Plant Cell Physiol. 46(10): 1635–1645 (2005)

doi:10.1093/pcp/pci179, available online at www.pcp.oupjournals.org

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The 14-3-3 Gene Expression Specificity in Response to Stress is Promoter-Dependent

Anna Aksamit 1, Alina Korobczak 2, Jacek Skala 1, Marcin Lukaszewicz 1 and Jan Szopa 2, *

1 Institutes of Genetics and Microbiology, Wroclaw University, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland 2 Biochemistry and Molecular Biology, Wroclaw University, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland

;

Genomic clone coding for the 16R isoform of 14-3-3

proteins from potato plants has recently been described.

This paper reports on 20R-gene isolation and analysis, and

compares two isoforms. The northern blot analysis of

mRNA of the 20R 14-3-3 isoform suggests its similarity to

16R. Vascular tissue-specific expression and age-dependent

synthesis in potato leaves has been detected in both promot-

ers. Screening of the potato genomic library using 20R

cDNA isoform resulted in identification and isolation of the

corresponding gene. This gene contains four exons and

three introns. Inspecting the promoter sequence of the 20R

isoform revealed several boxes important for the regula-

tion of gene expression. The strongest GUS expression in

transgenic potato plants transformed with the uidA

reporter gene under the 20R promoter has been found in

young leaf and stem vascular tissue, root tips, pollen and

ovules. Mature fragments exhibit a significant decrease in

GUS staining, which suggests age-dependent promoter

activity. The analysis of transgenic plants transformed with

20R-GUS in contrast to 16R-GUS has revealed strong acti-

vation of the 20R promoter by metal ions and NaCl. Instead

the 16R promoter is strongly affected by virus and salicylic

acid treatments. The only factor, which strongly induced

both promoters, was abscisic acid. It is thus suggested that

promoter domain composition is the main factor differenti-

ating the appearance of 14-3-3 isoforms.

Keywords: 14-3-3 gene — GUS staining — Promoter analysis

— Solanum tuberosum.

The nucleotide sequences reported in this paper have been sub-

mitted to the EMBL/GenBank databases under the following acces-

sion numbers: 14-3-3 20R gene [AY518222.]; 20R cDNA [X87370];

16R gene [Y070220].

Introduction

Recent investigations suggest the participation of 14-3-3

proteins in numerous biological processes of eukaryotic cells.

Members of the 14-3-3 protein family activate neurotransmit-

ter synthesis; activate ADP-ribosylation of proteins; and

regulate the activity of protein kinase C, sucrose phosphate

synthase (SPS), starch synthase (SS) and nitrate reductase

(NR). Moreover, they display phospholipase A2 activity and

associate with the products of proto-oncogenes, oncogenes and

the cdc 25 gene (Chung et al. 1999, Szopa 2002). The broad

spectrum of activities that are affected by 14-3-3 proteins sug-

gests their potential in modifying plant development and

metabolism (Finnie et al. 1999, Roberts 2003).

While in recent years there has been substantial progress

in the identification of diverse partners of 14-3-3 proteins, at

least two important questions remain to be answered: (i) is

there any specificity in the action of a particular 14-3-3 iso-

form on the biology of the plant; and (ii) why do cells contain

several isoforms and how is the expression of each particular

isoform regulated?

To answer these questions, we investigated a number of

14-3-3 isoforms, examining the tissue specificity of their

expression, and their age-related synthesis in potato plants. By

screening cDNA libraries from leaves, roots and epidermal

fragments, six cDNAs encoding 14-3-3 isoforms had been pre-

viously found in potato plants (Wilczynski et al. 1998).

To discover the significance of 14-3-3 isoforms for plant

metabolism, transgenic potatoes with repression of one, two

and six mRNAs were created. The selected transgenic plants

were analysed for enzyme activities known to interact with 14-

3-3 proteins, and metabolites, which are the products of the

interacting enzymes. A significant increase in NR, SPS and SS

activities was found for several of the analyzed enzymes (Zuk

et al. 2003). The changes in enzyme activity in transgenic

plants are reversible and can be complemented by heter-

ogonous recombinant 14-3-3 proteins either from Cucurbita

pepo or potato plants. It is interesting to note that the level of

all measured activity strongly depends on 14-3-3 protein con-

tent in the generated transgenic plants, thus suggesting that the

quantity of 14-3-3 protein rather than the particular isoform

controls the interacting enzymes. The questions now arise why

the genome of eukaryotes contains several 14-3-3 isoforms and

how the genes’ expression is regulated.

To try to answer these questions, the gene encoding the

16R isoform of 14-3-3 proteins has recently been isolated and

analysed (Szopa et al. 2003). Several motifs have been found

supporting the assumption that the promoter could be tissue-

specific and developmentally-regulated. Western and northern

analyses as well as the histochemical and fluorometric assays

* Corresponding author: E-mail, szopa@ibmb.uni.wroc.pl; Fax, +48-71-3252930.

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of various potato organs from 16R-uidA-transformed plants

revealed that the expression of the 16R isoform of 14-3-3

proteins is age-dependent. The highest expression was detected

in youngest organs or organ portions, leaves and stems in

particular. The analysis of GUS staining in transgenic plants

also showed the promoter tissue-specificity. Substantial GUS

expression was characteristic not only for meristems, but also

for the primary vascular tissues of minor vascular bundles. As

16R promoter activity seems to be induced by sucrose, 14-3-3

protein activity could be involved in the phloem loading. The

data obtained strongly suggests that 14-3-3 promoter investiga-

tion is the proper way to study the function of particular iso-

forms in cell biology.

In this study we first analysed by northern blot the expres-

sion pattern of mRNA which encodes the 20R isoform of 14-3-

3 protein in different plant organs and in leaves of different

ages. Then the genomic clone encoding 14-3-3 was isolated,

analysed in detail and compared to the database of accumu-

lated sequences. Finally, the promoter characteristic was stud-

ied in transgenic plants transformed with the reporter GUS

gene under the control of the 20R promoter and the data com-

pared to other 16R properties.

The promoters contain mostly the same motifs; however,

there are also several sequences that differentiate the genes’

activity. There is in 16R a unique motif activated by salicylic

acid and virus infection, which is absent in 20R. The latter

however contains unique sequences responsive to ethylene and

metal ions. This suggests that unique domains present in its

promoter distinctly regulate each isoform.

Results

14-3-3 gene organization

Screening of a genomic library with full length cDNA

encoding 20R isoform of 14-3-3 protein (EMBL/GenBank

Database Accession No. X87370), resulted in the isolation of a

genomic 3959 bp fragment [EMBL/GenBank database acc No.

AY518222] containing 1179 bp of the putative 5′ promoter

region, the complete coding sequence, and 246 bp of the 3′

noncoding element. There are four coding regions 1240–1716,

1973–2095, 2213–2329, 2423–2473 interrupted by three

introns. The transcription start site is 60 bp upstream of the

translation start codon. The amino acid sequence of 20R iso-

form is three residues shorter than that of the 16R isoform (Fig.

1), which has the highest homology (only 12 different amino

acids).

Analysis of promoter domains

The sequence analysis with the use of a Web Signal Scan

Program resulted in the identification of several core frag-

ments of great importance for promoter function. The 1239 bp

region upstream of the translation start site of 20R contains a

common CAAT box located at the –330 bp position. Inspec-

tion of downstream fragments reveals the expected TATA box

(TTATTT) at the –107 bp position.

Elements, which could potentially be recognised by tran-

scription factors, are good guidelines for design of experi-

ments aiming to reveal physiological processes in which a

given isoform could be involved. For example the AGAAA

motif (–1065, –217, –58, –49, –28) in the 20R promoter

sequence could be responsible for expression in pollen as was

shown for the late 52 gene in tomato (Bate and Twell 1998).

Analysis of putative targets for transcription factors is use-

ful for an experimental approach to test promoter activity. For

example, in both promoters (16 and 20R) eight E-box (CAN-

NTG) motifs have been found (Table 1). The E-box is recog-

nised as a Myc binding site that participates in ABA-induction

and binds a drought-inducible Myc homologue (Busk and

Pages 1998). The additional presence of Myb (–1113, –408)

and Myc (–233) binding sites in 20R promoter sequences, con-

comitant with the strong induction of these factors upon water

stress, may suggest the involvement of 14-3-3 protein synthe-

sis in the mechanism of ABA and water stress response of

potato plants.

In contrast to the protein sequence of 14-3-3 isoforms,

which is very conserved, their promoter sequences show sev-

eral important differences. For example, promoter 16R contains

two unique sequences TTGACC (–430) and TTWTWTTWTT

(–259). The first one is the core sequence of an elicitor respon-

sive element (ElRE), required for plant defence signalling

(Chen and Chen 2000). The second is a T-box found in a SAR/

MAR sequence. The presence of an E1RE sequence in a 16R

promoter might suggest the involvement of the 14-3-3 proteins

in plant defence mechanisms upon pathogen infection and sali-

cylic acid induction. The abundance of the 14-3-3 protein in

plant tissues may result from the presence of SAR/MAR

sequences in its promoter. It is suggested that this sequence is

responsible for chromatin attachment to the nuclear matrix and

genes within regions attached to this nuclear structure are

highly active.

Two very interesting promoter elements unique to 20R are

ethylene responsive at –1028 and metal responsive at –755.

The sequence AWTTCAAA was identified as an ethylene-

responsive region in the promoter of a fruit ripening gene and

as an enhancer element involved in the senescence-related

expression of the carnation and tomato glutathione-S-trans-

ferase gene (Hwang et al. 1998). Metals represent a class of

important transcription effector molecules which regulate gene

expression. The TGCRCNC sequence at –755 position in 20R

was earlier recognized as a metal-regulated element (MRE) of

the mouse metallothionein-I gene. Heavy metal ions effec-

tively induced metallothionein gene transcription and the

induction is MRE dependent. The transcription factor Sp1 and

a zinc-inducible factor MTF-1 bind to the MRE enhancer in

vitro (Thiele 1992).

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Tissue specificity of the promoters

The presence of many 14-3-3 protein isoforms in potato

plants may suggest their selective expression in tissue- or

developmental-dependent manner. To test this, the total RNA

was isolated from different plant organs, blotted onto nitro-

cellulose, and probed with the most divergent 3′-fragment of

16R and 20R cDNA. The results showed that both isoforms

Fig. 1 Amino acid alignment of 16R

and 20R 14-3-3 protein isoforms. Col-

our labels of amino acids: RED (small+

hydrophobic including aromatic -Y),

BLUE Acidic, MAGENTA Basic,

GREEN Hydroxyl + Amine + Basic. * =

identical residues in the alignment. : =

conserved substitutions according to the

colour label. · = semi-conserved substi-

tutions.

Fig. 2 A-G. Expression of 20R-GUS fusion in vegetative organs of

Solanum tuberosum cv Desiree. A. Adaxial surface of Solanum leaf.

Veins show blue-staining. B. Cross section of the leaf blade. Vascular

bundles and adjacent parenchyma cells are stained. C. Cross-section of

the leaf blade at higher magnification. Parenchyma cells surrounding

the vascular bundle and the bundle (here running in the section plane)

show GUS staining. D. Section through the tuber. Only vascular bun-

dles are blue stained. E. Cross-section of the tuber. Blue staining is

apparent in the vascular bundle and surrounding parenchyma. Storage

parenchyma are not stained. F. Roots. Root apices and vascular cylin-

ders are stained. G. Cross-section through the root hair zone of root.

Staining is apparent in the vascular cylinder. E – epidermis; V – vascu-

lar bundle; P – palisade mesophyll; S – spongy mesophyll; St – storage

parenchyma; R – rhizodermis. Bars = 50 µm.

Fig. 3 A-F. Expression of 20R-GUS in flowers of Solanum tuberosum

cv. Desiree. A. Flower bud. A portion of calyx and corolla were

removed so that the flower bud interior can be seen. Light blue stain-

ing is visible in the ovary. B. Stigma covered with blue-stained pollen

grains. C. Cross section through the anther. Pollen grains show the his-

tochemical reaction. D. Section through the stigma with pollen grains

attached to its surface. The grains are stained and some of them have

started to germinate. E. Stamen dissected from the flower bud, shown

from the adaxial side. Blue staining can be seen in the basal portions of

the anther. F. Cross-section of the flower bud. A portion of ovary with

placenta is shown. G. Ovules are blue-stained. Ov – ovary; Ant –

anther; Sep – sepal; St – stigma; Pl – placenta. Bars = 50 µm.

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encoded by 16R and 20R cDNAs are present in all the organs

analysed (Wilczynski et al. 1998).

Histochemical staining of transgenic potato plants trans-

formed with translational fusion of the 20R promoter with

GUS reporter protein revealed that tissue specificity is very

similar to the 16R isoform. In vegetative tissues 20R isoform

expression is localized mainly in the vascular tissue of young

organs (Fig. 2). In the leaf blade the strongest staining is lim-

ited to vascular bundles and adjacent parenchyma cells. In

tubers only vascular bundles and surrounding parenchyma are

blue, while storage parenchyma is not stained. In roots staining

is limited to vascular cylinders of root apices and the root hair

zone. In flowers (Fig. 3) GUS expression is limited to the gen-

erative organs: ovules and pollen grains which could result

from multiple AGAAA motifs.

Age-dependent analysis of 16R and 20R gene expression

Based on western analysis we have recently found that 14-

3-3 gene expression might be developmentally-regulated.

Indeed, northern blot analysis shows the highest quantity of

mRNA encoding 16R isoform in the middle stem sector. Anal-

ysis of consecutive leaves within one stem revealed the highest

mRNA level of 20R isoform in the youngest leaves. For com-

parison, the same blot when probed with 35G cDNA encoding

another 14-3-3 protein isoform showed that the expression of

this isoform was not age dependent (Wilczynski et al. 1998).

Table 1 Transcription factors binding sequences and tested factors on 14-3-3 promoters

Putative motifs recognised by transcription factors were found using Web Signal Scan Program.

+, induction of the promoter activity.

–, no effect on the promoter activity.

nd, conditions not analyzed.

TF 16R 20R

Sequence Effect Ref. Sequence Effect Ref.

ABA 1. ACGTG + 1. (Simpson et al. 2003) 1. ACGTC + 1. (Simpson et al. 2003)

2. ACGT 2. (Simpson et al. 2003) 2. ACGT 2. (Simpson et al. 2003)

3. CANNTG 3. (Stalberg et al. 1996) 3. CANNTG 3. (Stalberg et al. 1996)

4. WAACCA 4. (Abe et al. 2003) 4. WAACCA 4. (Abe et al. 2003)

5. TAACTG 5. (Urao et al. 1993) 5. YAACKG 5. (Abe et al. 2003)

6. CACATG 6. (Abe et al. 1997) 6. CTAACCA 6. (Busk and Pages 1998)

7. ACACNNG 7. (Kim et al. 1997)

Ethylene Not found – Not found 1. AWTTCAAA – 1. (Itzhaki et al. 1994)

Auxin 1. TGTCTC + 1. (Hagen and Guilfoyle 2002) 1. CATATG – 1. (Xu et al. 1997)

2. ACTTTA 2. (Baumann et al. 1999) 2. ACTTTA 2. (Baumann et al. 1999)

3. TGACG 3. (Terzaghi and Cashmore 1995)

Saliylic acid 1. TTGACC + 1. (Chen and Chen 2000) 1. TGACG – 1. (Redman et al. 2002)

Pathogen 1. GAAAAA + 1. (Park et al. 2004) 1. GAAAAA – 1. (Park et al. 2004)

2. TTGACC 2. (Chen and Chen 2000)

Metals Not found nd Not found 1. TGCRCNC + 1. (Wang et al. 2004)

NaCl 1. GAAAAA nd 1. (Park et al. 2004) 1. GAAAAA + 1. (Park et al. 2004)

Jasmonic acid 1. AACGTG – 1. (Boter et al. 2004) 1. AACGTG – 1. (Boter et al. 2004)

Light 1. GATA – 1. (Reyes et al. 2004) 1. GATA – 1. (Lam and Chua 1989)

2. GATAA 2. (Terzaghi and Cashmore 1995) 2. GATAA 2. (Terzaghi and Cashmore 1995)

3. GRWAAW 3. (Zhou 1999) 3. GRWAAW 3. (Terzaghi and Cashmore 1995)

4. GATAAG 4. (Giuliano et al. 1988) 4. TATTCT 4. (Thum et al. 2001)

5. CTCCCAC 5. (Ngai et al. 1997)

Hurt 1. AGATCCAA – 1. (Sugimoto et al. 2003) Not found – Not found

Low temp. 1. CANNTG – 1. (Chinnusamy et al. 2003) 1. CANNTG + 1. (Chinnusamy et al. 2003)

Gibberelic acid Not found – Not found 1. TAACAAR – 1. (Ogawa et al. 2003)

2. TAACAAA 2. (Gubler et al. 1995)

3. TGAC 3. (Zhang et al. 2004)

Sucrose AATAGAAAA + 1. (Grierson et al. 1994) 1. TAACARA – 1. (Huang et al. 1990)

2. TATCCAT 2. (Hwang et al. 1998)

3. TGACT 3. (Sun et al. 2003)

4. TATCCA 4. (Lu et al. 2002)

5. TATCCAY 5. (Toyofuku et al. 1998)

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Thus, the data from mRNA analysis strongly suggest the

ontogenetic regulation of 14-3-3 isoform expression. Quantifi-

cation of the 20R promoter-driven GUS expression in trans-

genic plants further supports age-dependent 14-3-3 promoter

activity (Fig. 6). The highest activity has been found in the

youngest analysed leaves.

Comparison of factors influencing 20R and 16R isoform

expression

Differences found between promoters during the analysis

of regulatory domains led to experimental verification of sev-

eral factors potentially influencing 16R and 20R promoter

activity (Fig. 4). The uidA gene under the control of 14-3-3

Fig. 4 GUS activity in 16R-GUS and 20R-GUS transgenic plants treated with various factors. Influence of ABA, salicylic acid, virus PVY, IAA

and sucrose on the 14-3-3 promoters activity. GUS activity was measured in leaves (about 8–12 plastochrons old) from 35S-GUS and 16R-GUS

and 20R-GUS transgenic potato plants. Tissues were harvested at noon from plants grown in the greenhouse and incubated on the MS medium

supplemented with ABA (100 µM), salicylic acid (100 µM), IAA (100 µM) and 2.5% sucrose. The mean values from eight independent measure-

ment ± confidence interval are presented.

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promoters was introduced via Agrobacterium infection into

potato plant genome. Transgenic plants were pre-selected by

means of PCR reaction with the primers specific for neomy-

cine phosphotransferase (kanamycin resistance) and then

selected by GUS activity measurements. The selected trans-

genic lines with the highest enzyme activity were used for fur-

ther analysis. Various factors were tested on young potato

leaves (about 9 plastochrons old) of in vitro-grown plants, cut

and incubated on MS medium. GUS activity was normally

measured after 0, 6, 12, 24 hours (up to 24 days for infection

experiments).

Although both promoters contain some common motifs,

which could potentially be involved in regulation by the same

factors, experiments have shown very different response pat-

terns to investigated factors (Table 1). In contrast to 16R, iso-

form 20R was not induced by sucrose and IAA. The only factor

strongly increasing the transcription of both isoforms was

abscisic acid. In the leaves of potato incubated on MS medium

supplemented with ABA after 6 hours a more than 3-fold activ-

ity increase was found in transgenic lines transformed with

20R-GUS construct. The expression remained almost constant

for 24 h. Northern blot on native transcript shows much higher

amount of 20R mRNA after 12 hours than 6 hours (Fig. 7). The

16R is also activated by ABA although the strongest promoter

Fig. 5 GUS activity in 20R-GUS transgenic plants treated with

heavy metals and NaCl. GUS activity was measured in leaves (about

8–12 plastochrons old) from 35S-GUS and 20R-GUS transgenic

potato plants. Tissues were harvested at noon from plants grown in the

greenhouse and incubated on the MS medium supplemented with

CdSO4 (100 µM), CuCl2 (100 µM), ZnSO4 (100 µM) and NaCl

(100 mM). The mean values from eight independent measurements ±

confidence interval are presented.

Fig. 6 Age-dependent GUS activity in 35S-GUS, 20R-GUS and

16R-GUS transgenic plants. The activity was measured in subsequent

leaves of plant from youngest to oldest. Tissues were harvested at

noon from plants grown in the greenhouse. The mean values from

eight independent measurements ± confidence interval are presented.

Fig. 7 20R mRNA accumulation in control potato and treated with

ABA, heavy metals and NaCl. Northern blot analysis of the accumula-

tion of the transcript 20R in control potato organs (Solanum

tuberosum) and treatments with ZnSO4 (100 µM), NaCl (100 mM),

ABA (100 µM), CuCl2 (100 µM), CdSO4 (100 µM) for 6 h and 12 h

before harvest. 40 µg of total RNA isolated from the control plant and

treated with ZnSO4, NaCl, ABA, CuCl2, CdSO4 was fractionated on

agarose formaldehyde gels, transferred onto membranes and hybrid-

ised with [32P]-labelled respective fragment cDNA of isoform 20R

(A). The size of ribosomal RNA is marked (B).

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activation appeared after 24 h of treatment. However as shown

by northern blot on native transcript (Fig. 8), promoter 16R

was also strongly induced by ABA after only 6 hours treat-

ment. Heavy metals (Zn, Cu, Cd) and NaCl treatment did not

result in significant increase of native 16R isoform transcript

detected on northern blot.

Virus infection activates 16R

Analysed promoters contain putative sequences which

could respond to salicylic acid and pathogen attack. The 16R

isoform contains an E1R domain, which could respond to both

salicylic acid and pathogen treatment (Chen and Chen 2000).

Two-fold increase was observed for the 16R isoform 6 hours

after salicylic acid treatment, while in 20R only slight induc-

tion after 24h was observed (Fig. 4). PVY infection of 16R-

GUS plants resulted after 12 days in eleven-fold increase of

GUS activity in directly infected leaves and seven-fold increase

in leaves on the same plant but not directly infected. The high-

est level of 16R native transcript has been also observed 14

days after PVY treatment (Fig. 9). In infected 20R plant, as in

the reference 35S-GUS plants, no significant change in GUS

activity was detected. Thus the analysed promoters differ in

their activity in response to pathogen attack and salicylic acid

treatment.

Metal ions affect 20R

Another factor discriminating both promoters is response

to metal ions, which affect only the 20R promoter. The treat-

ment of 20R plants with Zn, Cu and Cd resulted in an up to 9-

fold increase in GUS activity (Fig. 5). Induction with Zn seems

Fig. 8 16R mRNA accumulation in control potato and treated with

ABA, heavy metals and NaCl. Northern blot analysis of the accumula-

tion of the transcript 16R in control potato organs (Solanum

tuberosum) and treatments with ZnSO4 (100 µM), NaCl (100 mM),

ABA (100 µM), CuCl2 (100 µM), CdSO4 (100 µM) for 6h before har-

vest. 40 µg of total RNA isolated from the control plant and treated

with ZnSO4, NaCl, ABA, CuCl2, CdSO4 was fractionated on agarose

formaldehyde gels, transferred onto membranes and hybridised with

[32P]-labelled respective fragment cDNA of isoform 16R (A). The size

of ribosomal RNA is marked (B).

Fig. 9 16R mRNA accumulation in control potato and treated with

wirus PVY. Northern blot analysis of the 16R transcript in control

potato organs (Solanum tuberosum) and after (14 or 21 days) treat-

ment with PVY virus. 40 µg of total RNA isolated from the control

plant and treated with PVY virus was fractionated on agarose formal-

dehyde gels, transferred onto membranes and hybridised with [32P]-

labelled 16R cDNA fragment (A). The size of ribosomal RNA is

marked (B).

Fig. 10 Western analysis of proteins isolated from leaves of control

and treated S. tuberosum plants. 80 µg of protein was applied onto

each slot of SDS-polyacrylamide gel electrophoresis and on the same

blot was probed with antibody anti-recombinant 20R protein. The

plants (Solanum tuberosum) were exposed to ABA (100 µM), CdSO4

(100 µM), CuCl2 (100 µM), ZnSO4 (100 µM), NaCl (100 mM)

between 6–48 h. Control: plants harvested before onset treatment. The

ribulose-1–5 bisphosphate carboxylase/oxygenase (Rubisco) on the

same blot stained with Pounceau red is marked.

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to be slower and longer lasting than other metal ions. The acti-

vation of 14-3-3 20R isoform by ABA, metal ions and NaCl,

was also confirmed by northern (Fig. 7) and western (Fig. 10)

analysis on wild-type not transformed Desiree potato plants. In

all cases a significant increase in 14-3-3 20R mRNA and pro-

tein level was detected (Fig 7, 10) after treatment with ABA,

NaCl and heavy metals. The correlation coefficient between

induction observed on northern and western blots is in most

cases very high (0.98 for ABA, 0.99 for Cd and 0.84 for Zn).

Lower correlation for Cu results from maximal induction time

(6 h on western and 12 h on northern blot). Significant activa-

tion of nitrate reductase (NR) in transgenic potato plants with

repression of six 14-3-3 protein isoformes (G3 plants) has been

previously shown (Zuk et al. 2003). This activity could be

restored at control level by addition of recombinant 14-3-3 pro-

tein. Thus, to further study in vivo 14-3-3 expression induced

by heavy metals we have analysed NR activity in control and

G3 plants treated with 100 µM metal ions solution (Fig. 11).

Treatment with metal ions resulted in decreased NR activity,

both in control and G3 plants. This further supports hypothesis

that 14-3-3 protein 20R isoform is upregulated by metal ions.

Apart ABA, all tested factors induced GUS activity in a

promoter dependent manner and the results suggest the spe-

cific appearance of particular isoforms in response to environ-

mental stimuli. While isoform 20R is specifically induced by

metal ions, isoform 16R is induced in response to pathogen

attack salicylic acid treatment, sucrose, ABA and auxin.

Discussion

14-3-3 proteins are abundant eukaryotic proteins that

interact with many other proteins, thereby modulating their

function, and thus modulating cell metabolism (Chung et al.

1999). While there has been substantial progress in the identifi-

cation of the diverse partners of 14-3-3 in recent years their

role in regulatory pathways remains to be elucidated. Promoter

studies could provide helpful guidelines how to investigate the

specificity of 14-3-3 isoforms. To answer this question, investi-

gations were made on the two 14-3-3 gene promoters. It was

previously reported that plant and human 14-3-3s could be

replaced by yeast isoforms (van Heusden et al. 1992). Such a

result was not surprising since there is a very high homology

within 14-3-3 isoforms. Our own data also suggests that at the

metabolic level each potato isoform play the same role. The

repression of any isoform or all of them in potato transgenic

plants resulted in a significant increase in NR, SPS and SS

activities and thus affected carbohydrate and amino acids

metabolism (Szopa et al. 2001, Swiedrych et al. 2002, Zuk et

al. 2003). Thus the question arises why the eukaryotic genome

accumulated several genes coding for 14-3-3 isoforms? The

data presented in this paper demonstrates clearly for the first

time that promoters specify 14-3-3 genes expression. Two iso-

lated promoters 16R and 20R differ significantly when consid-

ering their response to environmental condition. The most

important differences are 16R activation by salicylic acid and

virus infection and activation of 20R by metal ions. It was ear-

lier demonstrated that an increase in 14-3-3 protein synthesis

results from pathogen treatment of the plant (Brandt et al.

1992) and now it is clear that it is due to the presence of the

specific domain in the isoform promoter. The increase in 14-3-

3 content resulted in NR and SPS inhibition, which subse-

quently redirected metabolism to the synthesis of other com-

pounds.

Thus we conclude that for 14-3-3 genes, the main differ-

ence is the promoter region containing specific inducible

domains, and not the coding region responsible for enzymatic

specificity. Recently we have reported on phenolic compound

synthesis affected by 14-3-3 protein. Overexpression of 14-3-3

cDNA in potato resulted in an increase of phenolic acid synthe-

sis, and a reduction of 14-3-3 content showed the opposite

effect (Lukaszewicz et al. 2002). Since the 14-3-3 genes pro-

moter contains a P box (Davies and Schwinn 2003) also char-

acteristic for genes coding key enzymes (CHS, CHI, DFR) of

flavonoid biosynthesis, it might suggest that in vivo both types

of genes are regulated in parallel. In this work we show that

expression of one 20R isofom may be restricted to very spe-

cific tissue like pollen and could be specifically stimulated by

heavy metals and NaCl. It is suggested that similar genes

respond to various environmental cues and stresses (Cooper et

al. 2003). Our results of promoter studies suggest that 14-3-3

protein could be such universal stress signal regulation mole-

cule. The need for the precise regulation of the response to var-

ious environmental cues could explain existence of several

isoforms with promoters dedicated to given stimulus set. We

have shown that 16R 14-3-3protein isoform could be special-

ised in response to pathogen attack and 20R isoform in

response to metal ions. In vivo we have shown that metal ions

affected NR activity. Other putative target in response to heavy

metal ions could be plasma membrane ATPase which is sup-

posed to affect plasma membranes fluidity and active transport

through membranes. These findings could be helpful in further

Fig. 11 NR activity in leaves of plants treated with heavy metals.

Leaves of Solanum tuberosum Desiree plants-control (D) and trans-

genic plants with repression of six 14-3-3 protein isoforms (G3) were

exposed to heavy metals (100 µM) between 6–24 h. Third and fourth

leaf from the top of the plant were frozen in liquid nitrogen and

extracted as described in materials and methods, then analysed and

compared to the control (D). Results are the mean ± SE (n = 6 different

plants from G3 transgenic line).

14-3-3 gene promoter analysis 1643

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deciphering the regulation of metabolic pathways by 14-3-3

proteins.

Materials and Methods

Plant material and bacterial strains

Potato plants (Solanum tuberosum L. cv. Desiree) were obtained

from “Saatzucht Fritz Lange KG” (Bad Schwartau, FRG). Plants in tis-

sue culture were grown under a 16 h light – 8 h dark regime on MS

medium (Murashige and Skoog 1962) containing 0.8% sucrose. Plants

in the greenhouse were cultivated in soil under 16-h light (22°)/8-h

dark (15°C) regime. Plants were grown in individual pots and were

watered daily.

Escherichia coli strain DH5a (Bethesda Research Laboratories,

Gaithersburg, USA) was cultivated using standard techniques

(Sambrook et al. 1989). Agrobacterium tumefaciens strain C58C1 con-

taining plasmid pGV2260 (Deblaere et al. 1985) was cultivated in

YEB medium (Vervliet et al. 1975).

Recombinant DNA techniques

DNA manipulations were done essentially as described by

Sambrook et al. (1989). DNA restriction and modification enzymes

were obtained from Roche (Germany) and New England Biolabs

(Beverly, MA). Escherichia coli strains DH5a and XL1-Blue were

used.

Construction of a 20R promoter GUS fusion

The 1084 bp SmaI–Asp718 fragment of the 5′ promoter region of

the 20R gene [EMBL/GenBank database acc No. AY518222.], from –

1239 to +1 (translation start site), was inserted in frame into the

BamHI/SalI site of pBI101 (Clontech), in front of the E. coli GUS

gene and the nos polyadenylation signal. The obtained vector, contain-

ing the 20R-GUS fusion, was introduced into the Agrobacterium

tumefaciens strain C58C1:pGV2260 as previously described (Rocha-

Sosa et al. 1989).

Transformation of potato plants

Young leaves of wild-type potato Solanum tuberosum L. cv.

Desiree were used for the transformation with A. tumefaciens by

immersing the leaf explants in the bacterial suspension. A.

tumefaciens-inoculated leaf explants were subsequently transferred

onto the callus induction and shoot regeneration medium (Rocha-Sosa

et al. 1989). Transgenic potato plants were screened by means of PCR

reaction with the use of the 20R promoter and kanamycin specific

primers as previously described (Wilczynski et al. 1998, Szopa et al.

2001).

Northern blot analysis

The expression of 20R and 16R 14-3-3 isoforms in potato plant

organs and developing leaves were performed by means of Northern

blot analysis. Total RNA was prepared from frozen plant material

using the guanidinium hydrochloride method. Following electro-

phoresis (1.5% [w/v] agarose, 15% [v/v] formaldehyde) RNA was

transferred to nylon membranes (Hybond N, Amersham, UK). The

membranes were hybridised overnight at 42°C in a 250 mmol sodium

phosphate buffer (pH7.2) containing 7% [w/v] SDS, 1% [w/v] bovine

serum albumin (BSA) and 1 mmol EDTA. A radioactively-labelled

hybridization probe of 16R (BamHI/PstI cDNA fragment) or 20R

(KpnI/PstI cDNA fragment) was used. The filters were washed three

times in 2× SSPE +0.1% [w/v] SDS for 10 min at 42°C; three times in

1× SSPE +0.1% [w/v] SDS for 10 min at 42°C; and three times in 0.1×

SSPE +0.1% [w/v] SDS for 10 min at 42°C.

Western blot analysis

An assessment of the expression of the 14-3-3 gene was

conducted by means of western blot analysis using the rabbit IgG

anti-recombinant 20R protein as described previously (Szopa and Rose

1986). Briefly, a solubilised protein was run on 12% SDS–poly-

acrylamide gels and blotted electrophoretically onto nitrocellulose

membranes (Schleicher and Schuell). After the transfer, the membrane

was sequentially incubated with a blocking buffer (5% dry milk)

and then with antibodies directed against the 20R recombinant protein

(1 : 2,000 dilution). The formation and detection of immune com-

plexes was performed as previously described (Szopa and Rose 1986).

Alkaline phosphatase-conjugated goat anti-rabbit IgG served as a

second antibody, and was used at a dilution of 1 : 1,500.

Histochemical analysis of the 20R promoter GUS fusion

Histochemical staining for GUS activity was performed with the

use of 5-bromo-4-chloro-3-indolyl β-D-glucuronide as a substrate

(Jefferson et al. 1987). Freshly-cut plant tissues were incubated in

GUS staining solution at 37°C, for 2–24 h depending on staining

intensity, and bleached with 70% ethanol. The material was examined

with a stereomicroscope Olympus SZX9, and anatomical sections

were prepared. The specimens were dehydrated and embedded in par-

affin (Johansen 1940). Minimal times were used for the treatment with

buthanol and buthanol–paraffin mixtures, in which the specimens were

kept in higher temperatures (60°C). Microtome paraffin sections, 25–

30 µm thick, were rinsed in xylene and ethanol for a short time.

Euparal was used as a mounting medium. The sectioned material was

examined using bright-field illumination microscopy Olympus BM50.

Fluorometric GUS assay

Potato transgenic lines with strong histochemical GUS staining

in leaves and stems were used for glucuronidase assay. The samples

were extracted with 50 mM Tris buffer, pH 8.0 containing 10 mM β-

mercaptoethanol, 10 mM EDTA, and centrifuged for 10 min at

13,000 rpm (Sigma Instrument). Aliquots of the supernatant were used

for enzyme assay with 4-methylumbelliferyl β-D-glucuronide as a sub-

strate (Jefferson 1987) and for protein determination with Bradford

reagent (Bradford 1976). The reaction product 4-methylumbelliferone

was measured fluorometrically (SFM 25 Fluorescence Spectrophotom-

eter, Kontron Instrument, Hamburg, Germany).

Treatment conditions

GUS activity was measured in leaves (about 8–12 plastochrons

old) harvested from plants grown in the greenhouse and incubated on

the MS medium supplemented with 2.5% sucrose, 100 µM ABA or

100 mM NaCl, 100 nM salicylic acid, 100 µM IAA, 100 µM CdSO4,

100 µM CuCl2, 100 µM ZnSO4. For virus infection entire greenhouse

plants were used.

Acknowledgements

The authors wish to thank Dr. G. Wilczynski for initial work on

14-3-3 gene isolation. This work is supported by grants No 2P06A 022

27 and PBZ-KBN-089/P06/2003 from National Research Committee

(KBN) and EC HPRN-CT-2002-00269.

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(Received February 11, 2005; Accepted July 24, 2005)