Fig. 2. EMSA with the S6C4 promoter fragment 430-bp and nuclear protein extracts from etiolated Sorghum leaves. Nu-
clear protein 2 mg and 15 fmol of the DNA probe were used in each assay. Lane 1, free probe; lane 2, probe with nuclear
protein extract from etiolated leaves; lane 3, nuclear protein extract from etiolated leaves were preincubated at 50°C in the
presence of 10 mg of proteinase K for 20 min, prior to EMSA; lane 4, nuclear protein extract from etiolated leaves were
boiled for 3 min prior to EMSA. The free probe and the DNA-protein complexes are indicated as FP, PC1 and PC2,
respectively.
ghum leaves. Two retarded bands, PC1 and PC2, the major one PC2 moving faster, were observed
in EMSA of the protein samples from etiolated leaves Fig. 2 lane 2. Pretreatment of the protein
extracts with proteinase K, or boiling, prior to electrophoresis caused the disappearance of the
retardation bands thus establishing that the shift resulted from protein-DNA interactions Fig. 2
lanes 3 and 4.
There was no detectable signal in EMSA with nuclear protein extracts from light-adapted leaves
for four days under photoperiodic conditions Fig. 3 lane 2. After the return of the greening plants to
darkness for 4 additional days, during which the
Fig. 3. Effect of alternated prolonged darkness and photope- riodic light on the binding activities to the 430-bp S6C4
promoter fragment. Sorghum seeds were germinated for 11 days in darkness thermoperiod, 27°C for 16 h and 17°C for
8 h stage 1, and then illuminated 4 days under photoperi- odic conditions 16 h of white light, 700 E m
− 2
s
− 1
, 27°C, 8 h of darkness, 17°C stage 2, returned to darkness as in
stage 1 4 days stage 3 and finally illuminated as in stage 2 4 days stage 4. Leaf and nuclear soluble proteins were
extracted at the indicated time. Specific activity of PEPC was determined in leaf protein extracts means value of three
independent experiments. The binding activities to the 430- bp probe were assessed with EMSA 15 fmol of the 430 bp
probe, 2 mg of nuclear proteins. Lanes a – d, probe + nu- clear proteins from stages 1 – 4, respectively. PC1, PC2 and
FP, as under Fig. 2.
mM leupeptin, 50 mM HEPES – NaOH pH 8. Other assays were supplemented with 500 U of
commercial, rat liver casein kinase II CKII or with 100 mM K252a general protein kinase in-
hibitor. After incubation for 20 min, 25°C, the reactions were blocked by 1 ml EDTA final con-
centration, 20 mM. The other components of the shift assay poly dI-dC and the labeled probe in
TE buffer were added and the mix total volume, 20 ml subjected to electrophoresis in agarose gel as
described above.
3. Results
3
.
1
. DNA-binding acti6ities for the S6C
4 430
bp promoter fragment in nuclear protein extracts
from etiolated Sorghum lea6es EMSA has been devised to detect the interac-
tion of nuclear proteins with the S6C4 promoter [25,26]. A S6C4 promoter fragment 430-bp, −
625 to − 195 bp from the translation start site was used in these experiments because a it con-
tains several consensus domains homologous to light dependent boxes found in other photoregu-
lated plant gene promoters [3] and Fig. 1 and b it was previously shown to direct the mesophyll-
specific expression of the marker gene uidA in transient expression experiments with Sorghum
leaves unpublished. The 430-bp probe was la- beled with digoxigenin-11-ddUTP and incubated
with nuclear protein extracts from etiolated Sor-
Fig. 4. Effect of Sorghum plant irradiation with red and far-red light pulses on the binding activities to the 430-bp
S6C4 promoter fragment. EMSA was performed as described in Fig. 2 using the S6C4 promoter fragment 430-bp as a
probe. The probe was incubated with 20 mg of proteins extracted from leaves of lane 1 etiolated plants 11 days of
continuous darkness; lane 2 green plants 11 days of pho- toperiodic conditions; lane 3, plants treated with red light;
lane 4 plants treated with far-red light; lane 5 plants treated with redfar-red light. C
4
PEPC specific activity U mg
− 1
of proteins in crude protein extracts from Sorghum leaves is indicated below each lane. PC1, PC2 and FP are as
given in Fig. 2.
3
.
2
. Binding acti6ities depend on the light quality Etiolated plants 8-day-old were irradiated by
pulses of red or far-red light for 3 consecutive days. Control plants were either maintained in
continous darkness etiolated plants, or illumi- nated with white light normal photoperiodic con-
dition; green plants. C
4
PEPC activity was measured in protein extracts from leaves of treated
and control plants. Compared to etiolated plants, white light- and red-treated plants showed a 7.2
and 5.8-fold enrichment in leaf C
4
PEPC activity, respectively Fig. 4 lanes 1 – 3. In far-red treated
plants, this C
4
PEPC activity increased to a lower extent 3.2-fold, and far-red given after red
blocked the red light effect; the redfar-red pho- toreversibility of this response was in good agree-
ment with data of previous reports [6,7]. With respect to EMSA, the retarded complexes PC1
and 2 were observed with protein extracts from etiolated leaves, but not from green leaves Fig. 4
lanes 1 and 2, as expected. Red and far-red treated plants behaved essentially as the green and
etiolated ones, respectively Fig. 4 lanes 3 and 4. In contrast, far-red given after red pulses, partially
but clearly restored the retardation signals Fig. 4 lane 5. Overall, the results support the view that
both the gain in C
4
PEPC activity and the loss in DNA-binding activities to the S6C4 promoter are
mediated by phytochrome during greening of the Sorghum leaf.
3
.
3
. The major nuclear binding acti6ity interacts with an AT-rich domain within the
430
-bp fragment of the S6C
4
promoter To further identify the protein binding se-
quences on the promoter DNA, four subfrag- ments covering most of the 430-bp probe of the
S6C4 promoter were prepared Fig. 1 and used in EMSA. Retarded bands with nuclear protein ex-
tracts from etiolated leaves were seen with the 430-bp probe Fig. 5 lane 2; positive control and
the ATL, ATC and R29R30 subfragments Fig. 5 lanes 4 and 6. Furthermore, ATC or ATL showed
only one retarded complex which, based on signal intensity, should correspond to the faster migrat-
ing band in the control PC2. The very faint retarded R29R30 band could be due to PC2, or
due to a very weak binding of PC1 to this probe. Therefore, one protein-binding domain was en-
Fig. 5. Identification of binding domains in S6C4 promoter fragments. EMSA was performed with subfragments of the
430-bp 15 fmol and nuclear protein extracts 2 mg from etiolated Sorghum leaves. Lane 1, 430-bp free probe; lane 2,
430-bp probe + nuclear proteins; lanes 3 – 10, subfragments of the 430-bp probe ATL, ATC, R36, R29R30 + − nuclear
proteins. PC1, PC2 and FP, as under Fig. 2.
activity of PEPC showed a 2.3-fold decrease, the DNA binding activities were not restored Fig. 3
lane 3. As expected, there was no detectable signal after another round of light treatment Fig.
3 lane 4. Therefore, the DNA-binding proteins lost their binding capacity during greening of the
C
4
leaf. Whether they were irreversibly inactived andor destroyed remains to be seen.
tirely located in the shorter ATC element of the S6C4 promoter. In a competition experiment, a
25-fold molar excess of the unlabeled ATC probe caused the retarded band to decrease severely Fig.
6 lane 3 and become almost non-detectable when higher concentrations of the competitor were used
Fig. 6 lane 4 – 6. In contrast, poly dI-dC had no effect on the intensity of the signal data not
shown. Thus, this DNA-protein complex, as de- tected in the EMSA, resulted from a specific
interaction.
3
.
4
. The major nuclear binding acti6ity PC
2
is modulated by phosphorylation
So far, reversible phosphorylation is the best documented posttranslational protein modification
shown to modulate the binding activity of trans- acting factors and transcriptional activity of pho-
toregulated genes [16,20,27 – 32]. To test this hypothesis, nuclear protein extracts from etiolated
leaves were preincubated in the presence of ATP and Mg
2 +
, to stimulate the activity of putative, endogenous protein kinases, and NaF, to simulta-
neously reduce the activity of protein-phos- phatases, before EMSA was performed. Under
these experimental conditions, the DNA binding activity PC2 to the ATL probe was considerably
reduced Fig. 7 lanes 2 and 3. It showed a further decrease if the pretreatment was performed in the
presence of commercial, rat liver casein kinase II Fig. 7 lane 5; however, it was essentially main-
tained when the preincubation medium contained the general protein serinethreonine kinase in-
hibitor K252a Fig. 7 lane 4. Similar results were observed when Mg-GTP was used during preincu-
bation of the nuclear protein extracts not shown. These findings demonstrated that a protein kinase
activity, possibly a CKII-type casein kinase [33], phosphorylating the protein factor is present in
nuclear protein extracts from etiolated Sorghum leaves.
4. Discussion
