Plant Physiol. (1999) 120: 599-604
Protection of Photosynthesis against Ultraviolet-B
Radiation by Carotenoids in Transformants of the Cyanobacterium
Synechococcus PCC79421
Thomas Götz,
Ute Windhövel,
Peter Böger, and
Gerhard Sandmann*
Lehrstuhl für Physiologie und Biochemie der Pflanzen,
Universität Konstanz, P.O. Box 5560, D-78434 Konstanz, Germany
(T.G., U.W., P.B.); and Botanisches Institut, FB Biologie, J.W. Goethe
Universität, P.O. Box 111932, D-60054 Frankfurt, Germany
(G.S.)
 |
ABSTRACT |
The cyanobacterium
Synechococcus PCC7942 was transformed with various
carotenogenic genes, and the resulting transformants either accumulated
higher amounts of 
-carotene and zeaxanthin or showed a shift in the
carotenoid pattern toward the formation of zeaxanthin. These
transformants were exposed to ultraviolet-B (UV-B) radiation, and the
degradation of phycobilins, the inactivation of photosynthetic oxygen
evolution, and the activity of photosystem II were determined. In the
genetically modified cells, the influence on destruction of phycobilins
was negligible. However, protection of photosynthetic reactions against
UV-B damage was observed and was dependent on the carotenoid
concentrations in the different transformants. Furthermore, it was
shown that endogenous zeaxanthin is more effective than -carotene.
Our results suggest that carotenoids exert their protective function as
antioxidants to inactivate UV-B-induced radicals in the photosynthetic
membrane.
| |
INTRODUCTION |
Because of the gradual depletion of ozone in the atmosphere, the
UV spectral region of sunlight is increasing at the earth's surface
(Caldwell et al., 1989
). Radiation in the UV-B range of approximately
300 nm interferes with various metabolic reactions, primarily by
generating free radicals and active oxygen species (Foyer et al.,
1994). These deleterious compounds are inactivated by
antioxidants. Several natural products have the potential to exhibit
antioxidative properties. Among them are the carotenoids, which protect
against photodynamic action in different ways: They are effective
quenchers of triplet-state photosensitizers and protect against singlet
oxygen and peroxy radicals (Krinsky, 1989). The photoprotective
function of carotenoids is essential for photosynthetic organisms.
Nonphotosynthetic organisms suffer from photooxidative stress caused by
light and near-UV radiation, which requires the presence of
antioxidative protection systems (Moradas-Fereira et al., 1996).
Protection against UV-B radiation by carotenoids was demonstrated in
several fungi and bacteria. Some strains of Ustilago violacea accumulate Cyt c and may or may not contain
carotenoids. Treatment of those strains with visible and UV radiation
showed that carotenogenic strains are more resistant to death caused by
light, demonstrating the effectiveness of carotenoids as protectants (Will et al., 1984). Similar studies carried out with the fungus Neurospora crassa gave comparable results (Blanc et al.,
1976). Studies with transformed Escherichia coli revealed
that effective protection against UV-B radiation depends either on the
chemical structure of the synthesized carotenoids or on their
accumulated amounts (Sandmann et al., 1998).
In photosynthetic organisms, UV-B radiation exerts direct effects on
photosynthetic light reactions and carbon reduction (Teramura and
Ziska, 1996). Several protection and repair mechanisms have been
elucidated, but little is known about how carotenoids alleviate UV-B
stress exerted on the cell and on the photosynthesis apparatus. Therefore, we modified the carotenoid content in
Synechococcus PCC7942 by the introduction of different
carotenogenic genes and examined the effects of UV-B radiation on these
transformants. Transformants were obtained in which either the amounts
of the endogenous carotenoids -carotene and zeaxanthin were
increased or the pigment pattern was shifted toward zeaxanthin. From
studies of UV-B irradiation in Synechococcus PCC7942, it is
well established that phycobilins decrease (Pandey et al., 1997), with
a concurrent loss of energy transfer from phycobilisomes to the
photosystems (Nedunchezhian et al., 1996). Impairment of photosynthetic
electron transport also occurs, which in particular affects PSII
activity (Campbell et al., 1998). Therefore, phycobilin contents and
electron-transport rates were used as parameters to evaluate the
protective effect of carotenoids in the transformants.
| |
MATERIALS AND METHODS |
Cultivation of the Cyanobacterium and Genetic Manipulations
The modified Synechococcus PCC7942 strain R2-PIM8 (van
der Plas et al., 1990) with an integration platform in the
metF gene for pBBR322-derived plasmids was used for
insertion of the genes crtB and crtZ from
Erwinia uredovora (Misawa et al., 1990) and pys
from Synechocystis PCC6803 (Martínez-Férez
et al., 1994). crtB and pys encode phytoene
synthase and crtZ encodes -carotene hydroxylase. All
transformants were cultivated for 2 d in BG11 medium (Rippka et
al., 1979) supplemented with 30 µg/mL methionin at 30°C. The
cultures were illuminated with a light intensity of 40 µmol
m
2 s1 and gassed with
air enriched to 1% to 2% (v/v) with CO2.
R2-PIM8 was grown with 10 µg mL1
streptomycin, and all resulting transformants were grown in the presence of 10 µg mL1 kanamycin and 1 µg
mL1 ampicillin.
Standard methods were used for genetic manipulations according to
Sambrook et al. (1989). DNA fragments were analyzed on agarose gels and restriction fragments were purified using Elu-Quick
(Schleicher & Schuell). Genomic DNA was isolated according to the
protocol described by Ausubel et al. (1995), and Southern
hybridization was carried out with a nonradioactive digoxygenin system
(Boehringer Mannheim) according to the supplier's instruction manual.
Transformation of Synechococcus PCC7942-PIM8 was carried out
according to the method of van den Hondel et al. (1980), with
modifications as previously described (Windhövel et al., 1994).
UV-B Treatment of Cells and Determination of Phycocyanin and
Carotenoids
Samples (45 mL) of 2-d-old cultures (chlorophyll adjusted to 3 µg mL1 cell suspension) were pipetted into a
Petri dish (12 cm in diameter), and the cells were exposed to UV-B
radiation as indicated. The radiation chamber was equipped with four
fluorescent UV-B lamps (TL40W/12, Philips, Eindhoven, The Netherlands),
which exhibited their emission maximum at 312 nm and had a cutoff at
270 nm. The samples were exposed to a fluence rate of 6.8 W
m2 as determined with a UV-B sensor (model IL
1700, International Light, Newburyport, MA).
For determination of phycocyanin, including allophycocyanin,
centrifuged cells from 2-mL samples were resuspended in 0.2 M Tris-HCl buffer, pH 7.5, and incubated with 1.5 mg of
lysozyme overnight at 30°C under constant shaking. The spectra were
recorded from the supernatant after centrifugation, and the amount of
phycocyanin was calculated as phycocyanin plus allophycocyanin (in
micrograms per milliliter) by their absorbance using the equation:
phycocyanin + allophycocyanin content = 0.273 E620 + 0.024 E650. The
carotenoid content of the Synechococcus PCC7942
transformants was determined after the 2-d growth period. Cells were
heated in methanol containing 6% KOH for 15 min at 55°C. The
carotenoids were partitioned into hexane:diethyl ether (10:1, v/v), and
the upper phase was collected. Carotenoids were separated by HPLC on a
25-cm Nucleosil C18, 3-µm column with
acetonitrile:methanol:2-propanol (85:10:5, v/v) at a flow rate of 1 mL/min (Ernst and Sandmann, 1988). Spectra were recorded on-line from
the elution peaks using a photodiode array detector (model 400, Kontron
Instruments, Eching, Germany).
Measurement of Photosynthesis Activities
Photosynthesis activities were determined with cells prior to or
following a 6-h treatment with UV-B. Oxygen evolution was measured with
a Clarke-type electrode under saturating white light of 400 µmol
m2 s1 (Böger et
al., 1981). For determination of PSII activity, benzoquinone and
potassium ferricyanide (1 mM each) were added as electron acceptors. All values were related to the chlorophyll content of the
cells. Chlorophyll was determined after extraction with methanol at
65°C from its absorbance and calculated as: chlorophyll a
(µg mL1) = 16.5 E665 
8.3 E650.
Differences in photosynthesis activities and other parameters between
the transformants and before and after radiation were analyzed by
Student's two-tailed t test. Results have a 5% level of
significance.
| |
RESULTS |
Construction and Genetic Analysis of Transformant Strains
The plasmid pFP1-3 was constructed by insertion of the 1.5-kb
HindIII/SalI fragment of the ntpII
gene (Putnoky et al., 1983) into pUC19 (Yanisch-Perron et al., 1985).
This plasmid was used to construct three integration vectors:
pFP1-3-crtB with the phytoene synthase gene from Erwinia
uredovora (Misawa et al., 1990), pFP1-3-crtZ with the -carotene
hydroxylase gene from the same bacterium, and pFP1-3-pys with the
phytoene synthase gene from Synechocystis PCC6803
(Martínez-Férez et al., 1994). pFP1-3-crtB resulted
from a 1.5-kb PvuII/HpaI fragment from pCar25
(Misawa et al., 1990), filled in with Klenow enzyme and ligated into
the SmaI site of pFP1-3. The plasmid pFP1-3-crtZ was
constructed using a 3.0-kb SnaBI/EcoRI fragment
from pCar25 after treatment with Klenow enzyme and ligation into the
SmaI site of pFP1-3. The resulting construct was then
digested with DraII to delete two other genes from E. uredovora and religated. The plasmid pFP1-3-pys was obtained by starting with a 3.3-kb BamHI/KpnI fragment from
plasmid pMF1041 (Martínez-Férez et al., 1994) and blunt-ending it
with Klenow enzyme to ligate it into the SmaI site of
pFP1-3. The resulting plasmid was then digested with ClaI to
delete a 1.3-kb region downstream of the pys gene and
religated.
Figure 1 shows the genomic region of the
integration platform of the four transformants. The control strain
PIM8-pFP1-3 carries only the kanamycin resistance gene together with a
functional bla gene for ampicillin resistance from pUC19. In
PIM8-pFP1-3-pys the phytoene synthase gene pys is
cotranscribed with nptII, which is under a strong promoter
and lacks a transcription stop signal. The same is true with
PIM8-pFP1-3-crtZ, in which the -carotene hydroxylase gene
crtZ is cotranscribed with ntpII. In
PIM8-pFP1-3-crtB the phytoene synthase gene crtB from
E. uredovora is also located downstream from the
nptII gene. However, orientation is in the opposite
direction, and it was not possible to obtain transformants in which
crtB had the same orientation as nptII.
Nevertheless, by northern analysis we could demonstrate that
substantial amounts of the crtB transcript were formed and
that in vitro phytoene synthase activity in PIM8-pFP1-3-crtB was almost
twice as high as in PIM8-pFP1-3 (data not shown).
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| Figure 1.
Genetic map of the integration platform of
Synechococcus R2-PIM8 transformants with inserted
crt genes (van der Plas et al., 1990). Integrated
plasmids were: pFP1-3 without a crt gene; pFP1-3-crtB, which includes
the coding region of the phytoene synthase gene from E. uredovora; pFP1-3-pys with the coding region of the phytoene
synthase gene from Synechocystis PCC6803; and
pFP1-3-crtZ with the coding region of the -carotene hydroxylase gene
from E. uredovora. MetF , Gene of the
methionin biosynthesis pathway; bla, ampicillin
resistance gene; nptII, kanamycin resistance gene;
ori, origin of replication; D, DraII; E,
EcoRV; H, HindIII.
|
|
Integration of carotenogenic genes into the Synechococcus
PCC7942 genome was confirmed by Southern hybridization (Fig.
2). The probes against crtB,
crtZ, and pys reacted specifically with the
corresponding genes in blots Figure 2, A to C, respectively. The
restriction pattern obtained for the EcoRV,
EcoRV/HindIII, and
EcoRV/DraII digests showed the expected bands
with the sizes calculated from Figure 1. The major part of the genes
were used to synthesize the individual probes. Accordingly, two bands
were detected in the EcoRV digest of the
crtB-containing transformant because an EcoRV
site is present in crtB. In the
EcoRV/HindIII double-digest, the larger 3.8-kb
fragment is cut to a size of 2.3 kb as calculated from Figure 1. The
EcoRV/HindIII digests of the
pys-containing transformant resulted in splitting of the 7.3-kb EcoRV fragment into two detectable bands. In the
crtZ-containing transformant digested with EcoRV,
EcoRV/HindIII, or
EcoRV/DraII, the probe detected a single band of
decreasing size.
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| Figure 2.
Southern genome analysis of
Synechococcus PIM8 transformed with pFP1-3, pFP1-3-crtB,
pFP1-3-pys, and pFP1-3-crtZ. Hybridization was with a
digoxygenin-labeled crtB probe (A), a pys
probe (B), or a crtZ probe (C). DNA was digested with
EcoRV (a), EcoRV and
HindIII (b), or EcoRV and
DraII (c). Sizes are indicated in bp.
|
|
Carotenoid Content of Synechococcus
PCC7942 Transformants
HPLC separation of extracted pigments after saponification showed
that Synechococcus PCC7942 contains -carotene and its
3,3-dihydroxy derivative zeaxanthin as major carotenoids. Depending on
growth conditions, trace amounts of -cryptoxanthin
(3-hydroxy--carotene) could be detected as an intermediate. Analysis
of the carotenoids from the control strain PIM8-pFP1-3 and from
PIM8-pFP1-3-crtZ with the inserted crtZ gene demonstrated a
much higher zeaxanthin peak in the latter at the expense of
-carotene. Growth at a light intensity of 50 µmol
m2 s1 of PIM8-pFP1-3
resulted in a total carotenoid content of 3.17 mg
g1 dry weight (Table
I) and a -carotene to zeaxanthin ratio
of about 2:1. Integration of one of the phytoene synthase genes, crtB or pys, resulted in an increase of the
carotenoid content of the transformants. In crtB, a value of
3.83 mg g1 dry weight represents a 20%
increase over the control, whereas PIM8-pFP1-3-pys with a 5.13 mg
g1 dry weight contained about 60% more
carotenoids than the control transformant PIM8-pFP1-3. Nevertheless,
the -carotene to zeaxanthin ratio did not change much in the
transformants with an additional phytoene synthase gene. In
transformant PIM8-pFP1-3-crtZ with the -carotene hydroxylase gene
crtZ from E. uredovora, total carotenoids were in
the same range as in the control. However, a stronger conversion of
-carotene to zeaxanthin was observed. The share of zeaxanthin
increased from 32% of total carotenoids in the control to 55% in
PIM8-pFP1-3-crtZ.
UV-B Treatment and Response
Treatment of the control transformant PIM8-pFP1-3 with UV-B
radiation of an intensity of 6.8 W m2 during a
6-h period decreased the phycocyanin content and photosynthesis activity (Table II). The latter was
measured under saturating light conditions to compare the entire
photosynthesis capacity of the transformants before and after UV-B
treatment. In all transformants the remaining phycocyanin concentration
was decreased by about one-half after exposure. Photosynthesis activity
of intact cells, measured as oxygen evolution involving PSII and PSI,
was very similar in the control and PIM8-pFP1-3-crtB. Untreated
transformants PIM8-pFP1-3-pys and PIM8-pFP1-3-crtZ exhibited lower and
increased photosynthesis activities, respectively. The higher rate of
oxygen evolution for PIM8-pFP1-3-crtZ was significant at a 5% level. The PSII activities of all transformants did not differ significantly. Protection of overall photosynthesis activity and PSII activity after
UV-B treatment was best with PIM8-pFP1-3-crtZ. Here, photosynthesis was
about 2-fold higher and PSII activity was almost 3-fold higher than in
the control (Table II). Residual activities were also higher in the
irradiated PIM8-pFP1-3-pys transformant. For UV-B-treated PIM8-pFP1-3-crtB a significant difference was found only in PSII activity, which was 2-fold higher than in the control.
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Table II.
Effect of UV-B radiation on phycocyanin content,
photosynthetic oxygen production by cells, and PSII activity
All values are means ± SD of at least five
determinations. The intensity of the UV-B radiation was 6.8 W
m2. Oxygen consumption rates in the dark were between
10% and 20% of the photosynthesis rates and did not differ among the
transformants.
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| |
DISCUSSION |
It was demonstrated previously that in higher plants the
phytoene-synthesis step is rate limiting for the entire
carotenoid-biosynthesis pathway (Fray et al., 1995; Kumagai et al.,
1995). Our results showing elevated carotenoid contents in
Synechococcus PCC7942 transformants expressing the foreign
phytoene synthase genes crtB or pys (Table I)
indicate that the same bottleneck exists in cyanobacterial carotenoid
biosynthesis. With the bacterial gene crtZ it was possible
to shift the carotenogenic pathway toward the synthesis of zeaxanthin.
Thus, Synechococcus PCC7942 transformants with a higher and
altered carotenoid content allowed us to investigate which carotenoids
are promising candidates in the protection of photosynthesis against
damage by UV-B radiation (Middleton and Teramuara, 1993).
The damage to photosynthesis by UV-B radiation is caused by the
generation of free radicals rather than singlet oxygen (Hideg and Vass,
1996). These radicals accumulate in the thylakoids and are responsible
for peroxidation reactions that destroy various components of the
photosynthesis apparatus (Malanga et al., 1997), among which are the D1
and D2 proteins of PSII, which are highly susceptible to peroxidative
conditions (Greenberg et al., 1989; Jansen et al., 1996). Protection
against UV-B can occur at different levels (for review, see Teramura
and Ziska, 1996). Plants can accumulate UV-B-screening compounds such
as flavonoids (Middleton and Teramura, 1993), the antioxidant system
can inactivate oxygen radicals, or the effective replacement of damaged
constituents can resist UV-B stress, as was shown for the D1 protein
(Campbell et al., 1998). In this context, carotenoids may have both a
screening and an antioxidant function. The latter has been established
for many carotenoid structures (Woodall et al., 1997). In
cyanobacteria, soluble carotenoid proteins (Diverse-Pierlussi and
Krogmann, 1988) or carotenoids in the cell wall (Jürgens and
Weckesser, 1985) could act as a filter for UV-B radiation.
Increasing amounts of zeaxanthin and -carotene in the transformants
(Table I) resulted in protection of photosynthesis against UV-B damage
(Table II). This effect was most pronounced on PSII activity. A
positive relationship between carotenoid content and relief from UV-B
inactivation was evident. The protection in the transformant in which
carotenoid biosynthesis is shifted toward formation of zeaxanthin
indicates that in Synechococcus PCC7942 zeaxanthin provides
the highest protective potential. It was shown recently that zeaxanthin
is the most effective protectant against UV-B radiation in E. coli transformants (Sandmann et al., 1998) and that it prevents
radical peroxidation processes in liposomes much better than
-carotene (Woodall et al., 1997). Furthermore, only destruction of
membrane-located processes such as photosynthetic electron transport by
UV-B could be prevented, but protection of (soluble) phycocyanins
against UV-B was not observed, since they are located outside of the
photosynthetic membrane and are not in contact with carotenoids (Table
II).
The genetic modification of Synechococcus PCC7942 made it
possible to elucidate the primary role of carotenoids in protecting photosynthesis as quenchers of radicals generated in the thylakoid membrane upon UV-B radiation. Light-saturation curves are available for
some of the Synechococcus PCC7942 transformants
(Windhövel et al., 1995); however, for a better understanding of
carotenoid function in the primary photosynthesis reaction, a more
detailed study including measurements of additional photosynthesis
parameters is needed. Because cyanobacterial species other than
Synechococcus PC7942 synthesize carotenoid structures that
are different from those shown in Table I, it may be possible to find
other carotenoids with even better protective properties than
zeaxanthin.
| |
FOOTNOTES |
1
This work was supported by a grant from the
Bundesministerium für Bildung, Wissenschaft, Forschung und
Technologie, Germany (grant no. 07UVB07).
*
Corresponding author; e-mail Sandmann{at}em.uni-frankfurt.d400.de;
fax 49-69-798-24822.
Received December 22, 1998;
accepted March 15, 1999.
| |
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Abstract
Introduction
