# Analysis of the Arabidopsis Mitogen-Activated Protein Kinase Families: Organ Specificity and Transcriptional Regulation upon Water Stresses

Authors: Khaled Moustafa; Delphine Lefebvre-De Vos; Anne-Sophie Leprince; Arnould Savouré; Christiane Laurière
Journal: Scholarly Research Exchange
 (2008)

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## Abstract

The Arabidopsis genome contains 20MPK, 10 MKK, 60 MAP3K, and 10 MAP4K genes, but the functions of the corresponding proteins are still poorly characterized. To research possible functional MAP kinase modules involved in water stress signalling, original macroarrays were developed for their sensitivity and specificity. The expression patterns of the MPK and MKK gene families, as well as selected key members of the MAP3K and MAP4K families, were studied in relation to organ specificity and to various water stresses. Several kinases of the scarcely studied MPK group containing a TDY activation motif were induced by water stresses. There are four different MPKs (MPK9, MPK11, MPK17, and MPK18), among which three contain the TDY activation motif, and MKK9 and MAP3K6 are induced at the transcriptional level by dehydration, salinity, and hyperosmolarity treatments. They probably constitute MAP kinase key elements involved in water stress signalling.


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## Body

## 1. Introduction

Mitogen-activated protein (MAP) kinase signalling is one of the most important and conserved
pathways in all eukaryotic organisms from plants to animals. MAP kinases are
involved in most cell activities, from cell division to death, including cell
differentiation and proliferation, cell growth, as well as environmental stress responses. MAP
kinase module is typically composed of at least three protein kinases, MAP
kinase kinase kinase (MAP3K), MAP kinase kinase (MAP2K, also called MKK), and
MAP kinase (MAPK, also called MPK), which are sequentially activated by
phosphorylation. Following a stimulus, MAP3K will activate an MAP2K by
phosphorylation on two serine/threonine residues in the conserved motif S/TxxxxxS/T,
which, in turn, activates an MAPK by phosphorylation on both threonine and
tyrosine residues in the T(E/D)Y motif [1]. MAP kinases with protein phosphatases
function as on or off switchers to regulate the activity of many downstream
substrates, such as transcription factors, cytoskeletal proteins, and other
protein kinases [2].In plants, the role of MAP linases remains poorly understood. Most studies were performed at protein and biochemical levels and
reported the involvement of only a few MAP kinases in response to environmental
stresses in many species like rice [3–5], tobacco [6], tomato [7], alfalfa [8, 9], and Arabidopsis [10]. Expression data are now available for a great number of Arabidopsis
genes through microarray databases like genevestigator
(https://www.genevestigator.ethz.ch/). However, results concerning signaling
proteins like MAP kinases are clearly insufficient, mostly due to their low
level of expression.The completion
and analysis of Arabidopsis genome
revealed the presence of 20MPK, 10 MKK, 60 MAP3K, and 10 MAP4K genes for which a new nomenclature was established [1]. Based on sequence alignment, plant MPK family is classified into four groups (A, B, C, D). Members in A, B, and C
groups have the TEY phosphorylation motif, while the D group members possess the
TDY motif. Some members of A and B groups in A. thaliana and
their orthologs in other species have been described to be activated in
response to biotic and abiotic stresses [11–16]. Arabidopsis MPK3 and MPK6 (A group) and their orthologs in tobacco (WIPK
and SIPK, resp.) are the most extensively studied. MPK3 and MPK6 kinases
are activated by abiotic [11, 15, 17] and biotic stresses [18, 19]. Low temperature, low humidity,
hyperosmolarity, salt stress, touch, and wounding induced a rapid, transient
activation of both Arabidopsis MPK4 (B group) and MPK6 without any significant
change in amount of mRNA or protein level [11].Compared to
human MAP kinase cascades [20], plant MAP kinase pathways are more complex
and only little information is available about their components. So far, only
one complete MAP kinase cascade, from receptor to transcription factor, was
identified in A. thaliana in response to biotic stress [18]. Potential components of MAP kinase cascade triggered
by abiotic stress were described using two-hybrid system, yeast mutant
complementation, and in-gel kinase assay. MAP3K8 is transcriptionally induced
by salt stress, drought, cold, and wounding [21] and can interact with MKK1 and MPK4 to form
potential MAP kinase module [22, 23]. Recently, using high-throughput
phosphorylation microarrays, 48 potential substrates of MPK3 and 39 of MPK6
were identified [24].The signalling
specificity via MAP kinases is an important feature to understand how a given
signal is directed to a particular target. One of the ways to ensure
specificity is the presence of scaffold proteins [25, 26] that form enzymatic complexes between the
different elements in a given MAP kinase pathway. Only very few published data
about plant scaffold proteins are available. Nevertheless, MAP kinase proteins
themselves might play the role of scaffold proteins to ensure signal
specificity [27]. Another feature for MAP kinase specificity is
the presence of compatible docking domains, which ensure the affinity between
all components of the same signalling pathway [28]. Surprisingly, some MAP kinase components appear
to act as positive regulators for abiotic stress but negative regulators for
biotic stress [4], as the overexpression of some MAP kinase
genes enhances abiotic stress tolerance whereas the knock-out of other MAP
kinases increases the plant resistance to pathogen.Many MAP kinases are activated by more than one stimulus, enhancing the complexity of
MAP kinase signalling networks. Regulation via modular and frequently cross-talking
MAP kinase modules are a common scheme in their organization. MAP kinases can
be regulated at transcriptional, translational, or post translational levels. Global
transcriptional analysis of MAP kinase cascade components under stress
conditions provides an overview of potential biological functions of MAP kinase
genes whose mRNA levels change in response to stress. Using an original
macroarray approach, we chose to analyze with a high sensitivity the transcript
levels of MAP kinase genes. Expression levels of wholeMPK and MKK gene families and of some candidate members of MAP3K and MAP4K gene families were analyzed under different osmotic and water stress conditions
together with known stress marker genes. Our results revealed specific patterns
of MAP kinase gene expression upon abiotic stresses and functional MAP kinase
modules may be suggested. Many of the noncharacterized MAP kinase genes showed
a significant induction indicating a potential role of these kinases in stress
adaptation. In addition to the gene expression analysis, the activity of some
MAP kinases proteins was also investigated.


## 2. Materials and Methods

### 2.1. Plant Materials

A. thaliana cells,
Columbia accession, were cultured in liquid medium as previously described [15] and subcultured after 5 days at
density of 100 mg fresh weight/mL. A. thaliana Columbia seeds were surface-sterilized in sodium
hypochlorite (12° chloride) for 5 minutes, rinsed 5-6 times in sterile water,
dried and sowed on Petri plates in 0.5x MS medium [29], 1% bacto-agar, and 0.5 g/L MES. Seeds were
then cold-stratified at 4°C for 24 hours and transferred to culture room at
22°C and light (125 μM m−2s−1) on a 16 hours light/8 hours dark cycle.
Plant organs were prepared from Arabidopsis plants cultured in soil for one
month at 22°C under 16 hours photoperiod. Hydroponic plants were also used for
the preparation of roots and rosette leaves. To obtain hydroponic plants, seeds
were treated as above and sowed in 0.5 mL Eppendorf tubes containing MS solid
medium, and half-cut to be adapted to custom-made polystyrene sheets as
support. Polystyrene sheets with tubes were floated in opaque plastic tank
containing autoclaved liquid culture medium [30] and then placed in culture box
at short day (9 hours light, 15 hours dark), 22°C and 50% relative humidity.
Culture medium was renewed twice a week to prevent contamination.


### 2.2.  Stress Treatments

Osmotic stresses were applied to cells equilibrated for 4 hours in their culture medium
containing 10 mM MES-Tris, pH 6.2, and
adjusted to 200 mOsm with sucrose. After equilibration, medium was replaced by
either the same volume of hypoosmotic medium, 20 mOsm of medium A composed of
10 mM MES-Tris, pH 6.2, 1 mM CaSO4, 10 mM sucrose or isoosmotic medium, 200 mOsm (medium A
plus 180 mM sorbitol), or hyperosmotic medium 500 mOsm (medium A plus 480 mM
sorbitol). Cells were maintained in these media under gentle shaking and
continuous light during all treatments. After treatments, cells were harvested
by filtration, frozen in liquid nitrogen, and stored at −80°C until use.
Osmolarity was monitored using a freezing point osmometer (Roebling, Berlin, Germany). Dehydration and osmotic
stress were applied to in vitro seedlings by transferring them onto Whatman
paper at room temperature (dehydration stress) or onto liquid media containing
200 mM NaCl or 320 mM mannitol for hyperosmotic stress treatments. Recovery
treatments were performed by transferring stressed seedlings onto a normal
growth medium. They were then harvested at different time points and stored at
−80°C until RNA extraction.


### 2.3. Preparation of DNA Macroarrays

When it was possible,
primers were chosen in the5′- or 3′-nontranslated gene regions. The size of
the amplified fragments ranged between 200 and 300 bp. Using specific primers (Table 1), 44 amplicons corresponding to the 20 MPK, 10 MKK, 12 MAP3K, and 2 MAP4K genes, were amplified by PCR from cDNA or genomic DNA and
then cloned in Escherichia coli. PCR reactions were performed in 50 μL
using Eurobio Kit and PCR Express machine (PerkinElmer) with the following PCR
program: 1 cycle of 1 minute 30 at 94°C for initial denaturing and 25 cycles of
45 seconds at 94°C (denaturing), 45 seconds at 55°C (annealing), 50 seconds at
72°C (elongation) followed by 7 minutes at 72°C. Amplicon quality and
specificity were verified in 1.2% agarose gel. Specific MAP kinase DNA
fragments were cut out, purified from gel using QIAquick Gel Extraction kit (Qiagen, France)
and cloned into pGEM-T vectors (Promega). After denaturation with 0.4 M NaOH,
30 μL (280 ng) of each PCR product were spotted onto 7.5×11 cm nylon membrane
(Nytran, Schleicher & Schuell) using the Dot Blot system (Fisher). The
spotted membranes were then cooked at 80°C for 2 hours and conserved at room temperature
until hybridization. To check the quantity of spotted PCR
products, membranes were first hybridized with polynucleotide kinase (PNK)
radiolabelled T7 primer (common to all inserts) and hybridization signals were
evaluated. T7 primer radiolabelling was performed in 50 μL containing 1 μL 100 μM T7 primer (200 pmol), 34 μL H2O, 5 μL 10X PNK buffer, 5 μL of PNK
(10 U/μL), and 5 μL [γ-P33]-ATP (3000 Ci/mmol; 10 μCi/μL) and
incubated at 37°C for 1 hour. Membranes were prehybridized with 10 mL Church
buffer (0.5 M phosphate buffer, 7% SDS, 1 mM EDTA, 1% BSA) at 65°C with
gentle shaking overnight, washed twice in 50 mL SSARC (600 mM NaCl, 60 mM
sodium citrate, 7.2% N-lauroyl sarcosine) at room temperature and twice at
4°C. Membranes were hybridized with radiolabelled T7 primer in 50 mL SSARC at
4°C overnight, washed twice 15 minutes in 50 mL precold SSARC at 4°C, wrapped
in Saran, exposed to phosphosensitive screen for 3–5 hours and scanned at 200 μm using PhosphorImager (GE Healthcare). 25 μg total RNA was incubated with
7.25 μg oligo dT(12-18) (Invitrogen, Cergy-Pontoise, France)
in total volume of 21 μL, heated to 70°C for 10 minutes and then cooled on ice
for ≥2 minutes. Tubes were briefly centrifuged and the following components
were added: 10 μl of first strand buffer, 5 μl DTT (100 mM), 5 μl dNTPs (10 mM
each of dATP, dGTP, and dTTP), 5 μl of [α-P32]-dCTP (10 μCi/μl, 3000 Ci/mol) and 2 μl of PowerScript Reverse Transcriptase (Clontech, St
Quentin-en-Yvelines, France). Reactions were incubated at 42°C for 1 hour, 2 μL
of cold dCTP were added and reaction was incubated further 1 hour at 42°C. The
synthesized labelled cDNA probes were purified using ProbeQuant G-50 Micro
Columns (GE Healthcare)
following the manufacturer protocol. One μL of each reaction was taken before
and after purification to calculate CPM (count per
minute) using scintillation counter to determine the percentage of incorporation.
Purified cDNA probes were denatured by adding 80 μL of 1X STE buffer (0.5X
final), 48 μL 1 M NaOH (0.2 M final), incubated at room temperature for 10 minutes
and then neutralized by 48 μL of 1 M HCl (0.2 M final) and 48 μL of 20X SSC.
Prehybridization was performed at 65°C for ≥3 hours in 10 mL Church buffer [31]
prewarmed to 65°C. Then, denatured cDNA probes
were added at equal CPM levels and hybridization was carried out at 65°C
overnight. Membranes were washed twice in 2X SSC, 0.1% SDS at room temperature
for 30 minutes, and twice in 1X SSC, SDS 0.1% at 65°C for 20 minutes, then
wrapped in Saran film and exposed to phosphor sensitive screens for 24–48 hours.
Phosphor screens exposed to macroarray filters were scanned with PhosphorImager
(GE Healthcare) at 200 μm resolution. After image acquisition, the scanned
images were analyzed using ImageQuant TL 2003 to quantify the radioactivity of
each spot. Background corresponding to nonspecific hybridizations (water or
empty spot intensity values) was subtracted from the signal intensity of each
spot. The signal intensities of each spot from three independent experiments
were averaged and normalized against the intensity of ACTIN2/8 or as a percentage of the total sum
of all spots on the membrane. Genes were considered as induced/repressed when
expression ratio (experimental/control) was ≥2.5-fold.Table 1
Oligonucleotides used to amplify the MAP kinase fragments from cDNA or genomic DNA.
F: forward; R: reverse.
Gene nameLocusPrimer sequences (5′–3′)MPK gene familyMPK1At1g10210F: GCCAAATCTCTCTCACTTCCACAGGTR: CCGTAAGCTCCACGACCAATAGGCMPK2At1g59580F: CCGTAAGCTCCACGACCAATAGGCR: CACACCGTACGCGCCTCGMPK3At3g45640F: ACCTTGCTAAATTGCACGACCCGAR: AGCCATTCGGATGGTTATTGGGTMPK4At4g01370F: TTGTTGTGAGGAATTTTGCTCCGR: GCTACCTCCTCTCCAGTCTCTGAGMPK5At4g11330F: CTCGAATCTGTGAAGTTCAATCCTCTGCR: GAAGAAACAGAACACTATCTAAGACATCCTGMPK6At2g43790F: GAGATGAAGATGAGATAAGGGTTR: GGTGGGCTATCATAACATAACTATTTCMPK7At2g18170F: GCCCGGTGCTTTGATCTCGTCTGR: GGCTAGCAAGTGGGGCAATGGCMPK8At1g18150F: GTTTTCTGGTGAAAAGAAATTGR: GCAAAGTGAATACTGAATAGMPK9At3g18040F: TTCCTCTACCAGCTTCTTCGTR: TACCATCTTGTAGCAACATAGTCAGMPK10At3g59790F: ATGGAGCCAACTAACGATGCTGR: CAAACGATGCCACAAGCACCTCMPK11At1g01560F: GTCAATAGAGAAACCATTCTTCGGTGATGR: GGCAATGACATTGTCATGATCCATATGCTMPK12At2g46070F: CCTTCTTCTTGGTTTCTCTCGTTCTCGGR: CCGCAGCACAGACAATACCGMPK13At1g07880F: GATGCTAGTTTTCGATCCTTCR: CTTGAAGTGTAAAGACTCTCTCCAGACMPK14At4g36450F: GGATCTCCAGTTCATAGATAACCAR: CTCCAAACTACTGACCGGAACAMPK15At1g73670F: CGAGTTCCAGCATCCAAGAATGR: CAATGTTGGTTTTAGATTGTACACCMPK16At5g19010F: AGTGTTACGCTATAACAACTGCR: TTAATACCAGCGACTCATTGMPK17At2g01450F: GTTGGCAAAGGAAGCTACGGR: GATGATGCTGAGGAGTGAGGMPK18At1g53510F: GAAGTTATTGGTAAAGGAAGCTATGGAGTTR: ATCTGATTCCATGAGCTCAAACACAACAMPK19At3g14720F: ACAGTACTCCCTCCACAGACCGTAR: CTAAGACATGCCATACCCAACAGCMPK20At2g42880F: GATATGCACCAGATGTAGCCR: GCCCAGCTTAGGTGGAGAACMKK gene familyMKK1At4g26070F: AAGATGTTTGAAGATTCGGAR: ATCCACCACTAGCAGAGAAAMKK2At4g29810F: AAATACGACTACTCGGGGATR: TTTTAAATAAACACTCTCATGTACCMKK3At5g40440F: GATGGAGTCCAAGCAGAAGGR: TCCGGCACTTTTTATGAACCMKK4At1g51660F: TCGTCCTCTGTCCTCGTCTTR: GGGGATACATGCACCATCATMKK5At3g21220F: TCCATAAGTTTTGCATACCCR: GCAAATCCAGCTAAAATCACMKK6At5g56580F: TTTGATTTGGCCTTAATGAAATGR: CTTGTTGGCACTCTGGAACCMKK7At1g18350F: ACCCTTTTCTCCGTGAAAGTR: TTCCTCTGTTTCTGTCGATTGMKK8At3g06230F: TGGACTGCTGTTTACGCAAGR: GTGGAGACCCTACATCTCGCMKK9At1g73500F: CCCAAAACTTATGTACACGATTGAR: CTTGCTCATCCTTTTCTCCGMKK10At1g32320F: GTGGAAGAGCTTCTTCGTCAR: CTGAACTCGAATCAAACCAAAMAP3K gene familyMAP3K1At1g09000F: TCACCAGGAAAATGACTTGCR: CTAGCAATTTGTAGCGCACTMAP3K2At1g54960F: GCATCTCCAGGCAAATGATCR: GGCTTAACACTCAATATGCAMAP3K3At1g53570F: CCCCTAGTTTTGGCCTTCTCR: AGGTTGAAAACAAATGGTTGAAMAP3K4At1g63700F: CCTCTTCATCCTCGAGCTGGAR: GCGATCCAGGGCTAACCGTAMAP3K5At5g66850F: GCAGCTCGAGAGGTTAACCAAR: GCTTCTTCTATCGATTCAACATTGMAP3K6At3g07980F: GTGAGTGTACATTTATTCCCR: GATCACATCACGTCCACATTAAMAP3K7At3g13530F: GTCCACCAAAACGATAATATCCCR: GAAGGCTAGCAAGAGCAGTATAMAP3K8At4g08500F: ATACACCCAAAAACCCCAAGR: AGCAGGCATAACCCGAAGATMAP3K9At4g08480F: CAACAACCAACCAATGAATGAR: CCTCCATAGGTGAAGCCTCTMAP3K10At4g08470F: CTGTACGGTGTACGGAGTCGR: TGCAATAGTATGGCGAAAACCMAP3K11At4g12020F: CCCGGTTATGTTTCCTTCAGR: CAGTACACCCAAGGCTCCATMAP3K12At3g06030F: GAGAATCTGCGACACGCTGGTGCR: CTATGAACCGTTGAAGAACTMAP4K
gene familyMAP4K1At1g53165F: CTCGGTCCGAAGAGATTATGTGCR: AAGCTGAGCCGTTACAATCTCCGMAP4K2At3g15220F: CCTGAGACAACCTACAGAGATR: GCCACACCCTTGATTATACAC


### 2.4. Protein Extraction

Plant materials
were ground in liquid nitrogen, homogenized at 4°C in extraction buffer  (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA,
10 mM orthovanadate, 10 mM NaF, 60 mM ß-glycerophosphate, 10 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, and 5 μg/mL antipain) and
centrifuged at 17.600×g at 4°C.
Soluble protein concentration was determined by the Bradford method with bovine serum albumin
as standard [32].


### 2.5. Immunoprecipitation

Immunoprecipitation
assays were performed with three polyclonal anti-MPK antibodies, which were
raised against the 16 N-terminal amino acids of Arabidopsis MPK3, MPK4, and
MPK6 [15]. Protein
extracts (400 μg for MPK4 antibody and 200 μg for the two others) were
incubated in 400 μL with 30 μg anti-MPK4, 10 μg anti-MPK3, or 10 μg anti-MPK6
in immunoprecipitation buffer at 4°C for 3 hours as previously described [15]. Then 30 μL of 50% protein A-Sepharose CL4B
(Sigma) was added and incubation continued for one additional hour. The
immunoprecipitate was washed several times and resuspended in SDS-PAGE sample
buffer before being electrophoresed in SDS-PAGE embedded with MBP for in-gel
kinase assay [15].


### 2.6. In-Gel Kinase Assay

Protein extracts (20μg) were electrophoresed in 10% SDS-PAGE embedded with 0.2 mg/mL
myelin basic protein (MBP) as substrate for the kinases. The gels were then
treated for washing and renaturing steps as described by Zhang etal. [33]. For the activity, the gels were pre-incubated
for 30 minutes at room temperature in kinase activity buffer (40 mM HEPES pH
7.5, 2 mM DTT, 20 mM MgCl2, 1 mM EGTA, 0.1 mM orthovanadate) then 1
hour in 8 mL of the same buffer supplemented with 25 mM cold ATP and 2.9 MBq [P33]
ATP per gel. The gels were then washed extensively in 5% TCA (w/v) and 1%
disodium pyrophosphate (w/v) solution and under vacuum dried. The protein
kinase activity was revealed on the dried gels by Storm imaging system (GE
Healthcare).


## 2.1. Plant Materials

A. thaliana cells,
Columbia accession, were cultured in liquid medium as previously described [15] and subcultured after 5 days at
density of 100 mg fresh weight/mL. A. thaliana Columbia seeds were surface-sterilized in sodium
hypochlorite (12° chloride) for 5 minutes, rinsed 5-6 times in sterile water,
dried and sowed on Petri plates in 0.5x MS medium [29], 1% bacto-agar, and 0.5 g/L MES. Seeds were
then cold-stratified at 4°C for 24 hours and transferred to culture room at
22°C and light (125 μM m−2s−1) on a 16 hours light/8 hours dark cycle.
Plant organs were prepared from Arabidopsis plants cultured in soil for one
month at 22°C under 16 hours photoperiod. Hydroponic plants were also used for
the preparation of roots and rosette leaves. To obtain hydroponic plants, seeds
were treated as above and sowed in 0.5 mL Eppendorf tubes containing MS solid
medium, and half-cut to be adapted to custom-made polystyrene sheets as
support. Polystyrene sheets with tubes were floated in opaque plastic tank
containing autoclaved liquid culture medium [30] and then placed in culture box
at short day (9 hours light, 15 hours dark), 22°C and 50% relative humidity.
Culture medium was renewed twice a week to prevent contamination.


## 2.2.  Stress Treatments

Osmotic stresses were applied to cells equilibrated for 4 hours in their culture medium
containing 10 mM MES-Tris, pH 6.2, and
adjusted to 200 mOsm with sucrose. After equilibration, medium was replaced by
either the same volume of hypoosmotic medium, 20 mOsm of medium A composed of
10 mM MES-Tris, pH 6.2, 1 mM CaSO4, 10 mM sucrose or isoosmotic medium, 200 mOsm (medium A
plus 180 mM sorbitol), or hyperosmotic medium 500 mOsm (medium A plus 480 mM
sorbitol). Cells were maintained in these media under gentle shaking and
continuous light during all treatments. After treatments, cells were harvested
by filtration, frozen in liquid nitrogen, and stored at −80°C until use.
Osmolarity was monitored using a freezing point osmometer (Roebling, Berlin, Germany). Dehydration and osmotic
stress were applied to in vitro seedlings by transferring them onto Whatman
paper at room temperature (dehydration stress) or onto liquid media containing
200 mM NaCl or 320 mM mannitol for hyperosmotic stress treatments. Recovery
treatments were performed by transferring stressed seedlings onto a normal
growth medium. They were then harvested at different time points and stored at
−80°C until RNA extraction.


## 2.3. Preparation of DNA Macroarrays

When it was possible,
primers were chosen in the5′- or 3′-nontranslated gene regions. The size of
the amplified fragments ranged between 200 and 300 bp. Using specific primers (Table 1), 44 amplicons corresponding to the 20 MPK, 10 MKK, 12 MAP3K, and 2 MAP4K genes, were amplified by PCR from cDNA or genomic DNA and
then cloned in Escherichia coli. PCR reactions were performed in 50 μL
using Eurobio Kit and PCR Express machine (PerkinElmer) with the following PCR
program: 1 cycle of 1 minute 30 at 94°C for initial denaturing and 25 cycles of
45 seconds at 94°C (denaturing), 45 seconds at 55°C (annealing), 50 seconds at
72°C (elongation) followed by 7 minutes at 72°C. Amplicon quality and
specificity were verified in 1.2% agarose gel. Specific MAP kinase DNA
fragments were cut out, purified from gel using QIAquick Gel Extraction kit (Qiagen, France)
and cloned into pGEM-T vectors (Promega). After denaturation with 0.4 M NaOH,
30 μL (280 ng) of each PCR product were spotted onto 7.5×11 cm nylon membrane
(Nytran, Schleicher & Schuell) using the Dot Blot system (Fisher). The
spotted membranes were then cooked at 80°C for 2 hours and conserved at room temperature
until hybridization. To check the quantity of spotted PCR
products, membranes were first hybridized with polynucleotide kinase (PNK)
radiolabelled T7 primer (common to all inserts) and hybridization signals were
evaluated. T7 primer radiolabelling was performed in 50 μL containing 1 μL 100 μM T7 primer (200 pmol), 34 μL H2O, 5 μL 10X PNK buffer, 5 μL of PNK
(10 U/μL), and 5 μL [γ-P33]-ATP (3000 Ci/mmol; 10 μCi/μL) and
incubated at 37°C for 1 hour. Membranes were prehybridized with 10 mL Church
buffer (0.5 M phosphate buffer, 7% SDS, 1 mM EDTA, 1% BSA) at 65°C with
gentle shaking overnight, washed twice in 50 mL SSARC (600 mM NaCl, 60 mM
sodium citrate, 7.2% N-lauroyl sarcosine) at room temperature and twice at
4°C. Membranes were hybridized with radiolabelled T7 primer in 50 mL SSARC at
4°C overnight, washed twice 15 minutes in 50 mL precold SSARC at 4°C, wrapped
in Saran, exposed to phosphosensitive screen for 3–5 hours and scanned at 200 μm using PhosphorImager (GE Healthcare). 25 μg total RNA was incubated with
7.25 μg oligo dT(12-18) (Invitrogen, Cergy-Pontoise, France)
in total volume of 21 μL, heated to 70°C for 10 minutes and then cooled on ice
for ≥2 minutes. Tubes were briefly centrifuged and the following components
were added: 10 μl of first strand buffer, 5 μl DTT (100 mM), 5 μl dNTPs (10 mM
each of dATP, dGTP, and dTTP), 5 μl of [α-P32]-dCTP (10 μCi/μl, 3000 Ci/mol) and 2 μl of PowerScript Reverse Transcriptase (Clontech, St
Quentin-en-Yvelines, France). Reactions were incubated at 42°C for 1 hour, 2 μL
of cold dCTP were added and reaction was incubated further 1 hour at 42°C. The
synthesized labelled cDNA probes were purified using ProbeQuant G-50 Micro
Columns (GE Healthcare)
following the manufacturer protocol. One μL of each reaction was taken before
and after purification to calculate CPM (count per
minute) using scintillation counter to determine the percentage of incorporation.
Purified cDNA probes were denatured by adding 80 μL of 1X STE buffer (0.5X
final), 48 μL 1 M NaOH (0.2 M final), incubated at room temperature for 10 minutes
and then neutralized by 48 μL of 1 M HCl (0.2 M final) and 48 μL of 20X SSC.
Prehybridization was performed at 65°C for ≥3 hours in 10 mL Church buffer [31]
prewarmed to 65°C. Then, denatured cDNA probes
were added at equal CPM levels and hybridization was carried out at 65°C
overnight. Membranes were washed twice in 2X SSC, 0.1% SDS at room temperature
for 30 minutes, and twice in 1X SSC, SDS 0.1% at 65°C for 20 minutes, then
wrapped in Saran film and exposed to phosphor sensitive screens for 24–48 hours.
Phosphor screens exposed to macroarray filters were scanned with PhosphorImager
(GE Healthcare) at 200 μm resolution. After image acquisition, the scanned
images were analyzed using ImageQuant TL 2003 to quantify the radioactivity of
each spot. Background corresponding to nonspecific hybridizations (water or
empty spot intensity values) was subtracted from the signal intensity of each
spot. The signal intensities of each spot from three independent experiments
were averaged and normalized against the intensity of ACTIN2/8 or as a percentage of the total sum
of all spots on the membrane. Genes were considered as induced/repressed when
expression ratio (experimental/control) was ≥2.5-fold.Table 1
Oligonucleotides used to amplify the MAP kinase fragments from cDNA or genomic DNA.
F: forward; R: reverse.
Gene nameLocusPrimer sequences (5′–3′)MPK gene familyMPK1At1g10210F: GCCAAATCTCTCTCACTTCCACAGGTR: CCGTAAGCTCCACGACCAATAGGCMPK2At1g59580F: CCGTAAGCTCCACGACCAATAGGCR: CACACCGTACGCGCCTCGMPK3At3g45640F: ACCTTGCTAAATTGCACGACCCGAR: AGCCATTCGGATGGTTATTGGGTMPK4At4g01370F: TTGTTGTGAGGAATTTTGCTCCGR: GCTACCTCCTCTCCAGTCTCTGAGMPK5At4g11330F: CTCGAATCTGTGAAGTTCAATCCTCTGCR: GAAGAAACAGAACACTATCTAAGACATCCTGMPK6At2g43790F: GAGATGAAGATGAGATAAGGGTTR: GGTGGGCTATCATAACATAACTATTTCMPK7At2g18170F: GCCCGGTGCTTTGATCTCGTCTGR: GGCTAGCAAGTGGGGCAATGGCMPK8At1g18150F: GTTTTCTGGTGAAAAGAAATTGR: GCAAAGTGAATACTGAATAGMPK9At3g18040F: TTCCTCTACCAGCTTCTTCGTR: TACCATCTTGTAGCAACATAGTCAGMPK10At3g59790F: ATGGAGCCAACTAACGATGCTGR: CAAACGATGCCACAAGCACCTCMPK11At1g01560F: GTCAATAGAGAAACCATTCTTCGGTGATGR: GGCAATGACATTGTCATGATCCATATGCTMPK12At2g46070F: CCTTCTTCTTGGTTTCTCTCGTTCTCGGR: CCGCAGCACAGACAATACCGMPK13At1g07880F: GATGCTAGTTTTCGATCCTTCR: CTTGAAGTGTAAAGACTCTCTCCAGACMPK14At4g36450F: GGATCTCCAGTTCATAGATAACCAR: CTCCAAACTACTGACCGGAACAMPK15At1g73670F: CGAGTTCCAGCATCCAAGAATGR: CAATGTTGGTTTTAGATTGTACACCMPK16At5g19010F: AGTGTTACGCTATAACAACTGCR: TTAATACCAGCGACTCATTGMPK17At2g01450F: GTTGGCAAAGGAAGCTACGGR: GATGATGCTGAGGAGTGAGGMPK18At1g53510F: GAAGTTATTGGTAAAGGAAGCTATGGAGTTR: ATCTGATTCCATGAGCTCAAACACAACAMPK19At3g14720F: ACAGTACTCCCTCCACAGACCGTAR: CTAAGACATGCCATACCCAACAGCMPK20At2g42880F: GATATGCACCAGATGTAGCCR: GCCCAGCTTAGGTGGAGAACMKK gene familyMKK1At4g26070F: AAGATGTTTGAAGATTCGGAR: ATCCACCACTAGCAGAGAAAMKK2At4g29810F: AAATACGACTACTCGGGGATR: TTTTAAATAAACACTCTCATGTACCMKK3At5g40440F: GATGGAGTCCAAGCAGAAGGR: TCCGGCACTTTTTATGAACCMKK4At1g51660F: TCGTCCTCTGTCCTCGTCTTR: GGGGATACATGCACCATCATMKK5At3g21220F: TCCATAAGTTTTGCATACCCR: GCAAATCCAGCTAAAATCACMKK6At5g56580F: TTTGATTTGGCCTTAATGAAATGR: CTTGTTGGCACTCTGGAACCMKK7At1g18350F: ACCCTTTTCTCCGTGAAAGTR: TTCCTCTGTTTCTGTCGATTGMKK8At3g06230F: TGGACTGCTGTTTACGCAAGR: GTGGAGACCCTACATCTCGCMKK9At1g73500F: CCCAAAACTTATGTACACGATTGAR: CTTGCTCATCCTTTTCTCCGMKK10At1g32320F: GTGGAAGAGCTTCTTCGTCAR: CTGAACTCGAATCAAACCAAAMAP3K gene familyMAP3K1At1g09000F: TCACCAGGAAAATGACTTGCR: CTAGCAATTTGTAGCGCACTMAP3K2At1g54960F: GCATCTCCAGGCAAATGATCR: GGCTTAACACTCAATATGCAMAP3K3At1g53570F: CCCCTAGTTTTGGCCTTCTCR: AGGTTGAAAACAAATGGTTGAAMAP3K4At1g63700F: CCTCTTCATCCTCGAGCTGGAR: GCGATCCAGGGCTAACCGTAMAP3K5At5g66850F: GCAGCTCGAGAGGTTAACCAAR: GCTTCTTCTATCGATTCAACATTGMAP3K6At3g07980F: GTGAGTGTACATTTATTCCCR: GATCACATCACGTCCACATTAAMAP3K7At3g13530F: GTCCACCAAAACGATAATATCCCR: GAAGGCTAGCAAGAGCAGTATAMAP3K8At4g08500F: ATACACCCAAAAACCCCAAGR: AGCAGGCATAACCCGAAGATMAP3K9At4g08480F: CAACAACCAACCAATGAATGAR: CCTCCATAGGTGAAGCCTCTMAP3K10At4g08470F: CTGTACGGTGTACGGAGTCGR: TGCAATAGTATGGCGAAAACCMAP3K11At4g12020F: CCCGGTTATGTTTCCTTCAGR: CAGTACACCCAAGGCTCCATMAP3K12At3g06030F: GAGAATCTGCGACACGCTGGTGCR: CTATGAACCGTTGAAGAACTMAP4K
gene familyMAP4K1At1g53165F: CTCGGTCCGAAGAGATTATGTGCR: AAGCTGAGCCGTTACAATCTCCGMAP4K2At3g15220F: CCTGAGACAACCTACAGAGATR: GCCACACCCTTGATTATACAC


## 2.4. Protein Extraction

Plant materials
were ground in liquid nitrogen, homogenized at 4°C in extraction buffer  (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA,
10 mM orthovanadate, 10 mM NaF, 60 mM ß-glycerophosphate, 10 mM DTT, 1 mM
phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, and 5 μg/mL antipain) and
centrifuged at 17.600×g at 4°C.
Soluble protein concentration was determined by the Bradford method with bovine serum albumin
as standard [32].


## 2.5. Immunoprecipitation

Immunoprecipitation
assays were performed with three polyclonal anti-MPK antibodies, which were
raised against the 16 N-terminal amino acids of Arabidopsis MPK3, MPK4, and
MPK6 [15]. Protein
extracts (400 μg for MPK4 antibody and 200 μg for the two others) were
incubated in 400 μL with 30 μg anti-MPK4, 10 μg anti-MPK3, or 10 μg anti-MPK6
in immunoprecipitation buffer at 4°C for 3 hours as previously described [15]. Then 30 μL of 50% protein A-Sepharose CL4B
(Sigma) was added and incubation continued for one additional hour. The
immunoprecipitate was washed several times and resuspended in SDS-PAGE sample
buffer before being electrophoresed in SDS-PAGE embedded with MBP for in-gel
kinase assay [15].


## 2.6. In-Gel Kinase Assay

Protein extracts (20μg) were electrophoresed in 10% SDS-PAGE embedded with 0.2 mg/mL
myelin basic protein (MBP) as substrate for the kinases. The gels were then
treated for washing and renaturing steps as described by Zhang etal. [33]. For the activity, the gels were pre-incubated
for 30 minutes at room temperature in kinase activity buffer (40 mM HEPES pH
7.5, 2 mM DTT, 20 mM MgCl2, 1 mM EGTA, 0.1 mM orthovanadate) then 1
hour in 8 mL of the same buffer supplemented with 25 mM cold ATP and 2.9 MBq [P33]
ATP per gel. The gels were then washed extensively in 5% TCA (w/v) and 1%
disodium pyrophosphate (w/v) solution and under vacuum dried. The protein
kinase activity was revealed on the dried gels by Storm imaging system (GE
Healthcare).


## 3. Results

### 3.1. Macroarray Design and Data Analysis

Completion of the Arabidopsis genome
has made possible to define the full content of MAP kinase family members in a
single plant species, disclosing more than a hundred genes clustered into
several families. To search for putative components of MAP kinase water stress
signalling, macroarrays were designed for expression profiling of the wholeMPK and MKK genes, as well as selected members of the MAP3K and MAP4K families. MPK (20 genes) and MKK (10 genes) families
display very close sequence similarity among their members, but the MAP3K family is represented by many more members with greater variety in primary
structures and domain compositions. For the MAP3K family, the 12 members
of the subgroup whose kinase domains have significant similarity to typical MAP3K were selected. Since the sequences of the different kinases inside each group
are very close, full length sequences could not be used and 200–300 bp
fragments were cloned, allowing both strong hybridization signal and high specificity.To optimize  the sensitivity of the macroarrays, manual
spotting was preferred, allowing to deposit a large volume (30μL) by spot,
corresponding to about 280 ng of PCR product. The homogeneity of the spotting
was verified by oligonucleotide hybridization using T7 primer (data not shown).To standardize the results, theACTIN2/ACTIN8 genes have been used, due to their demonstrated stable expression upon various
environmental stresses. Thus, relative transcript level of each MAP kinase or
marker gene standardized with the ACTIN 2/8 transcript level is
presented in the context of water stress experiments. In the case of the
expression profiling of the genes in Arabidopsis organs, ACTIN 2/8 expression
displayed a maximum of 4-fold variation, ranging from 4.2 (floral buds) to 16.5
(roots) when expressed in the percentage of the expression level sum for all
studied genes. Relative transcript level of each MAP kinase standardized with
the expression level of the total spotted genes will be presented in Section 3.2
concerning organs.


### 3.2. Relative Expression Profile of the MAPK Genes in Arabidopsis Organs

The relative transcript level of the 44 studied Arabidopsis MAP kinase genes in different organs is presented in
Figure1. In the growth conditions used, quite similar expression profiles are
observed from one organ to another and MPK3, MKK9, and MAP3K3 transcript levels were the highest in each gene family, respectively. Some
other kinase genes also displayed clear transcriptional activity in most
organs, in the absence of stress: the MPK: 6, 8, 16, 17, 19, and 20, the MKK: 1, 2, 4, 5, as well as the MAP3K: 5, 6, and 7. It is
also interesting that differences can be noticed in the expression level of
some genes, depending of the Arabidopsis organ. For example, a higher
expression of MPK17, MPK20, and MAP3K6 is observed in
roots in comparison with other tested organs.Relative expression profiles of the MAP kinase genes in different organs
ofA. thaliana. The transcript levels of the 20 MPK, 10 MKK,
the MAP3K 1 to 12, and the MAP4K 1 and 2 are represented. The
expression level is represented as a percentage of the sum
of the values for all genes.
(a)(b)(c)(d)(e)


### 3.3. Protein Kinase Activity of MPK3, MPK4, and MPK6 in Arabidopsis Organs

The three MPK3, MPK4, and MPK6
kinases were previously shown to be activated very early upon osmotic stresses
in cell suspensions [15, 34]. Therefore, it was interesting to
measure their corresponding activity in different Arabidopsis organs. The organ
protein extracts were submitted to SDS-PAGE followed by an in-gel kinase assay
using MBP as a substrate (Figure 2(a)). Three active kinase proteins were
observed in most organs, with apparent molecular weights around 44, 39, and 37 kDa. These values correspond to the apparent molecular weights reported for
MPK6, MPK3, and MPK4, respectively. To confirm the identification of the active
proteins detected in the organs, specific polyclonal antibodies directed
against MPK3, MPK4, and MPK6 were used to immunoprecipitate each kinase from
protein extracts of rosette leaves and roots (Figure 2(b)). The result clearly
indicates the identification of MPK6, MPK3, and MPK4 as the active proteins
corresponding to 44, 39, and 37 kDa in rosette leaves and roots and strongly
suggests that the same active kinases are detected in the other organs, with
the exception of siliques in which the activity of MPK3 could not be detected
(Figure 2(a)).Protein kinase activity assay in different organs ofA. thaliana. Organs
were prepared from plants grown in soil ((a), left part) or in hydroponics ((a),
right part: “Hydroponics” and (b)). Proteins present in crude extracts
(a) or immunoprecipitates (b) were analyzed by in-gel kinase assay using MBP as
a substrate. In (b), specific antibodies against MPK6, MPK3, and MPK4 were used
for immunoprecipitation and a crude extract (Ex) was compared. St: stems; Bu:
flower buds; CL: cauline leaves; RL: rosette leaves; Si: siliques; Ro: roots.
(a)(b)


### 3.4. Relative Expression Profile of the MAP Kinase Genes upon Water Stress

Expression profiles of the 44 kinase
genes as well as marker genes were investigated in different conditions of
abiotic stresses: hypoosmolarity, hyperosmolarity, salinity, dehydration, as
well as recovery after hyperosmotic or salt stresses.Cell suspensions were submitted to
hypo- or hyperosmotic stress (Table2). Significant variations of the marker
genes were observed, validating the stress protocol used. Classical markers of
hyperosmolarity were induced, RAB18 as early as 30 minutes and RD29A at 1 hour of hyperosmotic stress. It is interesting to note that these two
genes were also induced, although less strongly, from 30 minutes of hypoosmotic
stress and they thus appear as general markers of osmotic variations. The key
genes of the proline pathway behaved as expected with the inductions of P5CS1 from 1 hour of hyperosmotic stress and ProDH from 30 minutes of
hypoosmotic stress. For the kinase genes, rapid and transient induction of MPK20 by hypoosmolarity was observed, with similar kinetics than ProDH (Table 2). MPK7, MPK14, and MPK17 as well as MKK7 were transiently
induced from 3 hours of hyperosmolarity.Table 2
Differential expression of MAP kinase genes under
osmotic stresses in A. thaliana cell suspensions. The table presents the variations of transcript levels
during the treatment kinetics, compared with the corresponding control values
in the absence of stress. Among the 44 tested kinase genes,
only the genes displaying a transcript level variation are indicated. The
variations of markers genes are indicated at the bottom of the table. 0: Variation
less than 2.5 fold; +: increase; (n): n-fold increase.
Cell treatmentsGeneHypoosmotic stressHyperosmotic stress30 min1 h3 h6 h30 min1 h3 h6 hMPK genesMPK7000000+ (2.6)0MPK14000000+ (3.1)0MPK17000000+ (2.9)0MPK20+ (3.0)0000000MKK genesMKK7000000+ (2.6)0Marker genesRD29A+ (4.4)+ (3.2)000+ (5.1)+ (8.6)+ (3.3)RAB18+ (2.6)+ (3.4)00+ (2.9)+ (14.8)+ (151.2)+ (30.3)P5CS100000+ (3.2)+ (4.9)0ProDH+ (5.1)0000000Plantlets were also submitted to
dehydration, mannitol, or salt stress (Table3). RD29A and RAB18 were induced in each case as expected. On the contrary, P5CS1 was not
induced by dehydration, but only by mannitol or salt stress in the tested
conditions. For ProDH, induction was observed in the case of a return to
basal conditions after mannitol or salt stress (Table 3), coherently with the
previously noticed induction by hypoosmolarity (Table 2).Table 3
Differential expression of MAP kinase genes under different stress and recovery conditions
in A. thaliana plantlets. The table presents the variations
of transcript levels during the treatment kinetics, compared with the
corresponding control values in the absence of stress. Among the 44 tested
kinase genes, only the genes displaying a transcript level variation are
indicated. The variations of markers genes are indicated at the bottom of the
table. 0: Variation less than 2.5-fold; +: increase; (n): n-fold increase.
Plantlet treatmentsGeneDehydrationMannitolNaClStressRecoveryStressRecovery1 h3 h6 h3 h24 h1 h 305 h3 h24 h1 h 305 hMPK genesMPK2+ (2.6)0+ (4.0)00000000MPK3+ (3.1)+ (5.0)+ (5.1)00+ (5.5)000+ (4.6)0MPK4+ (2.6)0+ (5.8)00000000MPK5+ (3.1)+ (3.0)+ (3.5)00+ (3.4)000+ (2.9)+ (3.9)MPK7000+ (3.0)+ (8.5)00+ (3.0)+ (6.0)0+ (2.9)MPK900+ (4.6)+ (2.8)+ (8.8)+ (5.7)00+ (4.2)+ (5.1)0MPK100000+ (30.6)+ (2.9)00+ (20.7)+ (5.3)+ (2.7)MPK11+ (4.6)+ (4.8)+ (10.2)0+ (4.7)+ (4.0)00+ (4.3)00MPK1200+ (2.9)00+ (3.1)00000MPK1400+ (2.6)0+ (2.7)+ (4.4)000+ (8.3)+ (5.7)MPK17+ (2.9)+ (3.0)+ (6.2)+ (4.5)+ (2.6)+ (6.7)00+ (2.9)+ (4.3)+ (4.0)MPK180+ (2.5)+ (6.1)+ (2.5)+ (3.5)000+ (2.5)00MPK2000000+ (3.3)0000+ (2.9)MKK genesMKK30000+ (6.0)00+ (4.5)+ (6.5)00MKK4000+ (2.9)+ (3.3)+ (4.5)0+ (3.9)+ (3.6)0+ (2.5)MKK6000+ (4.3)0+ (2.9)0+ (3.1)000MKK7000+ (3.9)000+ (3.4)000MKK9+ (3.2)0+ (3.6)+ (4.0)000+ (3.5)+ (3.2)00MAP3K genesMAP3K3000+ (3.2)0+ (2.5)0+ (4.5)+ (3.0)00MAP3K400+ (3.9)00+ (4.5)0000+ (2.5)MAP3K5000+ (2.8)+ (3.1)00+ (2.9)+ (3.0)0+ (4.7)MAP3K600+ (2.9)+ (5.6)+ (2.8)+ (3.3)00+ (2.7)00MAP3K70000+ (3.1)+ (3.5)00+ (3.4)+ (3.1)+ (2.7)MAP3K900+ (3.4)+ (3.3)+ (2.8)+ (7.6)000+ (9.6)0MAP4K genesMAP4K20000+ (4.1)000+ (4.0)00Marker genesRD29A+ (5.3)+ (12.5)+ (11.3)+ (13.4)+ (6.2)00+ (11.4)+ (3.8)+ (3.8)0RAB18+ (2.9)+ (2.9)+ (5.0)11.60+ (10.2)0+ (11.5)+ (4.2)+ (7.2)+ (5.0)P5CS1000+ (19.3)+ (2.4)+ (3.8)0+ (17.9)+ (6.3)+ (8.0)+ (4.1)ProDH00000+ (6.3)000+ (6.2)+ (2.7)Genes belonging to each MAP kinase
family were induced in each case in response to dehydration, hyperosmolarity,
or salinity stresses. The increase of transcript levels by hyperosmolarity in
cell suspensions was confirmed in young plantlets, namelyMPK7, MPK14, MPK17, and MKK7, but more inductions by hyperosmolarity could be detected in
plants, with MPK9, MPK10, MPK11, MPK18, as well as MKK3, MKK4, MKK6,
MKK9, and MAP3K3, MAP3K5, MAP3K6, MAP3K7, MAP3K9. Surprisingly, no
specific induction by salinity was observed and most genes induced by mannitol
were also induced by NaCl treatment with the exception of MPK14 and MAP3K9. A set of genes was also induced by dehydration, some of them shared
by salinity and/or hyperosmolarity, the others specific for dehydration: MPK2,
MPK3, MPK4, MPK5, MPK12, and MAP3K4 (Table 3).


## 3.1. Macroarray Design and Data Analysis

Completion of the Arabidopsis genome
has made possible to define the full content of MAP kinase family members in a
single plant species, disclosing more than a hundred genes clustered into
several families. To search for putative components of MAP kinase water stress
signalling, macroarrays were designed for expression profiling of the wholeMPK and MKK genes, as well as selected members of the MAP3K and MAP4K families. MPK (20 genes) and MKK (10 genes) families
display very close sequence similarity among their members, but the MAP3K family is represented by many more members with greater variety in primary
structures and domain compositions. For the MAP3K family, the 12 members
of the subgroup whose kinase domains have significant similarity to typical MAP3K were selected. Since the sequences of the different kinases inside each group
are very close, full length sequences could not be used and 200–300 bp
fragments were cloned, allowing both strong hybridization signal and high specificity.To optimize  the sensitivity of the macroarrays, manual
spotting was preferred, allowing to deposit a large volume (30μL) by spot,
corresponding to about 280 ng of PCR product. The homogeneity of the spotting
was verified by oligonucleotide hybridization using T7 primer (data not shown).To standardize the results, theACTIN2/ACTIN8 genes have been used, due to their demonstrated stable expression upon various
environmental stresses. Thus, relative transcript level of each MAP kinase or
marker gene standardized with the ACTIN 2/8 transcript level is
presented in the context of water stress experiments. In the case of the
expression profiling of the genes in Arabidopsis organs, ACTIN 2/8 expression
displayed a maximum of 4-fold variation, ranging from 4.2 (floral buds) to 16.5
(roots) when expressed in the percentage of the expression level sum for all
studied genes. Relative transcript level of each MAP kinase standardized with
the expression level of the total spotted genes will be presented in Section 3.2
concerning organs.


## 3.2. Relative Expression Profile of the MAPK Genes in Arabidopsis Organs

The relative transcript level of the 44 studied Arabidopsis MAP kinase genes in different organs is presented in
Figure1. In the growth conditions used, quite similar expression profiles are
observed from one organ to another and MPK3, MKK9, and MAP3K3 transcript levels were the highest in each gene family, respectively. Some
other kinase genes also displayed clear transcriptional activity in most
organs, in the absence of stress: the MPK: 6, 8, 16, 17, 19, and 20, the MKK: 1, 2, 4, 5, as well as the MAP3K: 5, 6, and 7. It is
also interesting that differences can be noticed in the expression level of
some genes, depending of the Arabidopsis organ. For example, a higher
expression of MPK17, MPK20, and MAP3K6 is observed in
roots in comparison with other tested organs.Relative expression profiles of the MAP kinase genes in different organs
ofA. thaliana. The transcript levels of the 20 MPK, 10 MKK,
the MAP3K 1 to 12, and the MAP4K 1 and 2 are represented. The
expression level is represented as a percentage of the sum
of the values for all genes.
(a)(b)(c)(d)(e)


## 3.3. Protein Kinase Activity of MPK3, MPK4, and MPK6 in Arabidopsis Organs

The three MPK3, MPK4, and MPK6
kinases were previously shown to be activated very early upon osmotic stresses
in cell suspensions [15, 34]. Therefore, it was interesting to
measure their corresponding activity in different Arabidopsis organs. The organ
protein extracts were submitted to SDS-PAGE followed by an in-gel kinase assay
using MBP as a substrate (Figure 2(a)). Three active kinase proteins were
observed in most organs, with apparent molecular weights around 44, 39, and 37 kDa. These values correspond to the apparent molecular weights reported for
MPK6, MPK3, and MPK4, respectively. To confirm the identification of the active
proteins detected in the organs, specific polyclonal antibodies directed
against MPK3, MPK4, and MPK6 were used to immunoprecipitate each kinase from
protein extracts of rosette leaves and roots (Figure 2(b)). The result clearly
indicates the identification of MPK6, MPK3, and MPK4 as the active proteins
corresponding to 44, 39, and 37 kDa in rosette leaves and roots and strongly
suggests that the same active kinases are detected in the other organs, with
the exception of siliques in which the activity of MPK3 could not be detected
(Figure 2(a)).Protein kinase activity assay in different organs ofA. thaliana. Organs
were prepared from plants grown in soil ((a), left part) or in hydroponics ((a),
right part: “Hydroponics” and (b)). Proteins present in crude extracts
(a) or immunoprecipitates (b) were analyzed by in-gel kinase assay using MBP as
a substrate. In (b), specific antibodies against MPK6, MPK3, and MPK4 were used
for immunoprecipitation and a crude extract (Ex) was compared. St: stems; Bu:
flower buds; CL: cauline leaves; RL: rosette leaves; Si: siliques; Ro: roots.
(a)(b)


## 3.4. Relative Expression Profile of the MAP Kinase Genes upon Water Stress

Expression profiles of the 44 kinase
genes as well as marker genes were investigated in different conditions of
abiotic stresses: hypoosmolarity, hyperosmolarity, salinity, dehydration, as
well as recovery after hyperosmotic or salt stresses.Cell suspensions were submitted to
hypo- or hyperosmotic stress (Table2). Significant variations of the marker
genes were observed, validating the stress protocol used. Classical markers of
hyperosmolarity were induced, RAB18 as early as 30 minutes and RD29A at 1 hour of hyperosmotic stress. It is interesting to note that these two
genes were also induced, although less strongly, from 30 minutes of hypoosmotic
stress and they thus appear as general markers of osmotic variations. The key
genes of the proline pathway behaved as expected with the inductions of P5CS1 from 1 hour of hyperosmotic stress and ProDH from 30 minutes of
hypoosmotic stress. For the kinase genes, rapid and transient induction of MPK20 by hypoosmolarity was observed, with similar kinetics than ProDH (Table 2). MPK7, MPK14, and MPK17 as well as MKK7 were transiently
induced from 3 hours of hyperosmolarity.Table 2
Differential expression of MAP kinase genes under
osmotic stresses in A. thaliana cell suspensions. The table presents the variations of transcript levels
during the treatment kinetics, compared with the corresponding control values
in the absence of stress. Among the 44 tested kinase genes,
only the genes displaying a transcript level variation are indicated. The
variations of markers genes are indicated at the bottom of the table. 0: Variation
less than 2.5 fold; +: increase; (n): n-fold increase.
Cell treatmentsGeneHypoosmotic stressHyperosmotic stress30 min1 h3 h6 h30 min1 h3 h6 hMPK genesMPK7000000+ (2.6)0MPK14000000+ (3.1)0MPK17000000+ (2.9)0MPK20+ (3.0)0000000MKK genesMKK7000000+ (2.6)0Marker genesRD29A+ (4.4)+ (3.2)000+ (5.1)+ (8.6)+ (3.3)RAB18+ (2.6)+ (3.4)00+ (2.9)+ (14.8)+ (151.2)+ (30.3)P5CS100000+ (3.2)+ (4.9)0ProDH+ (5.1)0000000Plantlets were also submitted to
dehydration, mannitol, or salt stress (Table3). RD29A and RAB18 were induced in each case as expected. On the contrary, P5CS1 was not
induced by dehydration, but only by mannitol or salt stress in the tested
conditions. For ProDH, induction was observed in the case of a return to
basal conditions after mannitol or salt stress (Table 3), coherently with the
previously noticed induction by hypoosmolarity (Table 2).Table 3
Differential expression of MAP kinase genes under different stress and recovery conditions
in A. thaliana plantlets. The table presents the variations
of transcript levels during the treatment kinetics, compared with the
corresponding control values in the absence of stress. Among the 44 tested
kinase genes, only the genes displaying a transcript level variation are
indicated. The variations of markers genes are indicated at the bottom of the
table. 0: Variation less than 2.5-fold; +: increase; (n): n-fold increase.
Plantlet treatmentsGeneDehydrationMannitolNaClStressRecoveryStressRecovery1 h3 h6 h3 h24 h1 h 305 h3 h24 h1 h 305 hMPK genesMPK2+ (2.6)0+ (4.0)00000000MPK3+ (3.1)+ (5.0)+ (5.1)00+ (5.5)000+ (4.6)0MPK4+ (2.6)0+ (5.8)00000000MPK5+ (3.1)+ (3.0)+ (3.5)00+ (3.4)000+ (2.9)+ (3.9)MPK7000+ (3.0)+ (8.5)00+ (3.0)+ (6.0)0+ (2.9)MPK900+ (4.6)+ (2.8)+ (8.8)+ (5.7)00+ (4.2)+ (5.1)0MPK100000+ (30.6)+ (2.9)00+ (20.7)+ (5.3)+ (2.7)MPK11+ (4.6)+ (4.8)+ (10.2)0+ (4.7)+ (4.0)00+ (4.3)00MPK1200+ (2.9)00+ (3.1)00000MPK1400+ (2.6)0+ (2.7)+ (4.4)000+ (8.3)+ (5.7)MPK17+ (2.9)+ (3.0)+ (6.2)+ (4.5)+ (2.6)+ (6.7)00+ (2.9)+ (4.3)+ (4.0)MPK180+ (2.5)+ (6.1)+ (2.5)+ (3.5)000+ (2.5)00MPK2000000+ (3.3)0000+ (2.9)MKK genesMKK30000+ (6.0)00+ (4.5)+ (6.5)00MKK4000+ (2.9)+ (3.3)+ (4.5)0+ (3.9)+ (3.6)0+ (2.5)MKK6000+ (4.3)0+ (2.9)0+ (3.1)000MKK7000+ (3.9)000+ (3.4)000MKK9+ (3.2)0+ (3.6)+ (4.0)000+ (3.5)+ (3.2)00MAP3K genesMAP3K3000+ (3.2)0+ (2.5)0+ (4.5)+ (3.0)00MAP3K400+ (3.9)00+ (4.5)0000+ (2.5)MAP3K5000+ (2.8)+ (3.1)00+ (2.9)+ (3.0)0+ (4.7)MAP3K600+ (2.9)+ (5.6)+ (2.8)+ (3.3)00+ (2.7)00MAP3K70000+ (3.1)+ (3.5)00+ (3.4)+ (3.1)+ (2.7)MAP3K900+ (3.4)+ (3.3)+ (2.8)+ (7.6)000+ (9.6)0MAP4K genesMAP4K20000+ (4.1)000+ (4.0)00Marker genesRD29A+ (5.3)+ (12.5)+ (11.3)+ (13.4)+ (6.2)00+ (11.4)+ (3.8)+ (3.8)0RAB18+ (2.9)+ (2.9)+ (5.0)11.60+ (10.2)0+ (11.5)+ (4.2)+ (7.2)+ (5.0)P5CS1000+ (19.3)+ (2.4)+ (3.8)0+ (17.9)+ (6.3)+ (8.0)+ (4.1)ProDH00000+ (6.3)000+ (6.2)+ (2.7)Genes belonging to each MAP kinase
family were induced in each case in response to dehydration, hyperosmolarity,
or salinity stresses. The increase of transcript levels by hyperosmolarity in
cell suspensions was confirmed in young plantlets, namelyMPK7, MPK14, MPK17, and MKK7, but more inductions by hyperosmolarity could be detected in
plants, with MPK9, MPK10, MPK11, MPK18, as well as MKK3, MKK4, MKK6,
MKK9, and MAP3K3, MAP3K5, MAP3K6, MAP3K7, MAP3K9. Surprisingly, no
specific induction by salinity was observed and most genes induced by mannitol
were also induced by NaCl treatment with the exception of MPK14 and MAP3K9. A set of genes was also induced by dehydration, some of them shared
by salinity and/or hyperosmolarity, the others specific for dehydration: MPK2,
MPK3, MPK4, MPK5, MPK12, and MAP3K4 (Table 3).


## 4. Discussion

Since the
completion of the Arabidopsis genome sequencing project, about one hundred of
MAP kinase genes have been identified, annotated and classified into four
families [1]. So far, only very few of the identified
Arabidopsis MAP kinases were studied at biochemical levels in response to
biotic or abiotic stresses, but the majority of them need to be functionally
characterized. One of the early tasks to assign potential functions to genes is
their expression analysis in order to determine where, when, and how each gene
is switched on or off. The basic assumption underlying this approach is that
genes with similar expression patterns are likely to be functionally related [35]. In this way, genes without previous
functional assignments can be given a putative role in the biological process
based on the expression profile. Moreover, for genes known to be functional and
in cascades hieratically related (e.g., MAP kinases), this approach seems to be
of a particular interest, as it allows to have global insight to the
coregulated genes, suggesting that they may be involved in the same signalling
pathway.Based on this idea, a macroarray approach wasdeveloped in this study to measure MAP kinase expression levels in
different Arabidopsis organs without any stress treatment, and also to monitor
the impact of some environmental stresses (osmotic, salt, and dehydration) on
the accumulation of MAP kinase transcripts both
in Arabidopsis cell cultures and seedlings. A miniscale macroarray that
contained an important number of MAP kinase genes (44 genes) belonging to the
four identified MAP kinase families was constructed here. The choice of macroarray system using short DNA
fragments is based on its high sensitivity and ability to distinguish between hybridization signals corresponding to closely
related genes. The validity of this approach has been previously demonstrated
for many other gene families inSaccharomyces cerevisiae or plants [5, 36–39]. The reliability of this method was
ensured by a careful evaluation of the specificity of each amplicon, based on
sequence comparison and BLAST analysis. The main criteria in designing the MAP
kinase amplicons was that they should be as long and specific as possible
(200–300 bp), while showing minimum homology to each other or to any other
sequences in the Arabidopsis genome. The obtained data were averaged from three
independent experiments and normalized using Actin value or the sum of the
values for all spots on the membrane.To evaluate the validity of expression profiling under stress treatment using DNA macroarray,
we analyzed by real time RT-PCR (data not
shown), the expression of some selected stress-inducible MAP kinase genes (e.g.,MPK3, MPK18, MPK20 and MAP3K6). The results obtained by the two
methods were in good agreement.The MAP kinase gene expression profiles in five Arabidopsis organs,
namely stems, flower buds, cauline leaves, rosette
leaves, and roots, have shown that MAP kinase genes are generally not preferentially
expressed in a part of the plant. The transcript levels ofMPK3, MKK9, and MAP3K3, most highly expressed in each family, are detected in all
tested organs. Nevertheless, some of them seem
to be more expressed in certain organs than in others. Interestingly, MPK17,
MPK20, and MAP3K6 are more strongly expressed in roots. Some Arabidopsis
MAP kinase orthologous genes in other plants display a distinct localization in
comparative organs. For example, the MPK3 ortholog in poplar,
PtMPK3-1 (A group), displays a preferential expression profile in roots,
and PtMPK17 (D group) is preferentially expressed in roots and
leaves [40]. In rice, OsMPK4 has been shown to be
more expressed in mature than in young leaves [3].On the other hand, many MAP kinase genes
of each family, namelyMPK1, MPK9, MPK10, MPK13, MPK14, MPK18, MKK8, MKK10,
as well as MAP3K1, MAP3K2, MAP3K11, and MAP3K12 showed a very low
expression level in the studied organs. From the 12 MAP3K studied genes,
we found that MAP3K3 is highly expressed in the five Arabidopsis organs
and MAP3K6 transcripts are relatively abundant in flower buds and roots.
MAP kinase genes in D group appear to be expressed at a relatively high level
in the five tested organs, except MPK18 for which expression is hardly
detectable.No significant expression was observed forMKK8 and MKK10 in any organ or stress condition tested here. Indeed, MKK10 does not
contain the phosphorylation site (S/TxxxxxS/T) specific to MKK members [1] and no ortholog was found for MKK8 in
any other sequenced plant genome so far. This may reinforce the hypothesis
emitted by Hamel [41] that MKK8 and MKK10 may be
nonfunctional and the number of MKK genes in Arabidopsis genome is eight
instead of ten.On the other hand, the
accordance between the localization of kinase activity and mRNA transcript
profiles was investigated in plant organs using MBP as phosphorylable
substrate. Although MBP is a favorite substrate for MPK family members, only
three bands were observed in activity gel assay. This may be due to problems
related to protein denaturation/renaturation, or simply because these three MPK
proteins were the only temporarily and spatially activated MAP kinases at this
experimental time point.Using specific anti-MPK antibodies, the identity and ubiquity of these
proteins have been confirmed and shown to be MPK3, MPK4, and MPK6, present and
active in all studied organs (except MPK3 in siliques) (Figure2). It has been
previously reported that these proteins are involved in multiple biotic and
abiotic stress responses [11, 15, 17, 18, 34, 42–44]. Coherently with the detection of MPK3, MPK4,
and MPK6 activities in different Arabidopsis organs (Figure 2), the three kinases were previously
shown active at a basal level in the absence of stress, in cell suspensions [15]
and plantlets [34]. Their activities shown here in Arabidopsis
organs suggest that these three protein kinases may play roles in the
developmental processes in the absence of stress too. It is worthy of note that
MPK6 protein was activated in response to osmotic stress, but its mRNA
transcript level was unchanged, suggesting that post-transcriptional mechanisms
is required for MPK6 function. This is in agreement with previously reported
results [11].In both cell cultures and seedlings, the induction profiles of MAP kinase
genes in response to water stresses were comparable to those of stress marker
genes (RD29A, DREB2A, P5CS1, and ProDH), although the induction
factor was generally higher for these stress marker genes, indicating the
reliability of the macroarray technique for this MAP kinase expression
profiling study. The gene expression responses are dependent on the duration,
the intensity, and the type of stress. A previous transcriptional profiling in
response to abiotic stress treatments has shown that the majority of stress-induced
genes responded earlier than later stress time points [45]. The present analysis supports this point of view
where MAP kinase expression responses to stress were associated at early to
middle time points (1–6 hours) for dehydration treatments and 3 hours
until 24 hours for salt and hyperosmolarity.Based on Venn
diagram (Figure3), the analysis of overlapping regions shows
that MAP kinase genes are induced and can be classified, on the basis of their expression pattern, into four groups.
Firstly, a group of six previously noncharacterized genes was induced under the
three stresses. Genes induced only by salinity and hyperosmolarity form the
second group with the highest number of induced MAP kinase genes (10 genes). A
third and smaller group contains six genes which are induced only by dehydration.
The fourth and smallest group contains only two genes which are induced by dehydration
and hyperosmolarity. These results indicate that the cross-talking between
salinity and hyperosmolarity signalling processes may be greater than those between
hyperosmolarity and dehydration which, in turn, may be greater than those
between dehydration and salinity signalling processes. This cross-talk might correspond
to overlapping between the perception and signal transduction mechanisms of the
different abiotic stress sharing common effects [45–47]. Further functional analysis of the corresponding
overlapping-induced genes will be helpful to understand these complex stress
signal transduction pathways.Figure 3
Venn diagram of MAP kinase genes upregulated by water stresses. Names of
the genes with ≥2.5-fold change in their expression level in response to
dehydration, salinity or hyperosmolarity are indicated.K: MPK; 2K: MKK; 3K:
MAP3K; 4K: MAP4K.Interestingly, none of the 44 studied MAP kinase genes was specifically
induced only by salinity or hyperosmolarity conditions, which suggests common
signalling pathway between these two stresses. However, it was recently shown
that distinct signalling pathways are involved in the tight regulation of
proline metabolism with the involvement of phospholipases and calcium upon
ionic stress, but not in response to hyperosmotic stress [48].Most of the water
stress-dependant inductions of MAP kinases reported here were not detected in
the microarray analysis available in genevestigator (https://www.genevestigator.ethz.ch/) database. For example,
for the MKK family, five members were observed here transcriptionally induced, among
which only one (MKK9) was also observed in the database. Additionally to the
higher sensitivity already mentioned of the macroarrays used here, several
kinds of water stresses with several kinetics points were used here, allowing
this better detection of transcriptional regulations.We found that the
promoter region of some stress-induced MAP kinase genes, that is,MPK3,
MPK5, MPK7, MPK10, MPK18, MPK20, MKK9; MAP3K3, and MAP3K7,
contains the cis-acting proline-responsive-element sequence involved in
the proline and hypoosmolarity-inducible gene expression [49, 50], suggesting that these genes may also respond to
hypoosmolarity. However, high transcript level was only observed for MPK20,
which belongs to the MPK D group, in response to hypoosmolarity and
recovery conditions.It is worthy of note
that no significant repressed expression was observed for any of the studied
MAP kinase genes. In addition, genes having high levels of sequence similarity tend
to display similar expression patterns in organs or under stress conditions.
For example, all members of the B1 subgroup (MPK4, MPK5, MPK11, and MPK12) were induced by dehydration. However, like in the case of each gene
family, it is often supposed that there is a functional redundancy between the
gene family members, but this remains to be demonstrated using appropriate
techniques. In our conditions, we could not observed any induction of MAP3K8 (At4g08500), anciently known as AtMEKK1 and previously shown to be
induced and the corresponding protein to be the upstream activator of MKK in
the response to biotic and abiotic stress [18, 21]. Nevertheless, we could observe the induction
of MAP3K9 (Table 3) which is very similar in sequence to MAP3K8,
suggesting that MAP3K8 and MAP3K9 may be functionally redundant.The induction of MAP kinase genes under a given condition likely involves a
parallel induction or the activation of existing substrates to initiate an
appropriate MAP kinase cascade in response to stress. However, information
about plant MAP kinase substrates is still poor, but 48 potential substrates of
MPK3 and 39 of MPK6 were recently identified [24] indicating the complexity of  MAP kinase signalling.Understanding
the molecular and genetic basis of stress tolerance is an important
prerequisite step to increase plant tolerance through bioengineering
approaches. The stress-induced MAP kinase genes represent important targets for
further biochemical characterization and bioengineering approaches towards the
creation of stress-resistant genotypes.To our knowledge, this is the first thoroughly reported work on the expression of Arabidopsis
MAP kinase genes in relation to osmotic and dehydration stress. The
characterization of the corresponding proteins, the identification of MAP
kinase substrates, and the elucidation of the interaction network should supply
further information about the components and the role of the MAP kinase cascades
in plant adaptation to environmental stresses.



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*Source: 143656-2008-11-11.xml*