b) Relative transcript rates calculated from quantitative real-time PCR.
Note the RT-PCR product of SUC3 in the phloem shown in (a) represents 7%
of the expression in the mesophyll after quanti®cation by real-time PCR.
redox regulation (R ˆ19.4%), stress (S ˆ 11.0%), defence
(D ˆ 2.3%), metabolism (M ˆ 10.1%), transcription and
translation (TT ˆ 10.1%), hormones and signalling
(HS ˆ 9.1%), transport and membranes (TM ˆ 1.9%), cell
wall (CW ˆ 0.9%), photosynthesis (PS ˆ 0.9%) and cytoskeleton (CS ˆ 0.5%). Among the 563 genes analysed in
detail, 454 were singlets, 59 were doublets, 17 were triplets,
22 genes appeared 4±9 times and 11 genes appeared 10±40
times (Table 1). Six types of sequences were the most
abundant among the cDNAs analysed. They showed
homology with metallothionin 2b (40), a water stressinduced protein (26), thioredoxin h (18), a translation
initiation factor (18), dihydrofolate reductase (17), 12oxophytodienoate reductase (OPR1; 12), a low temperature and salt-responsive protein (11) and the heat shock
protein 17 (10).
In addition to the phloem markers SUC2, AKT2 and AHA3,
which were identi®ed by RT-PCR and did not yet appear in
the EST collection, we also searched for rare transcripts of
differentially expressed genes involved in signal transduction and allocation. These included auxin transporters,
ethylene and brassinosteroid receptors and a putative lipid
transfer protein defective in induced resistance (DIR1).
In the phloem-free mesophyll mRNA fraction, we found
ethylene insensitive4 (EIN4; Chang and Stadler, 2001, and
The companion cell EST collection did not contain ion
channel sequences (Table 1), so we used quantitative RTPCR to analyse the K‡ channel transcript pro®le in two
cDNA populations derived from non-cross-contaminated
companion and mesophyll cell protoplast samples. Among
the Shaker-like K‡ channel transcripts, KAT1 and AKT2
dominated the companion cell fraction, ATKC1 appeared
in mesophyll protoplasts and AKT1 transcripts were rare in
both cell types of the Arabidopsis ecotype C24 (Figure 5a). It
should be noted that in ecotype Col-0, KAT2 has also been
identi®ed as a phloem K‡ channel by KAT2 promoter-GUS
studies (Pilot et al., 2001). Further analyses are required to
clarify whether it is KAT1 or KAT2, which shares 72%
identical amino acids and exhibits identical electrical properties, that is expressed in the phloem of other Arabidopsis
ecotypes. Transcripts of the protein phosphatase AtPP2CA,
which has been shown to interact with the AKT2/3 channel
(Cherel et al., 2002; Vranova et al., 2001), were detected in
companion cells and also in non-AKT2 expressing cells
such as mesophyll, hypocotyl cortex, root hairs and
A. thaliana tumours (our unpublished data). The expression of KCO1, with small amounts of KCO5 and KCO6,
in mesophyll protoplasts was in line with the results of
SchoÈnknecht et al. (2002) for the dominant members of the
KCO family. KCO6, the only member of this channel family
in the phloem, showed the highest KCO expression level so
far measured in any tissue type (Figure 5a and our unpublished data). If the actin-based transcript abundance of
AKT2, KCO6 and KAT1 are compared for rosette leaves,
phloem-rich ¯ower stalks and companion cell protoplasts,
then AKT2 and KAT1 transcripts increased with the number
of companion cells in a given fraction (Figure 5b). KCO6
mRNA was also most abundant in the companion protoplast fraction, but was lower in the stalks than in the leaves.
Electrical properties of K‡ channels in the phloem
We characterized the electrical properties of K‡ channels
by performing patch-clamp measurements on KAT1-,
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
Isolation of companion cells for patch-clamp and expression profiling
935
Table 1 Representative genes selected from an EST collection of Arabidopsis companion cells grouped into 10 functional clusters
Singlets and contigs
ESTs
Putative gene identification
References
(a)
Cytoskeleton
ARAB.90.C1
A011-a12.TEx5_085.ab1
A012-e09.TEx5_067.ab1
A016-c10.TEx5_071.ab1
2
1
1
1
Actin de-polymerizing factor 6
Tubulin beta-2/beta-3 chain
Microtubule-associated protein
Dynein light subunit lc6
Cell wall
A010-b01.T3_009.ab1
ARAB.3.C1
ARAB.57.C1
2
5
2
Proline-rich protein M14
Arabinogalactan±protein
Pollen coat protein
Defence
ARAB.32.C1
ARAB.44.C1
ARAB.61.C1
A010-a01.T3_001.ab1
A012-b03.TEx5_025.ab1
A014-d09.TEx5_074.ab1
A016-c07.TEx5_051.ab1
ARAB.107.C1
A016-a07.TEx5_050.ab1
A015-g08.TEx5_056.ab1
ARAB.102.C1
A012-h01.TEx5_012.ab1
A001-f01.T3_011.ab1
2
2
1
1
1
1
1
3
2
2
1
2
2
Beta-glucosidase
Cystatin
Harpin-induced protein
Jacalin
Remorin
Bax inhibitor-1
AIG2
Myrosinase
Lectin
Lectin PP2
Component of aniline
Disease-resistance protein
Cysteine proteinase inhibitor B
Hormones and signalling
A007-d11.TEx5_091.ab1
ARAB.22.C1
ARAB.30.C1
A019-f07.TEx5_059.ab1
ARAB.40.C1
ARAB.31.C1
ARAB.47.C1
A011-g06.TEx5_040.ab1
ARAB.39.C1
ARAB.77.C1
A003-d09.T3_074.ab1
A002-h05.T3_044.ab1
A006-c03.T3_018.ab1
A003-a04.T3_021.ab1
A014-d01.TEx5±010.ab1
ARAB.93.C1
A017-c07.TEx5_051.ab1
A016-h07.TEx5_061.ab1
A004-b11.T3_089.ab1
ARAB.50.C1
A017-h05.TEx5_044.ab1
A013-a07.TEx5_049.ab1
A013-b05.TEx5_041.ab1
ARAB.91.C1
ARAB.63.C1
ARAB.65.C1
ARAB.75.C1
A007-f06.TEx5_048.ab1
1
3
7
1
5
2
12
1
4
2
2
1
1
1
1
2
1
1
1
2
1
1
1
2
11
4
3
1
ACC synthase (AtACS-6)
ACC oxidase
Gibberellin-responsive protein
Ethylene-type zinc finger protein
Ethylene-responsive transcriptional co-activator
Ethylene-responsive protein
12-oxophytodienoate reductase (OPR1)
12-oxophytodienoate-10,11-reductase
Ripening-related protein
Calcium-binding protein
Calmodulin-3
ENOD20
S-adenosylmethionine decarboxylase
S-adenosylmethionine synthase 2
Molybdopterin synthase (CNX2)
Subtilisin-like serine protease
Two-component phosphorelay mediator
Mitogen-activated protein kinase
Receptor Ser/Thr protein kinase
Protein phosphatase 2A
Protein phosphatase 2C
COP1-interacting protein CIP8
Symbiosis-related protein
Caltractin
Low temperature and salt-responsive protein
Cold and ABA inducible protein kin1
Pheromone receptor
NAC-domain protein
Metabolism
ARAB.9.C1
ARAB.83.C1
A006-b01.T3_009.ab1
ARAB.18.C1
ARAB.105.C1
A013-f04.TEx5_031.ab1
17
9
1
2
4
1
Dihydrofolate reductase
Steroid 5-alpha reductase
Tropinone reductase
Cytosolic triosephosphate isomerase
Peptidylprolyl isomerase ROC1
Anthranilate
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
(g)
(e), (f)
(c)
(a)
(c), (b)
(c)
(b)
(a)
(b)
(h)
(a)
936 Natalya Ivashikina et al.
Table 1 continued
Singlets and contigs
ESTs
Putative gene identification
References
A012-a12.TEx5_085.ab1
ARAB.25.C1
ARAB.74.C1
A017-d04.TEx5_030.ab1
A005-c07.T3_050.ab1
A019-c05.TEx5_034.ab1
A006-c06.T3_038.ab1
A018-a07.TEx5_049.ab1
A008-g08.TEx5_065.ab1
A002-f06.T3_047.ab1
A015-h11.TEx5_092.ab1
A002-h01.T3_012.ab1
A013-g11.TEx5_084.ab1
ARAB.52.C1
A002-d12.T3_094.ab1
A007-e04.TEx5_023.ab1
A007-f03.TEx5_027.ab1
A012-f03.TEx5_027.ab1
A017-h09.TEx5_077.ab1
A017-g07.TEx5_053.ab1
A012-d03.TEx5_026.ab1
A019-g04.TEx5_024.ab1
A003-f02.T3_015.ab1
A015-d12.TEx5_094.ab1
A012-d07.TEx5_058.ab1
1
2
2
1
1
1
1
1
2
2
2
1
1
2
1
1
1
1
1
1
1
1
1
2
1
Anthranilate synthase, alpha subunit
Nitrilase
Sucrose-UDP glucosyltransferase
Serine-O-acetyltransferase
Aspartate aminotransferase (Asp3)
Glycosyl transferase
Steroid-binding protein
Steroid sulfotransferase
Glyceraldehyde-3-phosphate dehydrogenase
Mitochondrial proline oxidase
Ubiquinol cytochrome-c
Anthocyanidin synthase
Mitochondrial ATP synthase delta chain
Epoxide hydrolase (ATsEH)
Flavanone 3-hydroxylase
Gamma glutamyl hydrolase
Pyruvate kinase
Phosphoribulokinase
Choline kinase GmCK2p
Fructokinase
Inorganic pyrophosphatase
Nicotianamine synthase
ADP-ribosylation factor
Acyl CoA-binding protein
Glutamate-/aspartate-binding peptide
Photosynthesis
ARAB.37.C1
A017-c05.TEx5_034.ab1
A018-h06.TEx5_048.ab1
ARAB.97.C1
2
1
1
2
Photosystem II reaction center (6.1 kDa)
PSI type III chlorophyll a/b-binding protein
Protochlorophyllide reductase
Ribulose bisphosphate carboxylase, SSU
Redox regulation
ARAB.1.C1
ARAB.0.C1
ARAB.60.C1
ARAB.88.C1
A018-h05.TEx5_044.ab1
ARAB.5.C1
A019-h09.TEx5_076.ab1
A011-h07.TEx5_060.ab1
A003-g02.T3_008.ab1
A012-h10.TEx5_080.ab1
A019-g12.TEx5_088.ab1
40
18
7
4
5
4
1
1
1
1
1
Metallothionein 2b
Thioredoxin
Glutathione-S-transferase (GST6)
Glutaredoxin
Quinone oxidoreductase
Blue copper-binding protein
Superoxidase dismutase
Copper/zinc superoxide dismutase
Cytochrome P450 monooxygenase
NADH dehydrogenase
Delta 9 desaturase
(b)
Stress
ARAB.28.C1
ARAB.43.C1
ARAB.27.C1
ARAB.27.C2
A014-h05.TEx5_044.ab1
A015-a08.TEx5_053.ab1
A017-b12.TEx5_094.ab1
A003-g07.T3_052.ab1
ARAB.99.C1
A014-g07.TEx5_052.ab1
ARAB.23.C1
26
5
10
3
2
2
1
7
3
1
2
Water stress-induced protein
Dehydrin Xero2
Heat shock protein 17
Heat shock protein 18
Heat shock protein DnaJ
Heat shock protein 70
Heat shock protein 81-2
Small heat shock protein
Cytosolic cyclophilin (ROC3)
GTP-binding protein GB2
Stress-induced protein
(b)
Transport and membranes
A004-a10.T3_069.ab1
A011-f10.TEx5_079.ab1
A011-e09.TEx5_067.ab1
2
1
1
Sugar transporter
Amino acid transport protein AAP2
Putative AAA-type ATPase
(b)
(b)
(b)
(b)
(b)
(a)
(c), (d)
(c)
(b)
(a)
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
Isolation of companion cells for patch-clamp and expression profiling
937
Table 1 continued
Singlets and contigs
ESTs
A011-f01.TEx5_011.ab1
A018-a12.TEx5_085.ab1
A008-g11.TEx5_085.ab1
A006-f06.T3_047.ab1
A011-c07.TEx5_050.ab1
A019-e10.TEx5_071.ab1
A007-b10.TEx5_078.ab1
A012-f02.TEx5_015.ab1
1
1
2
1
1
1
1
1
Transcription and translation
A002-c04.T3_022.ab1
A003-c07.T3_050.ab1
A008-b12.TEx5_094.ab1
A018-e01.TEx5_003.ab1
A011-f07.TEx5_059.ab1
ARAB.110.C1
ARAB.103.C1
A011-a10.TEx5_069.ab1
A001-d01.T3_010.ab1
A019-e01.TEx5_003.ab1
A008-e03.TEx5_019.ab1
A012-f10.TEx5_079.ab1
A013-a01.TEx5_001.ab1
A011-e03.TEx5_019.ab1
A008-a07.TEx5_050.ab1
A012-g12.TEx5_088.ab1
ARAB.10.C1
ARAB.45.C1
A003-b05.T3_041.ab1
A007-f02.TEx5_015.ab1
A003-e06.T3_039.ab1
A017-c02.TEx5_006.ab1
A012-d12.TEx5_094.ab1
A019-c06.TEx5_038.ab1
ARAB.96.C1
A010-g01.T3_004.ab1
A014-a01.TEx5_001.ab1
ARAB.80.C1
ARAB.67.C1
A002-b05.T3_041.ab1
A015-a01.TEx5_001.ab1
A012-f06.TEx5_047.ab1
A013-f03.TEx5_027.ab1
1
2
1
1
1
2
3
1
1
1
1
1
1
1
1
1
18
4
1
1
1
1
1
1
3
1
1
4
4
1
1
1
1
Putative gene identification
References
Mitochondrial phosphate translocator
Metal ion transporter
Outer membrane lipoprotein
Coated vesicle membrane protein
Coatomer delta subunit
Synaptobrevin
Snap25a
Geranylgeranylated protein
(d)
Transcription initiation factor TFIID-1
Transcription factor GT-3a
bZIP transcription factor
G-box binding bZIP transcription factor
Nucleic acid-binding protein
RNA polymerase II
Zinc finger protein (PMZ)
DNA helicase TPS1
Ribonuclease, RNS1
Histone H2B
Histone H2A
Histone H3.3
Histon H3
DNA damage-inducible protein
DNA-3-methyladenine glycosidase
Glutamyl-tRNA reductase
Translation initiation factor
Eukaryotic initiation factor 5A
Translation elongation factor eEF-1
Ribosomal protein S3a
30S ribosomal protein S20
60S ribosomal protein L36
40S ribosomal protein S20-like
50S ribosomal protein L33
60S ribosomal protein L10
60S acidic ribosomal protein P2
60S acidic ribosomal protein P0
Polyubiquitin (UBQ14)
Ubiquitin-conjugating enzyme E2
ATP-dependent protease, proteolytic Clp
PCI domain protein proteasome
Multicatalytic endopeptidase complex
Signal peptidase subunit
(a)
(b)
(b)
(b)
(b)
(c), (d)
(b)
EST numbers indicate identical cDNA clones within a contig. References confirming the phloem specificity of the genes listed in the table:
(a), Asano et al. (2002); (b), Nakazono et al. (2003); (c), Walz et al. (2002); (d), Hoffmann-Benning et al. (2002); (e), Husebye et al. (2002); (f),
Chen et al. (2001); (g), Sasaki et al. (2001); (h), Ruiz-Medrano et al. (1999).
AKT2- and KCO6-expressing companion cell protoplasts in
the whole-cell and outside-out mode. Among the companion cell protoplasts, there were two major populations:
some cells dominated by inward currents and others dominated by outward currents. When, in the whole cell con®guration, protoplasts were clamped at 48 mV, with
150 mM K‡ in the pipette and 30 mM K‡ in the bath,
hyperpolarizing voltages, negative to 108 mV, elicited
slowly activating inward currents (n ˆ 11; Figure 6a). Tail
K‡ currents reversed direction around the Nernst equilibrium potential for potassium (EK ˆ 41 mV; Figure 6b).
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
Time-dependent single channel ¯uctuations were observed
at hyperpolarizing voltages in outside-out patches excised
from protoplasts dominated by inward rectifying currents
(Figure 6c). With 150 mM K‡ in the pipette and 30 mM K‡ in
the bath, inward channels were characterized by a unitary
conductance of about 4 pS. Under these conditions,
the single channel current reversed direction at 40 mV,
close to the EK ˆ 41 mV (Figure 6d). When protoplasts
were exposed to increasing Ca2‡/K‡ ratios in the bath, a
pronounced voltage-dependent block of the inward recti®er
was observed (Figure 6e). Ca2‡-dependent decrease in K‡
938 Natalya Ivashikina et al.
Figure 4. Relative distribution of functional gene clusters.
Functional gene clusters of companion cell ESTs grouped by their putative
gene function: N, unknown function; R, redox regulation; S, stress; D,
defence; M, metabolism; TT, transcription and translation; HS, hormones
and signal transduction; TM, transport and membranes; CW, cell wall; PS,
photosynthesis; CS, cytoskeleton.
(a)
2487
Relative transcript number
2500
2000
1500
978
1000
792
500
294
3
KAT1
Relative transcript number
389
162
0
(b)
mesophyll cells
companion cells
1899
1 3
12 12
1
51 1
KAT2
AKT1
AKT2
ATKC1 AtPP2CA KCO1 KCO5
0
17 0
KCO6
3000
2500
AKT2
KCO6
KAT1
2000
1500
1000
500
0
Leaf
Stalk
CC
Figure 5. Phloem- and mesophyll-speci®c ion channel gene pro®le.
(a) Relative transcript number of Shaker and KCO channels, and phosphatase PP2CA quanti®ed by real-time-PCR on protoplast cDNA. GORK and
SKOR transcripts were not detectable. One representative of three separate
experiments is shown.
(b) Expression level of phloem channels quanti®ed by real-time PCR
obtained from RNA of whole leaves, phloem-rich in¯orescence stalks and
isolated companion cell protoplasts (CC).
current amplitudes at hyperpolarizing voltages was accompanied by a shift in voltage dependence of the inward
recti®er towards more positive potentials. Voltage-dependent Ca2‡ block increased when K‡ concentration was
lowered from 30 to 10 mM (Figure 6e). A similar block of
inward K‡ channels has previously been described for
Z. mays, V. faba, Solanum tuberosum, Nicotiana tabacum
and A. thaliana guard cells (Dietrich et al., 1998; FairleyGrenot and Assmann, 1992). However, when compared to
inward recti®ers from root hairs and guard cells, phloem K‡
channels exhibited an opposite pH-sensitivity. A change in
the pH of the external solution from 7.0 to 5.6 shifted the
voltage dependence of the phloem inward recti®er towards
more negative potentials (Figure 6f). A decrease in external
pH caused a 20 8 mV shift in half activation potential
(V1/2) of the Boltzmann curve. Under these conditions, the
voltage dependence of the guard cell inward recti®er shifts
towards more positive potentials (BruÈggemann et al., 1999).
Among the plant Shaker K‡ channels so far identi®ed, only
AKT2/3-type channels from Arabidopsis, maize and poplar
were blocked by protons (Bauer et al., 2000; Lacombe et al.,
2000; Langer et al., 2002; Marten et al., 1999). It should be
noted that proton-blocked inward recti®ers have also been
recorded in Samanea saman pulvinus protoplasts, which
also express AKT2/3-like channels (Moshelion et al., 2002;
Yu et al., 2001). The companion cell inward recti®er thus
seems to share properties with AKT2/3 (H‡ and Ca2‡ inhibition) and KAT1 (strong inward recti®cation).
When depolarizing voltages were applied to companion
cell protoplasts in the whole cell con®guration, an activation of time-dependent outward currents positive to
28 mV was observed (n ˆ 10; Figure 7a). Tail K‡ currents
reversed direction around EK ( 41 mV; Figure 7b). Single
channel ¯uctuations with a unitary conductance of approximately 19 pS were recorded in cell-free outside-out patches
excised from protoplasts with prominent time-dependent
outward currents (Figure 7c). The single channel currents
reversed direction around 40 mV, close to EK (Figure 7d).
Time- and voltage-dependent parameters, as well as the
unitary conductance of the phloem K‡ outward recti®er
(Figure 7a±e), were reminiscent of guard cell outward rectifying K‡ channel (GORK) expressed in Xenopus oocytes,
Arabidopsis guard cells and root hairs (Ache et al., 2000;
Ivashikina et al., 2001). In contrast to the latter Arabidopsis
cell type, phloem outward K‡ channels did not inactivate in
response to prolonged (10 sec) de-polarization (Figure 7f).
Inactivation of K‡ outward recti®er has previously been
described in Arabidopsis guard cells (Pei et al., 1998) and
root hairs, both being GORK-expressing cell types (Ache
et al., 2000; Ivashikina et al., 2001). RT-PCR analysis of
channel transcripts (Figure 5a), however, showed that
neither GORK nor SKOR was expressed in GFP-tagged
protoplasts. We may thus assume that the phloem outward
recti®er represents either the product of the KCO6 gene, or
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
Isolation of companion cells for patch-clamp and expression profiling
939
Figure 6. Electrical properties of the phloem K‡
inward recti®er.
(a) Time- and voltage-dependent whole-cell
inward K‡ currents in ¯uorescent protoplasts.
Voltage pulses were applied from a holding
potential of 48 mV in 20-mV steps in the range
from ‡52 to 188 mV, with subsequent pulse to
88 mV.
(b) De-activation (tail currents) in response to a
double-pulse protocol starting from a holding
potential of 48 mV to a pre-pulse voltage of
168 mV and followed by voltage steps from
‡12 to 148 mV. Pipette and bath solutions
contained 150 and 30 mM K-gluconate, respectively.
(c) Single channel ¯uctuations induced by
hyperpolarizing voltages in an outside-out
membrane patch excised from a protoplast with
a macroscopic inward current. O, open state of
the channel; C, closed state of the channel.
(d) Single channel amplitudes plotted versus
the membrane voltage. The data represent
mean channel amplitudes SD from three
patches.
(e) Voltage-dependent Ca2‡-block of the phloem
inward recti®er. Voltage dependence of inward
K‡ currents at different Ca2‡ to K‡ ratio. Current
amplitudes were sampled at the end of 1-sec
pulses to voltages in the range from 8 to
188 mV. External solutions contained: 20 mM
CaCl2 ‡ 10 mM K-gluconate (&), 20 mM CaCl2 ‡
30 mM K-gluconate (*), 1 mM CaCl2 ‡ 30 mM
K-gluconate ( ) and 0.1 mM CaCl2 ‡ 30 mM
K-gluconate (5).
(f) pH-dependent shift in voltage activation of
inward K‡ channels. Relative conductance at
different external pH values was ®tted by Boltzmann function. External solutions contained
1 mM CaCl2, 30 mM K-gluconate and 10 mM
Mes/Tris (pH 5.6) or 10 mM Hepes/Tris (pH 7.0).
Data points represent means SE for three
protoplasts.
another yet non-identi®ed K‡ channel involved in the repolarization of the phloem potential.
Discussion
Molecular mechanism of phloem K‡ loading and release
In our search for companion cell channels involved in K‡
retrieval and release in phloem cells, we focused on the
nine Shaker-like and six KCO channels encoded by the
Arabidopsis genome (reviewed by MaÈser et al., 2001). A
remarkable feature of Shaker channels is their ability to
promiscuously form heterotetramers with different a-subunits (Baizabal-Aguirre et al., 1999; Dreyer et al., 1997;
Paganetto et al., 2001; Pilot et al., 2001; Reintanz et al.,
2002). Inward rectifying channels result from heteromers
between KAT1 and AKT1, KAT1 and AtKC1 (Dreyer et al.,
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
1997), KAT1 and AKT2/3 (Baizabal-Aguirre et al., 1999), as
well as KAT1 and KAT2 (Pilot et al., 2001), but not between
KAT1 and the outward recti®er GORK (Ache et al., 2000).
Formation of heteromultimeric structures has been shown
to modify the sensitivity of K‡ channels to voltage, Ca2‡ and
pH (Dreyer et al., 1997; Paganetto et al., 2001; Reintanz
et al., 2002). Numerous K‡ channel subunits, KAT1,
KAT2, AKT1, AtKC1, GORK and AKT2/3 are expressed in
Arabidopsis guard cells (Szyroki et al., 2001), while root
hairs express just three Shaker K‡ channel a-subunits,
AKT1, AtKC1 and GORK (Reintanz et al., 2002). We also
found different K‡ channel transcripts in companion cell
protoplasts (Figure 5a). Among these channels, only AKT2/3
was characterized by a pronounced Ca2‡ sensitivity and
block by protons (Hoth et al., 2001; Lacombe et al., 2000;
Marten et al., 1999), pointing to a contribution by the AKT2/3
subunit to the Ca2‡-sensitive K‡ conductance of the
phloem. These properties, however, require further testing
940 Natalya Ivashikina et al.
Figure 7. Electrical properties of the phloem K‡
outward recti®er.
(a) Time- and voltage-dependent whole-cell outward K‡ currents in ¯uorescent protoplasts.
Voltage pulses were applied from a holding
potential of 48 mV in 20-mV steps in the range
from 108 to ‡52 mV, with subsequent step to
88 mV.
(b) De-activation currents in response to a double-pulse protocol starting from a holding
potential of 48 mV to a prepulse voltage of
‡52 mV and followed by voltage steps from
‡32 to 88 mV.
(c) Single channel ¯uctuations induced by depolarization in an outside-out patch excised
from a protoplast with a macroscopic outward
current. O, open state of the channel; C, closed
state of the channel.
(d) Single channel amplitudes plotted versus
the membrane voltage. The data represent
mean channel amplitudes SD from ®ve
patches.
(e) Voltage dependence of outward current
sampled at the end of 1-sec pulses in the range
from 108 to ‡ 52mV and normalized in respect
to ‡32 mV. Data points represent means SE
for three protoplasts. Iss, the (quasi) steadystate current.
(f) Comparison of time-dependent outward K‡
currents measured in protoplasts isolated from
companion cell and root hair protoplasts in
response to a 10-sec voltage pulse from 48
to ‡52 mV.
using direct genetic analyses such as loss-of-function
mutants or RNAi lines.
The Arabidopsis Shaker superfamily contains two outward rectifying K‡ channels: SKOR, localized in the root
pericycle and stelar parenchyma cells (Gaymard et al.,
1998), and GORK, expressed in guard cells (Ache et al.,
2000) and the root epidermis (Ivashikina et al., 2001). In
this paper, we suggest that the phloem outward recti®er
could be the product of the KCO6 gene (Figure 5a). The
latter is also expressed in mesophyll and hypocotyl cortex
cells, which lack GORK and SKOR transcripts, but contain a
non-inactivating delayed outward recti®er (our unpublished data). The phloem outward recti®er did not undergo
time-dependent inactivation (Figure 7f). The identi®cation
of the companion cell outward recti®er awaits the availability of GORK and KCO6 loss-of-function plants expressing GFP under the control of the SUC2 promoter.
Taken together, the inward and outward K‡ recti®ers
characterized in this study may provide for a mechanism
to control K‡ cycling, the membrane potential and, conse-
quently, H‡-driven assimilate translocation in the phloem.
Assuming that K‡ concentration in the phloem varies in the
range of 50±150 mM (Marschner et al., 1996) and apoplastic
K‡ from 1 to 10 mM, inward K‡ channels can activate
negative to 40 mV and outward K‡ channels positive to
120 mV. Both channels can therefore operate in the voltage range recorded for SE/CC (between 100 and
185 mV; van Bel and van Rijen, 1994; Deeken et al.,
2002). Future electrophysiological and molecular studies
will focus on the nature and regulation of phloem-localized
Ca2‡ and Cl± channels, as well as on electrogenic carriers
and pumps, in order to gain insights into the formation of
complex electrical signals travelling along the phloem.
Gene expression of companion cell protoplasts
Membrane transport. In addition to the well-known
phloem transporters SUC2, AKT2 and AHA3, two channels,
KAT1 and KCO6, were found differentially expressed
in companion cells, while ATKC1 and SUC3 appeared
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
Isolation of companion cells for patch-clamp and expression profiling
predominately in the mesophyll cell fraction. We studied
the expression of genes encoding plant signalling components in order to increase the number of potential
companion cell and mesophyll markers. In addition to
quantitative RT-PCR analyses with transporter-speci®c
primers, we, so far, found in the companion cell EST
collection, a sugar transporter, a metal ion transporter
and components of the membrane sorting/traf®cking
system (Table 1, `transport and membranes'), as well as
the previously identi®ed phloem-localized amino acid
transporter AAP2 (Okumoto et al., 2002).
Companion cell identity. In good agreement with the organelle composition of companion cells, the EST collection
contained chloroplastic (Table 1, `photosynthesis') and
mitochondrial genes, as well as a relative large number
of nuclear and ribosomal genes involved in transcription
and translation, together with genes required for protein
folding (HSPs) and protein degradation (ubiquitin). There
was even a translation initiation factor (contig of 18) among
the few high-copy genes (Table 1). This pro®le further
underlines the role of the companion cell as the `work
horse' of the SE/CC complex. Proteins residing in the
nucleus- and ribosome-free sieve tubes are produced in
the companion cells. The characteristic tubular structures
found in the phloem sap of sieve tubes and based on actin-,
tubulin-, dynein- and microtubule-associated proteins
(Schobert et al., 1998, 2000) might be gene products of
cytoskeleton genes expressed in companion cells
(Table 1, `cytoskeleton').
Signal transduction. Among the auxin transporter transcripts tested by RT-PCR analyses (data not shown), we
found PIN6 in companion cells and PIN3 in mesophyll cells.
Transcripts of the `so-called' auxin-binding protein ABP1
were found in both mRNA pools. This differential
expression points to the phloem as a bi-directional, longdistance pathway for auxin transport. Future studies on the
nitrilase found in the EST collection and other potential
auxin synthesis genes are still needed to con®rm that
companion cells are also sites of auxin production. In
this respect, it was somewhat unexpected that genes of
the M cluster were dominated by those with a known
function in secondary metabolism. Among them, the EST
collection harbours genes involved in ethylene, jasmonate,
ABA, gibberellin and steroid (possibly brassinosteroid)
synthesis, perception, transduction and response (for respective receptor kinases, calcium-binding proteins, protein
kinases and phosphatases and MAP kinase; see Table 1).
Future studies on candidate genes will help to link hormone
and phloem action.
Stress. Taken together, the genes encoding R, S and D
comprised up to 33% of identi®ed genes (Figure 4). This
raises the question as to whether this expression pattern
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
941
re¯ects stress imposed by protoplast isolation (including
loss of turgor), exposure to fungal cell wall-degrading
enzymes (and thus release of cell wall oligosaccharide
with potential elicitor-like function) or the companion cell
biology? Among the defence genes, we identi®ed a phloemlocalized myrosinase, which, together with phloem-mobile
glycosinolates, has been described before and linked to
pathogen defence (Chen et al., 2001; Husebye et al., 2002),
possibly directed against phloem feeding insects.
Furthermore, the lectin PP2 and cystatin were identi®ed
as major components of phloem exudates for all species
analysed so far (Schobert et al., 1998; Walz et al., 2002). The
stress gene fraction was dominated by water stressinduced proteins (cf. kin1, `hormones and signalling' in
Table 1) and heat shock proteins. Future loss-of-function
studies will clarify whether these genes are required for
protein folding and shuttling into the sieve elements and
therefore survival of the nucleus- and ribosome-free sieve
tubes. It should be noted that the large number of HSP
transcripts correlated with the large abundance of members of the TT gene cluster (Table 1). Similarly, some of the
defence gene members (e.g. lectin PP2, see `defence' in
Table 1) and of the R cluster have also been identi®ed as a
major protein fraction of the phloem sap (Walz et al., 2002,
and references cited). Among them, metallothionein,
thioredoxin, glutaredoxin and glutathione-S-transferase
appeared in contigs with up to 40 copies. Future studies
based on transgenic plants expressing promoter±reporter
gene constructs will have to clarify whether a `stress' gene
is constitutively expressed in companion cells or induced
upon interaction with pathogens/symbionts, meristem
development or fruit ripening (see, e.g. nodulin ENOD
40, symbiosis-related protein, ripening-related protein
and NAC-domain protein under `hormones and signalling' in Table 1).
Outlook. We are currently generating a saturating EST
collection with the present founder ESTs as a base.
Together with genome array data, future studies will take
advantage of a substantial phloem marker pool. Intact
phloem samples gained using LMPC (Figure 1) will allow
companion cell-speci®c genes to be distinguished within
the phloem-speci®c ones. Ongoing bioinformatic analyses
of the respective genes will provide the backbone for
genome-wide predictions about proteins involved in
phloem action, and improve our understanding of sink±
source regulation and control of ¯owering and ripening.
Experimental procedures
Plant material
Transgenic A. thaliana AtSUC2 promoter-GFP plants were grown
in soil in a growth chamber with a 8-h day/16-h night regime, 218C
942 Natalya Ivashikina et al.
day/168C night temperature and a photon ¯ux density of
120 mmol m 2 sec 1.
Laser microdissection and pressure catapulting
Arabidopsis in¯orescence stalks were cut into 5±15-mm pieces and
®xed for 4 h in 3 : 1 ethanol:acetic acid, and subsequently dehydrated and embedded in Paraplast plus (Sigma, Steinheim, Germany) according to Kerk et al. (2003). Cross-sections of 10±15 mm
were cut on a rotary microtome (RM2165, Leica, Bensheim, Germany), ¯oated in water on membrane-coated glass slides (PALM,
Bernried, Germany) at 428C and air-dried. Slides were de-paraf®nized two times in xylene for 5 min each and air-dried. LMPC of
phloem was carried out using the PALM Laser-MicroBeam System
(PALM, Bernried, Germany). One hundred and ®fty phloem
regions were collected in 10-ml DEPC-treated water containing
40 units RNase inhibitor (MBI, St Leon-Rot, Germany).
Protoplast isolation
Vascular strands were excised from fully developed rosette leaves
and incubated for 1.5 h at 308C in enzyme solution containing 0.8%
(w/v) cellulase (Onozuka R-10, Yakult Itorisha, Tokyo, Japan), 0.1%
pectolyase (Sigma), 0.5% bovine serum albumin, 0.5% polyvinylpyrrolidone, 1 mM CaCl2 and 10 mM Mes/Tris (pH 5.6). The osmolarity of the enzyme solution was adjusted to 630 mosmol kg 1
with D-sorbitol. Protoplasts released from vascular-enriched tissues were ®ltered through a 20-mm nylon mesh and washed two
times in 1 mM CaCl2 buffer (osmolarity 580 mosmol kg 1
(pH 5.6)). For isolation of mesophyll protoplasts, the osmolarity
of all solutions was adjusted to 400 mosmol kg 1 and protoplasts
were ®ltered through a 100-mm nylon mesh. The protoplast suspension was stored on ice, and aliquots were used for patch-clamp
measurements or separation of single protoplasts to generate
cDNA libraries.
Collection of contamination-free protoplasts
Individual phloem and mesophyll protoplasts were collected
under an epi¯uorescence inverted microscope (Axiovert 35 M Carl
Zeiss, Oberkochen, Germany) from 3-cm plastic dishes containing
leaf or stem protoplast suspension. Fluorescing protoplasts were
visualized by short-wave blue light. Protoplasts were transferred
by microcapillaries with a tip opening of approximately 50 mm (CC
protoplasts) and 200 mm (mesophyll) using a computer-controlled
micropump (dispenser/diluter, Microlab-M; Hamilton, Darmstadt,
Germany) as described by Koop and Schweiger (1985) and Kranz
(1999). For selection and washing, protoplasts were transferred
into 2000-nl microdroplets of washing solution (1 mM CaCl2 and
10 mM Mes/Tris (pH 5.6), osmolarity 580 mosmol kg 1), covered
by mineral oil. After washing, 145 phloem protoplasts and 150
mesophyll protoplasts were transferred with a microcapillary into
a 0.5-ml reaction tube.
As an alternative less time-consuming approach for isolating
mesophyll protoplasts, we examined the protocol of Cherel et al.
(2002), which was used by the authors to `semiquantify' the level of
expression of AKT2, KAT1, KAT2 and AtPP2CA in mesophyll cells.
In contrast to the approach by Cherel et al. (2002), we used AtSUC2
promoter-GFP plants to verify the purity of mesophyll protoplasts.
As a result, we found the mesophyll preparation contaminated by
green ¯uorescent protoplasts, and detected transcripts of the
phloem-speci®c marker genes SUC2 and AHA3 by RT-PCR (not
shown).
RNA extraction, cDNA synthesis and sequencing
RNA from LMPC samples was extracted using the Gentra-Purescript-RNA-Isolation-Kit (Biozym, Hess. Oldendorf, Germany).
Poly(A) RNAs from mesophyll and CC protoplasts were isolated
and puri®ed twice with the Dynabeads mRNA Direct kit (Dynal,
Oslo) to prevent contamination with genomic DNA. The SMART
cDNA Library Construction Kit (BD Biosciences Clontech, Heidelberg, Germany), which is designed for limited amounts of mRNA
and includes a PCR-based protocol, was used for cDNA synthesis
and ampli®cation. The resulting lTriplEx2 library was converted to
a plasmid library for preparation of the ESTs. Plasmid DNA was
subjected to a standard sequencing procedure using the lTriplEx
50 LD-Insert Screening Amplimer (50 -CTCGGGAAGCGCGCCATTGTGTTGGT-30 ), the Epicentre-SequiTherm-EXCEL II Kit (Biozym, Oldendorf, Germany) and the Li-Cor-dna-Analyzer-GeneReadir 4200 Sequencer (Li-Cor, Bad Homburg, Germany).
Sequencing of 2000 individual ESTs was performed in cooperation with Syngenta Biotechnologies Inc. (Research Triangle Park,
NC, USA) using an ABI PRISMj 3700 DNA Analyzer from Applied
Biosystems, Foster City, CA, USA. Alignment and BLAST procedures for the sequenced ESTs were also performed by Syngenta
following standard algorithms. All kits were used according to the
manufacturers' protocols.
RT-PCR experiments
First-strand cDNA was prepared with RNA of LMPC and protoplasts by using Superscript RT (Gibco/BRL, Karlsruhe, Germany).
Qualitative PCR was carried out using 1 ml of 1 : 10 water-diluted
cDNA in a standard 50 ml reaction. For quantitative real-time PCR,
the cDNA was diluted 20-fold in water and ampli®ed in a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) with
the LightCycler-FastStart DNA Master SYBR Green I kit (Roche
Molecular Biochemicals), according to the manufacturers' protocol. All primers were chosen to amplify fragments not exceeding
500 base pairs. Primers and gene accession numbers used are as
follows: KAT1fwd (50 -ACT TCC GAC ACT GC-30 ), KAT1rev (50 -CCC
AAA TGA CAT CTA A-30 ); KAT2fwd (50 -ATA TTG ATA TGG GGT CA30 ), KAT2rev (50 -ATC TAT TTC TGC GTT TT-30 ); AKT1fwd (50 -CCA
ACT GTT GCG TAT-30 ), AKT1rev (50 -CTG CGT GGT ACT CC-30 );
AKT2fwd (50 -AAA ATG GCG AAA ACA C-30 ), AKT2rev (50 -CGC TGC
TTC ACA TAG AA-30 ); AKT5fwd (50 -AGG CCA CAG TTG TTC-30 ),
AKT5rev (50 -CGC CAT TTT CTG ATA A-30 ); AKT6fwd (50 -GCC AGT
GCG GTT AC-30 ), AKT6rev (50 -GAC TCA ATC GCT TGG TA-30 );
ATKCfwd (50 -ATA TTG CGA TAC ACA AG-30 ), ATKCrev (50 -GAC
CTA ACT TCG CTA AT-30 ); GORKfwd (50 -CCT CCT TTA ATT TAG
AAG-30 ), GORKrev (50 -GCT CCA TCC GAT AG-30 ); SKORfwd (50 TGA AAC GGC TTC TTA-30 ), SKORrev (50 - GAG CCA CTC GGA AAC30 ); KCO1fwd (50 -GTT GGC ACG ATT TTC-30 ), KCO1rev (50 -GCT TCG
CAA GAT GAT-30 ); KCO2fwd (50 -GAT CGG GAC AAAGTG-30 ),
KCO2rev (50 -ACG CAG CCA TTA CAG-30 ); KCO3fwd (50 -CTT TAC
CAG AAC ACA ACG-30 ), KCO3rev (50 -GCA CAA TTA AAA AGC CAC30 ); KCO4fwd (50 -GCA AGA TAA GGT TAA AGT G-30 ), KCO4rev (50 CAT GAC AGT AGT ACG AT-30 ); KCO5fwd (50 -AGA CGA CAA AGA
AGA-30 ), KCO5rev (50 -CCG GTG AGA ATC ATA-30 ); KCO6fwd (50 ACC CAA TTC GTC AAA A-30 ), KCO6rev (50 -CCG CTT AGC AGA GTC
T-30 ); PP2CAfwd (50 -AAT TGT TGC TGA CTC C-30 ), PP2CArev (50 AAC TCT TAA CCA TCG T-30 ); SUC2fwd (50 -CTT ATG CTT AAC GCT
ATT-30 ), SUC2rev (50 -GAC AAT GGC TAG ATT-30 ); SUC3fwd (50 CAC TAT ATG TAC TCT TGT C-30 ), SUC3rev (50 -CAT CAA CGT AGG
TCT C-30 ); AHA3fwd (50 -GAG TCC ACT CTA CAA TC-30 ), AHA3rev
(50 -GTC TTT GTG TTT ACC GA-30 ); DIR1fwd (50 -TAT GTT GGT CGA
TAC ATC A-30 ), DIR1rev (50 -CAT GGA GAG TTC TTG TAA-30 );
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 931±945
Isolation of companion cells for patch-clamp and expression profiling
EINfwd (50 -GAA GTA ACT GCT GTC TCG-30 ), EINrev (50 -CTT TCC
CTA ACA TGA TCT-30 ); BRI1fwd (50 -CTT ACT ATG CTT ACG GA-30 ),
BRI1rev (50 -GTT AGC AGT TCT ATC GC-3); PIN3fwd (50 -GAA TGA
TGA TGC CAA C-30 ), PIN3rev (50 -GTT ACC CGA ACC TAA T-30 );
PIN6fwd (50 -ATC AAT GGA TCA GTG C-30 ), PIN6rev (50 -CCC ACG
ACT GTT AGT A-30 ). Fragment length: KAT1 ˆ 379 bp, KAT2 ˆ
392 bp, AKT1 ˆ 347 bp, AKT2 ˆ 353 bp, AKT5 ˆ 481 bp, AKT6 ˆ
428 bp, AtKC1 ˆ 373 bp, GORK ˆ 496 bp, SKOR ˆ 253 bp,
KCO1 ˆ 500 bp, KCO2 ˆ 401 bp, KCO3 ˆ 234 bp, KCO4 ˆ 281 bp,
KCO5 ˆ 456 bp, KCO6 ˆ 344 bp, PP2CA ˆ 431 bp, SUC2 ˆ
351 bp, SUC3 ˆ 239 bp, AHA3 ˆ 305 bp, DIR1 ˆ 174 bp, EIN4 ˆ
203 bp, BRI1 ˆ 360 bp, PIN3 ˆ 252 bp, PIN6 ˆ 197 bp. GenBank,
EMBL or MIPS-code numbers: KAT1 (X93022), KAT2 (CAA16801),
AKT1 (X62907), AKT2/3 (U40154), AKT5 (AJ249479), AKT6 ˆ SPIK
(CAC85283), AtKC1 (U81239), GORK (AJ279009), SKOR (AJ223358),
KCO1 (X97323), KCO2 (AJ131641), KCO3 (CAB40380), KCO4
(AT1G02510.1), KCO5 (AJ243456), KCO6 (AT4G18160.1), SUC2
(AY050986), SUC3 (AJ289165), PP2CA (P49598), AHA3 (AY072153),
DIR1 (AAL76110), EIN4 (NP_187108) and BRI1 (NP_195650), PIN3
(NP_177250), PIN6 (AAD52696). cDNA quantities were calculated
using LIGHTCYCLER 3.1 (Roche, Mannheim, Germany) and were
all normalized to 10 000 molecules of actin cDNA fragments (An
et al., 1996) ampli®ed by AtACT2/8fwd (50 -GGTGAT GGT GTG TCT30 ) and AtACT2/8rev (50 -ACT GAG CAC AATGTT AC-30 ). Each transcript was quanti®ed using an individual standard. To enable
detection of contaminating genomic DNA, PCR was performed
with RNA as template. These DNA-free RNA samples were subsequently used for cDNA synthesis.
Patch-clamp recordings
Patch-clamp recordings were performed in the whole-cell mode
using an EPC-7 ampli®er (List-Medical-Electronic, Darmstadt, Germany). Data were low-pass-®ltered with an eight-pole Bessel ®lter
(Compu Mess Electronic GmbH, Garching, Germany) with a cut-off
frequency of 2 kHz and sampled at 2.5 times the ®lter frequency.
Data were digitized (ITC-16, Instrutech Corp., Elmont, New York,
USA) and analysed using PULSE and PULSEFIT software (HEKA
Elektronik, Lambrecht, Germany), as well as IGORPRO (Wave
Metrics Inc., Lake Oswego, OR, USA). Patch pipettes were prepared from Kimax-51 glass capillaries (Kimble products, Vineland,
NY, USA) and coated with silicone (Sylgard 184 silicone elastomer
kit, Dow Corning GmbH, Wiesbaden, Germany). The command
voltages were corrected off-line for liquid junction potential
(Neher, 1992). The pipette solution (cytoplasmic side) contained
150 mM K-gluconate, 2 mM MgCl2, 10 mM EGTA, 2 mM Mg-ATP
and 10 mM Hepes/Tris (pH 7.4). The standard external solution
contained 30 mM K-gluconate, 1 mM CaCl2 and 10 mM Mes/Tris
(pH 5.6). Osmolarity of all solutions was adjusted to 580 mosmol
kg 1 with D-sorbitol. Modi®cations to solute compositions are
included in the ®gure legends. Chemicals were obtained from
Sigma-Aldrich (Taufkirchen, Germany).
Acknowledgements
We thank Bernd MuÈller-RoÈber for providing the KCO1 cDNA clone
and P. Dietrich, D. Becker and T.G.A. Green for critical reading of
the manuscript. This work was funded by DFG Grants to R.H.
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