REGULAR PAPER

Mutagenesis and phenotypic selection as a strategy towarddomestication of Chlamydomonas reinhardtii strains for improvedperformance in photobioreactors

Giulia Bonente • Cinzia Formighieri • Manuela Mantelli •

Claudia Catalanotti • Giovanni Giuliano •

Tomas Morosinotto • Roberto Bassi

Received: 8 October 2010 / Accepted: 24 April 2011

� Springer Science+Business Media B.V. 2011

Abstract Microalgae have a valuable potential for bio-

fuels production. As a matter of fact, algae can produce

different molecules with high energy content, including

molecular hydrogen (H2) by the activity of a chloroplastic

hydrogenase fueled by reducing power derived from water

and light energy. The efficiency of this reaction, however,

is limited and depends from an intricate relationships

between oxygenic photosynthesis and mitochondrial res-

piration. The way toward obtaining algal strains with high

productivity in photobioreactors requires engineering of

their metabolism at multiple levels in a process comparable

to domestication of crops that were derived from their wild

ancestors through accumulation of genetic traits providing

improved productivity under conditions of intensive culti-

vation as well as improved nutritional/industrial properties.

This holds true for the production of any biofuels from

algae: there is the need to isolate multiple traits to be

combined and produce organisms with increased perfor-

mances. Among the different limitations in H2 productiv-

ity, we identified three with a major relevance, namely:

(i) the light distribution through the mass culture; (ii) the

strong sensitivity of the hydrogenase to even very low

oxygen concentrations; and (iii) the presence of alternative

pathways, such as the cyclic electron transport, competing

for reducing equivalents with hydrogenase and H2 pro-

duction. In order to identify potentially favorable muta-

tions, we generated a collection of random mutants in

Chlamydomonas reinhardtii which were selected through

phenotype analysis for: (i) a reduced photosynthetic

antenna size, and thus a lower culture optical density; (ii)

an altered photosystem II activity as a tool to manipulate

the oxygen concentration within the culture; and (iii) State

1–State 2 transition mutants, for a reduced cyclic electron

flow and maximized electrons flow toward the hydroge-

nase. Such a broad approach has been possible thanks to

the high throughput application of absorption/fluorescence

optical spectroscopy methods. Strong and weak points of

this approach are discussed.

Keywords Green microalgae � Photosynthesis � Biomass

accumulation � Random mutagenesis � Chlamydomonas

Abbreviations

Chl Chlorophyll

Car Carotenoid

LHC Light-harvesting complex

H2 Hydrogen

Chl a/b Chlorophyll a to b ratio

Fo/Fv/Fm Basal/variable/maximum fluorescence

St State transition

PSI (II) Photosystem I(II)

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11120-011-9660-2) contains supplementarymaterial, which is available to authorized users.

G. Bonente � C. Formighieri � M. Mantelli � R. Bassi (&)

Dipartimento di Biotecnologie, Universita di Verona, Strada Le

Grazie 15, 37134 Verona, Italy

e-mail: roberto.bassi@univr.it

C. Catalanotti � G. Giuliano

Italian National Agency for New Technologies, Energy and

Sustainable Development (ENEA), Casaccia Research Center,

Via Anguillarese 301, 00123 Rome, Italy

Present Address:C. Catalanotti

Department of Plant Biology, Carnegie Institution for Science,

260 Panama Street, Stanford, CA 94305, USA

T. Morosinotto

Dipartimento di Biologia, Universita di Padova, Via U. Bassi

58/B, 34134 Padova, Italy

123

Photosynth Res

DOI 10.1007/s11120-011-9660-2

WT Wild type

DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea

NaN3 Sodium azide

Introduction

Biofuels production from microalgae is an interesting

perspective for contributing to global energy supply in next

decades since these organisms have the capacity of doing

photosynthesis with high efficiency to synthesize mole-

cules with high energy value. One option consists in

exploiting the green algae activity of producing hydrogen

(H2) using water and light energy. This represents the best

possible energy source in terms of cleanness and renew-

ability; nevertheless, major productivity limitations impair

large-scale application of this biological activity. Several

limiting steps affect the H2 productivity and different

research approaches must be pursued to overcome these

bottlenecks. This is generally true for the production of any

biofuel from algae, where multiple mutations must be

combined in order to redirect the metabolism in producing

high yield of desired molecules.

One of the major limitations for any large-scale algal

culture is the highly non-homogeneous light distribution

within the photobioreactor. This causes a strong limitation in

the solar light conversion efficiency. Indeed, many green

algae are equipped with a large antenna system, including

hundreds of chlorophyll (Chl) molecules per reaction center.

These pigments maximize the light-harvesting efficiency as

an evolutionary adaptation to a natural environment where

solar radiation is often limiting for growth and cell density is

very low (Kirk 1994), while mobility of algae in the water

column ensure escape from excess illumination. In large

capacity photobioreactors, however, algae are exposed to

very different conditions: biomass concentration is many

orders of magnitude higher and the strong optical density of

pigments limits light penetration. The consequent light

gradient along the photobioreactor optical path results into

an overall reduced photosynthetic yield and thus a lower

productivity (Neidhardt et al. 1998; Melis et al. 1998; Melis

2009). Light at the peripheral layers exceeds the photo-

chemical capacity of cells and leads to the activation of

photoprotective mechanisms, collectively known as non-

photochemical quenching (NPQ) that dissipate excess

energy into heat thus avoiding the formation of reactive

oxygen species. Therefore, the surface-located cells are

inefficient in photosynthetic light conversion into biomass,

far below the theoretical value of 8–10% (Melis 2009;

Posten and Schaub 2009). Consequently, cells in the inner

layers only receive a negligible amount of light, which

reduces photosynthetic rate and biomass accumulation.

Optimization of photobioreactor design and engineering,

although it offers a contribution to relieve this limitation, is

insufficient for a complete solution of this problem. A

strategy toward an improvement of homogeneity in light

distribution is to develop algal strains with a reduced

antenna size and a decreased optical density per cell (Chisti

2007; Melis 2009; Kok 1953). This approach is conceivable

because only approximately 50 and 90 of the 350 and 300

Chl molecules, respectively, found in each photosystem II

(PSII) or photosystem I (PSI) unit in Chlamydomonas

reinhardtii (Melis 1991) are strictly required for photo-

chemical activity, while the rest is bound to the light-har-

vesting complex (LHC) and are, in principle, dispensable.

Strains with a reduced number of Chl molecules per reaction

center are expected to be less efficient in absorption,

allowing for a better light penetration into the culture.

Successful demonstrations of this principle have been

obtained with strains from genetic manipulation such as

RNA interference of LHCI and LHCII genes (Mussgnug

et al. 2007) and isolation of insertion mutants (Polle et al.

2003). These strains showed a reduced antenna size of

photosystems, increased photosynthetic yield in high light

and saturation of photosynthesis occurring at higher light

intensities with respect to wild type (WT).

Strains with reduced antenna, however, might also

exhibit enhanced photosensitivity in high light since the

pigment binding complexes, besides light harvesting, are

also involved in photoprotection. Nevertheless, it is pos-

sible to distinguish, within the members of the LHC family,

polypeptides specialized in light-harvesting from others

devoted to photoprotection (Avenson et al. 2008; Horton

and Ruban 2005). Thus, the ideal truncated-antenna strain

should not be deleted in all antennas but it rather should

maintain those subunits active in quenching and/or scav-

enging mechanisms, while reducing light-harvesting

capacity and optical density.

A second major problem is the incompatibility between

oxygenic photosynthesis and H2 evolution. As a matter of

fact, hydrogenase activity is strongly inhibited by oxygen,

preventing contemporary H2O photobiolysis by PSII and H2

production. Upon illumination, dark-adapted green algae

cultures show high rates of H2 evolution, quickly declining

because of inhibition from PSII-produced O2 (Zhang and

Melis 2002). Since photosynthetic activity is needed in order

to provide reducing equivalents for hydrogenase activity, a

‘‘two stage’’ process allows temporal separation of O2 and H2

evolution (Melis et al. 2000): in a first stage, photosynthesis

is active and accumulates biomass. In a second stage, algae

are transferred to a sulfur-depleted medium where de novo

protein biosynthesis is inhibited particularly affecting the

synthesis of the fast turning-over PSII subunit D1. This

causes inefficient PSII repair and decline in oxygen evolu-

tion. In these conditions, oxygen consumption by respiration

Photosynth Res

123

is instead maintained, eventually leading to anaerobiosis

with induction of hydrogenase synthesis and H2 evolution

(Ghirardi et al. 2000). Reducing equivalents for H2 produc-

tion derive from the residual activity of PSII as well as from

the degradation of reserve molecules and sulfur deprivation

cannot be prolonged indefinitely and after a few days the

photosynthetic activity must be restored by sulfur repletion

in the medium (Chochois et al. 2009). Since sulfur depri-

vation is not applicable to large-scale industrial facilities,

alternative strategies for genetic control of PSII activity are

required to separate photosynthesis from H2 evolution. A

system for inducible control of PSII activity based on the

MBD1/CYC6 promoter has been reported (Surzycki et al.

2007). The NAC2 protein, encoded by the MBD1 gene, is

required for the stable expression and accumulation of the

PSBD mRNA; the repression of the CYC6 promoter has

been obtained by addition of copper ions. This interesting

inducible system opens the possibility of multiple cycles of

photosynthetic accumulation of endogenous substrates and

their anaerobic degradation to fuel hydrogenase activity

(Ferrante et al. 2008). While this system has been shown to work

in order to develop strains suitable for industrial applications, it is

important to have multiple gene products controlling PSII

activity to develop strains with the best productivity.

A third major limitation for bio-hydrogen production is

the presence of sinks of reducing equivalents which are

competing with hydrogenase. Consistently, inhibition of

cyclic electron transport leads to sustained H2 production

(Finazzi et al. 2002; Kruse et al. 2005).

Genetic manipulation of WT algae is required to make

biofuels production economically sustainable. Independently

from the final product chosen, there is the need of isolating

several positive traits to be combined to obtain a more

productive strain on a large scale. In order to select some of

these valuable mutations, we generated a C. reinhardtii

insertional mutant library and screened for phenotypes useful

to overcome the three above discussed bottlenecks. The

application of fluorescence/absorption techniques allows to

identify mutations affecting the antenna system, the PSII

reaction center activity or the reduction of plastoquinone

pool (PQ) (Elrad et al. 2002; Polle et al. 2003; Depege et al.

2003; Bellafiore et al. 2005). Association of these mutations

into a single strain might lead to an organism better suited

for cultivation in photobioreactors and production of H2 at

enhanced rates.

Materials and methods

Strains and growth conditions

WT cw15 (mt-) was used as a genetic background to

generate the insertion library. WT 8b (mt?), kindly

provided by J.D. Rochaix (University of Geneve) was

employed for backcross analysis. C. reinhardtii cells were

grown at light intensity of 60 lmol/s/m2, 45 rpm agitation,

25�C controlled temperature, 16 h light/8 h dark photope-

riod. C. reinhardtii strains were cultured in acetate and

sorbitol 1% supplemented TAP medium (Harris 1989).

Generation of the mutant library

EcoRI linearized pSL18 vector (Fischer and Rochaix 2001)

which carries the AphVIII gene (aminoglycoside 30-phos-

photransferase type VIII from Streptomyces rimosus) con-

ferring resistance to paramomycin sulfate, under the

C. reinhardtii HSP70 and RBCS2 promoter control (Sizova

et al. 2001), was used to produce transformants according

to the protocol described by (Kindle 1990). After trans-

formation, cells were plated on paromomycin 10 mg/l

supplemented TAP plates for the selection of transformant

lines.

In vivo fluorescence-based high throughput screening

Fluorescence kinetic curves were recorded with a home-

built video-imaging apparatus (saturating light is

1,000 lmol/s/m2). Since we expect the large majority of

strains to have unaffected fluorescence, we calculated

averages and standard deviations of all colonies for each

plate. Colonies showing significant differences with respect

to the WT were retained for further analysis. Further

analysis were performed with a PAM-101 (Waltz, Effel-

trich, Germany), saturating light was 4,080 lmol/s/m2.

High throughput pigment analysis

Each of the 96 wells of microtiter plates was inoculated

with a single insertion line in TAP medium and cultured in

control light (60 lmol/s/m2). In the late exponential

growth, phase pigment was extracted with 80% acetone.

Absorption spectra in the visible region between 350 and

750 nm were recorded with a SAFAS Xenius XL spec-

trophotometer. Different spectral regions were used to

quantify different pigment species: 660–665 for Chl a,

643–650 and 460–465 nm for Chl b, and 485–505 nm for

carotenoids (Car).

State transitions (St) screening and confirmation

The difference between maximum fluorescence (Fm) in

State 1 and in State 2 is an indicator of St capacity, which

can be calculated as: St% = (Fm state 1 - Fm state 2)/Fm

state 1%. Anaerobiosis in the dark is known to induce PQ

reduction in C. reinhardtii by reducing equivalents coming

from other cell compartments (e.g., mitochondrion) (Peltier

Photosynth Res

123

and Schmidt 1991; Peltier and Cournac 2002). As high

throughput screening, St was measured on TAP plates sup-

plemented with 3-(3,4-dichlorophenyl)-1,1-dimethylurea

(DCMU) 10-6M. A 15-min light exposure (100 lmol/s/m2)

induces State 1 while the sample plate, long dark incubated

in the presence of a N2 flux, switches toward anaerobiosis

and thus State 2 (Fleischmann et al. 1999). The confirma-

tion of primarily selected mutants was obtained according

to another protocol proposed by the same authors: State 1

was induced through vigorous shaking in the dark and by

adding DCMU 10-5M 1 min before the measurement,

while State 2 through a 20-min long dark incubation in the

presence of 250 lM sodium azide (NaN3).

Spectroscopic analysis

Absorption spectra were obtained with an AMINCO

DW2000 spectrophotometer, scan rate 2 nm/s, bandwidth

1 nm, optical path length 1 cm.

Pigment content determination

Pigments were extracted from pelleted cells, samples were

frozen in liquid nitrogen and resuspended in 80% acetone

buffered with Na2CO3, then the supernatant of each sample

was recovered after centrifugation (15 min at 15,0009g,

4�C); separation and quantification of pigments were per-

formed by HPLC (Lagarde et al. 2000). Chlorophyll a to

b ratio (Chl a/b) and Chl/cars ratio were corrected through

fitting analysis of the absorption spectrum (Croce et al.

2002).

SDS-PAGE electrophoresis and immunoblot

Denaturing SDS-PAGE was performed as described (Lae-

mmli 1970). Gels were transblotted onto a nitrocellulose

filter, against anti CP43, D2, PsaA, Cyt f, and LHCII sera

and developed through the alkaline phosphatase detection

system.

PSII and PSI functional antenna size estimation

Relative PSII antenna size has been estimated from Fm

saturation kinetic (1/t2/3) in the presence of DCMU 10-5M

(Cardol et al. 2008). The kinetic was measured with a

home-built apparatus. Fluorescence was excited using a

green LED with a peak emission at 520 nm (intensity

20 lmol/s/m2) and detected in the near infrared. Relative

PSI functional antenna size has been estimated from P700

photo-oxidation kinetic (Polle et al. 2000; Melis 1989)

upon application of a non-saturating light (82 lmol/s/m2,

10 s) and using a JTS-10 Joliot-type spectrophotometer

(Melis 1989). The analysis has been performed on 80 lg/

1 ml of stacked thylakoids. Stacked thylakoids preparation

has been performed accordingly to Bassi and Wollman

(1991), stopping the procedure before the fractionation of

grana membranes reported by authors. Briefly collected

cells were re-suspended in 20 mM Tricine pH 8, 0.4 M

sorbitol, 10 mM MgCl2, 0.2% BSA, 10 mM NaF, and

sonicated 5 s in the case of a cell wall-less strain. Broken

cells were centrifuged at 6,0009g and re-suspended in

20 mM Tricine pH 8, 0.1 M NaCl, 5 mM MgCl2, 0.2%

BSA, 10 mM NaF. After incubation on ice for 15 min,

thylakoids were collected by centrifugation at 8,0009g and

re-suspended in 20 mM Hepes pH 7.6, 5 mM MgCl2,

10 mM NaCl, 20% sorbitol, 10 mM NaF for PSI antenna

size measurement, with the addition of 60 lM DCMU that

inactivates PSII and 250 lM methyl viologen as PSI

electron acceptor.

Flanking region rescuing

Isolation of genomic DNA was performed according to the

manufacturer manual (Purification of total DNA from plant

tissue, mini protocol, QIAGEN).

In the Inverse PCR technique (Ochman et al. 1988), the

template for the PCR is a genomic restriction fragment

ligated upon itself, hence the resulting recombinant molecule

contains sequences from both the pSL18 vector and the

genomic flanking region. The primers for the following PCR

are oriented in the reverse direction of the usual orientation in

order to amplify an unknown region flanking a specific one.

Two enzymes among the endonucleases tested in the present

work were SchI and Tru9I, that cut 10 and 23 times respec-

tively in the pSL18 vector sequence; one of the sites is located

at position 635 or 699 respectively from the unique EcoRI

site. A first PCR was performed with oligos pSL18_543up

and pSL18_522dw (Electronic Supplemental Table 1), fol-

lowed by a second PCR with the nested oligos pSL18_594up

and pSL18_494dw (Electronic Supplemental Table 1). The

oligos pSL18_594up and pSL18_494dw were used to

sequence the 50 flanking region. Thermal asymmetric inter-

laced (TAIL)-PCR utilizes nested-specific primers in suc-

cessive reactions together with a shorter arbitrary degenerate

primer, so that the relative amplification efficiencies of

specific and non-specific products can be thermally con-

trolled (Dent et al. 2005; Liu and Whittier 1995). The nested-

specific primers in the present study were pSL18_706dw and

pSL18_522dw or pSL18_3847 up and pSL18_4340 up for

the 50- and 30-ends, respectively (Electronic Supplemental

Table 1). The short degenerate oligonucleotide chosen,

RMD227 (Electronic Supplemental Table 1), was that

already successfully used by Dent et al. (2005). Details of the

PCR reaction mix and thermal cycles have been described

(Dent et al. 2005). The restriction enzyme site-directed

Photosynth Res

123

amplification PCR (RESDA-PCR) strategy has been

recently reported in order to isolate flanking marker DNA in

insertional mutants (Gonzalez-Ballester et al. 2005). Briefly,

nested-specific primers (pSL18_522dw and pSL18_494dw

or pSL18_3847 up and pSL18_4340 up for the 50 and 30 ends

respectively, Electronic Supplemental Table 1) were uti-

lized in successive reactions together with a primer designed

to have a restriction site included in a low degenerated

sequence at the 30-end and a specific adapter sequence (Q0)

at the 50-end, with the two ends being linked by a polyinosine

bridge. The annealing of the primer depends on the random

distribution of the frequent restriction site in the genome.

Degenerate primers tested in the present work were Deg ApaI

and Deg Eco72I (Electronic Supplemental Table 1), with the

restriction sites ApaI and Eco72I not present in pSL18.

During the second round of the PCR, the Q0 primer (Elec-

tronic Supplemental Table 1) was used reducing non-spe-

cific products. Plasmid rescue (Gumpel and Purton 1994) is a

technique that does not depend on PCR reactions. Genomic

DNA was digested with KpnI, which has a unique site in

pSL18 at position 3467. Restriction fragments were self-

ligated. Again, the resulting recombinant molecule contains

sequences from both the pSL18 vector and the genomic

flanking region, and was amplified in E. coli (pSL18 has the

bacterial origin of replication and AmpR marker for selec-

tion) and finally sequenced. Chlamydomonas nuclear gen-

ome is GC-rich, often leading to problems for DNA

amplification. A DNA polymerase that has given us some

good results is AccuTaq (SIGMA).

Sequencing techniques

All the fragments obtained were purified and sequenced

using BigDye terminator Kit v3.1 (Applied Biosystem) on

an ABI 3730 sequencer with 50-cm capillaries. The

sequences, after trimming against the vector, were then

blasted on JGI Chlamy Genome center blast v 3.0 (http://

www.jgi.doe.gov). All the flanking sequences identified

were then confirmed by PCR.

Genetic crosses

Genetic crosses and progeny analysis to assess linkage of the

observed phenotype with antibiotic resistance were per-

formed accordingly to established methods (Harris 1989).

Results

Generation of an insertional mutant library

An insertional mutant library was generated by trans-

forming C. reinhardtii strain cw15 (Harris 1989) with the

linearized pSL18 plasmid (Fischer and Rochaix 2001). The

pSL18 insertion in the nuclear genome occurs randomly,

thus having probability of interrupting endogenous genes

and producing mutants. This approach is particularly suit-

able for the case of Chlamydomonas since nuclear trans-

formation has good efficiency, especially using cw15, a cell

wall-less strain. With this approach, we generated around

3,500 mutants, which were thereafter screened through

multiple approaches.

Screening for reduced antenna and PSII knock out:

fluorescence-based screening

In the first step, we exploited in vivo fluorescence, mea-

sured simultaneously in multiple colonies on TAP-agar

plates using a video-imaging apparatus. With such instru-

mentation, we measured fluorescence of dark-adapted cells

(Fo), and after a pulse of saturating light, we also deter-

mined Fmax (Fm), in conditions when all PSII primary

acceptors are reduced.

The fluorescence rise from Fo to Fm is observed only in

the presence of a functional PSII, while in its absence

fluorescence levels are constant (Fig. 1, mutant et3). This

kind of analysis of fluorescence kinetics allows identifying

mutants in PSII but also carrying other alterations in

electron transport chain. For instance, impairment in PSI or

Cytb6f produces alterations in the fluorescence induction

kinetic (Bennoun and Levine 1967). Colonies with a fluo-

rescence induction curve having a shape significantly dif-

ferent from the parental strain were thus retained for further

analysis (Fig. 1).

0 200 400 600 800 1000 12000

100

200

300

400

500

600

700

800

Flu

ores

cenc

e (a

.u.)

time (ms)

Non-selected mutant 1 Non-selected mutant 2 et3 as1 WT

Fig. 1 Induction fluorescence curves measured for transformant

colonies using a video-imaging apparatus. Mutants showing different

fluorescence intensity (mutant as1) or altered kinetics (mutant et3)

with respect to WT were retained for further analysis. On the

contrary, all mutants showing curves close to WT (±15%) were not

retained

Photosynth Res

123

The shape of the curve is not the only information these

measurements can provide. The maximal fluorescence Fm

depends from the number of Chl bound per PSII and its

value is therefore roughly indicative of PSII antenna size

(Polle et al. 2003). Consequently, mutants with reduced

antenna have a lower Chl fluorescence with respect to the

WT (e.g., Fig. 1, mutant as1). Clearly, in each plate, a

certain variability in maximal fluorescence is present

independently from the occurrence of mutations. This was

determined to be around 10–15% and we thus retained

mutants showing differences larger than 15% with respect

to WT. About 3.5% of the colonies (&120) were selected

in this first phase considering both selection criteria.

Screening for antenna size based on pigment content

Fluorescence in vivo is pre-eminently originating from

PSII since fluorescence yield of PSI is low at room tem-

perature; thus, the above-described method was effective

only for mutations affecting PSII antenna. An alternative

approach, sensitive to alteration in both PSI and PSII

antenna systems, can exploit the different pigment binding

of photosystems core complexes versus antenna systems.

As a matter of fact, antenna proteins bind Chl a, Chl b, and

xanthophylls, while core complexes only bind Chl a and

b-carotene. Therefore, mutants with altered antenna com-

position can be identified on the basis of changes in Chl

a versus Chl b content and/or Chl versus Car content. To

this aim, putative mutants were grown in 96-wells micro-

titer plates and their pigments extracted with 80% acetone.

Since Chl a, b, and Car have different absorption proper-

ties, spectra of acetone extracts provide information on the

relative pigment content (Fig. 2). This screen led to iden-

tification of 2.5% (&90) of the colonies with acetone

extract spectra significantly different with respect to WT.

Screening for impaired State 1–State 2 transitions

Video-imaging fluorescence measurements can be exploi-

ted to screen the insertion library also for St mutants, as

shown in Fleischmann et al. (1999). Since fluorescence

intensity is directly proportional to the size of PSII asso-

ciated antenna, cells adapted to State 1 will have higher

fluorescence yield than cells adapted to State 2, and the

capacity for performing St can be quantified by the fluo-

rescence difference in cells adapted in the two states. The

transition between the two states depends on the redox state

of PQ and we induced State 1 and State 2 by illuminating

cells in the presence of DCMU 10-6M and fluxing N2 in

the dark, respectively. C. reinhardtii stt7 mutant has been

used as negative control, since it lacks the STT7 kinase

protein responsible of LHCII phosphorylation and thus is

permanently in State 1 (Depege et al. 2003). Figure 3

reports an example of St measurement, where strains str4

and 5 show significantly reduced St% values with respect

to the WT. A first, single repeated, screening allowed the

selection of about 2% colonies (&70).

Confirmation of phenotypes

Results from the different screening methods are largely

dependent on colony/culture growth in agar plates/

350 400 450 500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

Abs

orpt

ion

(a.u

.)

wavelength (nm)

WT nonselected as1 as2 as3

Car

Chl bChl a

Fig. 2 Absorption spectra in the visible region obtained for WT and

some transformants. Data are normalized at Chl a absorption the Qy

region (660 nm). Absorption regions where signals from Carotenoids

(Car), Chl a and b are detected are also indicated. Spectra evidence

differences in pigment content in some analyzed mutants (as1, 2 and

3), while most of them are very similar to WT and were not retained

(see example of non-selected mutant)

0

2

4

6

8

10

12

14

16

18

20

Non selected mutant

str5str4stt7

ST

%

WT

Fig. 3 State transition analysis of colonies plated on TAP-agar

containing DCMU 10-6 M. Accordingly to the protocol from

(Fleischmann et al. 1999), State 1 is induced in white light

(100 lmol/s/m2), while State 2 in anaerobiosis fluxing N2 in the

dark. WT constitutes the positive control, while stt7 mutant the

negative one. For the definition of ST%, see ‘‘Materials and methods’’

section

Photosynth Res

123

microtiter wells, which could thus produce a large number

of false positives. For this reason, all selected mutants were

subjected to a second round of screening in order to reduce

the number of false positives.

Colonies selected from the fluorescence imaging

screening were then further analyzed using more sensitive

methods. For the identification of mutants affected in PSII

activity, we exploited a PAM fluorometer, which has a

better time resolution and sensitivity. The composition of

the photosynthetic apparatus of candidate mutants was also

evaluated by western blotting using antibodies against

CP43, D2, PsaA, Cyt f, and LHCII subunits. PSI, PSII,

and Cyt f are all multisubunit complexes with a strong

post-transcriptional control of assembly (Rochaix 1996;

Choquet et al. 2001) and any alteration in expression/

accumulation of one subunit would result into a strong

downregulation of all proteins belonging to these com-

plexes. Thus, western blotting data are indicative not only

of the absence of one specific subunit, but also of the

concentrations of PSI, PSII, and Cyt b6f complexes. Such

analysis was effective in discriminating mutants affected at

different steps of the electron transport chain also down-

stream from PSII.

The selection for PSII mutants led to the isolation of five

mutants clearly depleted in PSII activity (Table 1), as con-

firmed by the absence of variable fluorescence (Fv), the

absence of anti CP43-reacting bands (Fig. 4a and b, analysis

of et3 and et4 is shown as an example) and the inability to

grow in minimal medium (not shown). Interestingly, the

screening also resulted in the selection of mutants altered in

other steps of the photosynthetic chain (Table 1); in par-

ticular, we obtained two mutants depleted in Cytochrome f

(et6 and et7) and one mutant depleted in PSI (et8).

In the case of mutants selected for a different fluores-

cence yield or pigment content, pigments extracted from

cells grown in control conditions were analyzed by

absorption spectroscopy and HPLC. The selection for

colonies with altered pigment content with respect to WT

led to the isolation of 11 confirmed mutants. However, only

two of them (as1 and as2, Table 1) displayed a phenotype

clearly suggesting a reduction of antenna content, as shown

by their high Chl a/b ratio, 6.4 and 3.5, respectively, versus

2.7 in the WT. This suggestion was confirmed by the

observation that as1 and as2 also showed increased

b-carotene content, consistent with this Car being specifi-

cally bound to reaction centers (Table 2). We verified

that functional PSII antenna size, as estimated from the

kinetics of fluorescence induction in the presence of

DCMU (Cardol et al. 2008), was indeed reduced especially

in the case of as1. PSI functional antenna size was esti-

mated from P700 oxidation kinetics, according to an

established method (Polle et al. 2000; Melis 1989), and is

reduced as well, especially in as1 mutant (Fig. 6).

State 1–State 2 transition measurements were repeated

on the first lot of selected mutants by applying a more

accurate method (Fleischmann et al. 1999). State 1 was

induced by vigorously shaking the liquid cultures in the

dark, followed by addition of DCMU 10-5M 1 min before

the measurement. State 2 was obtained by dark incubation

in the presence of NaN3, a chemical known to inhibit

mitochondrial cytochrome oxidase, resulting in the

increase of the glycolytic reactions in order to synthesize

ATP and enhance the non-photochemical reduction of the

PQ pool (Rebeille and Gans 1988). Fm in State 1 and State

2 was measured using a PAM fluorometer. The difference

Table 1 Mutants selected combining the different screening methods

and grouped in three main classes of interest

Electron transport/PSII KO

et1 PSII KO

et2 PSII KO

et3 PSII KO

et4 PSII KO

et5 PSII KO

et6 Cyt F KO

et7 Cyt F KO

et8 PSI KO

et9 Reduced Cyt F

et10 Increased D2

et11 Reduced D2, PsaA

Antenna size/pigment composition

as1 Higher a/b ratio, reduced Chl content

as2 Higher a/b ratio, reduced Chl content

as3 Reduced Chl content

as4 Reduced a/b ratio, higher fluo

as5 Lower fluo, reduced a/b ratio,

higher Car content

as6 Reduced Car content

as7 Reduced Car content

as8 Reduced Car content

as9 Slightly pale green, lower fluo,

altered fluo kinetic

as10 Pale green only in the dark

as11 Pale green only in the dark

State transitions

str1 Altered state transitions

str2 Altered state transitions

str3 Altered state transitions

str4 Altered state transitions

str5 Altered state transitions

str6 Altered state transitions

str7 Altered state transitions

str8 Altered state transitions

The mutants with interesting phenotypes for hydrogen production/

growth in photobioreactors are highlighted in bold

Photosynth Res

123

between Fm in state 1 and in state 2 is an indicator of St

amplitude. The analysis allowed the confirmation of eight

mutants impaired only in St (Table 1). Some of the mutants

mentioned above (as1 and as5 for example) also displayed

a St phenotype but this most likely was a secondary effect

of mutations on electron transport chain, photosystems, or

antenna systems. Moreover, among these eight State

1–State 2 mutants, it is possible that the phenotype is due to

other mutations which have secondary effects on St. For

instance, str1 also displayed a complex phenotype, being

affected not only in St but also in the recovery of PSII

activity (Fv/Fm) after a high light stress (Electronic Sup-

plementary Fig. 1).

Overall, in the first phase of the screening, we retained

around 200 strains, many of them being selected by more

than one screening method. In total, we were able to con-

firm the phenotype of 30 of them, thus with a 15% yield,

showing that in the first round of screening we had retained

a large number of false positive colonies. This figure was

particularly high in the case of methods aimed at selecting

antenna size mutants, while it was lower in the case of

screening for St.

Identification of the genomic regions flanking

the insertion vector

Confirmed mutants were analyzed through molecular

techniques in order to identify the genomic region (flanking

region) interrupted by the exogenous DNA insertion and

thus the gene responsible for the phenotype. Different

sequencing strategies were attempted, such as Plasmid

rescue (Gumpel and Purton 1994), Inverse PCR (Ochman

et al. 1988), TAIL-PCR (Dent et al. 2005), and RESDA-

PCR (Gonzalez-Ballester et al. 2005). These techniques,

however, showed a low success rate, a common feature

when applied to C. reinhardtii templates. In Table 3,

sequences obtained and confirmed through PCR on geno-

mic DNA are listed. We succeeded in obtaining informa-

tion on the DNA sequence flanking one of the insertion

ends in six cases only (out of 30 mutants listed in Table 1).

Moreover, we started the backcrosses and genetic analysis

of these mutants in order to verify the co-segregation

between the marker selection resistance and the observed

phenotype, indicative for the presence of single or multiple

insertions (Table 3).

Discussion

In this work, we screened a C. reinhardtii mutant collection

for the presence of phenotypes which may be helpful for

increasing the capacity of this alga of producing H2. The

search for algal strains capable of producing biofuels at

high yield requires the isolation of several positive traits

which must be combined to achieve the maximal produc-

tivity and finally exploit the potential yield for the organ-

ism under analysis.

Fig. 4 a Fluorescence induction analysis on mutants et3 and et4 with a PAM fluorometer, b western blots against CP43, LHCII, PsaA and Cyt

f on WT and mutant thylakoids (loaded on an equal chlorophyll basis, 3 lg)

Table 2 Pigment content (picomoles) of antenna mutant as2 and as1with respect to WT

b-car Chl/Car ratio a/b ratio Cars Chl a ? b

WT 7.3 3.2 2.7 30.9 100.0

as2 25.7 1.7 3.5 58.5 100.0

as1 34.4 1.5 6.4 66.6 100.0

Data are normalized to 100 pmol of chlorophyll a ? b. Standard

deviation within 15%. Data refers to three independent experiments

Photosynth Res

123

Mutants with a reduced antenna size

As stressed by several authors in many reports, a way to an

increased algal productivity in photobioreactors requires

selecting strains with reduced antenna size. In our screen-

ing, we did not find mutants from insertion into individual

genes encoding subunits of the LHC superfamily, but we

did isolate two interesting mutants showing a large deple-

tion in antenna complexes, namely as1 and as2. In both

mutants, a large reduction in antenna complexes was

accompanied by reduced Chl content per cell (‘‘pale-

green’’ phenotype), particularly evident in as1. Biochemi-

cal and physiological data confirmed that antenna size of

both photosystems is reduced in these mutants: the Chl a/b

ratio is increased up to 3.5 and 6.4 for as2 and as1,

respectively, versus 2.7 in WT (Table 2) and b-carotene

content is increased as well (Table 2). Spectroscopic esti-

mation of PSII antenna size, through the kinetic of Fm

saturation in the presence of DCMU (Cardol et al. 2008),

confirmed the functional PSII antenna reduction in both

mutants, particularly large in as1 (Fig. 5). PSI antenna size

was also significantly reduced in both mutants (Fig. 6).

All experiments described confirm that the antenna

system of as1 is more severely affected with respect to that

of as2. However, both mutants grow photoautotrophically

even with strong illumination at 400 lmol/s/m2 (not

shown), suggesting these strains might be useful for

improved growth in high light-exposed photobioreactors.

Unfortunately, we did not succeed in obtaining information

about the insertion flanking regions in as1 and as2 with the

molecular techniques described in this article. However,

backcrosses and progeny analysis in both cases demon-

strated co-segregation between the antenna phenotype and

the paromomycin resistance cassette. As a matter of fact,

no recombinant colonies were observed in both cases

among progeny (132 and 121 colonies analyzed respec-

tively for as2 and as1 backcross), thus supporting the

hypothesis that a single insertion, responsible for the

Table 3 List of mutants whose flanking region was identified through molecular techniques and confirmed by PCR on nuclear DNA

Mutant Phenotype Flanking (Genome Chre4) Cosegregation

with paroR

Recombinants/

progeny

Sequencing

technique

as3 Reduced

chlorophyll

content

50 Flanking: chromosome 5, 3081998:3082413 (416 bp). Gene

model: GUN4 tetrapyrrole binding protein.

Yes 0/75 Inverse/Tail/

Resda-PCR

et2 PSII KO 50 Flanking: chromosome 13, 1177049:1177453 (404 bp). No 47/82 Plasmid

rescue

et6 Cyt F KO 50 Flanking: chromosome 9, 4472552:4472915 (364 bp). Gene

model: TCA1 translation factor for chloroplast petA RNA.

Not verified – Inverse PCR

str4 State transitions 30 Flanking: chromosome 6, 169829:170020 (192 bp). Gene model:

DHC1b Cytoplasmic dynein 1b heavy chain.

Not verified – Inverse PCR

str5 State transitions 50 Flanking: chromosome 8, 2080692:2081201 (510 bp). Not verified – Plasmid

rescue

str1 State transitions 50 Flanking: chromosome 1, 5488277:5488408 (132 bp). Gene

model: DNJ9 dnaJ-like protein.

No 44/84 Inverse PCR

4000200000.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

as1as2

Rel

ativ

e P

SII

ante

nna

size

Genotype

B

WT

Flu

ores

cenc

e (a

.u.)

time (ms)

WTas2as1

A

Fig. 5 Relative PSII functional antenna size of as2 and as1 mutants

estimated through fluorescence induction kinetics in the presence of

DCMU (Cardol et al. 2008). a Fluorescence curved normalized to Fm,

b data shown in panel A plotted as (t2/3)-1. Error bars refer to five

replicates

Photosynth Res

123

phenotype and the antibiotic resistance, is present in both

mutants, likely to be identified by future analyses.

as3 mutant is knocked out in the GUN4 gene

A third mutant with a ‘‘pale-green’’ phenotype was isolated

and screened because of an altered Chl/Car ratio (as3).

Pigment analysis confirmed a reduced Chl content per cell

(Table 1), accompanied by a photosensitive phenotype.

The insertion site was mapped through multiple molecular

approaches (Inverse PCR, Tail PCR, RESDA-PCR) in the

second exon of the GUN4 gene sequence in chromosome 5.

Backcrosses and progeny analysis also confirmed the

co-segregation between the observed phenotype and the

paromomycin cassette (Table 3), strongly suggesting that a

single insertion, in the GUN4 ORF, is responsible for the

observed phenotype. A mutant in the homologous gene in

Arabidopsis thaliana has been isolated in a screen for

genes affecting the Plastid-to-Nucleus retrograde signaling

(Genome Uncoupled) (Larkin et al. 2003). In Arabidopsis,

the GUN4 protein has been localized in the chloroplast and

has been shown to be involved in the Chl biosynthesis

pathway, interacting with the Mg-chelatase enzyme and

regulating its activity. The ‘‘pale-green’’ phenotype of the

Chlamydomonas as3 mutant is consistent with a mutation

in a Chl biosynthesis regulatory factor. This mutant,

although not useful for the purposes of our screening

because of its light sensitivity, may prove a useful tool for

the study of photosystem organization under limited Chl

supply and the role of Chl precursors in controlling nuclear

gene expression (retrosignalling).

Other mutants with altered pigment content

Next to the antenna and the gun4 mutants, eight other

strains were selected for alterations in the pigment content.

In particular, four of them were screened for an altered Chl/

Car content. Mutant as5 showed higher Car content, beside

a lower Chl a/b ratio and a lower in vivo fluorescence

yield. On the opposite, mutants as6, as7, and as8 showed

reduced Car content. The high number of mutations

affecting Car accumulation suggests the presence of many

factors affecting Car biosynthesis and/or accumulation. An

alternative origin for these mutants could be an altered

composition of Car-binding proteins.

Interestingly, two selected mutants (as10 and as11)

displayed the same phenotype: they were yellow in the

dark, while under illumination they displayed WT green

pigmentation. Chl biosynthesis is light-regulated in all Chl-

synthesizing photosynthetic organisms due to the light-

dependent activity of the NADPH:protochlorophyllide

oxidoreductase enzyme (Reinbothe and Reinbothe 1996).

In addition to this, light-dependent mechanism for proto-

chlorophyllide reduction, photosynthetic bacteria, cyano-

bacteria, and members of algae, mosses, ferns, and

gymnosperms are capable of reducing Pchlide to Chlide by

a light-independent pathway and thus synthesize significant

amounts of Chl in the dark (Li et al. 1993; Fujita et al.

1998). Most probably, mutants as10 and as11 carry

mutations in this light-independent Chl biosynthetic path-

way, which is active in C. reinhardtii. Similar C. rein-

hardtii mutants, known as ‘‘yellow in the dark’’ or

y mutants, have been previously isolated and characterized

(Cahoon and Timko 2000). They all resulted to be blocked

in the translation initiation or elongation of the plastid CHL

L transcript, encoding one subunit of the light-independent

protochlorophyllide reductase enzyme. Post-transcriptional

processes are well known to play a key role in the control

of chloroplast gene expression (Rochaix 1996; Choquet

et al. 2001), and the isolation of two mutants affecting this

step in a small size mutagenesis also suggests that several

nuclear factors are involved in the control of expression of

plastid-encoded CHL L.

Electron transport chain mutants

The selection of mutants affected at different steps of the

photosynthetic electron transport chain was more success-

ful with respect to the screening for antenna size strains,

due to the reliability of fluorescence kinetics in vivo which

reduced the number of false positive. In particular, we

selected five mutants lacking PSII activity (i.e., Fv). This is

a highly desired phenotype useful for development of

genetic strategies for the control of the aerobiosis/anaero-

biosis state of the cultures. Biochemical characterization

-250 0 250 500 750 1000 1250 1500 1750 2000 2250 2500

-0.8

-0.4

0.0ΔA

705n

m

time (ms)

WTas2as1

Fig. 6 P700 photo-oxidation kinetic of WT, as2 and as1 at low light

intensity accordingly to (Melis 1989; Polle et al. 2000). The kinetic

provides an estimation of PSI functional antenna size. The measure

has been performed on thylakoids in the presence of MgCl2 to

maintain membrane stacking. n = 4, error bars within 10%

Photosynth Res

123

validated the absence of a correctly assembled PSII in this

group of mutants (Fig. 4b), together with the observation

that they were unable to grow autotrophically (not shown).

Information on the genes responsible for these PSII KO

phenotypes is strictly required for the proposed application

of complementing mutants with a gene carrying an

inducible promoter to facilitate the shift of the culture from

aerobiosis to anaerobiosis. Unfortunately, we were able to

recover the information on the site of insertion in the

genome in the case of the et2 mutant only. The 50-end

flanking sequence is located in a gene model encoding a

putative nuclear receptor. However, this putative gene

appears not to be actively transcribed, since no ESTs have

been detected in any database, questioning the possible

correlation between this gene and the phenotype. More-

over, backcrosses and progeny analysis indicated the

presence of multiple insertions, and supporting the idea that

a different, unknown, mutation is likely responsible for the

phenotype.

As a side-product of the search for PSII mutants, we

found other mutations affecting the photosynthetic chain,

which are also easily selected because they all result in

highly fluorescent colonies due to impairment of photo-

chemical reactions. Two mutants without a correctly

assembled cytochrome b6f were selected, et6 and et7. For

one of them (et6), we were able to recover the sequence

flanking the insertion site, which was mapped in the TCA1

gene, in chromosome 9. This gene is a factor required for

the correct translation of the PETA plastid transcript,

encoding the cytochrome f subunit. The identified insertion

is thus fully consistent with the observed phenotype, and an

allelic mutant has been previously selected and character-

ized (Wostrikoff et al. 2001). Two insertions in the same

gene occurring in independent mutagenesis experiments

could indicate a high frequency insertion region in the

TCA1 locus, due to unknown reasons. This finding opens

the question of whether mutagenesis in C. reinhardtii is

truly random or rather occurs in hotspot regions of the

genome, as previously suggested (Barakat et al. 2000;

Sessions et al. 2002; Alonso et al. 2003) or could be

somehow guided by endogenous DNA sequences which

are present on the foreign DNA used for transformation

(for example, the PSAD promoter/terminator cassette and

the RBCS/HSP70 fusion promoter which are present in the

pSL18 vector).

State transition (St) mutants

The screening for impaired St yielded several mutants and

in the case of eight of them we confirmed the phenotype

without identifying any other observable alteration in

components of the photosynthetic electron transport chain.

We can attribute this high number both to the application

of a well-established screening method (Fleischmann et al.

1999) and to the fact that many metabolic processes may

influence the cell redox potential and thus the PQ reduction

state, both at the chloroplast and at the mitochondrion level

(Peltier and Schmidt 1991; Cardol et al. 2003).

We were able to get information about the insertion site

in three cases. In mutants str4 and str5, the insertion is

flanking, respectively, a gene model predicting a putative

cytoplasmic dynein in chromosome 6 and an unknown

gene in chromosome 8. No information about the transgene

copy number is yet available for these mutants, thus we

cannot exclude that the observed phenotypes are due to

multiple insertions.

In mutant str1, the 50-end was located in a gene model

coding a putative DNAJ-like gene, belonging to the HSP40

chaperone family, actively transcribed. This mutant also

displayed a parallel phenotype, being unable to repair PSII

efficiently upon high light treatment as shown by the delay

in recovering the initial Fv/Fm value with respect to WT

(Electronic Supplementary Fig. 1). In this case, back-

crosses with WT and progeny analysis showed that colo-

nies carrying the insertion in the DNAJ-like gene

(identified through PCR) also displayed the PSII repair

deficiency, thus corroborating the hypothesis that mutation

in DNAJ-like genes is responsible for the PSII repair

deficiency phenotype. The alteration of St ability, however,

did not co-segregate with dnaJ and the repair phenotype,

suggesting the presence of at least two insertions in this

mutant.

Evaluation of different screening methods efficiency

The screening methods were chosen at the beginning of

this project on the basis of their high potential sensitivity.

This is especially true for the analysis of acetone extracts

by absorption spectroscopy, which allows the detection of

very small differences in pigment content (Croce et al.

2000) and thus is able to detect even small alterations in the

antenna composition. This sensitivity, however, was

strongly affected by the inhomogeneous growth of different

colonies in microtiter wells, which caused a large number

of false positives and thus complicated the identification of

small differences in the absorption spectra. This is also due

to the fact that during exponential growth, Chl a/b content

changes and thus even a small de-synchronization of

growth between cultures can affect the Chl a/b ratio to

produce false positives or leads to the loss of potentially

interesting mutants.

Culture of colonies in TAP-agar plates yielded more

homogenous results. However, in this case, the estimation

of antenna content from fluorescence in vivo was less

sensitive due to self absorption from high local concen-

tration and differences in colony size. Because of these

Photosynth Res

123

factors of variability, we have not been able to isolate

mutants affected in the accumulation of individual antenna

complexes, an expected event on the basis of the large

number of genes belonging to the LHC family (Elrad and

Grossman 2004). These limitations are clearly increasing

the number of false positives, which could be excluded

only after multiple rounds of selection. Even more nega-

tively, mutants with inconspicuous phenotypes were easily

discarded as negatives and only mutants with strong phe-

notypes were retained through all selections.

Furthermore, the high level of redundancy in C. rein-

hardtii genes encoding LHCA (9 genes) and LHCB (11

genes) (Elrad and Grossman 2004; Stauber et al. 2003;

Teramoto et al. 2001; Merchant et al. 2007) can also sug-

gest that mutants affected in a single LHC gene might have

an inconspicuous phenotype, because of a compensatory

effect from homologous LHCs. This is in agreement with

the observation that the npq5 mutant, knocked out in the

LHCBM1 gene, encoding one of several polypeptides

composing the LHCII major trimeric antenna, and the one

showing the highest level of transcription, is only slightly

affected in the Chl a/b ratio (2.55 vs. 2.30) (Elrad et al.

2002).

Screening for St mutants was instead more efficient,

since the State 1 and State 2 fluorescence are measured on

the same colony and thus eventual differences in colony

growth can be compensated. Similarly, the analysis of

fluorescence induction kinetics was also more successful,

since mutant selection does not depend on signal intensity

but rather on the shape of kinetics curve which is inde-

pendent from colony shape and growth.

We conclude that, while methods for the identification

of mutants affected in St or blocked in the electron trans-

port chain are efficiently employed, spectral analysis of

acetone extracted pigments and the fluorescence yield

parameter do not allow the efficient selection of mutants

affected in antenna size, while allowing for the identifica-

tion of several strains with an altered Car content.

The recovery of the sequences flanking insertions

In addition to the screening system, one additional factor

complicating insertion mutagenesis approaches in Chla-

mydomonas is the difficulty in generating mutants with a

single insertion and the successful identification of the

insertion site in the genome.

At present, only the broad application of multiple

molecular approaches (Inverse PCR, Tail PCR, RESDA-

PCR and Plasmid Rescue) allowed to get sequence infor-

mation on selected mutants, with a frequency around 20%.

This frequency is still very low and does not overcome the

possibility that multiple insertions or big deletions/rear-

rangements occurs, calling for supplemental work in order

to demonstrate the co-segregation between an interesting

phenotype and the gene disruption event responsible for it.

A frequent problem is the presence of concatameric

insertions of the vector cassette (as in the case of PSII

mutant et3 and pigment mutant as7) and sequencing of the

products obtained by applying the aforementioned molec-

ular techniques yields sequences of the plasmid itself as

previously reported (Dent et al. 2005).

Alternative molecular techniques for the targeted dis-

ruption of target genes have not yet been described in

literature, despite the fact that homologous recombination

has been demonstrated in C. reinhardtii, even if at very

low efficiency (Sodeinde and Kindle 1993; Zorin et al.

2009). Additional possibilities are RNAi (Rohr et al.

2004) and microRNA (Molnar et al. 2009) silencing

techniques, successfully applied in many reports. Never-

theless, these techniques result in the down-regulation,

rather than in the disruption, of the expression of the

target genes. Moreover, a major drawback of the latter

techniques is to maintain stable expression of the silenc-

ing constructs long enough for useful experimentation, not

to mention the use of resulting strains for industrial

applications.

Conclusions

In this work, we describe a mutant screening approach,

applying the use of selection methods for different phe-

notypes, exploiting multiple, although simple spectroscopic

methods. One of these screenings was aimed at selecting

mutants in antenna systems. Despite the methods employed

were sensitive enough, the screen failed to detect mutants

with a weak phenotype because of the unavoidable heter-

ogeneity in growth during the cultivation of a large number

of colonies. We expect that mutants selected by this

method would be affected in genes encoding for factors

involved in the assembly of multiple members of the LHC

family, or in pigment biosynthesis genes like CBS3

(Tanaka et al. 1998) or in components of the machinery

involved in the insertion of LHC precursors into the thy-

lakoid membranes such as ALB3 (Ossenbuhl et al. 2004) or

CHAOS (Klimyuk et al. 1999).

On the contrary, selection of PSII and St mutants was

more efficient since the desired phenotype had a low

dependence on the growth stage of the colonies. All

screening methods, however, faced an intrinsic limitation

generally found in Chlamydomonas mutant screens: the

difficulty to identify the insertion site and the occurrence of

multiple insertions in some cases.

Acknowledgments This study was supported by the Italian Minis-

try of Agriculture, Project IDROBIO.

Photosynth Res

123

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