morphological changes in the life cycle of the green _kobayaky

8/15/2019 Morphological Changes in the Life Cycle of the Green _kobayaky

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JOURNALOF FERMENTATIONND BIOENGINEERING

Vol. 84, No. 1, 94-97. 1997

Morphological Changes in the Life Cycle of the Green

Alga

Haematococcus pluvialis

MAKIO KOBAYASHI,‘* YOSHIRO KURIMURA,’ TOSHIHIDE KAKIZON0,2

NAOMICHI NISH10,2

AND

YASUNOBU TSUJI’

Research Laborat ory of Hi gashimaru Shoyu Co. Lt d., 100-3 Tomi naga, Tatsuno, Hy ogo 679-41’ and Department of

Fermentat ion Technology, Facult y of Engineeri ng, Hi roshima Uni versit y, Hi gashi- Hi roshima 724,2 Japan

Received 10 January 1997IAccepted 28 April 1997

A 2-week model life cycle of the green alga Haematococcuspluvialis was constructed, consisting of four cell

stages: vegetative cell growth, encystment, maturation, and germination. Each algal cell stage could be distin-

guished by the ratio of pigments (carotenoid/chlorophyll) and the intracellular protein content. Using the

culture system developed, light was shown to be essential for both carotenogenesis and cell differentiation

(encystment and germination). The results also suggested that

H. pluv ia l i s

has a novel photosynthesis-depen-

dent system of carotenogenesis regulation.

[Key words:

astaxanthin, encystment, germination, Haematococcus luv ial is, ife cycle, maturation]

Astaxanthin (3,3’-dihydroxy-P, p-carotene-4,4’-dione),

a red ketocarotenoid, is not only used as a pigmentation

source in marine fish aquaculture (l), but also has poten-

tial clinical applications due to its higher antioxidant

activity than p-carotene and vitamin E (2, 3). The green

unicellular alga Haematococcus pluvialis is a potent

producer of astaxanthin (4, 5). A number of recent

studies have suggested that astaxanthin biosynthesis in

H. pluv ia l is

is associated with the formation of cyst

cells (encystment) in the resting stage under autotrophic (4,

6-8) and mixotrophic conditions (5, 9-11). The morpho-

logical change from vegetative to enlarged thick-walled cyst

cells has been reported to take several weeks under auto-

trophic conditions (4, 7), and in nitrogen-deficient (10)

and phosphate-deficient (11) acetate media. However, we

previously showed (12) that a high acetate content in the

medium induced encystment within several days under

mixotrophic conditions. We have also examined various

environmental factors influencing growth and carotenoid

biosynthesis in

H. pluvialis,

including media composi-

tion (5), growth kinetics (13), light conditions (14), and

environmental stress (15). In the present study, we con-

structed a 2-week model life cycle of

H. pluv ia l i s

and in-

vestigated the mechanisms of the morphological changes

that occurred.

The freshwater alga H. pluv ia l i s Flotow NIES-144 was

obtained from the National Institute for Environmental

Studies, Tsukuba, Japan. An acetate (15 mM) basal

medium was used as described previously (5). For the

basal culture, 10ml of a 4-d-old culture was inoculated

into 100 ml fresh basal medium in a 200-ml Erlenmeyer

flask. The flask was incubated at 20°C under a 12-h light/

12-h dark illumination cycle at 25 pmol quanta-mp2. s-l

(white fluorescent lamp) as described previously (5).

The 4-d culture (vegetative cells in the exponential

growth phase) was used for the subsequent two-stage

supplementation culture. Sodium acetate solution (2.25

M, pH 7.0) was first added to the 4-d basal culture at a

final concentration of 45 mM to prepare immature (carot-

enoid-poor) cyst cells (12). Then, after a further 2 d, fer-

rous sulfate solution (22.5 mM, pH 1.5) was added to

*

Corresponding author.

the acetate-supplemented culture at a final concentration

of 450,uM to prepare mature (carotenoid-rich) cyst cells

(15). The supplementation cultures were incubated at

20°C under continuous illumination (125 pmol quanta.m‘-2+

s-l) as described previously (15).

After another 3 d, mature cyst cells in the Fe2+-sup-

plemented culture were harvested by centrifugation at

2,OOOxg for lOmin, and washed twice in fresh basal

medium. The washed mature cysts were resuspended in

100 ml fresh basal medium. Then, a lo-ml portion of the

mature cysts was transferred to 1OOml fresh basal medi-

um in a flask. The flask was incubated at 20°C under a

12-h light/lZh dark illumination cycle (25 ,umol quanta.

rnp2.

s- ‘)

as the basal culture of vegetative cells.

To investigate the regulation of the life cycle of

H.

pluvialis,

the following inhibitors obtained from Sigma

Chemical Co. (St. Louis, MO, USA) were used as de-

scribed previously (13, 15): a transcriptional inhibitor

(actinomycin D) at a final concentration of 10 ,ug.mlVl;

two translational inhibitors (cycloheximide for cytoplas-

mic protein synthesis and chloramphenicol for organellic

protein synthesis, at 0.3 /-lg.ml-’ and 50 pg+rnl-‘, respec-

tively); 3-(3,Cdichlorophenyl)-1,1-dimethylurea (DCMU),

a specific inhibitor of electron flow from photosystem

(PS) II to the plastoquinone pool (16), at 20 PM; and 2,5-

dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB),

a specific inhibitor of electron flow from plastoquinone

to cytochrome (Cyt) 16), also at 20 ,QM.

The algal cell number was counted with a hemacytome-

ter. For the protein assay, algal cells were suspended in

2 M NaOH for 1 h on ice as described by Whitelam and

Codd (17) and the alkaline-solubilized protein was deter-

mined by the Bradford method (18) with bovine serum

albumin as the standard. The intracellular pigments

carotenoid and chlorophyll were extracted with 90%

(v/v) acetone, and assayed as described previously (5,

15). All analyses were performed in triplicate cultures,

and the means are shown.

Morphological changes of H. pluv ia l i s

The model

life cycle of

H. pluv ia l i s

using acetate media was divided

into four cell stages, as illustrated in Fig. 1: I, vegetative

cell growth; II, encystment (vegetative to immature cyst

cells); III, maturation (immature to mature cyst cells);

94


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VOL. 84, 1997

NOTES 95

Vegetative Cell

Immatu% Cyst

FIG. 1. Schematic diagram of the model life cycle of

H pluviulis

IV, germination (mature cyst to vegetative cells). The

changes in the intracellular contents and cell number in

the algal life cycle are shown in Fig. 2. Ellipsoidal vegeta-

tive cells were capable of actively swimming with two

flagella and of increasing in number (O-4 d culture). Sup-

plementing the vegetative cell culture with a high level of

acetate at 4 d induced the algal cells to become spherical

immotile cyst cells (4-6 d culture). During maturation

(6-9 d culture), carotenoid biosynthesis in cyst cells was

significantly enhanced by the addition of FeZ+ at 6 d. When

mature cysts were transferred to fresh medium at 9 d,

intracellular daughter cells were released from the mature

cyst cells into the medium, and vegetative cells regenerat-

ed from daughter cells grew mixotrophically (9-14 d

culture). In this way, the life cycle of H.

pluvialis

was

completed within only 2 weeks in our experimental culture

system.

During the life cycle of the alga, vegetative cells con-

tained high levels of chlorophyll and protein but had

very low carotenoid contents, whereas encystment was

accompanied by the degradation of chlorophyll and pro-

tein. The maturation of cyst cells was accompanied by

enhanced carotenoid biosynthesis and accelerated protein

degradation. Germination coincided with chlorophyll and

protein syntheses, and carotenoid degradation. Encystment,

0

stage I

II

111

IV I

P

a

=

Ireah medium

7 2

6 g

: /

1 o

0 e

a

6 =

25

5

2

4

15

3

Iii

5

1

2 E

1

8

5

if

f

1

2 3 4 5 6 7 8 9 1 11121314 g

Culture time(d)

FIG. 2. Changes in the contents of protein

,

carotenoid (a),

and chlorophyll

A),

cell number (A) and the ratio of carotenoid/

chlorophyll (0) in the life cycle of

H pluviulb

(A) Cell number and

pigment ratio; (B) intracellular contents. Each cell stage (I-IV) in

the life cycle of

H pluviulis

is shown in Fig. 1. Culture times: O-4 d,

vegetative cell growth (I); 4-6 d, encystment (II); 6-9 d, maturation

(III); 9-14d, germination and vegetative cell growth (IV-rI). The

three arrows indicate the times of acetate addition (45 mM), Fe2+

addition (450,uM), and transfer to fresh medium, respectively.

Light conditions: O-4 d, 12-h light/l2 h-dark illumination cycle (25

pmol quanta-m-*.sst); 4-9 d, continuous illumination (125 pmol

quanta.m-2.s~1); 9-14 d, 12-h light/l2 h-dark illumination cycle (25

,nmol quanta. m-2.

SC’).

The means of three replicate experiments I

standard deviation as shown.

maturation, and germination are usually assessed by

microscopic observation. In this study, we determined

each algal cell stage using the intracellular protein and

pigments contents. Since carotenoid accumulated in cyst

cells only on maturation, the ratio of intracellular carot-

enoid/chlorophyll was considered a good parameter for

distinguishing among vegetative (green), immature cyst

(brown), and mature cyst (red) cells. The pigments ratios

were about 0.5, 1.0, and 7.0 in vegetative, immature, and

TABLE 1. Effects of light and inhibitors on each cell stage in the life cycle of

H pluvialis

Cell stage&

I (0-4d)

II (4-6 d) III (6-9 d)

IV-*1 (9-14 d)

Culture timea at 4d at 6d

at 9d at 14d

Protein

Car/Chlb

Protein

Car/ChP

Protein

@g/cell)

(pglcell)

(pg/cell)

Car/ChP

Protein

(pg/cell)

Car/Chlb

Lightc

None

270*23

0.4kO.l 150+15 l.lkO.2 13*5

7.OkO.7 281+25

0.3i-0.1

+Actinomycin D

NDd ND* 251+30 0.3LO.l 25t9

6.OkO.5

15c5

5.520.5

+ Chloramphenicol

250&25 0.5t0.1 145+20 l.lkO.2 29f5 7.OkO.6

20F4 5.9kO.6

+ Cycloheximide

NDd

NDd

244+25 0.4kO.l

33+8

6.5kO.5

13+5

5.7kO.5

+DCMU

254124 0.3kO.l 255?28 0.4t0.1 145+-20

0.9t0.3 45+10 5.510.5

+ DBMIB

260 + 30 0.4fO.l 240? 19 0.4fO.l 20*5

7.520.8 40t9 5.2kO.4

Dark None

250220 0.520.1 248&30 0.5kO.l 140+ 15 0.7kO.3

5OilO 5.310.5

a Each cell stage and indicated times are as shown in Fig. 2.

b Ratio of carotenoid/chlorophyll.

c Cell treatments: light conditions were as shown in Fig. 2. In each cell stage, cells were cultured for the indicated times in the presence of

inhibitors. Inhibitor concentrations: actinomycin D, 10 pg.ml-t; chloramphenicol, 50 ,ug.ml-‘; cycloheximide, 0.3 /-‘g.ml- r; DCMU, 20 pM;

DBMIB, 20 /IM.

d Not determined because of no growth.


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96

KOBAYASHI ET AL.

mature cyst cells, respectively.

Effects of light and inhibitors on the algal life cycle

We have already shown that vegetative cells of H. pluv iu-

Zis can grow heterotrophically on acetate in the dark

(13). We investigated the light requirement and regula-

tion of each cell stage during the model life cycle of H.

pluv ia l is using various inhibitors. The results are shown

in Table 1. Green vegetative cells grew heterotrophically

on acetate in the dark (about 3 x lo5 cells/ml), as well as

mixotrophically on acetate in the light (about 5.5 x 10’

cells/ml). Although photosynthesis was completely in-

hibited by either DCMU or DBMIB, algal vegetative cell

growth was retained at the heterotrophic level in the

light. Actinomycin D and cycloheximide completely in-

hibited vegetative cell growth in the light, whereas vegeta-

tive cells grew at the heterotrophic level in the presence

of chloramphenicol.

Neither cell differentiation (encystment and germina-

tion) nor maturation (carotenogenesis) occurred in the

dark. With addition of acetate to H. pluv ia l is in the

vegetative growth phase, cysts were formed within 48 h

in the light. This encystment was blocked by actinomy-

tin D or cycloheximide, but was unaffected by chloram-

phenicol, suggesting that encystment is regulated at the

transcriptional level of algal chromosomal genes, and

probably does not involve organellic genes. No encyst-

ment was observed in the presence of either DCMU or

DBMIB, indicating that the induction of encystment by

a high C/N ratio might require photosynthesis.

Carotenogenesis in cyst cells was enhanced by FeZ+

both in the presence and absence of transcriptional or

translational inhibitors in the light. To further inves-

tigate photo-dependent carotenogenesis in the cyst cells,

two electron flow inhibitors of photosynthesis were used

(16). Fe2+-enhanced carotenogenesis in cyst cells was in-

hibited by DCMU but not by DBMIB, suggesting that

plastoquinone might be involved in carotenoid biosynthe-

sis of

H. pluvialis.

Recently, it was reported that plasto-

quinones can be substituted for molecular oxygen as a

terminal electron acceptor in the carotenoid biosynthetic

pathway in phytoene desaturation in chromoplasts of the

daffodil, Narcissus pseudonar cissus (19, 20). Moreover,

Cyt has been shown to be selectively lost in red cyst

cells of H. pluv ia l i s (ll), while selective destabilization

of Cyt occurred during gamete induction in Chlamydo-

monas 21). Therefore, in cyst cells of H. pluvialis, in-

stead of impairment of the linear electron flow between

PS II and PS I, the plastoquinone pool may function as

an electron crossover point between photosynthesis and

carotenoid biosynthesis.

When mature cyst cells prepared within only 5 d (in-

duction for 2 d followed by activation for 3 d) were

transferred to a fresh basal medium, intracellular daugh-

ter cells were rapidly released from the mother cells (cyst

cells) into the medium in the light. We observed the

same results regarding germination using 2-month-old

cyst cells as described by Lee and Ding (22). Vegetative

cells regenerated from daughter cells grew mixotrophical-

ly or heterotrophically on acetate in the light or dark

(data not shown). Germination was inhibited by the

three antibiotics tested, actinomycin D, cycloheximide,

and chloramphenicol. These results suggest that, unlike

the case with cyst formation, cell differentiation from the

resting to vegetative growth stage is regulated at the

transcriptional level of both chromosomal and organellic

genes. Germination as well as encystment was inhibited

J. FERMENT. IOENG.

by the two photosynthesis inhibitors.

From these results, it is suggested that light is essential

for the life cycle of H. pluvialis, particularly for cell

differentiation (encystment and germination) and matu-

ration (carotenogenesis). The fact that these metabolic

events were all inhibited by DCMU suggests that photo-

synthesis is an absolute requirement for their occur-

rence. Since light-dependent carotenoid formation has

been reported in many microorganisms (23), such photo-

synthesis-dependent induction and activation indicate

that

H. pluv ia l i s

has a novel photo-dependent system for

the regulation of carotenogenesis.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

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morphological changes in the life cycle of the green _kobayaky

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