Conditions of Synechocystis sp PCC
The glucose-tolerant strain of
Synechocystis sp.PCC 6803, designated GT-O1 as
WT , the control which was removed the psb
DII copy of psb D gene as DIC+/DII- and the strains which introduced the point
mutation were grown in BG-11 media which contains 5mM glucose,20uM
atrazine,10mM TES-NaOH (pH8.2) , 0.3% sodium thiosulfate and appropriate antibiotics. Then, the liquid cultures were performed
in a BG-11 media mixotrphically. In both media, antibiotics such as kanamycin
and spectinomycin at 25 ug/ml were used . Photoautotrophic growth measurements were done according to
To assess the oxygen evolution for
these mutants, Clark-type oxygen electrode (Hansatech, UK) was used at 30’C by
a recirculating water bath. Samples were measured at a chlorohyll a
concentration of 10ugml-1 in the presence of artificial electron
acceptors such as 0.2mM 2,6 dichloro-1-4 benzoquinone (DCBQ), 0.2mM 2,5
dimethyl-p-benzoquinone (DMBQ) and 1mM potassium ferricyanide (K3(Fe(CN)6).
For whole chain oxygen evolution measurement, 15mM
sodium bicarbonate was used.
damage and recovery measurement
To determine the light sensitivity
for these mutants, photo damage and recovery assays were carried out. Samples
at 10ugml-1 of chlorophyll concentration were exposed to high white
light intensity at 2000umol photons m-2s-2 using a Kodak
Ektalite 1000 slide projector for 45 minutes followed by low white light
intensity at 30 umol photons m-2s-2 for 135 min provided
by metal halide bulbs as in . Then Oxygen evolution was measured as
describe above at 15 minutes intervals.
Variable chlorophyll a fluorescence
3000 fluorometer (PS I instruments ), Brno, Czech Republic was used to measure
variable chlorophyll a flurescent induction and decay .For fluorescence induction,
samples at 5 ugml-1
of chlorophyll concentration which were dark adapted for 5minutes prior to procedure
were measured under blue measure flash (455nm) or red measuring flash( 625nm) .
To analyze the back electron ransfer, 3-(3-4 dichlorophenyl)1-1 dimethylurea
(DCMU) ,40uM was added . For fluorescence decay, a series of BMF spaced at 1s
and 200ms intervals for single flash, double flashes, triple flashes and five
flashes were used.
temperature (77K) fluorescence emission spectra
A modified MPF-3L fluorescence
spectrometer( Perkin Elmer, USA) equipped with a custom made liquid nitrogen
Dewar was used to measure the fluorescence emission at 77K . Samples at 2.5 ugml-1 of
chlorophyll concentration were frozen using liquid nitrogen and data was
collected at an excision peak of 440nm and 580nm respectively. To normalize of
the spectra , the basic of baseline subtraction and normalization to the maxima
of photosystem I emission at 725nm was performed.
of thylakoid membranes
Cells are harvested and resuspended
in 50mM HEPES-NaOH (pH7.5), 10mM MgCl2, 20mM CaCl2, 1mM 6 amino caproic acid,
1mM phenylmethyl suffonyl fluoride and 2mM benzamidine. Then, cells were disrupted
by using a mini-bead beater( Biospec, Bartlesville, OK, USA) for five breaking
cycles. Glass beads and unbroken cells were removed by centrifugation at 8000g
for 5minutes. Thylakoid membranes were collected by centrifugation at 25 000 rpm for 1 hour at 4’C followed by resuspended
in disruption buffer and extracted at 25000 rpm for 30mins at 4’C. Isolated
thylakoids were frozen in liquid nitrogen and stored at-80’C.
Native polyacrylamide gel electrophoresis and Western Blotting
Samples from thylakoid isolation at
0.5 mgml-1 of
chlorophyll a concentration were solubilized in solubilization buffer which
contains 3% b dodecyl maltoside and incubated on ice
for 5 minutes in 2 times and 15 minutes respectively followed by centrifugation
at 15 000g for 15 minutes to remove insoluble materials. Samples at 0.2 mgml-1 of chlorophyll
a concentration were loaded onto precast 3-12% Bis-Tris gradient gels
(Life-Technologies,USA) and run at 4’C . On completion , proteins were transferred to polyvinylidene
difluoride membrane and probe with protein
specific antibodies (D1,D2 and CP43) . Secondary antibodies (anti-rabbit IgG
peroxidase) was incubated and visualized by enhance chemiluminescence (ECL)
using a CCD detector system (Fuji imager PS 3000).