ABBS 2005,38(03): Survival of Human Metallothionein-2 Transplastomic Chlamydomonas reinhardtii to Ultraviolet B Exposure

Received:
December 2, 2005

Accepted:
December 15, 2005

*Corresponding
author. Tel, 86-10-62751842; Fax, 86-10-62751842; E-mail, [email protected]

Abstract        Solar ultraviolet (UV)
radiation has a great influence on green organisms, especially plankton like Chlamydomonas.
A human metallothionein-2 gene, which is generally considered to have an
anti-radiation function by its coding product, was transferred into the
chloroplast genome of Chlamydomonas reinhardtii. To dynamically measure
the UV effects on Chlamydomonas cells grown in liquid
tris-acetate-phosphate medium, a new method was developed based on the
relationship between the chlorophyll content of an algal culture and its
absorbance at 570 nm after the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this
experiment, both the wild-type and the transplastomic C. reinhardtii
cells were cultivated in 96-well microplates containing liquid
tris-acetate-phosphate medium in the absence or presence of zinc, copper,
cadmium and cysteine. The transgenic C. reinhardtii showed a higher
resistance than wild-type to UV-B exposure under all the examined conditions.
Metals in the medium had positive impacts on both types of cells, but had
significant influence only on the transplastomic cells. However, the high cell
viability of the transgenic alga at the end of the 8 h UV-B treatment
disappeared after a 20-h recovery culture. Cysteine did not protect cells from
UV-B damage, but clearly enhanced the growth of both wild-type and transgenic C.
reinhardtii.

Key words        ultraviolet B; MTT; transplastomic Chlamydomonas;
metallothionein

The penetration of
increased amounts of ultraviolet B (UV-B; wavelength of 280–320 nm) threatens the health of living organisms
on Earth, including the planktons that populate the top area of water [1].
Studies showed that UV-B radiation resulted in many deleterious effects:
inhibition­ of photosynthetic processes, degradation of proteins­ and DNA and
increased oxidative stress [2]. The natural circadian clocks are considered to
be an adaptation­ of organisms to the environment. During cell division cycles,
many important cellular events, such as DNA replication­ and cell division,
occur during the night to escape­ from deleterious wavelengths during the
daytime [3]. UV sensitivity rhythm was reported in the unicellular alga Chlamydomonas,
which was considered to be related to nuclear division [4]. Wild-type Chlamydomonas
in the natural­ environment shows more sensitivity to UV-B during­ the night
than during the daytime.

A codon-optimized human
metallothionein-2 (hMT-2) gene was integrated into the chloroplast
genome of Chlamydomonas­ reinhardtii to generate a transplastomic C.
reinhardtii. As metallothionein (MT) is believed to be an efficient
oxyradical scavenger [5], this study was carried­ out to validate if the
expression of mammal MT in a photosynthetic­ organelle could lessen the cell
injuries caused by UV-B exposure.

Colony counting is the
currently used measure for the study of UV effects on Chlamydomonas [5].
Cells spread on solid medium were exposed to UV radiation and visible colonies­
were counted 5–7 d later.
This method is not applicable to observe the influences of UV on Chlamydomonas­
cells grown in an aqueous environment. Moreover, it can not monitor algal
growing situations moment-by-moment. Chlorophyll content is an important
parameter to evaluate the biomass of algal culture. Optical absorbance at 645
nm and 663 nm can be easily measured with spectrophoto­metry at any time during
the culture. Because chlorophyll is quite sensitive to UV exposure [6], this
method is useless­ when algae are exposed to UV radiation. The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is the
most frequently used method for quantitating cell viability in mammal cell
culture [7]. The redox activity of living cells can convert MTT into
purple-colored­ MTT formazan, but dead cells do not. However, MTT assay is not
feasible for an intravital study of algal culture, because cells are usually
dead after the reaction.

In order to accomplish a
dynamic observation of UV-B effects on both wild-type and transplastomic C.
reinhardtii cells, a standard curve-based method was set up, by integrating­
the optical absorbance method and the MTT assay as well as the microscale algal
culture technique. Using this method, we effectively studied the survival of
the hMT-2 transplastomic C. reinhardtii cells to UV-B exposure­
under different culture conditions.

Materials and Methods

Plasmids, reagents and
alga strains

The enzymes and
chemicals used for DNA manipulation­ were from New England Biolabs ( 

Construction of C.
reinhardtii chloroplast expression vector

The hMT-2
fragment was amplified from the clone vector pET 

Generation of
chloroplast transgenic C. reinhardtii

Foreign DNA was
delivered into the chloroplast by biolistic bormbardment (PDS100/He; Bio-Rad,  

PCR analysis

Total DNA was extracted
from C. reinhardtii cells as described previously [9]. The presence of
the hMT-2 gene in the transformed cells was detected by PCR using the hMT-2
primers mentioned above. Integration platform primers (chlL primer1, 5‘-GTTTTATTCCTGGAGTTTG-3‘
and chlL primer2, 5‘-GAAAGTATTTAAAGCTGC-3‘) were also
employed to study the homogeneity of C. reinhardtii transformants. The
agarose gels (1%) were used to separate the PCR products.

Western blotting
analysis

Whole cells were
collected by centrifugation at  

Cell culture

Stock cultures of both
the wild-type and the pCS-MT-2 transformed C. reinhardtii cells were
maintained in continuous light in liquid TAP medium (pH 6.0) as described
previously [9]. In preparation for UV-B experiments, a 100- ml flask containing
60 ml TAP medium was inoculated at A750=0.2. After
growing for 48 h in light, the cells reached a middle exponential phase, then
were cultured for 8 h in the dark to get synchronization. Microculture was
carried out on sterile 96-well microplates (Costar, 0.45 ml, flat-bottom;  

Chlorophyll content
assessment

The absorbance of
microscale algal cultures at 645 nm or 663 nm was measured using a microplate
reader (Spectra Rainbow; Tecan,  

UV treatment

The cultured microplate
was uncapped and placed below­ a UV-B lamp ( 

MTT assays

MTT was dissolved in
phosphate-buffered saline (pH 7.4) at a concentration of 5 mg/ml and stored at
4 ºC. When measuring the living cell ratio, MTT stock solution was added to
each sample on the 96-well microplate so that the final concentration of MTT in
the medium was 0.5 mg/ml. Then the cells were incubated for 4 h at 25 ºC.
Because cells were precipitated to the bottom of the wells, the supernatants
were carefully removed away with a pipettor. Dimethyl formamide (100 ml) was added to each well. After 10 min incubation
on a shaker (140 rpm), the absorbance was measured with a microplate reader at
a test wavelength of 570 nm.

Results

Construction of
chloroplast expression vector

Based on the C.
reinhardtii chloroplast-specific vector p64D [11], a chloroplast expression
vector, pCS-MT-2 was constructed [Fig. 1(A)]. The hMT-2 gene was
put under the control of a strong psbA promoter and an rbcL 3‘
untranslated region from the C. reinhardtii chloroplast genome. The
aminoglycoside 3‘-adenylyltransferase (aadA) was a selectable
maker gene from Escherichia coli, which conformed the transformed cells
spectinomycin and streptomycin resistances. The flanking sequences, clpP–trnL–petB–chlL5′
and chlL3′-rpl23-rp12-rps19, allowed the site-specific integration of
the foreign DNA into the C. reinhardtii chloroplast genome. This
insertion would disrupt­ the native chlL gene, encoding
light-independent protochlorophyllide reductase, and lead to a
“yellow-in-the-dark” phenotype [12].

Identification of
transplastomic C. reinhardtii

Chloroplast-transgenic C.
reinhardtii were obtained as described by Kindle et al. [13]. After
10 rounds of selection­ with 100 mg/ml
spectinomycin, approximately 60 colonies­ were obtained from two bombardments.
One of these colonies, named CM2, was used for this study.

DNA integration into the
chloroplast genome was determined by two sets of PCR primers. Taking total
cellular­ DNA from both the CM2 transformant and the wild-type as PCR
templates, an approximately 200 bp MT-2 fragment could be amplified from
the tansformant CM2, just like that of the positive control (pCS-MT-2). As
expected, neither the untransformed C. reinhardtii nor the spectinomycin
mutant­ (caused by the spontaneous mutation of the 16S rRNA) showed any
products [Fig. 1(B)]. Another primer set, chlL1 and chlL2,
annealed to the native chloroplast genome­ region adjacent to the insertion
point, was also used. This primer set generated a 1 kb fragment in the
wild-type cells or the spectinomycin mutant. Because the insertion of the
foreign gene had disrupted the natural chloroplast chlL gene and one of
the chlL primer target sites as well, only several­ unspecific weak
bands were obtained in the transformed colony [Fig. 1(C)]. These results
confirmed the integration of the foreign DNA fragment into the C.
reinhardtii chloroplast genomes, and also confirmed that an approximate
homoplasmy had been reached. In addition­ to PCR analysis, the
“yellow-in-the-dark” phenotype of CM2 further confirmed the
successful transformation of C. reinhardtii chloroplasts. The insertion
of foreign DNA into the chlL gene had inactivated the dark-dependent
pathway­ of chlorophyll biosynthesis. After incubation for 10 d in the dark,
the CM2 transformant colony appeared a yellow color, but the wild-type remained
green (data not shown).

Western blotting

The expression of hMT-2
protein in transplastomic C. reinhardtii was examined using the specific
rabbit MT-2 antiserum. By Western blotting, a protein band of approximately 13
kDa was detected in the CM2 sample but not in the wild-type, which was quite
near to the standard dimer of MT-2 (Fig. 2). However, the nonspecific
reaction between­ the native C. reinhardtii protein and the polyclonal
rabbit MT-2 antibodies interfered with the exact assessment­ of hMT-2
expression level.

Standard curve for
measurement of living cell ratio in a C. reinhardtii culture

To study the
relationship between the chlorophyll content­ of Chlamydomonas culture
and the living cell ratio, we carried out an experiment to generate a standard
curve. Wild-type C. reinhardtii culture at the late exponential phase
was prepared for this study. The culture was serially diluted­ on a 96-well
microplate with fresh liquid TAP medium, to a total volume of 300 ml per well with three replicates for each
concentration. After determining the chlorophyll content­ by measuring optical
absorbance at 645 nm and 663 nm, these samples were subsequently measured by
MTT assay. A relation curve was then derived from the absorbance of MTT
formazan at 570 nm and the chlorophyll content was calculated by the A645 and A663 values of the serial algal dilutions
according to Equation 1 [14] (Fig. 3).

Eq. 1

Then we obtained Equation
2 to calculate the theoretical­ value of A₅₇₀ of a living algae system without killing the
cells:

Eq.
2

By this means, the
living cell ratio of a C. reinhardtii liquid culture, before and after a
lethal treatment, could be conveniently compared.

Living cell ratio after
UV-B treatment

The relation derived from
the standard curve was applied­ to test the lethal effect of UV-B on C.
reinhardtii. Wild-type and the hMT-2 transplastomic C.
reinhardtii were grown in mediums containing different supplements on two
parallel 96-well microplates. Before exposure to UV-B radiation, the A645 and A663 values of these cultures were measured, which
were used to calculate the theoretical A570 value. MTT assay was immediately carried out
on one of the parallel microplates after the 8 h UV-B treatment, and 20 h later
on the other microplate for a recovery culture. Taking the A570 value obtained from MTT assays as numerators­
and the calculated A570 from the chlorophyll content as denominators, the obtained ratios
reflected the cell viabilities after each treatment. The survival ratio after
the UV-B treatment is shown in Fig. 4(A). The results showed that living
transplastomic C. reinhardtii cells increased­ in most of the mediums,
except one containing cadmium; the wild-type cells decreased in all of these
tested conditions. However, the 20 h recovery culture eliminated the raised
living cell ratio in the transgenic cells. Under this circumstance, the living
cell ratio of the transgenic cells was quite similar to that of the wild-type [Fig.
4(B)].

Chlorophyll content
variations during UV-B exposure

Chlorophyll contents of
the wild-type and transgenic C. reinhardtii cells were quantitated by
measuring A₆₄₅ and A₆₆₃ values at different time points. Differences between the two
strains at each time point are shown in Fig. 5. It was quite obvious
that the chlorophyll content of the wild-type cells decreased in almost all
circumstances, but that of the transgenic cells slightly increased in most of
the situations.

Influence of metals and
cysteine

During the first 3.5 h
UV-B exposure, significant decreases­ in chlorophyll content were observed in
both wild-type and transgenic cells grown in pure TAP medium or medium
containing 10 mg/ml
cysteine. But the dimi­nishments­ were relatively light in media containing
metals, and a slight increase was even observed in the transplastomic cells
grown in the medium containing 20 mM of zinc. In
the metal-containing mediums, the chlorophyll content of the transgenic cells
slowly increased as the UV-B radiation was prolonged, however, the chlorophyll­
content of wild-type C. reinhardtii was lower than before UV-B exposure.
As far as the recovery is concerned, cysteine­ showed a prominent enhancement
to the growth of both types of cell, but none of the three metals showed such
function. The effects of the metals on both kinds of cell fell into two
categories: zinc and copper protected cells during the UV-B exposure process,
but the living cell ratio in these mediums decreased during the recovering
progress; cadmium lessened the UV-B damage in the course of both UV-B exposure
and the 20 h recovery in a relatively weak way. After 8 h UV-B exposure, cell
viability with 50 mM zinc was
less than that with 20 mM, indicating
that the concentration of metal is also important for its function.

Discussion

In the present study, an
optimized hMT gene was integrated­ into the chloroplast genome of C.
reinhardtii and successfully expressed in the transplastomic alga.
Considering­ the anti-radiation function of MT [5], we mainly studied the
survival capability of the transplastomic alga cells to UV-B exposure.

By this method, we
studied the survival of wild-type and hMT-2 transplastomic C.
reinhardtii cells to UV-B exposure. hMT-2 expression in the
chloroplast of C. reinhardtii could protect cells from the deleterious
effects of UV-B radiation. This conclusion was supported by two facts: (1) the
transplastomic algae had relatively higher viability after 8 h of UV-B
treatment; and (2) during the UV treatment, the chlorophyll content kept
increasing in transgenic cells, while it dropped in wild-type cells. As
chlorophyll is very sensitive to UV-B exposure, the increased­ chlorophyll
content in transgenic cells might be due to the cell division or the maturing
of progeny cells. However, when the UV-treated cultures were recovered for 20 h
in dim lights, the living cell ratio in the transgenic cells decreased to a
similar level to the wild-type cells. Such decline might be explained by the UV
sensitivity rhythm of Chlamydomonas. C. reinhardtii cells showed
the daily sensitivity to UV-B: cells were slightly affected by UV-B during the
day, but were much more sensitive during the night. In a synchronized Chlamydomonas
cell culture, maximal UV sensitivity occurred within the first few hours of the
light-to-dark transition (time of DNA synthesis). C. reinhardtii cells
are more sensitive to UV irradiation during the exponential phase than the
saturation phase [4]. As described above, the transgenic cell cultures might
contain more young cells than the wild-type cultures. When these progeny cells
were exposed to UV irradiation in their early growing phase, the damage was
much more serious. Though they survived after UV-B treatment, they could hardly
last for a long time.

The spectrophotometric-MTT
microscale assay is especially­ suited to studies where the cell viability is
influenced­ by numerous factors. In the present work, survival­ abilities of
wild-type and transplastomic C. reinhardtii cells to UV-B exposure were
examined in the presence of zinc, copper, cadmium and cysteine. The chemicals
were selected­ according to the cysteine-rich and hyper-metal binding
properties of The
spectrophotometric-MTT microscale assay introduced here is an easy and
effective way to analyze cell viability of multiple samples simultaneously. It
is a very promising method for characterizing the sensitivity of C.
reinhardtii to some other lethal effects, which could be applied to many
other algae too.

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