Indian Journal of Biochemistry & Biophysics Vol. 37, December 2000, pp. 477-485

--' ! C./'" ; .

s

" Characterization of a phototolerant mutant of nechocystis s (PCC 803

created by random mutagenesis of PSlt. gene

Munna Singh"'* and Kimi yukl Satoh" ..-

~"Deparlment of Biology, Okayama Uni versity, Okayama 700, Japan,.)

*Plant Ph ysiology Depilrtmen t, CBS H, G.B. Pant University of Agriculture and Technology, Pant nagar 263 145 (U .P. ), India

Received 8 September 2000; accepted 25 October 2000

Photosensiti vit y and photosynthetic characteristi cs have been analyzed in wild type (KC) and its psbA lJ mutant (16) of ynechocysti s having three point amino acid substitutions, i.e., N3221, I326F and F328S, which are locali zed in the C­

terminal ex tension of D I protein of the photo system II reaction center. Wild type and mutant cell s show almost an identi cal growth pall ern under normalllow li ght (30 ~mo l m-2s-I

, 30°C) liqu id culture (BG-II ) condition. However, upon shifting the cultures to hi gh li ght (500 ~mo l m-2s-I

, 30°C), these two types of cell s ex hibit entirely different growth characteri sti cs, i. e. , the mutant cell s continue to grow normall y whereas, the control cell s fail to adapt the li ght stress and eventuall y resulting in complete loss of the photosynthetic pi gments. On the other hand , a quick loss in the Fv/Fm value wi th half - decay time of abou t 30 min is observed in the mutant , in contrast to 120- 130 min in case of control , upon shifting to hi gh li ght cond iti ons. In spite of this, mutant cell s are able to adapt and grow well under prolonged hi gh light exposure even aft er losing a major part of the variable yield of chlorophyll flu orescence (Fv/Fm). The hi gh li ght treatment also induced decrease in the level of D I protein in the mutant. However, half-decay time for D I is much longer ('" I 0 hr) than that of vari able tluorescence. Thus. the mutalll cell s have shown an unique way for cell growth and maintenance under high light even after losing Fv/Fm and photosynthetic oxygen evo lving capaci ty as well as D I content to a great extent. Therefore, these results cou ld extend an interes ti ng insight to understand the coordination of physiolo ical, biochemical and molecul ar mechani sms regulating pho-totolerance of the photosynthetic organi sms. ( ~

./ The 0 I protein is encoded by psbA gene which exi sts as a single copy gene on the plastid genome in higher plants and al gae while, it belongs to a small multigene family in cyanobacteria with two to seven genes l

. In cyanobacterium Synechocysti s PCC6803 pshA has three variant forms, namely , pshA/, pshAII and psbAfII (ref. 1-3). However, psbA/ gets rarely ex­pressed while other two forms i.e., pshAII and pshAfII share 99% nucleotide identity and also encode an identical 0 I protein product4.5 . Interestingly, pshAII accounts for almost 90% of the produced pshA tran­script' . The photosystem II reaction center consists of two structurall y similar proteins 0 I and 0 2 along with cy tochrome b559 (ref.6). 0 I and 02 polypep­tides ex ist as hetero-dimers within the thylakoid membrane assoc iated with major components that mediate primary charge separation and water ox ida­tion. D I protein is highl y li ght sensiti ve in contrast to other photosyntheti c reacti on centre core proteins. It possesses a rapid turn-over potenti al under strong

*Author for correspondence. Fax: +91-5944-33473 Email: munna-s ingh@123 ind ia.com

irradiance and al so plays an important role in energy transformation. The inherent rapid turn-over tendency of 0 I protein sustains photosynthetic efficiency un­der photoinhibitory irradiance7

. Several in vesti gation ­have already been made in the last few years to reveal the molecular mechani sm of photoinhibition which enables photosynthetic organi sms to cope up with strong photon flux densities8

- ' \ PFDs). The cyanobacterium Synechocystis sp. PCC6803 is

unicellular, obligate phototrophic in nature and re­quires only inorganic nutrients and light for growth . The major photosynthetic pigments of SYllechocystis are chlorophyll a (ChI a) and phycob iliproteins (PBS). The PBS are blue-green pigmented constitu­ents of the thylakoid membrane, form about 40% of the total cellular proteins, are water so luble and work as light-harvest ing complex similar to hi gher plants' 6

.

The abundance of these pigments and other compo­nents of the photosyntheti c apparatus varies with en­vironmental variables such as li ght quality, quantity and nutrients 17 . Our laboratory has generated many mutants by in vitro random mutagenes is of the part of pshAII gene cod ing 178 amino ac ids of the carboxy-

478 INDI AN J. BIOC HEM. BI OPHYS. , VOL. 37, DECEM BER 2000

terminal porti on of the 0 I polypeptide in Synecho­cyslis PCC 6803, which form blue-green co loni es phenotypicall y, if placed under low or strong PFDsIS

.19. The phenotypic appearance gets changed in

wild type cell s under high irradiance due to photo­bleaching of their light-harvesting pigments, having unaltered 0 I polypeptide. Consequentl y, it appears that mutational consequences in 0 I prote in have ex­tended phototolerance to the mutant ce ll s with the ability to retain photosynthetic pigments and unaf­fected spectral properties l9 . The mutant showing phototolerance has tripl e amino ac id substituti ons viz., N322I, I326F and F328S, all locali zed in the E­loop of the 0 I polypeptide, near C-terminal extru­sion, oriented towards lumenal side of the thylakoid membrane. It was therefore chosen to elucidate the integrated phys iological, biochemica l and molecul ar means regul ating phototolerance under strong illumi­nati on.

Materials and Methods Experill1enlal organism and cullllre condiliuns

The cyanobacterium Synechocysti s PCC 6803 having mUltiple copies of psbA (psbA l, psbA fI and psbA fll) gene2 was chosen as an experimental organ­ism. Synechocysti s PCC 6803, Cm 4~-1 abbreviated as KC (cont ro l strain) was kindl y gifted by Dr Debus, UC, Rivers ide, USA and maintained in Pro f. Kimi­yuki Satoh's laboratory, having psbA l and psbA lIl gene fo rms inacti va ted by inserti on mutagenes is using anti bioti c cassettes2

. The va ri ous psbA fI mutants fro m control KC strain were generated by random PCR IS.

Mutant 16 was se lected to find out psbA fI mutational consequences on photosyntheti c charac teri stics fa­vouring phototolerancy. The mutant has shown three point amino acid substitutions i.e., N322I, I326F and F328S as confirmed by psbA fI gene sequencing through colony PCR (Table I ) . The wild type (KC) and its psbA II mutant were grown on either BG I I so lid (Bacto-agar 1.5%, Difco) or in BG II liquid cultures20.21, always contai ning antibiotics kanamyc in (S ~lg/ml ) , spectinomyc in (S ~g/ml ) and chl oram­phenicol (2.S fl g/ml ). The solid BG II medium was supplemented with I.S % (w/v) agar , 10 mM TES­KOH pH (8.0) and 0.3% (w/v) sodium thiosulphate. BG II liquid culture contained S mM HEPES-NaOH (pH 7.S). The host strain KC and mutant 16 cell s were grown in liquid BG 11 at 30°C with continuous bub­bling by using sterile atmospheric air containing 1% CO2 suppl y for effi cient mixing of the growing cell s.

Tab le I-Nucleotide and ami no acid substitutions in 0 1 poly­pepti de encoded by IJsbA II gene of cY:1Il0bacteri ul1l SYllcchocys­slis PCC 6803 [Mu tational consequences affect photosensitivit y,

Cyano-bacteriu l1l strai n

Wild Type (KC) Mutant(l6)

as ind icated ]

Nature o f substitut ions Photo­sensitivity

--A-nl-i n-o-a-c-id------N-l-Ic-I e-o~tid~e-s-

Unaltered 0 I polypep ti de

N322 1 (Asn-lIe) 1326F (lie-Phe) F328S (Phe-Ser)

AAC-ATC ATC-TIC TIC-TCT

Photo­sensitive Phototo lerant

The cell cultures were illuminated wi th low PFDs (white flu orescent bulb, 30 ~lIno l photons m·2

S· I) or high light (heat filtered halogen lamp, SOO ~mo l

photons m'2 S· I). For all studi es ce ll s were grown as a continuous culture to obtain mid-logarithmic growth phase (Am "'0.8) unless menti oned otherwise.

Absorption and chlorophyll mcasllrements The ce ll turbidity was monitored spectropho­

tometri call y (Mil ton-Roy, Spectronic 3000) by meas­uring optical density at A7.10 nm in a cuvette with I cm light path21. Chlorophyll content of the cell s and thylakoids was estimated as by using 80% acetonell .

In vivo photoinhibitory trealmenl Synechocysti s sp. PCC 6803 cell s were harvested

(S OOO g, 10 min at room temperature) at mid­logarithmic growth phase (ADo ",0.8) and resuspended in BG II mediurn23 to a fin al concent rati on of I 0 ~l g chUrnl. These cell s were illuminated under PFDs of SOO flmol photons m·2s,1 using halogen lamp as a li ght source. The temperature was controll ed so as not to exceed 30°C and aliquots fo r the va ri ous measure­ments were withdrawn at spec ific time peri od2

.1. After

photoinhibitory treatment both cont ro l and mutant cells were immedi ateley re-kept under growth PFDs (30 ~mo l m·2

S' I or darkness at 30°C) to all ow recov­ery process. The photoinhibi tion was monitored by measuring oxygen evolving capac ity and flu orescence induction kinetics (Fv/Fmax).

In vivo measurements of oxygen evo/ul ion The oxygen evolution was measured under satu­

rating light with an oxygen electrode (Hansatec h) at 2SoC using li ght projector (Hilux-HR 210, Japan) as a light source. All measurements were made by using 2 ml cell suspension23 corresponding I 0 ~g chUml

SINGH & SATOH: CHARACTERIZATION OF A PHOTOTOLERANT MUTANT OF SYNECHOCYSTIS SP. 479

under saturating white light (3000 I1mol photons m-\- I). The measurements were performed in fresh BG-II, either without artificial electron acceptor or in the presence of I mM dichloro-p-benzoquinone (DCBQ), I mM dimethyl-p-benzoquinone (DMBQ) with 3 mM potassium ferricyanide to keep the qui­nones in the oxidized form.

Preparation of cell extracts and thylakoid membranes Cyanobacterial cells were harvested (10,000 g, 10

min at 4°C), and the pellet was resuspended in thy la­koid buffer ( 1/1 OOth of the original culture volume) containing 20 mM MES-NaOH (PH 6.5), 5 mM MgCh , 5 mM CaCh and 25% glycerol (w/v). Cell suspension (:::1.0 ml) was transferred in ice-cooled screw-cap tube (2 ml) containing glass beads (1 .5 g). The cells were broken using Mini Bead Beater (Bio­spec Product, USA) by six breaking cycles (20 sec shaking at 5000 rpm), each followed by 3-5 min cooling on ice. The broken cells along with debris and glass beads were transferred into a screw-cap plastic tube (50 ml) and then diluted by adding thyla­koid buffer (5 ml), mixed gently and subsequently supernatant was collected in a centrifuge tube. This was repeated three times to extract thylakoids . After,

centrifugation (2000 g, 10 min at 4°C) to remove un­broken cells and cell debris, the suspension was fur­

ther centrifuged (15,000 g, 30 min at 4°C) to get puri­fied thylakoid pellet. The pellet was suspended in minimum thylakoid buffer containing CaCh (20 mM) , and used for Western blot analysis24.

SDS-PAGE and Immunodetrction of DI protein Thylakoid membranes were isolated as described

earlier24. The polypeptides of the thylakoid mem­

branes equivalent to 2 I1g of chi were fractionated on SDS-polyacrylamide gel (16%), containing urea25

.

The fractionated polypeptides were transferred onto

0.2 11m nitrocellulose membrane (Schleicher .and Schuncll) us ing semi-dry blotting apparatus (AHo, Japan) for 90 min at 200 rnA constant current. The blots were probed with antibody specific for the pri­mary 0 I prote in (kindly gifted by Prof. lkeuchi, To­kyo University , Japan) followed by secondary anti­rabbit antibody (Amersham), subsequently treated by chemiluminescence kit (ECL, Amersham ) to detect signals on X-ray film (Fujifilm, Japan). The 01 pro­tein was quantified by scanning the X-ray immu­noblots with laser densitometer (Molecular Dynam­ics, PO II 0) .

Chlorophyll fluorescence measurements The measurements of Fv/Fmax from Synechocystis

cells were made with PEA (Plant Efficiency Ana­lyzer, Hansatech, UK) . For recovery experiment cells were photoinhibited to about 50% of the Fv/Fmax (variable fluorescence over maximum fluorescence) value at the beginning of the experiment. This was accomplished in :::30 and::: 120 min with mutant and control cells respectively, after imposing photoin­

hibitory treatment (500 I1mol m-2 S-I, 30°C). The 2 ml cell suspension was used in BG II culture medium

containing 10 I1g chi for all measurement, after 15

min dark adaptation at 30°C, which allow relaxation of fast fluorescence-quenching components

l9

Results Growth characteristics

The growth rates and phenotypic appearance of mutant (16) having three point mutations i.e., N322I, I326F and F328S, localized in the E-Ioop of the 0 I polypeptide (Table I) and its control (KC) cells were almost identical (Fig. I A and Table 2) under normal growth light (30 I1mol photons m-2 S- I, 30°C). In spite of similar growth trends, wild type cells showed higher photosynthetic efficiency (Fv/Fmax), oxygen evolving capacity along with 0 I content (Table 2). However, both cell types showed typical blue-green colour under BG II liquid/solid culture medium, if grown under low light. While, blue-green colour of the wild type (KC) cells get photo-bleached quickly upon exposure to high irradiance (500 I1mol m-2 S- I,

30°C), mutant cells did not show any sign of photo­bleaching and grew well continuously, even if placed for a longer duration under strong light (Fig. I B). De­spite displaying a yellow-green colour, the wild type cells exhibited a slightly faster growth rate in the be­ginning of high light illumination. However, these cells were not able to sustain their growth rate be­cause of severe photooxidation of the PBS and chlo­rophyll as well. But, thcse photobleached cells did not lose their viability, as confirmed by placing them back under normal growth light (data not shown). In contrast to this, mutant cells started with slow rise-up followed by continuous increase in their growth trend (Fig. I B) with the ability to retain photosynthetic pigments . These observations are also supported by unaffected absorbance spectra of mutant cells while, dramatic de­cline in the absorbance at A620 nm has been observed in wild type cells due to loss in the major light-harvest ing pigment protein complex i.e ., PBS (data not shown).

480 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, DECEMBER 2000

Photoinhibition of PSII in vivo The oxygen evolving activity was monitored for

the wild type and mutant cells at mid-logarithmic

growth phase (A730 ",,0.8) . These cells (10 Ilg chllml) were subjected to photoinhibitory treatment for two hours. Subsequently, their oxygen evolution patterns were measured by using DCBQ or DMBQ as an arti­ficial electron acceptor. Nearly, 50% higher oxygen evolution (H20-DCBQ) values were found in control cells with almost identical whole chain electron transport activity a ' monitored in the absence of electron acceptor if grown under normal growth irra­diance. The photoinhibitory irradiance induced about 50% inhibition of PS II activity in wild type cells in

",, 140 min whereas, it happened in four times less du-

2.5 -,--------------, A

--0- KC

2 --<r- 16

1.5

1

0.5

E c::

0 0 ("f)

r-- 100 120 140 160 180 <t: 3 B

2

o 20 40 60 80 100 120 140

Time (hr) Fig. I-Growth characteristics of wild type (KC) amI its psbAIIID I mutant strain (16) of cyanobacterium S\'lleChOC.l'Slis PCC 6803. (A): Cells grown under low light (30 ~mol m·2s·'). (B): Cells grown under low light upto the early log phase (ADO '" 0.35) and then shifted under high light (500 ~mol m·2s-') [The cultures were supplied sterile atmospheric air mixed with I % CO2

for continuous stirring. Temperature was maintained at 30n C throughout. Cell growth was monitored spectrophotometrically by observing culture turbidity at optical density ADo, The experiment was repeated more than five times usi ng different cell cultures under low and hi gh PFDsJ.

ration (30-33 min) in the mutant cells (Table 2). A rapid declining tendency of PSII activity has also been adapted throughout the photoinhibitory illumi­nation by mutant cells while, wild type cells followed a gradual and slower declining pattern . Consequently, about 60% and 20% PSI! activity was maintained af­ter two hour of the photoinhibitory treatment in KC and mutant 16 cells respectively, as measured by us­ing an artificial electron acceptor (DCBQ) to monitor the status of photoinhibition at specific time intervals (data not shown). Generally, DCBQ has been ob­served as a better electron acceptor (Table 3) , but it also becomes limiting for stimulating electron trans­port with mutant cells particularly, if treated long time under strong light. This indicates towards QA perturbation21

, while DMBQ reflects inhibition for

Table 2-Photosynthetic characteristi cs of wi ld type (KC) and its psbAl/ mutant strain (16) of cyanobacterium S.l'nechocystis PCC 6803 [Values in parenthesis are the mean values of Iluorescence variablelfluorescence maximum ratio (Fv/Fmax) obtained from at least 5-7 experiments using different cultures].

Strainl mutant

Doubling Fv/Fmax time or

"PI ",50%

Auto- Fv/Fmax H20-trophi c DCBQ growth (hr) (min)

Wild type 16-18 0.550 (100) 125 ± 5 140 ± 10 (KC) Mutant (16) 16-18 0.400 (73) 30 ± 2 32 ± 3

'Cells grown upto the mid-log phase (ADO ",0.8 ) under growth light (30 ~mol m-2s-') were shifted under photoinhibitory irradi­ance (500 ~mol m-2s-l

) to create ca. 50% photoinhibition. The temperature was maintained at 30nC throughout.

Table 3-Steady state rates of oxygen evolution from wild type (KC) and D 1 mutant (16) cells of SYllechocystis PCC 6803 grown under normal growth conditions (30 ~mol m-2s-', 30°C and 1% CO2 mixed with air [The rates are shown in arbitrary units usi ng the 100 value for the rate from wild type cells without adding acceptors (210 ± 15 ~mole O2 mg-' chi 11('). Values in parenthe­sis are the per cent values of the oxygen evolution rates of mutant cells (16) relative to that of the wild type (KC) in presence or ab­sence of same acceptor (either DCBQ or DMBQ with FeCy). All measurements were made under saturating white light (3000 ~mo l

m-2s- ') at 25°C. The data is based on three to fi ve independent experiments using different cell cultures]

Nature of O2 evolution Wild type (KC)

No addition 100 I mM DCBQ + 3 mM FeCy 270 I mMDMBQ+3mMFeCy 130

Mutant (16)

100 (100) 134 (50) 90 (69)

SINGH & SATOH: CHARACTERIZATION OF A PHOTOTOLERANT MUTANT OF SYNECHOCYSTIS SP. 481

PSII e lectron transport activity significantly (Table 3).

We have further tried to reveal photosusceptibility of photosynthetic efficiency (FvlFmax) of PSII. The mutant cells showed phototolerance even after high light-induced faster inactivation of PSI! activity under photoinhibitory irradiance. In spite of this, mutant cells have always been able to express their photo­synthetic potential -;,;73 % of the control cells (Table 2) under normal growth condition . However, declining trend of Fv/Fmax has been shown by both cell types, when placed under strong photoinhibitory irradiance. Mutant cell s show a rapid loss of their ch lorophyll fluorescence variable yield (Fv/Fmax) . Consequently,

-;,;50% loss in photosynthetic efficiency of PSII was monitored within 30-33 min in mutant cells as com­pared to wild type cells which reached -;,;50% loss in about two hour under similar light exposure (Fig. 2).

Recovery from photoinhibition Our results indicate rapidly losing trends of PSII

electron transport activity and photosynthetic effi­ciency by mutant cell s as compared to control ce ll s during photoinhibitory irradiance (Fig. 2 and Table

0 --+-KC

'-....... ---16 c 0 0

~ 0 50 -x til E

LL 25 --->

LL

o -r---.----.----r---,----,--~ o 20 40 60 80 100 120

Time (min) Fig. 2-Photoinh ibition of photosyntheti c effi ciency (Fv/FmClx) of PS II in wild type (KC) and its pshA IIIDI mut ant (16) of cya­nobacterium SYllechocystis PCC 6803 [The photoinhibitory li ght was 500 ~rnol 1ll-

2{I, I % CO2 mixed with air was supplied to the cell cultures to maintain continuous stirring. Cell s were grown (ABO ",0.8) under growth li ght prior to shifting under photoin­hibitory PFDs. 2 ml cell suspension was used for all measure­ments containing I 0 ~g chi , aft cr dark adaptati on ( 15 min) pri or to measurcment s, which all ow rel axation of fast flu oresccncc quenchin g components. The temperatu re was maintained at 30°C throughout. Values represent the mean of ti ve measurements us­ing different cell cultures l.

2) . To clarify and correlate these differential photo­synthetic characteristics with restoration process, both cell types were illuminated (500 Ilmol m-2

S- I ,

30°C) in such a manner as to create -;,;50% photoin­hibitory loss, and immediately these photoinhibited cells were transferred to recovery under growth light (30 Ilmol m-2

S- I, 30°C) with the simultaneous addi­tion of translational inhibitor lincomycin to prevent protein synthesis-dependent repair process of PSII reaction centre (data not shown). In spite of differen­tial photo-susceptibility for losing PSII activity and photosynthetic efficiency (Fv/Fmax) both cell types indicated similar intrinsic abi li ty to overcome the ir photoinhibitory losses (Fig. 3), if allowed to recovery after PI 50%. In fact, the initial one hour of recovery phase has played a very crucial role for repair . In view of this , -;,;66% of the total restoration occurred within the first hour of recovery duration . Afterwards, it became rather slow and could recover about 60% of the total photoinhibited loss in five hours (Fig. 3) . Darkness did not show any pos itive role for improv­ing recovery process . However, presence of prote in synthesis inhibitor (lincomycin) during recovery in­duced further loss in the res idual chlorophyll flu ores­cence variables parallel with the incubation time (data not shown).

D I degradation and resynthesis during recovery The mutant cells were abl e to divide even under

prolonged high light exposure (Fig. I B). Surpri singly,

100 -,--------------------------,

~ 75 0

>. ... (j) 50 > 0 u (j)

25 a::

0

0

~KC -+-16

2 3 Recovery Time (hr)

4 5

Fi g. 3-Restoration of photoinhi bi ted photosyntheti c elTiciency (Fv/Fmax ) of PSII in wild type and it s D I mutant (I 6) [Cell s were illuminated under recovery Igrowth li ght (3 0 ~mo l m·\ ·I ) up to ti ve hours immediately after creating ",50% pholoi nhi bition in both cell types using strong light (500 ~mo l m-~s - I). The meas­urements were made with 2 ml cell suspension in BGII culture medium (equivalent to 10 ~g chI) at specitic time intervals as indicated, after dark adaptati on ( 15 min. ). Each point represents the mean va lue of thrce observati ons usi ng different cell cultures. Temperature during the experiment was maintained at 30°CJ .

482 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, DECEMBER 2000

thi s kind of growth behaviour is just sustained by re­sidual (::,,20%) electron transport (data not shown) and chlorophyll flu orescence vmiable (Fig. 2). Mutant cell s lost almost 80% of their photosynthetic potenti al within two hours as compared to control ce ll s, which retained more than 50% PSII activity under similar photoinhibitory conditions. Keeping this in account, D I photodegradation was al so analysed for providing further possibilities associated with D I turnover linked with photoinhibition (Fig. 4). The levels of D I photodegradati on do not overlap with loss in their photosynthetic efficiency and electron transport. This may be due to an inseparable assoc iation of acti ve and inac tive forms of DI. Immunoblot analys is corre­sponds to the sum of active and photodamaged D I protein at all spec ific points of photoinhibitory meas­urements. The wild type cell s always showed slower D I photodegradati on for los ing electron transport acti vity and chl orophyll fluorescence yield. However, the half-decay time fo r 0 I was much longer == I 0 hr

KC~

() 5 10 2() (h)

Dl ~ .. --~;

o D1

16 ) 10 20 (11 )

Fig. 4-lmmunob lots showing photodegradati on kinetics of {lsbA l/ encoded thylakoid membrane DI protein from wi ld type (KC) and mut an t (16) of SYllec/lOcyslis 6803 [Cell s were grown under low li ght (30 ~mo l m·2s· l

) to the mid- log growth phase (ABo "'0.8) prior to shifting undcr strong li ght (500 ~mo l m-2s-I). Tempcrature was maintained 30DC throughout. Cell s were har­vested at 0, I , 5, 10 and 20 hr after high light exposurc. Thylakoid membranes were iso latcd as described in 'Materi al and Methods' . Thylakoid membrane protei ns were reso lved by SDS­polyacrylamidc gel ( 16%) containi ng urea (6 M) and subsequcntly transferred onto nitrocell ulose membranc. Levels of D I protcin were detected fro m the cross reaction with specifIc D I ant ibody. The level of D I protein decreased as a fun cti on of high irradiancc cxposure time (1-20 hr). The samples were applied on chlorophyll bas is (2 pg/lanc). D I signals were quanti lied by using laser dcn­sitometer].

and more than 20 hr in mutant and control cell s (Fig. 4) as compared to the PI 50% of their chlorophyll fluorescence yield achieved in ==30 min and == 120 min respectively (Table 2).

Furthermore, D I photo-degradation and its light and dark dependent re-synthes is patterns were also investigated. A loss in photosynthetic efficiency ==50% was chosen as a criteri a, as stated earlier. The immunoblots of D I indicate differenti al D I photo­degradation as shown by mutant and control cell s. It also indicates that D I gets re-synthes ized sufficientl y after photoinhibition, if photoinhibited cells shifted under recovery or growth light (30 Ilmol m-2

S- I) for longer duration (5 hr) at optimal growth temperature (30°C). Hence, intensity of 0 I signal after recovery under growth li ght appears hi gher than the control, indicating nascent D I synthes is during restorati on process as well as its associati on with res idual D I (damaged and acti ve), and also favouring that D I translational acti vities are strictl y governed by light. Eventually, the sum of these three forms of D I makes D I quantitati vely higher in totality under growth li ght recovery process, as detected immmunologicall y (Fig. 5). However, darkness seems to be uncoupled fo r any nascent D I protein synthes is and rather promotes post-i lluminati on degradati on of photodamaged fo rm of the DI. This may be due to light-induced protease involvement promoting post-illuminat ion proteolytic degradati on of D I content (S ingh el a / ., data under publication).

Discussion

The amino ac id sequences of 0 I po lypeptide be­tween D and E loop have many properties essenti al for the functi on and turn-over of PSII under li ght stress26

. Therefore, these amino ac id sequences are expected to affect the photosynthetic efficiency and phototolerance. In view of thi s, Synechocysti s 6803 DI mutant (16) containing engineered pshA II gene l S

with three point amino acid substititi ons viz., N3221, 1326F and F328S in the E-Ioop of the D I polypeptide, near C-terminal (Table I) was analysed. Low and high li ght growth characteri stics of both cell types were observed to evaluate the role of amino ac id sub­stituti ons, which extended di ffe renti al photosynthetic properties and phototolerance under normal and photoinhibitory conditi on (Table 2). Almost an iden­tical growth pattern 21 along with electron transport acti vity was shown by both cell types under growth light (Fig. lA, Table 3). However, upon shi fting fro m

SINGH & SATOH: CHARACTERIZATION OF A PHOTOTOLERANT MUTANT OF SYNECHOCYSTIS SP. 483

KC C PI I,R DR

Dl

16 C PI LR DR

D1

Fig. 5-Immunodetection of D I protein after photoinhibition and after recovery [Immunoblot demonstrating total D I protein con­tent and its nature of turnover in wild type (KC) and mutant (16) strai ns of Synechoc),slis 6803. The cell s were grown to the mid­logari thmic phase (A 730=0.8) under growth light (30 )..tmol m-2s- l

)

pri or to photoinhibitory treatment. Lane PI . cell s were shifted from growth li ght to high light (500 )..tmol m-2s-l

) to create ca. 50% photoinhi biti on in both types of cells. Lanes LR and DR cells were allowed recover ei ther in growth light or in darkness fo r 5 hI' immediately after photoinhibition. Temperature was maintained 30°C throughout. Cells were harvested at each specific step and subsequent ly used for thyl akoid isolation as stated in ' Materi al and Methods'. Thylakoid membranes were fractionated on SDS-PAGE (16% with urea 6 M) on chlorophyll basis (2 )..tg/ lane). Reso lved po lypepti des were transferred onto nitrocellulose membrane and immunologically detected with D I-specific pri­mary antibody fo ll owed by anti-rabbit secondary antibody using ECL kit. An increase or decrease in the levels of D I signals are reflected by environmental vari ables as indicated].

low to high irradiance (30 to 500 Ilmol photons m-2

s- ), 30°C) at an early logarithmic growth phase (Am

::: 0. 35) wild type cells exhibited an early rising with faster cell multiplication up to 24 hr in the beginning followed by continuous gradual decline, representing typical bell shaped growth pattern (Fig. I B) due to severe photob leaching. Mutant cells started with slightl y slow ri se-up in comparison to wild type cells initia ll y, but later showed an increase in their growth tren·d (S-shape) and a lso retained blue-green appear­ance throughout. Thi s indicates that wild type cell s suffered photobleaching (both phycobilisomes and chi a ), whi ch impaired process ing of sufficient pho­tosynthase to keep cells photosynthetically dynami c as seen in the ir absorbance spectra (data not shown). The PBS degradation mi ght have contributed to a small ex tent for ce ll survival in the beginning of li ght stress in KC cell s, and it also appears to be criti cal

with mutant cells for conferring their survival and phototolerance under high PFDs. The reduced PBS levels become more critical when cells experience higher light intensities or exposed to the combination of stresses)7. Furthermore, phototolerant mutant cells were also analysed for PSII electron transport rates, chlorophyll fluorescence variables and photodegra­dation of D 1 to correlate physiological basis favour­ing high light adaptation. Surprisingly, an early loss in PSII O2 evolving capacity, Fv/Fmax and D I photo­degradation were found in mutant cells whereas , wild type cells indicated rather slower way for losing all

these intrinsic properties. Noticeably, :::50% 1.0ss in photosynthetic efficiency occurred within 30-33 min in mutant cells while, it takes almost 120-130 min in wild type cells (Table 2). To the best of our knowl­edge, it is the first pshAlI mutant reported so far showing typical nature of photo-tolerance27 with an early loss in their PS II electron transport (H20-DCBQ), photosynthetic efficiency, and D I photode­gradation in comparison to the wild type cells . Even­

tually, mutant cells lost :::80% PSII electron transport (data not shown) and Fv/Fmax values within two hours of continuous photoinhibitory illumination (Fig. 2) . In spite of this, mutant cell s sustained their growth potential by retaining normal phenotypic ap­pearance along with photosynthetic pigments, and spectral properties (data not shown). This may be in­terpreted as that wild type cells kept on losing all these intrinsic characteristics gradually till the end (Fig. I B) while, mutant cells chose biphasic 'strate­gies . A rapid loss followed by steady state around

:::20% residual photosynthetic efficiency could sup­port cell growth sufficiently under strong irradiance with lower photosynthetic abilit/ 7.28

. Oquist et al.,29 have also suggested that photoinhibited PSII reaction centres (with photodamaged D I protein) as they ac­cumulate, extend an increased photo-protection for the remaining functional PSII by controlled non­photochemical diss ipation of excess exc itati on energy to establish down-regul ation of PSJI under sustained strong PFDs, which regulate res istance against irre­versible photodamage'O in shade and low li ght grown plants.

So far no report exists to our knowledge stating such a kind of phototolerance behavi our in Synecho­cysti s. However, reports do ex ist on amino acid sub­stitutions in D I that favour e ithe r more or equally photosusceptible D I mutants')·" . Whereas, a condi­ti onal i.e., higher or equa l leve ls of phototolerance

484 INDIAN J. B10CHEM. B10PHYS ., VOL. 37, DECEMBER 2000

has also been observed in Synechocystis2.1. The loss

in photosynthetic efficiency (FvlFmax) under photo­inhibitory irradiance has been correlated with the in­hibition of PSII electron transport due to donor or acceptor side limitation or perturbation9

,21. In our ex­periments, because of three point amino acid substi­

tutions, mutant cells reflected ==50% photosynthetic oxygen evolving capacity under normal growth con­dition, when assayed using DCBQ as an electron ac­ceptor (Table 3) as compared to the wild type. How­ever, PI 50% duration of electron transport activity was slightly higher with the similar pattern to the chlorophyll fluorescence values (Table 2) as moni­tored with DCBQ, which accepts electrons directly from Q/4.36.

Apart from D I mutational consequences, we could not get specific clue of phototolerance as shown by mutant 16 cells based on their sustainibility towards cell multiplication, ability for retaining photosyn­thetic pigments, rapid loss in electron transport and chlorophyll fluorescence variables. Generally, D I mutants represent modified acceptor side of PSII, in­cluding herbicide-resistant mutants31.37, the pshH de­

letion mutaneS.39

, the ~PEST mutant40 and all have shown intrins ically either equal or higher susceptibil­ity towards photoinhibitory damage of PSII than the wild type. However, restoration of photoinhibited photosynthetic efficiency was observed more rapidly under normal growth light after photoinhibition (Fig. 3), while darkness did not favour restorat ion (data not shown), The rapid first phase of recovery occurred within the first hour of the restoration process, and

contributed to ==66% of the total recovery achieved in five hours. The light dependent recovery indicates strict translational regulation of D I, and accumulation or stabilization of full length D I protein4 1

.44

, although the exact mode of regul ation is not known .

Furthermore, loss in photosynthetic effici ency (Fv/Fmax), and PSII photoinactivation were accom­panied with s low D I photo-degradation process in both cell types . However, mutant cells showed faster D I photo-degradation with the half-decay time == I 0 hr

than the wild type cells hav ing ~ 20 hr, which could be due to poss ible conformational changes after amino acid substitutions (Table I, Fig. 4) . In contrast to this, s lower D I degradation in mutants was ob­served under strong irradiance23

, However, light de­pendent recovery process focused a better D I signal, as detected by using specific D I antibody , possibly due to an integrated immunodetec tion of D I which

includes nascent, damaged, and already ex isting ac­tive forms of D 111

.45 while , de fecti ve synthesis or sta­bilization of the DI protein has a lso been thought to be an underlying reason for the fa ilure to repair dam­aged PSII reaction centres during darkness (Fig. 5). The light induced inhibiti on of photosynthetic effi­ciency of PSII also generates protease, which renders D 1 degradation in vivo and in vitro during pos t­illumination under darkness46

-48 (Singh and Satoh, data under publication), Further studies to e)(p lore a better insight regulating phototolerance with sustain­able cell growth and maintenance endowed with unaf­

fected spectral properties along with res idual (==20%) psn efficiency under prolonged photoinhibitory irra­diance as opted by phototolerant mutant are war­ranted .

Acknowledgement JSPS, Japan and G .B. Pant University of Agri cu l­

ture and Technology, Pantnagar, Indi a are duly ac­knowledged for providing JSPS Young-Scienti st Fellowship and granting leave respective ly to MS . We thank Dr. Yuchiro Takahasi for helping us in fluorescence measurements, and Profs . Yasusi Ya­mamoto and Norio Murata for he lpfu l discussions.

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