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Acta Physiologiae Plantarum ISSN 0137-5881Volume 32Number 6 Acta Physiol Plant (2010)32:1103-1111DOI 10.1007/s11738-010-0502-1

Identification and characterization of twogenes encoding plasma membrane H+-ATPase in Cucumis sativus L.

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ORIGINAL PAPER

Identification and characterization of two genes encoding plasmamembrane H+-ATPase in Cucumis sativus L.

Ewa Młodzinska • Anna Wdowikowska •

Gra _zyna Kłobus

Received: 25 January 2010 / Revised: 9 March 2010 / Accepted: 29 March 2010 / Published online: 8 May 2010

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010

Abstract Two genes (CsHA2, CsHA3) homolog to the

plasma membrane H?-ATPase encoding sequences have

been identified in cucumber cells. The full-length cDNA of

these sequences comprise an open reading frame that

encodes about 950 amino acid polypeptide with several

potential transmembrane domains. Both sequences share a

high homology with putative plasma membrane-associated

proton pumps in higher plants, ranging from 84% with that

of Arabidopsis thaliana to 90% with Sesbania rostrata.

Phylogenetic analysis grouped the two isoforms into one

group (subfamilies II) together with Kosteletzkya virginica

KVATP1, Sesbania rostrata SRHA4, tobacco PMA4 and

rice OSA7. The expression of CsHA isoforms at the mRNA

level showed they different organ pattern. Transcript level

of CsHA2 was detected in all vegetative organs, but more

abundantly in leaves than in roots, while CsHA3 expression

was limited only to the roots in immature as well as in

mature plants. Moreover, the RT-PCR analysis of genera-

tive organs (flowers), fruits and dry seeds revealed differ-

ential expression pattern for CsHA2 and CsHA3 suggesting

that these proteins could be involved in separate physio-

logical processes.

Keywords Cucumber � Gene expression � Isoforms �Plasma membrane � H?-ATPase

Abbreviation

PM Plasma membrane

Introduction

H?-ATPase belongs to the P3A-type ATPase (P-type

ATPase Database at http://biobase.dk/-axe/Patbase.html)

and is the major electrogenic pump of the plasma mem-

brane in higher plants and fungi (Axelsen and Palmgren

2001). This protein couples ATP hydrolysis with proton

pumping out of the cells, generating an electrochemical

gradient of plasma membrane (Palmgren et al. 1991;

Portillo 2000; Kasamo 2003), which contribute to many

physiological processes, such as nutrient uptake, regulation

of ion homeostasis and intracellular pH, cell turgidity and

related phenomena, such as the cell growth or stomata and

organ movements (for review, see Sussman 1994; Palm-

gren 2001; Lefebvre et al. 2003). The H?-ATPase also

plays a crucial role in resistance to stress factors such as

salinity (Niu et al. 1993; Virart et al. 2001; Palmgren 2001;

Lefebvre et al. 2003; Klobus and Janicka-Russak 2004),

mechanical stress (Ouffatole et al. 2000), changes in

nitrogen availability (Santi et al. 2003) and phosphorus

starvation (Shen et al. 2006) and such wide variety of

physiological functions linked to the H?-ATPase implies a

need for complex regulation of its activity at both tran-

scriptional and post-translational levels (Michelet et al.

1994; Noubhani et al. 1996; Astolfi et al. 2003; Camoni

et al. 2006). It is well documented that the plasma mem-

brane H?-ATPases are encoded by a multi-gene family: 12

genes in Arabidopsis thaliana (AHA1–AHA12), 10 genes in

Oryza sativa (OSA1–OSA10), 9 genes in Nicotiana

plumbaginifolia (PMA1–PMA9). All of the identified genes

are classified into five subfamilies according to their amino

acid sequence identities and expression profiles (Arango

et al. 2003). Genes belonging to the first and second sub-

family are highly expressed in all plant tissues, while genes

from other three groups are more specialized and their

Communicated by L. A. Kleczkowski.

E. Młodzinska (&) � A. Wdowikowska � G. Kłobus

Plant Physiology Department, Institute of Plant Biology,

Wrocław University, Kanonia 6/8, 50-328 Wrocław, Poland

e-mail: ewamloda@biol.uni.wroc.pl

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DOI 10.1007/s11738-010-0502-1

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expression is restricted to the particular tissues and/or to

the specific conditions, such as salt stress, mechanical

damage, heat and cold treatment (Arango et al. 2003;

Baxter et al. 2003). For example, the AtAHA10 classified

as a member of the subfamily III has been localized only in

the developing seeds (Arango et al. 2003), whereas the

AtAHA9, belonging to the subfamily IV, only in anthers

and NpPMA5—the other member of subfamily IV—in

pollen tube (Lefebvre et al. 2005). Strong dependence of

expression level of particular genes encoding H?-ATPase

isoforms, especially those belonging to the subfamily II, on

environmental factors as well as hormones was also found.

It was shown that external nitrate distinctively induced

MHA4 expression in maize roots (Santi et al. 2003). In

addition, salinity increased accumulation of specific

mRNA in tomato (LHA8), as well as in Arabidopsis

(AtAHA4) (Kalampanayil and Wimmers 2001; Virart et al.

2001). Auxin application enhanced mRNA level of MHA2

in maize (Frıas et al. 1996).

Limited information is available on the proton pump

isoforms in cucumber. Santi et al. (2005) identified

fragments of two genes (CsHA1 and CsHA2) and showed

their different dependency on iron availability. Therefore,

the aim of the presented work was to identify and char-

acterize a complete cDNA encoding a PM proton pumps

in cucumber plants and to describe this sequence to

find the tissue-specific expression patterns in Cucumis

sativus. These approaches will be particularly useful for

the explanation of molecular and biochemical basis of

the H?-ATPase adaptation to different environmental

conditions.

Materials and methods

Plant material

Cucumis sativus L. cv. Krak F7 was grown in nutrient

solution as previously described by Kłobus (1990); 5 days

and 3-week-old seedlings were divided for cotyledons,

leaves, leaf petioles, shoots, primary and lateral roots.

Flower and fruits were collected from 5-week-old plants

and immediately frozen in liquid nitrogen and stored at

-70�C and used for RNA isolation.

RNA isolation

RNA was isolated from 50 mg of plant tissues by TRI

Reagent (Sigma) according to the manufacturer’s proto-

cols, resuspended in DEPC water and stored at -70�C until

analysis. The integrity of total RNA was checked by

electrophoresis in 1% denaturing formaldehyde–agarose

gels.

cDNA synthesis

First-strand cDNA was synthesized using 5 lg of root

RNA in pre-amplification system for first-strand cDNA

synthesis (Invitrogen). The full-length cDNAs of CsHA2

and CsHA3 for cucumber H?-ATPase were obtained with

RNA ligase-mediated rapid amplification of 50 and 30

cDNA ends (RLM-RACE) method, using GeneRacer Kit

(Invitrogen Co., Carlsbad, CA). The gene-specific primers

were designed complementary to the specific fragment of

partial nucleotide sequences of cucumber deposited under

the accession numbers AF289025 and AJ703811. Primers

AJ147 rev (50-GGCTTCACCCTGTCCTTTGAGATGG

TC-30) and 50-Gene Racer were used to amplify the missing

50-end of CsHA2 cDNA (AJ703811) by PCR (25 cycles,

Tm = 65�C). To obtain both missing 50 and 30 ends of

cDNA for AF289025 sequence, specific primers AF310 rev

(5-CGCCCTCGAGAAGACGAGCATCCGCCGG-30) and

AF1076 for (50-GGCATCGAGCAAGCAAAGGAGCTCC

CGAGC-30) were used, respectively. PCR parameters for

50 end were 23 cycles and Tm 68�C, whereas for 30 end

were 23 cycles and Tm = 66�C.

Cloning and analysis of construct

Gel-purified PCR products were cloned into pCR4-TOPO

plasmid (TOPO TA Cloning Kit, Invitrogen) and propagated

in E. coli TOP 10, according to manufacturer’s instruction.

Transformants were selected on LB-agar plates supple-

mented with 50 lg/ml ampicillin and identified by blue/

white screening. Plasmid DNA was isolated with Wizard

Plus SV Minipreps DNA Purification System (Promega),

analyzed by EcoRI digestion and sequenced with internal

primers (M13 for and M14 rev described in the protocol for

TOPO Kit) by the Dideoxy Chain Termination Method. The

cDNA sequences have been submitted in the GenBank with

accession number EU735752 for CsHA2 and EF375892 for

CsHA3. In addition, the fragments were aligned and edited

in Chromas program (version 2.23).

Full-length cDNA sequences CsHA2 and CsHA3, as

well as amino acid sequences were analyzed to determine

the degree of similarity to previously published sequences

in GenBank resources (http://www.ncbi.nlm.nih.gov).

Homology searches were calculated using BLASTN and

BLASTP algorithms. Protein sequence alignment and

phylogenetic tree analysis were performed with ClustalW

program (http://www.ebi.ac.uk/clustalw) and calculation of

molecular weight with ProtParam (http://www.expasy.org/

tools/protparam.html). Transmembrane protein topologies

were predicted using TMHMM 2.0 server (http://www.

cbs.dtu.dk/services/TMHMM-2.0/) and two-dimensional

model of proton pump was created by TMRPres2D

(http://biophysics.biol.uoa.gr/TMRPres2D/). Conserved

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characteristic regions of P-type ATPase were provided by

InterProScan (http://www.ebi.ac.uk/InterProScan/).

Gene expression

The expression levels of CsHA2 and CsHA3 in different

parts of cucumber were determined with semi-quantitative

RT-PCR (Titan One Tube RT-PCR System, Roche) with

specific primers. As a template in RT-PCR reaction,

0.150 lg RNA was used. A 263-bp-long fragment of

CsHA2 was amplified with two specific primers: 50-ACC

CGAGTCGACAAACATCT-30 (forward) and 50-CTTGG

CACAGCAAAGTGAAA-30 (reverse) (Santi et al., 2005).

For amplification of 393-bp-long region of the CsHA3

50-AAGTTTCTGGGGTTCATGTGGAAT-30 (forward) 50-GTAACAGGAAGTGACTCTCCAGTC-30 (reverse) were

used as the primers. As an internal standard, 18s RNA gene

was used. RT-PCR reactions were performed with Tm at

57�C and 22 cycles for CsHA2 and 18 cycles for CsHA3,

which corresponded to the log-linear phase of the ampli-

fication reaction. For the cDNA of internal standard, nine

cycles were chosen. The products were visualized on

ethidium bromide-containing 1.5% agarose gels and ana-

lyzed using the BioCapt version 99 software.

Results

Two complete sequences encoding plasma membrane

H?-ATPases were obtained on the basis of partial cDNAs

corresponding to different CsHA genes. The missing frag-

ments of both genes were isolated and amplified by 30 and

50 RACE method. After cloning and sequencing, the

700-bp and 1,800-bp cDNA products were obtained as the

30 and 50 missing fragments of CsHA3 (Fig. 1b). These

fragments together with the original, partial template

cDNA, deposited earlier in GenBank with the accession

number AF289025 gave the complete 3,305-bp-long

CsHA3 gene. The 50 end of the partial cDNA (AJ703811)

was amplified resulting in 2,300 bp and assembled full

3,223-bp-long gene, which was named CsHA2 (Fig. 1a).

Full nucleotide sequences of CsHA2 and CsHA3 were

deposited in the GenBank database under accession num-

ber EU735752 and EF375892, respectively. The CsHA2

and CsHA3 cDNAs encode 954 and 953 amino acid

polypeptides, respectively, with calculated molecular

masses about 105 kDa. Both proteins show high similarity

(about 95%) to each other (Fig. 2).

The deduced amino acid sequences of CsHA2 and

CsHA3 were used in a BLASTP (basic local alignment

search tool) search of the National Center for Biotechnology

Information (NCBI) database. Alignment of the top-scoring

matches with CsHA2 and CsHA3 sequences demonstrated

that all proteins are highly similar. CsHA2 and CaHA3 share

the highest similarity, more than 84%, with SRHA4

(Sesbania rostrata), KVATP1 (Kosteletzkya virginica),

PMA4 (Nicotiana plumbaginifolia) and AHA5 (Arabidopsis

thaliana) proteins. This polygenetic analysis has also shown

that both proteins are the members of plasma membrane

H?-ATPases belonging to the subfamily II of P-ATPases

(Fig. 3). Furthermore, the computational analysis of CsHA2

and CaHA3 revealed all characteristic structural features of

the plasma membrane proton pumps. Both proteins posses

ten transmembrane segments, small and large central

hydrophilic loops, as well as the N-terminal and C-terminal

tails located at the cytoplasmic side of the membrane

(Fig. 4). As would be expected, both proteins contain the

phosphorylatable penultimate residue (Thr) in the C-termi-

nal domain, which can be involved in the regulation of their

activities. Moreover, some conserved regions that are

characteristic for P-type ATPases are also present in CsHA2

and CaHA3 proteins (Fig. 4). The KGAP motif is involved

in ATP binding, the DKTGT sequence in which the aspartate

becomes reversibly phosphorylated during catalysis and the

GDGVNDA motif which plays a role in the hydrolysis of

acylphosphate intermediate (Kasamo 2003).

To examine the precise tissue- and development-specific

expression profile of CsHA2 and CsHA3, the levels of

transcripts in different parts of plants (immature leaves,

mature leaves, leaf petioles, shoots, flowers, fruits, seeds,

primary and lateral roots) and different developmental

stages (5-day-old or 3/5-week-old plants) were determined.

CsHA2 transcripts were found in all organs of both

immature and mature plants, but were absent in dry seeds

(Fig. 5a–c). Generally, its expression pattern differs in

young and mature cucumbers. CsHA2 transcript level in all

parts of young seedlings was equal (Fig. 5a), whereas it

varied in particular organs of 3-weeks old plants (Fig. 5b).

Lowest expression of CsHA2 was observed in the lateral

roots, whereas the highest transcript level was found in the

Fig. 1 Amplifying cDNA of CsHA2 and CsHA3 by RACE. The

following fragments were obtained: 50 end of CsHA3 = 700 bp. and

30 end = 1,800 bp. 50 end of CsHA2 = 2,300 bp. M-DNA ladder

(1,000–10,000 and 100–10,000 bp, Qiagen)

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leaves of mature plants. The expression for CsHA3 was

restricted to the roots and, in comparison with CsHA,2 its

expression pattern was totally different. CsHA3 transcript

was found only in the roots of seedlings as well as in the

lateral roots of mature plants. No CsHA3 mRNA was

detected in the other examined organs (Fig. 6a–c).

Fig. 2 Alignment of the

predicted amino acid sequences

of plasma membrane proton

pumps CsHA2 and CsHA3 from

cucumber using Clustal W

program. ‘‘*’’ means that the

amino acid residues in that

column are identical in

sequences in the alignment;

‘‘:’’ means that conserved

substitutions have been

observed; ‘‘.’’ means that semi-

conserved substitutions are

observed. Amino acid

sequences of CsHA2 and

CsHA3 across their entire

length show 95% similarity

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Discussion

Plasma membrane H?-ATPase has been localized in all

living plant cells. However, some types of cells and organs,

for example the epidermis, root hairs, phloem or guard

cells are much more abundant in this protein than others

(Arango et al. 2003; Harper et al. 1990; De Witt et al. 1996;

Nakajima et al. 1995). Therefore, the aim of this research

was to identify two full-length CsHA genes and to char-

acterize their structure and organ localization in immature

and mature cucumber plants.

Based on the partial nucleotide sequences of HA genes,

which have been found in cucumber, we amplified whole

cDNAs of two genes encoding plasma membrane proton

pumps named CsHA2 and CsHA3. Multiple alignments of

protein sequences can be very useful in identification of

new members of protein families. Alignment of the

nucleotide and amino acid sequences has shown high

similarity between CsHA2, as well as CsHA3 and many

other plant H?-ATPases (Figs. 2, 3). In addition, the

presence of the characteristic structures and the conserved

regions specific for H?-transporting ATPase (Fig. 4)

confirmed that these genes belong to the large group

of the plasma membrane proton pumps family and

encode the H?-ATPase isoforms. As demonstrated by the

phylogenetic tree (Fig. 3), both isoforms from cucumber

are more closely related to the H?-ATPases from Sesbania

rostrata and Kosteletzkya virginica than to the other spe-

cies. The great similarity of amino acid sequences between

CsHA and PMA4, OSA7 and AHA1, 2,3,5 (Fig. 3) seems to

imply that CsHA2 and CsHA3 isoforms belong to the

subfamily II of PM H?-ATPases (Arango et al. 2003).

Genes from this subfamily show high levels of expression

throughout the plant under normal conditions. The results

from CsHA2 gene expression analysis confirmed the pre-

sumption, because CsHA2 was expressed in all plant

organs, both in seedlings and adult plants, except dry seed

(Fig. 5a–c). This finding indicated that CsHA2 could

appear to function as a housekeeping gene required for

fundamental metabolic processes in cells. Thus far these

housekeeping genes have been well characterized in other

species, i.e. AHA1 and AHA2 in Arabidopsis thaliana,

PMA2 and PMA4 in Nicotiana plumbaginifolia, MHA2 in

Zea mays, LHA1, 2, 4 in Lycopersion esculentum (Ewing

and Bennett 1994; Morsomme and Boutry 2000; Arango

et al. 2003; Gaxiola et al. 2007). However, despite CsHA2

being a ubiquitous gene in cucumber, its expression level

in particular organs of adult plants was different. CsHA2

was preferentially expressed in the above-ground parts,

whereas in lateral roots lower abundance of mRNA was

observed. Conversely, CsHA3 was expressed only in roots

for both types of plants (Fig. 6a, b) and such differences in

the expression pattern of both genes, despite the similarities

in amino acid sequence, does not clearly group CsHA2 and

CsHA3 within subfamily II. Differential expression pattern

of mentioned genes could be a direct result of the regula-

tion by specific transcriptional factors and future work is

required to explain this inconsistency.

Roots, especially root epidermis, play an important role

in the uptake of nutrients from spoil and translocation of

these nutrients to other parts of the plant (Palmgren 2001).

In roots, plasma membrane proton pumps have been

shown in the epidermis and vascular tissues (Michelet and

Boutry 1995). It has been evidenced that in Arabidopsis,

AHA2 is strongly expressed in roots. This was verified by

experiments with gene construct AHA2::GUS, which

revealed a strong signal in epidermal and root cortex cells,

as well as in phloem, xylem and root hairs (Fuglsang et al.

2007). These results suggested that AHA2 participates in

nutrient uptake and long-term transport in plants. Our

analysis by RT-PCR demonstrated that CsHA3 isoform

may also be required in the nutrient acquisition, because

high expression of this gene was detected predominantly

in lateral roots of adult plants as well as in seedling roots

and such strong signal of the CsHA3 transcript in root

cells suggests a possible role for this isoform of plasma

membrane ATPases in energizing the massive ion fluxes

taking place there. While relatively lower level of CsHA2

Fig. 3 Phylogram of sequence similarities between proton PM

H?-ATPase from Cucumis sativus (CsHA2, 3), Kosteletzkya virginica(KVATP1), Sesbania rostrat (SRHA4), Nicotiana plumbaginifolia(PMA1, 2, 3, 4, 5, 6, 8, 9), Oryza sativa (OSA1, 2, 3, 4, 6, 7, 8, 9, 10)

and Arabidopsis thaliana (AHA1–AHA10). Amino acid sequences of

several H?-ATPases were compared with the program Clustal W and

divided into five subfamilies (I–V)

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Fig. 4 Schematic model of a

cucumber H?-ATPase. The

predicted topology represents

the most widely proposed model

for H*ATPases in plant. The

plasma membrane proton pump

has ten membrane—spanning

regions and the C- and

N-terminal tails on the

cytoplasmic site. The prediction

of transmembrane segments was

performed using TMHMM 2.0

server and two-dimensional

model of proton pump was

created by TMRPres2D

Fig. 5 Semi-quantitative RT-PCR analysis of CsHA2 expression in

cucumber 5-day-old seedlings (a) mature plants (b) and in flowers,

fruits and dry seeds (c). CsHA2 mRNA levels were normalized with

respect to the internal control 18s RNA. Each lane was loaded with

150 ng of total RNA from the cucumber organs. All experiments were

repeated two times independently with comparable results

Fig. 6 Semi-quantitative RT-PCR analysis of CsHA3 expression in

cucumber 5-day-old seedlings (a) mature plants (b) and in flowers,

fruits and dry seeds (c). CsHA3 mRNA levels were normalized with

respect to the internal control 18s RNA. Each lane was loaded with

150 ng of total RNA from the cucumber organs. All experiments were

repeated two times independently with the comparable results

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Fig. 7 Alignment of the

deduced nucleic acid sequence

of plasma membrane proton

pump CsHA3 and partial

protein sequence of CsHA1

from cucumber using Clustal W

program. ‘‘*’’ means that the

amino acid residues in that

column are identical in

sequences in the alignment;

‘‘:’’ means that conserved

substitutions have been

observed; ‘‘.’’ means that semi-

conserved substitutions are

observed. Amino acid

sequences of CsHA3 and

CsHA1 show 100% identity

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transcript in comparison with higher amount CsHA3 in

lateral roots vote a co-operation between these two iso-

forms. Beyond the expression of CsHA3 on the higher

level in roots can indicate that it is regulated under specific

environmental conditions, not tested by us. It is known

that acidification of the rhizosphere serves to drive iron

uptake under deficient conditions. Presumably, a proton

ATPase pumps proton across the plasma membrane in

response to iron limitation. Recent studies identified the

cucumber CsHA1 gene encodes an H?-ATPase that

functions in iron deficiency induced rhizosphere acidifi-

cation (Santi et al. 2005; Santi and Schmidt 2008). Fur-

thermore, in Arabidopsis, it was confirmed that expression

of the AHA7 gene is also upregulated in response to iron

deficiency and contributes in strategy I response to solu-

bilize and transport iron into roots (Colangelo and

Guerinot 2004). Making a comparison between nucleotide

sequences of CsHA1 partial cDNA (AJ703810) and full-

length cDNA of CsHA3 (EF375892) we have noticed

surprisingly a high degree of similarity 99 and 100%

identity in amino acid sequences (Fig. 7) (data not pub-

lished). Furthermore, the genome of cucumber has been

recently sequenced and published (Huang et al. 2009) and

based on an alignment of cDNA CsHA3 and partial cDNA

CsHA1 with whole genome shotgun sequence (contig

8888, accession number ACHR01008888.1), we can sug-

gest that CsHA1 can be a part of complete CsHA3 gene.

Taken together, we cannot rule out the possibility that

more than two isoforms can exist in cucumber roots,

which have not been previously characterized.

It is well known that H?-ATPases can be regulated by

reverse phosphorylation, resulting in enzyme activation

or inhibition (Duby and Boutry, 2009). This mechanism

involves phosphorylation of the penultimate residue (Thr)

on the C-terminus region of H?-ATPase and is essential

for the binding of 14-3-3 proteins, which modulate the

activity of the enzyme (Arango et al. 2003). The results

of the C-terminal ends alignment confirmed the strong

conservation of the phosphorylatable Thr in CsHA2,

CsHA3 and the rest of the analyzed sequences. It was

demonstrated earlier that salt stress in cucumber roots

enhanced activity of plasma membrane proton pump

and that post-translational modification, such as a rever-

sible phosphorylation, is involved in the stimulation

H?-ATPase under salt conditions (Kłobus and Janicka-

Russak 2007). An understanding of this regulation at the

molecular level will require a more detailed analysis of

the CsHA genes by hybridization in situ and genomic

library construction for isoforms of H?-ATPases in

cucumber. These approaches will be particularly useful

in explanation of the molecular and biochemical basis of

the H?-ATPase adaptation to the different environmental

conditions.

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