Light Regulation of Phycobilisome Structure and Gene Expression ...

Plant Cell Physiol. 40(12): 1194-1202 (1999)JSPP © 1999

Light Regulation of Phycobilisome Structure and Gene Expression inSpirulina platensis Cl (Arthrospira sp. PCC 9438)

Pinthipya Nomsawai '•2-3, Nicole Tandeau de Marsac2, Jean Claude Thomas3, Morakot Tanticharoen '•4

and Supapon Cheevadhanarak'1 School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand2 Unite de Physiologie Microbienne, Institut Pasteur, 75724 Paris Cedex 15, France3 Laboratoire de Photorigulation et Dynamique des Membranes Ve'gitales, CNRS URA 1810, Ecole Normale Superieure, Paris Cedex

05, France4 National Center for Genetic Engineering and Biotechnology^ Bangkok 10400, Thailand

Light intensity is the most significant environmentalfactor influencing the light harvesting complexes (phyco-bilisomes) in cyanobacteria. Spirulina platensis Cl, a fila-mentous cyanobacterium, was grown in batch cultureunder low (50 f& m"2 s"1) and high (500 fjE m~* s"1) pho-tosynthetic photon flux density. Phycobilisomes were iso-lated, purified and analyzed by spectrophotometry, gelelectrophoresis and electron microscopy. The absorptionspectra in conjunction with the electron micrographs rev-ealed that they are hemidiscoidal with three allophycocya-nin cylindrical cores and six phycocyanin rods radiatingfrom the core. The 33 kDa linker polypeptide normal-ly found in low light cells disappeared under high lightresulting in smaller phycobilisomes. The decrease in thenumber of phycobilisomes correlated well with the de-crease in phycocyanin and allophycocyanin content fromcells shifted from low to high light. The phycobiliproteintranscripts exhibited different pattern in response to differ-ent light intensities. Three species of phycocyanin mRNAof sizes 1.4, 1.5 and 3.5 kb and two species of allophycoc-yanin of 1.4 and 1.7 kb were found in low light grown cells.However in high light, the 3.5 kb phycocyanin transcriptdisappeared while the others remained with subtle changes.This observation indicates that the largest transcript en-coding for phycocyanin subunits and linker polypeptidesand the phycobilisome structure are modulated by lightintensity.

Key words: Arthrospira — Light harvesting complexes —Light regulation — Phycobilisome — S.platensis Cl.

Spirulina, or Arthrospira, is a multicellular filamen-tous cyanobacterium and has been used as human food forcenturies (Venkataraman 1983). It is not only rich in pro-

Abbreviations: AP, allophycocyanin; Chi a, chlorophyll a;Gly, glycogen; ftE m~2 s~', micro Eistein x square meter per sec-ond; Lc. core linker protein; LR, rod linker protein; LRC, rod-corelinker protein; LiDS, lithium dodecyl sulfate; PBP, phycobili-protein; PBS, phycobilisome; PC, phycocyanin; SDS, sodiumdodecyl sulphate; TP, total proteins; SP, soluble proteins.

teins (50-70% of dry weight) but also in vitamins (partic-ularly B12), lipids (y-linolenic acid) and pigments (phyco-biliproteins and carotenoids) (Richmond 1987, Vonshak1990). Currently, Spirulina is highly valuable commerciallyas dietary supplement of human food and animal feed. Inaddition to its high commercial value, it is scientificallyattractive as a model organism for an outdoor mass culti-vation. In the United States, Spirulina is under investiga-tion for revitalization of air, waste processing and foodproduction for Space Program Controlled Ecological LifeSpace Support System (CELSS) (Tadros et al. 1993). Inaddition, hundreds of publications reveal that its uniquephytonutrients (including glycolipids, polysaccharides, pig-ments and other growth factors) and extracts are capableof raising the immune system and improving health. Forexample, they are thought to have anti-cancer property,potent anti-viral activity, and can reduce cholesterol levelin serum (Richmond 1987, Henrikson 1997).

Up to 50% of Spirulina's total proteins are phyco-biliproteins (PBPs) i.e. allophycocyanin (AP) and phyco-cyanin (PC). These fluorescent accessory photosyntheticpigments have the most commercial potential because theyprovide useful reagents for multicolor FACS (fluorescenceactivated cell sorter) analysis (Jung and Dailey 1989).When PBPs are associated to specific linker polypeptides,they constitute the light harvesting structure, so called phy-cobilisomes (PBSs) which are localized on the thylakoidmembrane at the photosystem II (Gantt 1981). In cyano-bacteria, PBS structure and composition are dependenton various environmental parameters (for reviews seeGrossman et al. 1994, Bryant 1987). Currently, Spirulina isthe only cyanobacterium extensively grown outdoor on alarge industrial scale. As such light intensity is of particularimportance to the outdoor cultivation since it is largelyvariable in natural environment and S. platensis must ac-climate their photosynthetic apparatus to that change. Inthe present study it was intended to understand the conse-quences of this acclimation on the quantity and qualityof the potential production of light harvesting pigmentsand also to improve the fundamental knowledge of thephotoacclimation process in S. platensis. We examinedthe precise physiological, morphological and biochemical

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Light regulation of Spirulina platensis phycobilisome 1195

characteristics of its PBS and the constituent of PBPs inS. platensis. Due to the lack of knowledge on the regulationof the light harvesting complexes at the molecular level ofthis organism, we also examined the effect of light intensityon the expression of S. platensis genes encoding for itsPBPs.

Materials and Methods

Culture and growth conditions—Axenic cultures of Spirulinaplatensis Cl (Arthrospira sp. PCC 9438) were obtained from thePasteur Culture Collection. A batch culture was grown in modi-fied Zarrouk's medium (no addition of TiSO4) (Zarrouk 1966) at30° C and agitated by a magnetic stirrer with continuous air bub-bling. The cultures were illuminated with "Durolite" lamps at 50(low light) and 500 (high light) fxE m~2 s"1 throughout the study.To study the effect of light shift conditions on the acclimation ofthe cells to light intensity, cells in log phase from continuous lowlight or high light intensity were subjected to opposite conditions.Cell growth was determined with a Klett Summerson colorimeterat KUeeonm-

PBP determination—PBPs were isolated following the meth-od of Tandeau de Marsac and Houmard (1988). Cells were har-vested by high speed centrifugation at 8,000 xg, for 15 min at20°C, and resuspended in 20 mM sodium acetate buffer, pH 5.5,and broken by passing through a French pressure cell at 135 MPa.PBPs were precipitated from crude cell-free extracts with \%(w/v) streptomycin sulfate for 30 min at 4°C. After precipitation,the PBPs were collected by centrifugation at 18,000xg, for 30min at 4°C. The amounts of PC and AP were calculated from themeasurement of their absorption spectra at 620 and 650 nm, re-spectively.

Chlorophyll a (Chi a) determination—Chi a was extractedfrom crude cell-free extracts with 90% (v/v) methanol for 1 h at4°C in dim light, followed by centrifugation at 10,000xg, for 10min at 4°C. The supernatant was used for the determination ofthe Chi a content while the pellet was further extracted until Chia was not detected. The Chi a content was calculated from theabsorbance of the methanolic extract at 665 nm as described byTandeau de Marsac and Houmard (1988).

Protein determination—The protein content of crude cell-free extracts was determined by the Lowry method (Lowry et al.1951).

Glycogen (Gly) determination—Glycogen was analyzed asdescribed by Liotenberg et al. (1996). Culture suspensions fromeach condition were harvested by cetrifugation at 8,000 x g, for 15min at 20°C and stored at -20°C until analysis.

Isolation of phycobilisomes—PBSs were isolated and sepa-rated by sodium dodecyl sulfate polyacrylamide gel electrophore-sis (SDS-PAGE) as described by Gamier et al. (1994) with theexception that sodium dodecyl sulfate was substituted for lithiumdodecyl sulfate (LiDS).

Electron microscopy—Purified PBSs were prepared forTransmission Electron Microscopy and uranyl acetate was used asa negative stain according to Yamanaga and Glazer (1980).

Northern hybridization—Total RNA isolation was adaptedfrom the method described by Glatron and Rapoport (1972). The400 bp of cpcA specific for the alpha subunit of phycocyanin(GenBank Accession No. Y09074) and 900 bp of cpcAB specificfor the alpha and beta subunits of AP from S. platensis (GenBankAccession No. X95898) were used as probes. Verification of in-tegrity and equivalence of RNA loading was made after transfer

by comparing the intensities of the ethidium bromide stained 23Sand 16SrRNA bands in each lane.

Primer extension mapping—The 5' end of the transcripts ofthe ape and epe operons were mapped by primer extension usingoligonucleotide primers generated from the sequences of ape andepe operon as follows. The oligonucleotide used for the ape ope-ron primer extension was 22 bases long and located 42 basesdownstream of the first translation codon (5'-CAC CAG GGCTCA GAT AAC GCG C-3'). The oligonucleotide used for the epeoperon primer extension was 22 bases long and located 40 basesdownstream of the first translation codon (5'-GCT GTA CTCAGC ATT TCG CCG C-3'). This process was composed of 4consecutive steps: (1) end-labeling of the oligonucleotide, (2) co-precipitation, (3) annealing and (4) primer-extension as describedby Golden et al. (1986).

Results

In order to study the effect of light intensity on thePBS structure and gene expression of S.platensis Cl, fourculture conditions were used, i.e. continuous low light (50yuEm~2s-1), continuous high light (500^E m~2s~') andlight shift conditions from continuous high to low lightintensity and vice versa. Cells from these conditions wereharvested at various time courses and their alterations incell morphology, cell composition and structure and PBScomposition, were investigated. To study the regulation ofPBS by light intensity at the level of gene expression, therelative levels of messenger RNA of both PC and AP fromcells grown under these conditions were analyzed in corre-lation with the amount of PC and AP.

Morphology of S. platensis Cl under high and lowlight intensities—S. platensis Cl grown under high and lowlight intensities had distinctly different characteristics. Thefirst appearance that could be noticed was the color of theculture. Cells under low light intensity were blue-greenwhile those under high light were yellow green (result notshown). This color change was also apparent when S. pla-tensis Cl grown under low light condition was subjected tohigh light and vice versa, i.e. the blue green cells initiallygrown under low light turned to yellow-green upon reach-ing the high light levels and the reverse color change wasobserved when cells were transferred from high light to lowlight conditions. In addition, large increase in the amountof gas vacuoles was clearly seen. They appeared as redspots along the filaments of the cells grown under high light(Fig. 1A), while gas vacuoles were rarely observed in lowlight grown cells (Fig. IB).

Effect of light intensities on growth and cell com-position—Another distinctive feature between high andlow light grown cells was their growth patterns. Cells incontinuous high light as well as those shifted from contin-uous low to high light grew faster than those in low lightcondition (result not shown). Physiological responses ofS. platensis C1 to continuous low and high light intensitieswere summarized in Table 1. The most obvious character-


Light regulation of Spirulina platensis phycobilisome

B

cells was always 3-3.4 times less than that found in con-tinuous low light grown cells.

In order to observe the global changes during lightintensity acclimation, we studied some cellular parametersassociated with growth in S.platensis Cl under variouslight conditions. These parameters are PC (Fig. 2A), AP(Fig. 2B) and Chi a (Fig. 2C). The results suggested that thecultures in continuous low and high light intensities ex-hibited their own specific steady levels of the biosynthesisof PC, AP, Chi a. Upon transfer from either low to high orhigh to low light intensity, the cells acclimated to the newenvironmental conditions by rapidly adjusting their pig-ment composition to the native steady level according tothe respective light intensity. These acclimation processeswere seen more rapidly when cells were shifted from highto low light than from low to high light intensities. Thesetypical characteristics of acclimation processes could applyfor other cell components i.e. soluble protein, total proteinexcept for glycogen which had a reversed effect (result notshown).

In addition, we found that the PC, AP, and Chi acontent per cell (as expressed with Klett units) decreased

Fig. 1 S.platensis Cl under phase contrast microscope. (A)Culture under high light intensity (500^E m" 2 s" ' ) . Red spotsalong the filament correspond to the gas vacuoles. (B) Cultureunder low light intensity (50/iE m" 2 s" ' ) . Using OLYMPUS In-verted System Microscope 1X70 with PhL ring slit. Bars represent12/im.

istic of high light grown cells compared to that of low lightones was the lower amount of cell components, i.e. Chi a,PBPs, soluble protein, total protein except the glycogencontent. The PBP content of continuous high light grown

Table 1 Biochemical composition, pigment content andgrowth of S.platensis Cl under continuous high light (500

2 ' l) intensities~2 s~'m 2 s ' ) and low light (50/iE m2 s~2

ChicPCAPPBPSPTPGly

i (jjg/KU)

(ug/KU)Oig/KU)Oig/KU)fag/KU)Oug/KU)Oig/KU)

PC/APDoubling time

High Light(5OOfiEm-2s~l)

0.025 ±0.0010.082±0.0020.049 ±0.0010.138 ±0.0080.280 ±0.0280.642±0.0250.359±0.0151.7 ±0.011

21-24 h

Low Light(50 /xE m s~')

0.055 ±0.0010.280±0.0190.148±0.0100.427 ±0.0290.586±0.059l.OO3±O.O710.212±0.0051.9 ±0.009

31-34h

All values are given as means ± 1SD of 3 independent experiments.Chi a, chlorophyll a; PC, phycocyanin; AP, allophycocyanin;PBP, phycobiliproteins (PC+APC); SP, soluble protein; TP,total protein; Gly, glycogen.

3 0.25U.

"S> 0.2

0.15

0.1

" 0.05

0

0.08

0 20 40 60 SO 100 120

Time (Hours)

Fig. 2 The amount of phycocyanin (A), allophycocyanin (B)and chlorophyll a (C) from cells acclimated to various light in-tensity conditions. O, continuous high light; A, continuous lowlight; D, shift from high to low light and • , shift from low to highlight intensities. KU: Klett Units.


Light regulation of Spirulina platensis phycobilisome

immediately and rapidly during the first 24 h after shifting within 48 h (Fig. 2A-C). One hundred and twenty hoursthe culture from low to high light intensity. Then these after shifting, the PC, AP and Chi a contents decreasedcomponents slowly declined to reach a new steady level 2.4, 2.5 and 2.3 times of the initial state, respectively. In

A

-••^>'--V/.

al a2 a3 a4

B

~^:l?'-':•-:#$2*^-

bl b2 b3Fig. 3 Hemidiscoidal phycobilisomes isolated from S.platensis Cl cells grown under continuous low light (A) or continuous high light(B) (162,000 x) . al-a4, bl-b4: enlargements of those from low light and high light, respectively. Bars represent 50 nm.


1198 Light regulation of Spirulina platensis phycobilisome

contrast, when shifting cells from high to low light condi-tions (Fig. 2A-C), the AP and Chi a content per cell in-creased immediately and rapidly during 24-48 h after theshift. They potentially reached to a new higher steady level.after 120 h. In an attempt to determine the mechanism thatregulated the alteration in the pigment content and cellcomposition, we subsequently measured their rate of syn-thesis. It was found that these pigment changes resultedfrom the modulation in the rates of their biosynthesis.When cells were shifted from low to high light intensity,the rates of PC, AP and Chi a biosynthesis were reduced2.3, 2.0 and 1.7 times compared to those grown in contin-uous low light, respectively. Alternatively, these rates were2.6, 1.9 and 1.9 times higher compared to those grown inhigh light condition (results not shown).

Effect of light intensity on the ultrastructure andcompositions of S. platensis Cl PBSs—The ultrastructureof the PBSs of S. platensis was first studied by vanEykelenburg (1979). He suggested, based on ultra-thinsections, that PBS were in pairs on the outer surface of thethylakoid membranes and had a diameter of 22 nm perPBS unit. However, the complete structure of the PBS ofS.platensis has not been demonstrated. Here, we reportelectron micrographs and a detailed analysis of the com-position of the PBSs of S. platensis from cells grown underlow and high light intensities, as well as from cells adaptedto both light intensities.

In this study, S. platensis Cl PBSs isolated from ex-ponentially growing cells freshly harvested were analyzedby light absorbance, electron microscopy, and gel electro-phoresis.. From the absorption spectra, it appeared thatPBS isolated from both low and high light intensitiesexhibited only two absorption peaks at 620 and 650 nm,corresponding to PC and AP, respectively. No absorptionfor phycoerythrin (PE) was found. SDS-PAGE also sug-gested the presence of only two PBPs (results not shown).Altogether our absorption spectra and SDS-PAGE supportthe result reported by Babu et al. (1991) that S. platensisdoes not belong to complementary chromatic adapters.Electron micrographs of PBS isolated from both low andhigh light intensities suggested that it was hemidiscoidal inshape as commonly found in many cyanobacteria (Fig. 3A,B). A closer analysis of its structure allows its division intotwo subdomains. The first one is a tricylindrical core ele-ment, which is always located at the base of the PBS. Thesecond one consists of six rods radiating from the corewhich form a crown. The result from absorption spectrasuggested that the core contained AP and the rods PC.Each rod is composed of mostly two, sometimes three,stacked elements, typical of hexamer PBP complexes. Inaddition, we found that PBSs under high light intensityhad a lower sedimentation coefficient on a sucrose gradientthan that of the low light (Fig. 4). Electron microscopyrevealed that the PBS structure from cells grown under

Fig. 4 Discontinuous sucrose gradient of the phycobilisomesisolated from S. platensis Cl from continuous low light (LL, 50^Em~2s~') and continuous high light intensities (HL, 500/iEm^s"1) .

high compared to those grown under low light intensitydiffered by the number of rods per PBS, the number ofdiscs per rod and the number of discs per PBS (Table 2).PBSs of cells adapted to high light intensity showed anaverage of 4.5 rods per PBS, 1.7 discs per rod and 7.7 discsper PBS (Table 2). In contrast, in cells adapted to low light,PBSs had an average of 5.8 rods/PBS, 2.2 discs/rod and12.9 discs/PBS. The results indicated that PBSs from highlight grown cells were smaller than those from low lightgrown cells.

The purified PBSs of S. platensis Cl from low andhigh irradiance were also analyzed by SDS-PAGE alongwith those of S. maxima. As shown in Fig. 5A, PBSs fromlow light grown cells contain five colorless polypeptides(34.5, 33, 31, 15 and 13 kDa) and colored tetrapyrole-bearing polypeptides, the core membrane linker (LCM. 97kDa) and the a and P subunits of the PBPs, PC and AP.The colorless polypeptides were apparent only after Co-

LLHL

Fig. 5 SDS-PAGE of phycobilisomes isolated from S. platensisCl grown under continuous low light (LL, 50/iE m~2s~') andcontinuous high light intensities (HL, 500 |/E m~2 s~'). (A) Co-omassie blue staining, (B) Zinc acetate staining.



Table 2 Ultrastructure analysis of phycobilisomes with various numbers of rods and discs apparent in S. platensis Clgrown under continuous high light (500//E m~2 s"1) and low light (50/*E m~2 s"1)

A.

Rl,

B.

Characteristics of

Condition

Low light

High light

R2, R3, R4, R5 and

Characteristics of

Condition

Low light

High light

phycobilisomes

R0 Rl

0 0

6 4

with various numbers of

R2

0

4

R3 R4

0 4

3 18

R6: number of phycobilisomes with 1, 2, 3, 4,

rods with various numbers of discs

Dl D2

52 340

149 290

D3

195

14

Total discscount

1,317

771

rods

R5 R6

17 81

30 .35

5, and 6 rods.

Means ofdiscs/PBS

12.91

7.71

Total rodcount

587

453

Means ofdiscs/rod

2.24

1.70

Means ofrods/PBS

5.76

4.53

Total PBScount

102

100

Dl, D2, D3: number of rods with 1,2, and 3 discs, respectively.

omassie blue stain was added while the major subunit ofPBPs were directly detected on the SDS-PAGE. The core-membrane linker protein could be detected by staining withzinc acetate (Fig. 5B). Based on the polypeptide distribu-tion of the PBS from S. maxima (Gamier et al. 1994) andother cyanobacteria, Synechococcus sp. PCC 6301 (Glazeret al. 1983) and PCC 7002 (de Lorimier et al. 1990), we canidentify those colorless polypeptides of S. platensis PBS asLR

345, LR33, LRC

31, LR1 5 and Lc

13; i.e. 34.5 kDa rod-rodlinker protein, 33 kDa rod-rod linker protein, 31 kDa rod-core linker protein, 15 kDa rod linker protein, and 13 kDacore linker protein, respectively. In comparison to the PBSpolypeptide profile of S. maxima, most of S. platensispolypeptides migrates to nearly the same position as thosefrom S. maxima except for the disappearance of the 30 and47 kDa polypeptides in S.platensis Cl (results not shown).

Effect of light intensity on the PBP transcripts—Twooperons, cpcBAHID encoding the /? subunit, a subunit,33 kDa, 34.5 kDa and 15 kDa rod linker polypeptides ofphycocyanin (GenBank Accession No. Y09074, AF164139)and apcABC encoding the a subunit, /? subunit and 13 kDacore linker polypeptides of allophycocyanin (GenBank Ac-cession No. X95898), govern the PBP synthesis in S. pla-tensis Cl were isolated. To study the effect of light intensityon these operons, total RNA from culture grown under'variety of conditions were isolated and analyzed by North-ern hybridization. The PC specific probe corresponding to400 bp of cpcA hybridized to three transcripts of 3.5, 1.5and 1.4 kb (Fig. 6). The two smaller species were the mostabundant and were always expressed in both continuouslow and high light intensities, while a much less abundanttranscript species of approximately 3.5 kb was apparentonly from the low light culture. After shifting the culturefrom low light to high light intensity, the amount of the

1.5 and 1.4 kb transcripts did not show any significantchange when compared to the control (continuous lowlight) (Fig. 6A). However, the biggest transcript speciesdisappeared within 24 h after light shifting (results not

B

•-'•?.'•

I

. 3.5 kb

1.5 kb1.4 kb

LL HL HL LL

C Di

LL HL HL LL

1.7 kb1.4 kb

Fig. 6 Northern hybridization of total RNA isolated fromS.platensis Cl grown under different conditions. (A) Cells grownin continuous low light (LL, 50 fiE m~2 s~') then shifted to highlight intensities (HL, 500//E m~2 s~'); (B) grown in high light in-tensities (HL, 500^Em"2s" ') then shifted to continuous lowlight (LL, 50//Era"2s" ') , hybridized with cpcA specific probe.(C) Cells grown in continuous low light (LL, 50/uE mshifted to high light intensities (HL, 500 uE min high light intensities (HL, 500/JE m'2 s"1) then shifted to

2 1

~2s"')then~ 2 s" ' ) ; (D) grown

continuous low light (LL,specific probe.

m~2 s"1), hybridized with apcAB



shown). In contrast, all PC transcripts from cells shiftedfrom high to low light intensity increased significantly(Fig. 6B). Analysis of AP mRNA using the 900 bp ofapcBA gene fragment as a probe revealed 1.4 and 1.7 kbtranscripts. Both transcripts were expressed in all cultureconditions regardless of light levels, although the smallertranscript species was generally more prominent (Fig. 6C,D).

The transcriptional organization of the PBS rod(cpc) and core (ape) encoding genes in S. platensis Cl arepresented in Fig. 7A and 7B. From primer extension anal-ysis of cpc operon, we found that there were three possibletranscription start points at 176, 186 and 282 nucleotidesupstream of the first translation codon. The 1.4 and 1.5 kbtranscripts, contained only cpcBA and possibly differed inthe location of their start sites or in one of two possible3-end points. Based on the size, location and characteris-tics of the cpc operon of S.platensis Cl and other cyano-bacteria from other studies (de Lorimier et al. 1990, 1992,Tandeau de Marsac et al. 1988, Kalla et al. 1993), the 3.5kb transcript was expected to be cpcBAHID and the in-tergenic region in addition to its transcription start sitebelonged to one of three possible 5'-end points. This tran-script was affected strongly by high light intensity as men-tioned above. From the primer extension of AP, it wasfound that a single transcription start site was 271 nucleo-tides upstream of the first translation codon. This resultsuggests that the difference in length within two AP tran-

. 500 bp

cpc B cpc A cpc H cpc I cpc D

B500 bp

1.4

1.7

apcA apcB apcC

Fig. 7 Physical map and transcriptional organization of the (A)cpcBAHID operon encoding the /? subunit, a subunit, 33 kDa,34.5 kDa and 15 kDa rod linker polypeptides of phycocyanin and(B) ape ABC operon encoding the a subunit, /? subunit and 13 kDacore linker polypeptide of allophycocyanin. Horizontal arrowsrepresent transcripts of the genes in each operon. Vertical arrowsrepresent the possible 5' or 3' ends of the transcripts. The width ofthe arrows represents relative amount of the transcripts.

scripts (1.4 and 1.7 kb) was at the possible 3'-end points.This 0.3 kb difference was nearly equal to the proposed sizeof the apcC fragment extending downstream from 3'-endof the apcBA genes. The 1.7 transcript species was thuslikely to be the cotranscription of apcAB and apcC.

Discussion

In order to understand the overall processes of theresponses to light intensity, we initially focused on themorphological, physiological, biochemical characteristicsof S.platensis Cl PBS and their constituent PBPs, as wellas on the phenotypic morphology of the cell response tolight intensity. Consequently, we examined the effect oflight intensity on the expression of PBP genes encoding PCand AP.

The influence of light intensity on PBP content hasbeen reported in different organisms (Yamanaka andGlazer 1980, Lonneborg et al. 1985, Muller et al. 1993,Gamier et al. 1994). One obvious characteristic of theresponse to light intensity of S. platensis Cl was the colorchange. The culture was yellow-green in high light and bluegreen in low light. This difference in color resulted from themodulation of PBP content (see earlier results) in con-junction with the alteration in carotenoids as reported inS. platensis UTEX 1928 (Olaizola and Duerr 1990) andAnacystis nidulans (Muller et al. 1993). Another significantchange to light intensity was the increase in the amount ofgas vacuoles when S.platensis Cl was subjected to highlight condition. Our results correspond with the findings ofvan Eykelenburg (1979) who also reported an increase inthe number of gas vacuoles from cells grown under highlight intensity. The increase in gas vacuoles and their lo-cation on the periphery of cell membrane could indicate anadaptation for light shielding rather than for buoyancy asin Nostoc muscorum (Waaland et al. 1970, Walsby 1972,1994), Trichodesmium erythraeum (van Baalen et al. 1969)and some Anabaena strains (Waaland et al. 1970). Thisactivity might help in reducing the amount of lightpenetrating into the cells, thus preventing light harvestingcomplexes from photobleaching.

The results of the time-course measurements of cellcomposition accumulations in S.platensis Cl culturesgrown under different light conditions are the firm evidenceof the acclimation ability of this cyanobacterium. Rapidchanges in light intensity from high to low light caused adramatic increase in the accumulation of PC, AP, and Chia. Whereas in response to high light intensity, S. platensisCl underwent a significant change in pigmentation mainlydue to the reduction of PC and Chi a synthesis. However,in contrast to other cyanobacteria, S.platensis Cl exhibit-ed a substantial decrease in AP content following growthin high light condition. In comparison with S. maxima, itcould be suggested that the PC of S.platensis Cl was not



only affected by light intensity but also by temperature. Inthe study of the PC/AP ratio, we found that there was asignificant difference in this ratio between S. platensis Cland S. maxima. This difference was possibly due to thedifference in growth temperature. S.platensis Cl grownin high and low light at 30° C had slightly different PC/AP ratios (Table 1) i.e. 1.7 and 1.9, respectively, whileS. maxima grown at 20°C, had the ratios of 1.2 and 2.4,respectively (Garnier et al. 1994). It was also found that thePC/AP ratio of S. platensis Cl grown under low light in-tensity at 30°C closely resembled that of S. maxima grownat 40° C (1.8) (Garnier 1995). This suggests that even underthe same light intensity the different responses of PBSmight occur in conjunction with other environmental fac-tors such as temperature. This is in agreement with Miilleret al. (1993) who showed that apart from light intensity,growth conditions and cyanobacterial species also influencePBP content.

Furthermore, shifting from low to high irradiance isknown to cause an immediate increase in photosyntheticactivity and growth rate, inducing an initial accumulationof glycogen as found in S. platensis (de Philippis et al.1992), Anabaena variabilis (Ernst and Boger 1985) andOscillatoria agardhii (Post 1987). The response of S. pla-tensis Cl to a rapid shift from low to high light intensitywas consistent with this model.

The changes of S. platensis PBS ultrastructure inresponse to light intensity was first documented by vanEykelenburg (1979). He investigated the cell inclusions andnumbers of PBS by EM but his interpretation could notsufficiently explain the acclimation process of S. platensisto different light intensities. Generally, PBS structures re-sponded to the change of light intensity in several wayssuch as changing the size and/or the number of PBSs(Bryant 1987, Muller et al. 1993, Garnier and Thomas1993, Post 1987, Glazer 1981, Lonneborg et al. 1985, Rapset al. 1985, Williams et al. 1980), including the alteration inthe type of chromophore (Yu et al. 1981). In our study,electron micrographs demonstrated that PBS were hemi-discoidal in shape and that in high light grown cells theywere smaller than those in low light grown cells. Thisphenomenon occurred due to a reduction in the rod length,which was caused by a decrease in PC content in conjunc-tion with the disappearance of the LR

33. Fujita et al. (1989)suggested that this smaller size under high light allows thelight harvesting system to escape from an excess photonflux and the larger size under low light intensity provides anefficient trap for photons. Using SDS-PAGE, we foundthat high light intensity induced a new 29 kDa polypeptides(P29) of unknown function which co-purified with the PBS.This protein has the same molecular weight as that foundin the PBS preparation of S. maxima under high light. Ithas been reported that P29 was not the degradation productof LR

33 and it had no immunological relationship with

LR33 (Garnier et al. 1994). In addition to the alteration of

the PBS size, S.platensis Cl also varied the number ofPBSs, i.e. the amount of PBP per Chi a varied inverselywith light intensity. This variation in the number of PBSwith respect to light intensity was the same as suggested byde Lorimier et al. (1992). Thus light intensity modulatesS.platensis Cl PBSs both in sizes and numbers.

In order to investigate the regulatory mechanisms thatcontrolled such changes in PBS components, we also exa-mined the relative levels of mRNA encoding PC and AP asa function of light intensity. In S.platensis Cl, it is likelythat the increase in the content of PBPs from cells shiftedfrom high light to low light conditions resulted from anincrease in transcription levels of the cpc and ape operons.When cells were shifted from low to high light, the 3.5 kbtranscript species corresponding to the cpcBAHID operondisappeared in concomitant with the disappearance of the33 kDa linker polypeptide within 24 h (data not shown).However, the other two rod linker polypeptides, 34.5 kDaand 15 kDa, still associated with the intact phycobilisome(as shown in Fig. 5). Thus, it seems that in the high lightcondition, there was an unequal translation efficiencyof the proteins in the cpcBAHID operon responsible forthe rod structure of phycobilisome in conjunction withthe rapid degradation of its transcript. However, if theefficiency of translation of every gene in cpcBAHID operonwas unchanged, the incoporation rate of the 33 kDa in thephycobilisome must have been altered or its half-life wasshorten by the influence of high light. The disappearance ofthe 33 kDa and the shorter rod of phycobilisomes mightexplain the function of this protein as the governance ofthe length of the rods. Furthermore, the cpcBAHID tran-scripts including the cpcBA genes were induced when cellswere shifted from high to low light. In contrast, the steadystate level of the transcripts corresponding to the apcABCoperon was not affected by high light intensity. We foundthat the abundance of both the 1.4 and 1.7 kb ape tran-scripts did not change when the light intensity was shiftedup but it increased when the light was shifted down. Thisresult suggested that under a downward shift in light in-tensity, the regulation of both the cpc and ape operons iscontrolled at least at the transcriptional level. In contrast towhat Kalla et al. (1993) found in Synechococcus PCC 6301,S. platensis Cl cells shifted from low to high light condi-tions showed no alteration in the amount of the cpc andape transcripts, whereas there were less PC and AP foundin the PBS. The difference in the amount between the cpcand ape transcripts and the corresponding proteins impliesthat there are additional levels of control. We propose thateither a post-transcriptional control as described in Syne-chococcus PCC 6301 by Kalla et al. (1993) or translational(Houmard et al. 1995) and/or post-translational ones asdescribed in Synechococcus PCC 6301 (Klotz et al. 1986)and Mastigscladus laminosus (Riimbeli et al. 1987), may



reponsible for the light regulation of PBS structure andgene in S. platensis Cl .

The authors would like to thank Dr. Gerard Guglielmi for hiscritical comments and valuable discussion and Dr. George A. Galefor reading and editing the manuscript. This work was supportedby a grant from the National Center for Genetic Engineering andBiotechnology (BIOTEC), Thailand. Pinthip Nomsawai was sup-ported by a fellowship from BIOTEC.

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(Received January 21, 1999; Accepted October 12, 1999)


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