3.1.1 Screening conditions set-up

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RESULTS

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RESULTS

3 Results

3.1 Physiological Results

3.1.1 Screening conditions set-up

Analyses were performed to set–up optimal work-conditions and plant parameters. The description of A. thaliana ecotype Columbia as a freezing-tolerant species was reported by various authors (Thomashow, 1990, 1999; Lang et al., 1994; Warren et al., 1996; Gilmour et al., 1998, 2000). We used ecotype Columbia-0. Freezing tolerance parameters in Arabidopsis were characterized in order to select age, temperature and timing for the subsequent proteomic analysis. The aim was to find the optimal conditions for freezing intact specimens of A. thaliana, that proved to be the most resistant ones after a short time of high intensity freezing treatment, either in the presence or absence of a previous acclimation at 4 °C for 1 week.

Cross experiments were performed using the following parameters:

• Plants age

• Cold acclimation treatment

• Freezing temperature

• Time of exposition to each temperature

We performed the analyses on the range of 5 days-old to 35 days-old plants (5, 10, 15, 20, 25, 30 and 35 days). Plants of each age were transferred at temperatures ranging from –4

°C to –12 °C (4°, 6°, 8°, 10° and 12°) and from 0 hours treatment (only the cooling-time) to 24-hours treatment.

3.1.2 Experiments to establish the best growth conditions for freezing tolerance studies

Freezing tolerance was estimated as: 1) the lethal temperature killing 50% of plants

surviving after 7 days of recovery under unstressed conditions (Fig.1); 2) cellular leakage

of ions out of the cell (Fig.2); and 3) chlorophyll contents (Fig.3). The combination of

thermal analysis and LT has been widely used to describe frost resistance strategies in

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RESULTS

plants (Neuner and Bannister, 1995; Pearce, 2001). Electrolyte leakage assay was

performed because the plasmalemma may be the primary site of freezing injury in non

acclimated plants (Steponkus, 1984). The leakage of electrolytes from frozen and thawed

tissues is a sensitive indicator of loss of integrity by the plasmalemma, and it has been

commonly used to assay freezing injury (reviewed by Calkins and Swanson, 1990). We

applied this assay to determine whether freezing sensitivity was manifested by increased

sensitivity of the plasmalemma in leaf tissues. The state of the photosynthetic apparatus was

evaluated by determining pigments content, as indicator of the physiological status of the

plants during cold acclimation and freezing stress (Steponkus, 1984).

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RESULTS

Fi g. 1. Th e leth al temp erature k illin g 50 % o f th e p lan ts (LT50 ) afte r reco v ery u n d er un stressed con d iti o n s for 1 week . An aly sis were per fo rme d on 5 da ys -ol d t o 3 5 da ys -ol d pl an ts t aki n g sam p le s eve ry 5 d ays. Pl an ts o f vari ous a g e we re ke pt at – 4 ° C t o – 12 °C f o r 2 4 h o u rs o f treatment. Plant s subj ect ed t o co ld accl im at ion were put i n a c o ld c h am ber ( 4 °C ), 1 6 /8 h l ight /d ar k da y l engt h an d a p h o to n fl u x densi ty of 1 50 µmol m

–2

s

–1

, for 1 week. Val u es corres pond to the ave rage of three LT d etermin atio ns using 30 p lan ts p er temp erat ure t reatment. N.A.= non acclimat ed pla n ts; A.= acclimated pla n ts.

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RESULTS

Fig. 2. Cellu lar leak ag e o f i o n s ou t of th e cell co rresp ond ing to leth al temp erat u re k illin g 50 % of th e p lan ts (LT5 0) after freezin g tr eatme nt . Electrolyte lea k age was meas ure d by HANNA in st rume nt – HI 8733 CONDUCTIVITY METER a nd re ported as perce n tage of the total am oun t prese n t. Anal y si s were per fo rme d o n 5 da y s-ol d t o 35 da ys- o ld pl ant s. Each a g e pl an ts was t est ed f ro m – 4 °C t o –1 2 °C d u ri n g 24 ho ur s o f treatment. Plants subjected to cold acclima ti on were p u t i n a col d chamb er (4 °C ), 1 6 /8 h l ight /d ar k d ay l engt h an d a ph ot o n fl ux d ensity o f 150 µmol m

–2

s

–1

, for 1 week. Values corres pond to the av era g e o f fi ve det ermi n at io n s usi n g 10 pl ant s per t em p er at ure t reat me n t. N.A .= no n acclimated p lan s; A.= acclimated p lan ts.

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RESULTS

Fig. 3. Pigme n ts content correspondi ng to l ethal te mperat ure killing 50% of the pla n ts (LT50) a fter freezing treatment. Analysis a re pe rfo rm ed on 5 day s- o ld t o 35 da ys -ol d pl ant s. Eac h age pl ant s was t est ed f ro m – 4 °C t o – 1 2 °C du ri n g 2 4 ho u rs of t reat me n t. Pl ant s s u bj ec te d t o col d accl im at ion w ere put i n a c o ld c h am ber ( 4 °C ), 1 6 /8 h l ig ht /d ar k da y l en g th an d a ph ot on fl ux densi ty of 15 0 µmol m

–2

s

–1

, for 1 week. Values cor res po n d t o t h e av era g e o f f ive det ermi n at ions usi n g 10 p lants pe r temperature treatme nt. N. A.= non acclimated pla n ts; A.= ac climated plants. Mg . Pi gmen ts /g. F .W. = millig rams o f p ig m en t on a fresh weigh t b asis.

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RESULTS

From integrated and comparative data analysis of Figures 1, 2 and 3 it was possible to observe that:

• Plants with ages ranging from 15 to 25 days-old showed the best tolerance to freezing temperatures.

• LT 50 (the lethal temperature killing 50% of the plants after recovery under unstressed conditions for 1 week) of cold acclimated plants decreased from 24 to 12 hours when receiving the -10 °C treatment. Below -8°C plants survival remained close to 100%. This result was interesting considering we were studying the effect of freezing temperatures during short period of time.

• LT 50 of non acclimated plants remained close to 12 hours at lower freezing T (4-8 ºC), when receiving the -10 °C treatment it decreased to 9 hours; after 24 hours of treatment all the plants died.

• In plants underwent to -10 °C treatment during 12 hours, electrolyte leakage was lower than the loss at other temperatures, except for the -8 °C treatment.

• N.A. plants after the -10 °C treatment during 12 hours lost less electrolyte than A.

ones. On the contrary chlorophyll content was higher in A. plants than in N.A. ones.

As shown in Figure 4, the time course of the experiments performed on 20 days-old plants treated at -10°C was particularly interesting. The regrowth rate (Fig. 4a) remained close to 100% until 6 hours after the beginning of the treatment. After 9 hours-treatment, A. plants seemed to survive less than N.A. ones (Fig. 4a) and showed an higher electrolyte leakage (Fig. 4b), but increased their chlorophyll content (Fig. 4c). After 12 hours-treatment, the A.

plants showed a survival rate close to 50% (Fig. 4a) and a decrease in electrolyte leakage

when compared to the 9 hours-treatment (Fig. 4b), maybe due to plasmalemma acquired

tolerance. Chlorophyll content was similar to 20d C plants (Fig. 4c). The same trend was

observed after 24 hours of treatment, although at lower levels. On the contrary, N.A. plants

after 12 hours-treatment showed a lower survival rate (25%) (Fig. 4a), an increase in

electrolyte leakage (Fig. 4b) and a decreased in chlorophyll content (Fig. 4c); after 24 hours

of treatment the survival rate went down to 0% (Fig. 4a).

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RESULTS

0 25 50 75 100

0 h 3 h 6 h 9 h 12 h 24 h

Timing

% R E GR OW T H

20d N.A. 20d A.

a

0 25 50 75 100

0 h 3 h 6 h 9 h 12 h 24 h

Timing

% L E AKAG E

20d N.A. 20d A.

b ff

Fig. 4. Regrowth (a), Electrolyte Leakage (b) and Pigments Content (c) of 20d non- acclimated plants (N.A.) and 20d acclimated plants (A.) after treatment at - 10 ºC.

0 0.5 1 1.5 2 2.5

0 h 3 h 6 h 9 h 12 h 24 h

Timing

M G . P IG M /G . F .W .

20d N.A. 20d A.

c

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RESULTS

Cross experiment results (data only partially shown in Fig. 1, 2, 3 and 4) led us to the conclusion that the experiment should be carried out using the following parameters, that allow to obtain a good plants survival with reduced leakage and not irreversible injury to the photosynthetic apparatus.

Plants age 20 days-old

Cold acclimation treatment 1 week at 4°C

Freezing temperature - 10 °C

Time of exposition to freezing temperature 12 hours

Confirming the goodness of the chosen parameters is the observation that they were in agreement with parameters used in data previously published (Reyes-Diaz et al., 2006;

Warren et al., 1996). This would make it possible to detect proteins of interest in both conditions (freezing treatment on plants either previously acclimated or not-acclimated).

3.1.3 Freezing tolerance studies on 20-days-old plants

Before starting the proteomic analysis, we had to optimize our experimental design (Fig. 5) considering the following aspect:

• It is known that acclimation leads to a decrease in the growth rate of plants (see also Fig. 5 B). The delay in growth was also visible 1 week after the restore of the control conditions but not as pronounced as directly after the cold-stress treatments.

Beside the reduced plant size no others evident morphological changes were observed under the growth conditions used. To avoid finding proteins related to this different vegetative status and not to the freezing treatment of our interest, we used a double control:

- 20 days-old plants (20d C) versus 20 days-old plants acclimated for 1 week at 4°C (20d Accl): to have a physiological control.

- 13 days-old plants (13d C) versus 20 days-old plants, acclimated for 1 week at 4°C (20d Accl. 4°C): to have a morphological control.

• Since we did not know the neo-synthesis rate for putative target proteins related to

frost resistance, so we collected the material immediately after the end of freezing

treatment, but also after a day of regrowth at control condition (Fig. 5).

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RESULTS

Seven different conditions have been analyzed:

20d C 20 days-old plants (Fig. 5Ba)

N.A. 20 days-old plants , non acclimated and treated at -10 °C for 12 hours (Fig.

5Bb)

N.A.+R. 20 days-old plants, non acclimated, treated at -10 °C for 12 hours and collected after one day of regrowth at control condition (Fig. 5Bc)

13d C 13 days-old plants (Fig. 5Bd)

Accl. 4°C 20 days-old plants acclimated 1 week at 4 °C (Fig. 5Be)

A. 20 days-old plants, acclimated and treated at -10 °C for 12 hours (Fig. 5Bf) A.+R. 20 days-old plants, acclimated, treated at -10 °C for 12 hours and collected

after one day of regrowth at control condition (Fig. 5Bg)

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RESULTS

Fig. 5 Arabidopsis thaliana plants cultivated under different temperature regimes. (A) Overview of

experimental design. (B) Plant status at sampling time. 20-days-old Arabidopsis plants were growth as

described in material and method. Half of the plants were harvested (a) and half were submitted to freezing

treatment at -10°C for 12 hours. Half of the stressed plants was harvested at the end of freezing treatment (b)

and the other half was shifted back to the control conditions for a day and then harvested (c). 13-days-old

Arabidopsis plants were growth as described in material and method and harvested (d). 13-days-old

Arabidopsis plants were shifted to cold-stress conditions (4 ºC). After a cultivation time of 1 week under these

conditions half of the stressed plants was harvested (e), and half were submitted to freezing treatment at -10°C

for 12 hours. Half of the stressed plants was harvested at the and of freezing treatment (f) and the other half

was shifted back to the control conditions for a day and then harvested (g).

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RESULTS

On each of these conditions we repeated freezing tolerance analysis and we evaluated also chlorophyll a, chlorophyll b, carotenoid, glucose, fructose, sucrose and total soluble sugar contents, in order to clearly define some of the most important physiological parameters involved in plant response to low temperature (Fig. 6).

Photopigments content

0,00 0,50 1,00 1,50

20d C NA NA+R 13d C Accl 4Cº

A A+R

pigment mg/g F.W.

Total pigments Chl a Chl b Carotenoid

Glucose

0 10 20 30 40

20d C N. A . N. A .+ R 13d C A ccl 4 C A. A. +R

[u m o l/g . F .W .]

Fructose

0 1 2 3 4 5

20d C N. A . N. A .+ R 13d C Ac c l 4 C A. A. + R

[u m o l/g . F .W .]

Sucrose

0 5 10 15 20 25

20 d C N. A . N. A .+ R 13 d C Ac c l 4 C A. A. +R

[u m o l/g . F .W .]

Total sugars

0 20 40 60 80

20d C N. A . N. A .+ R 13d C Ac c l 4 C A. A. + R

[u m o l/ g . F .W .] [μ mol/g. F.W.]

[μ mo l/ g . F.W .] [μ mo l/ g . F .W. ] [μ mo l/ g. F. W. ]

Fig. 6 Chlorophyll content expressed as mg of pigment / g of fresh weight; sugar content expressed as μmol of sugar / g of fresh weight. 20d C: 20 days-old plants; N.A.: 20 days-old plants , non acclimated and treated at -10°C for 12 hours; N.A.+R: 20 days-old plants , non acclimated, treated at -10°C for 12 hours and collected after one day of regrowth at control condition; 13d C: 13 days-old plants; Accl 4C:

20 days-old plants acclimated 1 week at 4°C; A.: 20 days-old plants , acclimated and treated at -10°C

for 12 hours; A.+R: 20 days-old plants, acclimated, treated at -10°C for 12 hours and collected after one

day of regrowth at control condition.

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RESULTS

No injury of the photosynthetic apparatus was shown by measuring pigments (chlorophyll and carotenoid) after cold acclimation. A decreased in chlorophyll a and carotenoid content appeared after the freezing treatment in both acclimated and non acclimated plants.

Chlorophyll b decreased in non acclimated plants (from ~ 0.35 mg/g F.W. to ~ 0.20 mg/g F.W.), while remained constant in acclimated ones. One day of recovery at control condition (after freezing treatment) led to slight increase in pigments content (chlorophyll and carotenoid) of non acclimated plants (total pigments from ~ 0.75 mg/g F.W. to ~ 0.90 mg/g F.W.; chl a from ~ 0.5 mg/g F.W. to ~ 0.6 mg/g F.W.; chl b from ~ 0.25 mg/g F.W. to

~ 0.30 mg/g F.W.; carotenoid from ~ 0.15 mg/g F.W. to ~ 0.19 mg/g F.W.) , while no changes were observed in chlorophylls content of acclimated plants. Only carotenoid content slightly decreased in acclimated plants (from ~ 0.15 mg/g F.W. to ~ 0.12 mg/g F.W.). From our analysis it seemed that acclimation led to a decrease in cellular soluble sugars, particularly with regard to sucrose (from ~ 8μmol/g F.W. of the 20d C to ~ 3μmol/g F.W. of the Acc 4 ºC). Only fructose content increased during acclimation (from ~ 1 μmol/g F.W. of the 20d C to ~ 1.8 μmol/g F.W. of the Acc 4 ºC). After freezing stress however acclimated plants showed considerably higher sugar levels in comparison to non acclimated ones (glucose from ~ 31 μmol/g F.W. to ~ 11 μmol/g F.W.; sucrose from ~ 19 μmol/g F.W. to ~ 5 μmol/g F.W.; fructose from ~ 2.3 μmol/g F.W. to ~ 2.1 μmol/g F.W.;

total sugars from ~ 56 μmol/g F.W. to ~ 18 μmol/g F.W.) and to control plants (glucose

from ~ 31 μmol/g F.W. to ~ 14 μmol/g F.W.; sucrose from ~ 19 μmol/g F.W. to ~ 8 μmol/g

F.W.; fructose from ~ 2.3 μmol/g F.W. to ~ 1 μmol/g F.W.; total sugars from ~ 56 μmol/g

F.W. to ~ 22 μmol/g F.W.). The freezing stress treatment involved a loss of soluble sugar in

plants not previously acclimated, but a substantial increase in acclimated ones. However,

fructose content increased after freezing stress in both acclimated and non acclimated plants

(from ~ 2.1 μmol/g F.W. of the N.A. and from ~ 2.3 μmol/g F.W. of the A. to ~ 1 μmol/g

F.W. of the 20d C). The levels of glucose, sucrose and total sugar in acclimated plants

treated at -10°C for 12 hours showed a small decrease if the plants were recovered for a day

at control condition (glucose from ~ 31 μmol/g F.W. of the A. to ~ 30 μmol/g F.W. of the

A+R; sucrose from ~ 18 μmol/g F.W. of the A. to ~ 16 μmol/g F.W. of the A+R.; total

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RESULTS

sugars from ~ 56 μmol/g F.W. of the A. to ~ 54 μmol/g F.W. of the A.+R.), while non acclimated plants submitted to the same treatment showed a significant increase after the day of recovery (glucose from ~ 11 μmol/g F.W. of the N.A. to ~ 26 μmol/g F.W. of the N.A.+R.; sucrose from ~ 5 μmol/g F.W. of the N.A. to ~ 12 μmol/g F.W. of the N.A.+R.;

total sugars from ~ 19 μmol/g F.W. of the N.A. to ~ 40 μmol/g F.W. of the N.A.+R.). In both acclimated and non acclimated plants, treated at -10°C for 12 hours and recovered for one day at control condition, fructose levels increased remarkably (from ~ 2.3 μmol/g F.W.

of the A. to ~ 3.9 μmol/g F.W. of the A.+R. and from ~ 2.1 μmol/g F.W. of the N.A. to ~ 3 μmol/g F.W. of the N.A.+R.).

• To be sure to work only with plants surviving the freezing treatment and since the

interest was to investigate the late events of adaptations at the protein level

following initial signaling events, we chose to perform proteomics analysis only on

plants recovered for a day at control condition (NA+R: 20 days-old plants, non

acclimated, treated at -10 ºC for 12 hours and collected after one day of regrowth at

control condition; A+R: 20 days-old plants, acclimated, treated at -10 ºC for 12

hours and collected after one day of regrowth at control condition).

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RESULTS

3.2 Proteomic Results

Based on the results obtained and on the need to set-up a feasible proteomic study design, we reduces our samples conditions, from seven to the most relevant four:

20d C 20 days-old plants (Fig. 5Ba)

N.A.+R. 20 days-old plants, non acclimated, treated at -10°C for 12 hours and collected after one day of regrowth at control condition (Fig. 5Bc)

Accl. 4°C 20 days-old plants acclimated 1 week at 4°C (Fig. 5Be)

A.+R. 20 days-old plants, acclimated, treated at -10°C for 12 hours and collected after one day of regrowth at control condition (Fig. 5Bg)

To detect differentially expressed proteins associated with freezing tolerance, with or without previous acclimation, 2-DE patterns from control and the corresponding stress treatment were compared. Extracts were prepared from rosette leaves of Arabidopsis thaliana control and treated plants and proteins resolved by 2-D gel electrophoresis as described in the Materials and methods. To ensure the reproducibility of protein patterns, at the beginning we resolved protein samples on pH 3-10NL (18 cm) IPG gel strips, performed in triplicate biological repeats. In addition, two iterations of one harvest, for technical replication, were run. In Fig. 7, a representative image from a silver stained 2-DE gel is shown. Silver staining, as known, proves generally the best spots staining sensitivity, but this staining is not compatible with further identificative analysis by mass spectrometry.

For this purpose we used colloidal Coomassie G 250, but the gel staining in 3-10 pH range was too low to permit discrimination of differentially expressed protein spots, even if a greater amount of protein was loaded (1000 μg) (see Fig. 8.I and 10). Thus, to improve the separation and resolution of proteins we applied the zoom-in gel approach and an increased amount of total proteins (800 μg) were further separated by 2-DE in the following pH range (Fig. 8.I, 11, 12 and 13):

• pH 3-5.6 NL (18 cm), IPG strips

• pH 4-7 (18 cm) IPG strips

• pH 6-11NL (18 cm) IPG strips.

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RESULTS

10 15 20 25 37 50 75 100 150 250

MW

3 4 4.8 5.4 5.6 6 6.2 6.8 8 9 10

pI

10 15 20 25 37 50 75 100 150 250

MW

3 4 4.8 5.4 5.6 6 6.2 6.8 8 9 10

pI

IEF IEF SDS SDS

Mw (kDa)

Fig.7. Representative image of 20 days-old Arabidopsis thaliana cellular soluble proteins in the pH 3-10NL range. Proteins (100 μg) were resolved by 2DE and silver-stained. Mw in kDa and pI of proteins are indicated on the right and on the top of the image.

The zoom-in separation resulted in a better proteins resolution and in the appearance of new spots (Fig. 8.I). New protein spots probably were already present in samples, but in low abundance to be detectable on 3-10 pH range (Fig. 8.II ). In particular, the mean of the detected spots in the gels (three replicates of each pH range) are reported below:

pH gel range Number of spots 3 -10 NL 506 +/- 22 3 - 5.6 NL 482 +/- 18 4 – 7 962 +/- 58 6 - 11 596 +/- 55

The amount of the detected spots in the gel with narrower pH range, without considering

spots repeated in more than one gel, equals 1210 +/- 25. In contrast, in the gel with the pH

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RESULTS

varying from 3 to 10, the amount of the spots was 506 +/- 22. This shows an increase in the number of the detected spots equal to 139 %.

The quality and goodness of our analysis was confirmed comparing the gels produced with the SWISS 2D Arabidopsis thaliana gel on line database (http://www.expasy.org/swiss- 2dpage). Computer-assisted 2D gel differential analysis was performed by using the PDQuest version 7.1 software (BioRad), to compare differential protein expression between control and treatments. We found 8 and 6 protein spots, respectively in the non- acclimated (NA+R) and acclimated (A+R) plants exposed to freezing compared to control conditions, showing an expression increase ranging from 1.2 to 3 folds; while 5 and 7 spots showed a decrease (range from 1.3 to 2.8 folds) compared to control conditions (see later Tab. 2); others 10 spots showed an expression increase ranging from 1.2 to 4 folds in the acclimated plants (4 ºC Accl.); while 2 spots showed a decrease (1.6 and 1.8 folds) compared to control conditions (see later Tab. 2). The protein spots resulted to be relevant from the image analysis and the most abundant ones were chose for protein identification.

Preparative gels in the pH range 3-10NL, 3-5,6NL and 4-7 were run with a protein loading of 1000 μg, stained with colloidal Coomassie G 250 and spots of interest were picked out.

It was more difficult to separate proteins in basic pH range; after several attempts we were

able to obtain good results loading and running gels with 400 μg of protein and staining

with Vorum’s silver protocol because their detection was not appreciable with Coomassie

staining.

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RESULTS

pH 3 -1 0 N L pH 3-5.6NL pH 4-7 A

pH 3 -1 0 N L pH 3 -1 0 N L pH 3-5. 6NL pH 3-5. 6NL pH 4-7 A B

pH 3-10NLpH 3-5.6NLpH 6-11NLpH 4-7pH 3-10NLpH 3-10NLpH 3-5.6NLpH 3-5.6NLpH 6-11NLpH 6-11NLpH 4-7

p H 6-11 NL p H 6-11 NL

75 50 37 25 20 15

Co omas s ie p H 3- 10 NL

344.85.45.666.26.88910 75 50 37 25 20 15 1010015020044.85.45.666.27

Coomas sie p H 4 -7L Co omas sie p H 3-5. 6 N L

75 50 37 25 20 15 10100150200344.85.45.66.567.5 25 20 15 1050 37

11

Vor um S ilve r Staining p H 6-11

75 50 37 25 20 15

Co omas s ie p H 3- 10 NL

344.85.45.666.26.88910 75 50 37 25 20 15 1010015020044.85.45.666.27

Coomas sie p H 4 -7L Co omas sie p H 3-5. 6 N L

75 50 37 25 20 15 10100150200344.85.45.66.567.5 25 20 15 1050 37

11

Vor um S ilve r Staining p H 6-11

6.567.5 25 20 15 1050 37

11

Vor um S ilve r Staining p H 6-11

A A A

B B B B

NL Fig. 8.I: I m p rove ment i n t h e sepa rat io n a n d resol u ti o n of p rot ei n as res u lt o f nar ro w in g t h e p H gra d ie nt on 18 c m gel s, st ai ne d wi th col lo id al C o o m assi e G 25 0, an d Vo ru m ’s si lv er. Se para ti on of t o tal cellu lar so lub le pro tein s fro m Arab id op sis t h alian a was do n e bet wee n pH 3 -10 , p H 3- 5. 6, p H 4- 7 a nd p H 6- 11 . 8.II : Exam pl e of t h e hi g h er re sol u ti o n o f s p ot s in t h e na rr ow pH g ra d ie nt g el s i n co mp ariso n wi th th e pH 3-10 g el.

8.I 8.II

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RESULTS

3.2.1 Spot identification by mass spectrometry analysis

All the spots marked on the 2D-gels in Figures 10-13 were excised, submitted to tryptic digestion and mass spectrometry analysis as described in Material and Methods. A total of 105 spots were analyzed, among these, 83 were successfully identified corresponding to 46 different proteins. Peptide mass fingerprinting was also used to confirm the identity of the spots picked from independent gels.

In Table 1 are reported the identification parameter for each spot. Five spot were identified using MS/MS analysis (in collaboration with Dr. Alpi – Scuola Normale Superiore, Pisa).

Among all the identified 40 spots are not already annotated in the SWISS 2D Arabidopsis thaliana gel on line database (http://www.expasy.org/swiss-2dpage). The identified proteins belong to several different metabolisms like photosynthesis (17%), protein biogenesis, folding, modification and degradation (23%), antioxidant activity (4%), carbon metabolism (8%), stress response and signal transduction (15%), transport (4%). In particular, in Fig. 9 is reported the biological process involvement of the identified proteins.

Fig. 9. Biological process involvement of the identified proteins.

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RESULTS

Several of the proteins found to exhibit different accumulation patterns after stress treatment could be identified using MALDI-TOF-MS or MS/MS. In particular 26 spots showing change in protein expression were identified, corresponding to 21 different proteins (Tab.2). In Table 2 are also indicated the results of transcript RNA level expression evaluated by microarrays reported previously (www.genevestigator.ethz.ch).

S7

S8 G2

G11/T9 28

L1

7308 2206

L8

50 75

37

25

20 A 15 S2

S3 S4

3 B C

D E F

L5 L4

L2

20 G13

3 4 4.8 5.4 5.6 6 6.2 6.8 8 9 10

S7

S8 G2

G11/T9 28

L1

7308 2206

L8

50 75

37

25

20 A 15 S2

S3 S4

3 B C

D E F

L5 L4

L2

20 G13

3 4 4.8 5.4 5.6 6 6.2 6.8 8 9 10

Mw (kDa)

pI

Fig. 10. Solubilized proteins analyzed by 2D-PAGE over the pI range 3-10 NL and stained with colloidal

Coomassie G 250. Out of the 24 spots marked, 22 were identified by mass spectrometry. Among these

proteins, no ones seemed to changed in quantity after cold acclimation or freezing treatment. The protein

identity and MS analysis data of these spots are summarized in Table 1.

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RESULTS

3

2 20 19

21

15 8

M 33

3

6

7

8 11

13 22 14

23

24 31

35

41 40

L8 S8

G11

L5

38

27

18 30

37 39

S3 S2

S4 F

29 L4 27bis G2

28bis

28

4 4.8

Fig. 11. Solubilized proteins analyzed by 2D-PAGE over the pI range 3-5,6 NL and stained with colloidal Coomassie G 250. Out of the total of 46 spots marked, 36 were identified by mass spectrometry. The protein identity and MS analysis data of these spots are summarized in Table 1. Among these, 23 spots that changed in quantity in treated plants are highlighted (their data are shown in Table 2).

Fig. 12. Solubilized proteins analyzed by 2D-PAGE over the pI range 4-7 and stained with colloidal Coomassie G 250. Out of the 63 spots marked, 48 were identified by mass spectrometry. The protein identity and MS analysis data of these spots are summarized in Table 1. Spots that changed in quantity in treated plants are highlighted (their data are shown in Table 2).

50 37

25

20 15 10 100 5,6

L1 B

75 L2

36

3

2 20 19

21

15

4 4.8

50 37

25

20 15 10 100 5,6

L1 B

L2 8

M 33

3

6

7

8 11

13 22 14

23

24 31

35

41 40

L8 S8

G11

L5

38

27

18 30

37 39

S3 S2

S4 F

29 L4 27bis G2

28bis

28

36

32

34

75 Mw (kDa)

pI

N

pI

4

P 84

71

75 7

16 20

43 44 45 42

41 39 40 46

47

61 S8

65

S2 S4

50 100

37

25

20 15 10

A R

37 L5

L4 28 27

29 30 83

34 85 32 31

68 69 70 67

64

S3

G11

L8

L1 M

36 24

7 33

35

20 E D

L2 3

1 2

B S7 F 8 G2

27bis 28bis

C

4.8 5.2 5.6 6 6.2 4

P 84

71

75 7

16 Z

43 44 45 42

41 39 40 46

47

61 S8

65

S2 S4

50 100

37

25

20 15 10

A R

37 L5

L4 28 27

29 30 83

34 85 32 31

68 69 70 67

64

S3

G11

L8

L1 M

36 24

7 33

3

S7

27bis 8 G2

28bis

4.8 5.2 5.6 6 6.2

E D 1

2

B F C

35

20

Mw

(kDa)

(22)

RESULTS

6 6.5 7.5

25 20 15

10 50

37 11

94 92

97

96

90 91

93

99

A

S7 98

93

6 6.5 7.5

25 20 15

10 50

37 11

94 92

97

96

90 91

99

A

S7 98

Mw (kDa)

pI

Fig. 13. Solubilized proteins analyzed by 2D-PAGE over the pI range 6-11NL and stained with Vorum’

silver. All the 11 spots marked were identified by mass spectrometry. Protein identity and MS analysis data of

these spots are summarized in Table 1. Three spots that changed in quantity in treated plants are highlighted

(their data are shown in Table 2).

(23)

RESULTS

ble 1 . Ide n ti fi cat ion o f pr ot ei n sp ot s. Ide n ti fi cat ion was d o n e by d at abase search a g ai nst “ Arab idop sis th a liana ” pr ot ei n. Ma scot 2. 1 www.matri xs cience.c o m ) was used to interrogate the integrat ed into UniProtKB/Swiss- Prot database. T h e accession numbe rs and corre spondin g SC OT sco re are l is te d bel o w . Tw o mi sse d cl eava g es we re allowe d for trypsin s p ecific it y . C arba m id o m et hyl at io n of cyst ei ne was se t as fi xed (c o m pl et e) ificatio n wh ile meth ion in e ox id ation was set as v ariab le (p artial) mo d ificatio n . Mass to leran ce fo r mo no iso topic d ata was se t to 50 , 10 0 o r 2 0 0 pp m. ei n i d ent if icat ion was co mpl et ed b y si gni fi cant P < 0. 05 pr oba bl y base d M o wse Sco res. M w ( k Da): M o lecul ar Wei g ht . p I: Isoel ectric poi nt. Obs.: rv ed . Th .: Theo ret ical val u es. S pot s n u m b er a nd t h ei r gel s-l ocal iz at ion, Acce ssi o n n u m b er on Uni P rot K B /Sw iss-Pr ot dat aba se, Pr ot ei n na me, A G I d e (Arab idop sis Gen o me In itiativ e co d e), calcu lated and th eo retical M w and pI, M o wse sco re, p ercen tag e sequ en ce cov erage o f t he pr ot ei n b y t h e ed pep tides, Numb er o f M A LDI-TOF match ed pep tides an d Number of MAL D I-T OF sea rc h ed peptides were indicated. Sp ot a n d posi ti on Ac c ess io n nu mb er Name AGI c o de Obs T h O bs Th Score Se quenc e coverage (% )

Matched pepti d e Sear ched pepti d es ot S2 P1 089 6 Ribulos e b isphosphate carboxylase/oxyge- n ase activase

At2g 397 30 46 47 5.4 5 .0 193 60 20 63 ot 8 g el pH 5. 6 N L 38 47 4.4 5 .0 131 35 12 20 ot 3 1 g el 3-5.6 N L 37 47 5.3 5 .0 101 19 11 20 ot G2 59 47 4.5 5 .0 197 65 20 60 ot 3 5 g el 3-5.6 N L O03042 Ribulos e b isphosphate carbox ylase large ch ain [Precursor ]

AtCg00490 25 53 4.9 5 .9 192 34 23 44 ot 06 04 g el d p H 3 - NL 50 53 5.9 5 .9 76 40 14 59 ot S7 50 53 5.8 5 .9 205 53 29 72 ot 22 06 g el 3-10 NL 50 53 6.0 5 .9 74 31 18 94 ot T9/Spo t 1 g el pH 3- NL 15 53 4.7 5 .9 69 22 10 20

Mw kDa p I

(24)

RESULTS

Sp ot a n d posi ti on Ac ce ss io n nu mb er Name AGI c o de Obs T h O bs Th Score Se quenc e coverage (% )

Matche d pepti d e Sear ched pepti d es Sp ot B Mixt ure: O03 042 Ribulos e b isphosphate carbox ylase large ch ain [Precursor ]

AtCg 004 90 50 53 5.5 5 .9 368 66 44 83 P2 569 6 En ola se At2g 365 30 50 48 5.5 5 .5 119 50 20 83 P1 936 6 ATP synthase subuni t b et a AtCg 004 80 50 54 5.5 5 .4 93 46 20 83 Sp ot D Mixt ure: O03 042 Ribulos e b isphosphate carbox ylase large ch ain [Precursor ]

AtCg 004 90 50 53 5.4 5 .9 145 40 22 5 9 P1 936 6 ATP synthase subuni t b et a AtCg 004 80 50 54 5.4 5 .4 108 45 21 5 9 Sp ot 20 ge l p H 3-10 NL O03 042 Ribulos e b isphosphate carbox ylase large ch ain [Precursor ]

AtCg 004 90 50 53 6.2 5 .9 259 47 31 5 4 Sp ot G13 ge l p H 3-10 NL 15 53 4.7 5 .9 76 27 17 8 2 Sp ot S7 ge l p H 4-7L 50 53 6.2 5 .9 311 63 44 1 00 Sp ot L8 O03 042 Ribulos e b isphosphate carbox ylase large ch ain [Fra g ment ]

AtCg 004 90 19 48 5.0 6 .1 92 21 8 1 9 Sp ot C O03 042 Ribulose bisphosphate carbox ylase large ch ain [Precursor]

AtCg 004 90 50 53 5.5 5 .9 72 14 7 1 3

Mw kDa p I

(25)

RESULTS

Sp ot a n d posi ti on Ac ce ss io n num. Name AGI c o de Obs T h O bs Th Score Se quenc e coverage (% )

Matche d pepti d e Sear ched pepti d es ot A P1 079 5 Ribulos e b isphosphate carbox ylase small ch ain 1A

At1g 670 90 15 21 6.2 7 .6 203 60 15 4 0 ot 73 08 g el H 3-10 NL 15 21 5.9 7 .6 132 64 17 9 3 ot 9 8 g el H 6-11 L Q9SCU8 B eta-gal act o si dase 14 p recursor At4g 385 90 50 10 2 8.0 8 .4 59 11 10 1 6 ot S8 Q42 029

Oxygen- evolving enh ancer protein 2 -1 , chlorop last p recursor ) (OE E2 (2 3 k D a sub unit)

At1g 066 80 23 25 5.4 5 .2 58 50 10 8 2 ot 3 gel pH 10 NL 23 25 4.7 5 .2 165 49 12 2 1 32 32 4.9 5 .9 203 66 22 5 0 ot L5 P2 332 1

Oxygen- evolving enh ancer protein 1 -1 , chlorop last p recursor ) (OE E1 (3 3 k D a subu nit)

At5g 665 70 31 31 5.0 5 .0 148 50 14 2 0 ot 2 7 g el H 4-7L 31 35 5.0 5 .6 161 44 12 2 1 ot 2 8 g el H 4-7L 31 35 4.9 5 .6 169 56 15 3 6 ot F P8 348 3 ATP synthase subunit be ta-1, m itochondrial p recursor

At5g 086 70 63 60 5.4 6 .6 240 41 19 4 0

Mw kDa p I

(26)

RESULTS

Sp ot a n d posi ti on Acc. num. N ame A GI c o de Obs T h O bs Th Score Seq. coverage (% )

Matc. pep. Search. pep . Sp ot 6 gel pH 3- 5. 6 N L Q9 LR 75 Copr oporphyrinogen II I o x id ase, chlo roplast p recursor

At1g 034 75 42 41 5.4 7 .9 201 41 17 4 1 Sp ot 2 gel pH 3- 5. 6 N L O04 157 Ras-related p rotein Rab7 At1g 227 40 25 23 4.3 5 .3 52 21 6 1 7 Sp ot 3 6 g el p H 3-5.6 N L 33 23 5.4 5 .3 61 20 6 2 1 Sp ot 2 3 g el p H 3-5.6 N L 15 23 4.4 5 .3 64 26 7 1 9 Sp ot 4 0 g el p H 3-5.6 N L 23 23 5.1 5 .3 63 21 6 1 8 Sp ot 2 2 g el p H 3-5.6 N L Q9 F H J2 DRL3 4 , Pro b able d isease resistan ce pr otein At5g45440

At5g 454 40 16 40 4.5 5 .2 63 35 11 2 0 Sp ot 2 4 g el pH 4 -7 O23 403 Psb P -related thy lako id lumenal p rotein 1, ch lor o pl as t pr ec ur so r

At4g 155 10 20 32 4.9 8 .9 101 21 11 2 0 Sp ot 27b is g el pH 4 -7 Q 9 XEX2 Peroxiredoxin-2B At1g 659 80 41 18 4.1 5 .2 51 14 4 1 1 Sp ot 2 9 g el p H 3-5.6 N L Q9 MA B 3 Proba b le nucleolar pr ot ei n NO P5- 2 At3g 050 60 38 59 4.4 9 .2 131 27 11 2 3 Sp ot 3 2 g el pH 4 -7 O80 934 Un characterized protein At2g37660, chloroplast p recursor At2g 376 60 30 30 5.3 5 .3 106 31 10 2 0 Sp ot 3 3 g el pH 4 -7 P4 275 2 Cyclin-D2-1 At2g 224 90 45 41 4.5 5 .0 62 21 9 3 6

Mw kDa p I

(27)

RESULTS

Sp ot a n d posi ti on Acc. num. N ame A GI c o de Obs T h O bs Th Score Seq. coverage (% )

Matc. pep. Search. pep . ot 7 gel pH 7 Q03 250 Glycin e-rich RNA- bi ndi n g pr ot ei n 7 At2g 216 60 17 17 5.4 5 .9 115 65 11 2 0 ot 30 pH 3- 6 N L Q9GCB9 Mitochondrial ribosoma l protein S2 At3g 036 00 25 25 4.2 9 .3 57 15 4 1 0 ot N gel pH 5. 6 N L Q43 349 ROC2 , 29 kDa ribonucleopr otein, ch lor o pl as t pr ec ur so r

At3g 534 60 25 36 5.2 4 .8 106 30 13 4 4 ot 4 1 g el -7 Q9FM89 F-box/FBD/LRR-repeat pr otein At5g56420 At5g 564 20 22 49 5.2 8 .4 53 14 6 1 5 ot 8 5 g el -7 Q9 M8L 5 FDL1 3, putative F - box/FBD/LRR- repe at pr otein At1g80470

At1g 804 70 35 54 4.4 4 .9 61 10 6 1 3 ot 4 g el p H 3 - 6 N L Q3 E9 B8 P rob ab le wh ite-b rown complex homolog pr ot ei n 24

At5g 194 10 30 70 5.4 6 .3 76 12 9 1 9 ot 2 9 g el -7 Mixt ure: O81 149 PSA5A , P roteaso m e subunit alpha type-5-A At1g 538 50 25 26 4.2 4 .7 80 45 9 4 0 Q43 349 ROC2 ,2 9k D a ribonucleopr otein, ch lor o pl as t pr ec ur so r

At3g 534 60 25 36 4.2 5 .2 140 35 14 3 4

Mw kDa p I

(28)

RESULTS

Sp ot a n d posi ti on Acc. num. N ame A GI c o de Obs T h O bs Th Score Seq. coverage (% )

Matc. pep. Search. pep . Sp ot 3 9 g el p H 3-5.6 N L Q9 Z U U 4 ROC1 , Put ati ve ribonucleopr otein, ch lor o pl as t pr ec ur so r

At2g 372 20 23 29 4.1 4 .7 62 26 7 2 8 Sp ot 3 8 g el p H 3-5.6 N L 22 29 4.1 4 .7 87 24 7 1 8 Sp ot 3 7 g el p H 3-5.6 N L 22 .5 29 4.0 4 .7 99 27 9 2 1 Sp ot P gel 20d C p H 4-7 25 29 4.5 4 .7 108 30 9 1 9 Sp ot 34 g el 20 d C pH 4 -7 25 29 4.5 4 .7 129 33 11 2 5 Sp ot 3 1 g el 20 d C pH 4 -7 25 29 4.5 4 .7 92 27 8 1 6 Sp ot 8 3 g el pH 4 -7 25 29 4.5 4 .7 113 35 10 2 2 Sp ot E P1 936 6 ATP synthase subunit be ta AtCg 004 80 50 51 5.5 5 .3 142 30 9 1 7 Sp ot L1 P3 479 1 P ep tidyl-proly l cis- tra n s isome rase CYP20- 3

At3g 620 30 20 22 5.5 5 .4 85 48 13 3 4 Sp ot L2 17 28 5.5 8 .8 93 31 9 2 3 Sp ot 1 gel pH 4- 7 P3 770 2 Myr o si na se pr ec ur sor At5g 260 00 62 63 5.4 5 .2 151 34 19 4 0 Sp ot 2 gel pH 4- 7 62 63 5.5 5 .3 142 45 24 6 0 Sp ot 3 9 g el pH 4 -7 Q9 LDT3 Axial regulator YABBY 4 At1g 234 20 25 26 6.4 5 .9 53 28 7 3 5

Mw kDa p I

(29)

RESULTS

Spot and po sitio n Acc. num. N ame A GI c o de Obs T h O bs Th Score Seq. coverage (% )

Matc. pep. Search. pep . ot 8 4 g el -7 Q9FF W2 FBD1 7, FB D- associated F-b o x pr otein At5g38590

At5g 385 90 25 48 4.5 5 .5 55 14 6 1 5 ot 4 2 g el -7 Q96 266 Gluta thione S- tran sferase 6 At2g 477 30 22 29 6.3 8 .5 132 41 10 2 0 ot 4 4 g el -7 Mixture: Q9 LEZ 3 Tran scriptio n facto r BIM1 At5g 081 30 22 59 6.5 0 .3 86 21 9 2 4 Q9SLC4 RING-H2 fing er pr ot ei n AT L 2K At2g 423 50 25 25 6.4 6 .0 60 19 5 1 5 ot S4 Q944 G9 P rob ab le fru ctose- bisphosphate aldolase 2, ( E C 4. 1. 2. 13)

At4g 389 70 35 43 5.6 7 .3 93 32 12 3 3 ot 1 6 g el H 4-7L P9 381 9 Malate dehydrogena se, cytoplasmic 1 At1g 044 10 35 36 6.2 6 .1 130 37 10 2 0 ot 91 gel pH 11 L Q9SJU4 P rob ab le fru ctose- bisphosphate aldolase 1 At2g 213 30 45 43 6.5 6 .2 106 36 12 2 8 ot 9 0 g el H 6-11 L Q9 4B 40 Zinc finger A20 At3g 528 00 40 19 6.1 9 .1 59 46 4 1 0 ot 9 2 g el H 6-11 L P9 296 0 Potassium channel KAT3 At4g 326 50 28 76 6.1 7 .3 66 22 9 1 7

Mw kDa p I

(30)

RESULTS

Spot and po sitio n Acc. num. N ame A GI c o de Obs T h O bs Th Score Seq. coverage (% )

Matc. pep. Search. pep . Sp ot 9 9 g el p H 6-11 L P2 818 6 Ras-related p rotein ARA-3 At3g 460 60 25 24 7.5 7 .7 62 42 6 1 7 Sp ot 9 3 g el p H 6-11 L P8 262 1 Un characterized protein SCRL2 [Precursor] At1g 651 13 25 11 6.3 8 .9 55 58 4 9 Sp ot 9 4 g el p H 6-11 L Q9 F H 27

KCS20, Pr obable 3- k etoacyl-Co A syn thase 20. A lso known as: Very lo ng-chain fatty acid condensing enzy m e 20

At5g 490 70 25 53 6.4 9 .2 54 15 6 1 0 Sp ot 9 6 g el p H 6-11 L P5 679 2 Chlor o pl as t 5 0S ribosoma l protein L14 AtCg 007 80 15 14 9.5 9 .4 75 64 6 1 4 Sp ot 9 7 g el p H 6-11 L Q9 LX 08 Annexin D6 At5g 102 20 16 37 9.5 7 .7 62 26 7 1 4 Sp ot R g el pH 4 -7 P9 300 2 Regulatory p rotein NPR1 At1g 642 80 25 67 4.6 5 .7 89 25 12 2 4 Sp ot 3 6 g el pH 4 -7 P8 228 1 Putative L-ascor b ate peroxidase 4, chlo roplast precursor At4g 090 10 25 38 5.0 8 .6 118 36 12 3 1

Mw kDa p I

(31)

RESULTS

t number and lo calisa tio n

Ac c. nu mb er Pr otei n name AGI c o de Obs T h O bs Th ot 4 2 g el d C pH 4 -7 Mixt ure: Q96 266

GSTF6_ARATH , Gluta thione S- tran sferase 6 , ch lor o pl as t pr ec ur so r

At2g 477 30 22 29 6.3 8 .5 Q8 L6 Y 1 UBP1 4_ARATH , Ubiquitin carboxyl- ter m inal hydrolase 14

At3g 206 30/ At3g 206 25 22 89 6.3 5 .1 ot Z gel pH 7L Q9LKA3 Malate dehydr o genase 2 At3g 150 20 32 36 6.4 8 .3 ot M g el pH 5. 6 N L Q94CI6 EDL18, Suga r tra n sporter E RD6-like 18

At5g 273 60 45 53 4.4 8 .7 ot 9 1 g el H 6-11 L Q9SJU4 P rob ab le fru ctose- bisphosphate aldolase 1 At2g 213 30 47 43 6.5 6 .2 ot 3 6 g el H 4-7L Q9C 6 Y3 Cyclin-A1-1 At1g 441 10 23 53 5.1 8 .4

Mw kDa p I K.FEMTAPTTK.C Ox. (M) (Ions score 24) Scor es > 22 in dicate p < 0.05

Ident ified peptides (MS/MS) R .T AAYY QQGAR .F (Ions score 3 3) K .E A AW GL A R .Y (Ions score 35) Sc or es > 1 9 i n di cat e p< 0.0 5

K.MAIIL G R R.K (Ions score 27) Sc or es > 2 3 i n di cat e p< 0.0 5 K.ALE GADL VIIPAGVPR .K (Io ns score 69) R .D D LFNINAGIVK.N (Io ns score 43) K.KLFG VTT LDVV R.A (Ions score 40 ) K.LFGV TTL DVVR .A (Io ns score 49)

R .A ITQ YLAE EYSE KGE K.L ( Io ns s cor e 36 ) K.VLD VYEA R.L (I ons score 23) Sc or es > 2 2 i n di cat e p< 0.0 5 K.QKAG KGLE END MR .S Ox. (M) (Ions score 24) Sc or es > 2 2 i n di cat e p< 0.0 5

(32)

RESULTS

Table 2 Protein spots that increased or decreased in treated plants compared with control plants. Spots number and their gels-localization, Accession number on UniProtKB/Swiss-Prot database, Protein name and AGI code (Arabidopsis Genome Initiative code; www.arabidopsis.org) were indicated. Modulation:

upregulation (range 1.2-4) (⇑) or downregulation (⇓) (range 1.3-2.8)of expression level in treated plants compared with the control; (x) expression level comparable to control. Transcript level ratio (4ºC/Control):

Log(2) ratio (4ºC/control), data by three different experiments annotated on Gene Vestigator (www.genevestigator.ethz.ch).

Accession Function

Spot number and

localisation 20d C 4 gradi

Trascript level ratio (4ºC/Control)

NA+R A+R

S2 pH 3-5.6 P10896

Ribulose bisphosphate carboxylase/oxygenase activase PRECURSOR

x x 0.79 and 1.05

and 0.79 ⇓ ⇓

8 e 31 pH 3-5.6 P10896

Ribulose bisphosphate carboxylase/oxygenase activase FRAGMENT

x x x ⇓

S7 pH 4-7 O03042 LARGE CHAIN RuBisCO,

PROTEIN x ⇑ ^{2.13 an} _2.38 ^3.7

1.52 1.94 an 2.17

3.52 1.64 1.32

d

and ⇑ ⇑

35 pH 3-5.6 O03042 LARGE CHAIN RuBisCO,

FRAGMENT x ⇓ A x

L5 pH 4-7 P23321

Oxygen-evolving enhancer protein 1-1, (OEE1), PROTEIN

x ⇑ 1.01 and 0.72

and 0.91 ⇑ ⇑

S8 pH 3-5.6 Q42029

Oxygen-evolving enhancer protein 2-1, (OEE2), PROTEIN

x x 0.97 and 0.63

and 0.82 ⇓ x

6 pH 3-5.6 Q9LR75 Coproporphyrinogen III

oxidase. PROTEIN x x 0.6 and 0.99

and 0.85 ⇓ ⇓

27bis pH 3-5.6 e 4-

7 Q9XEX2 Peroxiredoxin-2B,

PRECURSOR x ⇑ ^{0.59 and} _{and 1.02} ⇑ ⇑

22 pH 3-5.6 Q9FHJ2

DRL34, Probable disease resistance protein, FRAGMENT

x ⇑ ⇑ ⇑

7 pH 3-5.6 e 4-7 Q03250 Glycine-rich RNA-binding

protein 7, PROTEIN x ⇑ _and ^{d 0.95} A A

93 pH 6-11 P82621 Uncharacterized protein

SCRL2. PRECURSOR x ⇑ ^{no data} ⇑ ⇑

34 pH 3-5.6 Q3E9B8

Probable white-brown complex homolog protein 24. FRAGMENT

A ⇓ 0.9 and 1 and

A x

30 pH 3-5.6 Q9GCB9 Mitochondrial ribosomal

protein S2. PROTEIN A ⇑ ^{0.89 and} _and ⇑ x

Modulation

(33)