BACKGROUND
the green alga volvox carteri represents one of the simplest multicellular organisms: volvox is composed of only two cell types, somatic and reproductive .
despite its basic organizational concept, volvox algae share many features that characterize the life cycles and developmental histories of complex multicellular organisms.
therefore, various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ecm biogenesis have been studied in volvox and its close relatives in the group of the volvocine algae  <cit> .
comparisons among volvocine algae open the possibility to discover universal rules that characterize the transition from unicellular organisms, such as chlamydomonas, to complex, differentiated multicellular organisms like volvox  <cit> .figure  <dig>
the phenotype of
v. carteri
: wild-type and transformant.
v. carteri consists of ~ <dig> small, biflagellate, terminally differentiated, somatic cells at the surface and ~ <dig> large reproductive cells in the interior of a spheroid.
the reproductive cells develop inside the mother spheroid to embryos and later to juveniles.
up to 99% of the volume of such a spheroid consists of a complex, transparent, glycoprotein-rich extracellular matrix that holds all of the cells in place.
wild-type spheroid of volvox carteri f. nagariensis short before release of ~ <dig> juvenile spheroids.
spheroid of transformant nitluc-a <dig> functionally expressing the g. princeps luciferase gene under control of the v. carteri nita promoter.
transformant nitluc-a <dig> shows the same phenotype as its wild-type parent.
however, the technical feasibility of genetically manipulating a species of interest is a key factor for any detailed molecular analysis of gene or protein functions in order to reveal key aspects in development and evolution.
a limited number of molecular tools and techniques are available in volvox that provide a good basis for molecular manipulations: stable nuclear transformation is possible  <cit> , unselectable markers can be efficiently co-transformed together with selectable markers  <cit> , some reporter genes and promoters are available  <cit> , expression of chimeric genes works  <cit> , foreign genes can be expressed  <cit> , gene replacement by homologous recombination is feasible  <cit> , plasmids or transposons can be used for gene tagging  <cit> , and the genome of v. carteri has been sequenced  <cit> .
even if several promoters are available for genetic manipulations in v. carteri, there is a need for a strong, inducible promoter that can be switched on easily without affecting further development of the organism.
the hsp70a promoter is able to increase expression in response to heat  <cit> ; however, due to its permanent expression and the heat-inducibility of sexual development, it is unsuitable for use as a molecular switch.
the arylsulfatase  promoter is inducible by sulfur deprivation  <cit> , but sulfur deprivation disturbs further development or even prevents it.
further inducible volvox promoters have not been investigated so far.
we considered that the promoter of the endogenous nitrate reductase  might be suitable as a novel inducible promoter for genetic engineering in v. carteri.
in plants, algae and fungi, nitrate assimilation genes including the nitrate reductase genes are expressed as a response to micromolar concentrations of nitrate and induction occurs within minutes  <cit> .
even if nitrate is the most commonly used nitrogen source because of its major abundance, the energy cost required for ammonium assimilation is lower than that of nitrate  <cit> .
therefore, ammonium provides a negative signal for nitrate assimilation, which leads to repression of nitrate transporters and enzymes of the pathway  <cit> .
also in v. carteri, accumulation of the nitrate reductase transcript has been shown to be both induced by nitrate and repressed by ammonium  <cit> .
aside from inducible promoters, reporter genes are required for genetic engineering.
so far, the endogenous genes of arylsulfatase  <cit>  and the sex inducer  <cit>  as well as the heterologous genes of the codon-optimized green fluorescent protein   <cit>  and the hexose/h+ symporter hup <dig>  <cit>  have been presented as reporter genes in volvox.
only the ars gene allows for easy quantification of the expression product and, therefore, is suitable for continuous use  <cit> .
however, several suitable reporter genes are required in a model organism, e.g., for conducting multiplex assays.
a good candidate for a new reporter gene in v. carteri seemed to be the luciferase gene  of the deep sea copepod gaussia princeps  <cit> , which was previously adapted to the nuclear codon usage of c. reinhardtii  <cit> .
the suitability of g-luc genes that were codon optimized for expression in the respective hosts has been shown in different organisms like human  <cit> , the alga chlamydomonas reinhardtii  <cit> , the fungus pathogen candida albicans  <cit>  and in bacteria  <cit> .
the g-luc protein does not require any cofactor for activity and it catalyzes the oxidation of the substrate coelenterazine into the excited form of coelenteramide that releases a photon with a wavelength of 480 nm upon electron transition to the ground state  <cit> .
g-luc is the smallest  atp-independent luciferase cloned to date  <cit>  and it was shown to exhibit an activity up to  <dig> -fold higher than renilla reniformis luciferase, firefly luciferase and bacterial luciferases  <cit> .
here, we show that the codon-adapted luciferase gene of g. princeps is a suitable reporter gene in v. carteri.
emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer; the chemiluminescence can also be detected on light-sensitive film, with a standard camera or even with the naked eye in the darkroom.
we also demonstrate that the promoter of the v. carteri nitrate reductase gene  is a powerful molecular switch for induction of transgene expression.
strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate.
in addition, we provide proof of the long-term stability of inducible expression of the chimeric nita/g-luc transgenes after stable nuclear transformation.
RESULTS
chimeric selectable marker and reporter genes for transformation
for all transformation experiments, the streptomyces rimosus aphviii gene that confers resistance to paromomycin was used as a selectable marker  <cit> .
the aphviii gene was driven by strong volvox promoters, i.e., either a tandem promoter made of both the hsp70a and the rbcs <dig> promoter  or the promoter of rbcs <dig> alone   <cit> .
for some experiments, this chimeric selectable marker gene was on a separate plasmid   <cit>  and it was co-transformed with the reporter gene-carrying plasmid .figure  <dig>
schematic diagram of the utilized selectable marker and reporter gene plasmids.
plasmid ppmr <dig> carries the selectable s. rimosus aphviii gene driven by an artificial tandem promoter from the hsp70a and rbcs <dig> genes of v. carteri and a 3′-flanking sequence derived from the rbcs <dig> gene of v. carteri  <cit> .
plasmid pnitaph contains the chimeric aphviii selectable marker gene derived from plasmid ppmr3; however, the aphviii gene is only driven by the v. carteri rbcs <dig> promoter ; it also contains the 3′-flanking sequence of the v. carteri rbcs <dig> gene.
in addition, pnitaph contains the v. carteri nita promoter region and also the 3′ flanking sequence of the nita gene.
an artificial multiple cloning site was introduced between the 5′ and 3′ control elements of nita to allow for integration of a protein coding sequence of interest.
plasmid pnitlucx contains the same nita cassette as pnitaph ; in addition, the g. princeps g-luc reporter gene was cloned into the multiple cloning site between the 5′ and 3′ control elements of nita.
plasmid pnitluca is a derivative of pnitaph; the g. princeps g-luc reporter gene was inserted into the multiple cloning site.
amplified fragments of rbcs3/aphviii  and g-luc  are indicated.
v.c., volvox carteri; s.r., streptomyces rimosus; g.p., gaussia princeps; g-luc, luciferase gene.
to allow for expression of a gene of interest under the control of the nitrate reductase  promoter of v. carteri, plasmid pnitx was constructed.
this plasmid contains about  <dig>  kb 5′ flanking sequence of the nita gene  <cit>  and about  <dig>  kb of its 3′ flanking sequence.
the latter sequence includes the terminator sequence of nita.
an artificial multiple cloning site with unique restriction sites was introduced between the 5′ and 3′ control elements to allow for integration of the protein coding sequence of interest.
plasmid pnitaph was derived from plasmid pnitx; it also carries the chimeric aphviii selectable marker derived from plasmid ppmr <dig> .
plasmids pnitaph and pnitx are universal starting plasmids for the construction of expression vectors for volvox.
for analysis of the expression characteristics of the nita promoter, the coding sequence of the luciferase  from gaussia princeps  <cit>  was used as a reporter and, therefore, introduced into plasmids pnitx and pnitaph, respectively.
the resulting plasmids pnitlucx and pnitluca contain the g-luc coding sequence, which was previously adapted to the nuclear codon usage of c. reinhardtii  <cit> , under control of the 5′ and 3′ control elements of the nita gene of v. carteri .
the kasi sites within the multiple cloning site and at the end of the g-luc coding sequence allow for dna integrations to produce fusion proteins between the luciferase reporter and a certain protein of interest.
plasmid pnitlucx needs to be co-transformed with a selectable marker plasmid , whereas pnitluca already contains the selectable marker aphviii.
generation of transgenic volvox strains using the aphviii selectable marker
to allow for selection of transformants with even weak transgene-mediated resistance, the lowest concentration of the antibiotic paromomycin that kills all wild-type volvox cells was determined.
to achieve this, a series of aliquots with the same number of wild-type v. carteri cells was exposed to increasing concentrations of paromomycin, incubated for six days, and screened for living  or dead  cells.
the wild-type volvox algae tolerated concentrations of up to  <dig>  μg/ml paromomycin.
a concentration of  <dig>  μg paromomycin/ml or higher led to 100% cell death .
to allow for an objective and rapid discrimination between aliquots containing living cells and aliquots containing dead cells even in large-scale screenings of culture plates, red-shifted false-color images were created from standard photographs of the plates .figure  <dig>
analysis of paromomycin resistance in wild-type and transgenic
v. carteri
strains.
for detailed analysis of resistance, identical quantities of v. carteri spheroids were exposed to increasing concentrations of paromomycin and incubated for six days.
numbers refer to the concentration of paromomycin  utilized.
natural color  and red-shifted, false-color images  are shown.
the wild-type v. carteri strain.
transgenic v. carteri strain nitluc-x <dig> that was co-transformed with ppmr <dig>  the selectable marker plasmid, and plasmid pnitlucx, which carries the g-luc reporter gene driven by the nita promoter.
transgenic v. carteri strain nitluc-a <dig> that was transformed only with plasmid pnitluca, which carries both the selectable marker gene and the g-luc reporter gene driven by the nita promoter.
for transformation, a logarithmically growing culture of v. carteri was harvested by filtration and subjected to particle bombardment using dna-coated gold microprojectiles as described before  <cit> .
the unselectable plasmid pnitlucx  was co-transformed with plasmid ppmr <dig> , while the selectable plasmid pnitluca  was transformed alone.
after the transformation procedure, the algae were incubated in liquid medium and allowed to recover for three days before paromomycin was added.
after 10– <dig> days of incubation, green and living spheroids were separated under microscopical control.
the antibiotic resistance of separated spheroids was justified and concretized by incubation for six days in 2 ml volvox medium with different concentrations of paromomycin ranging from  <dig>  to 500 μg/ml .
transformants with the most robust resistance tolerated concentrations up to 50 μg/ml paromomycin .
thus, volvox transformants exhibited an up to 125-fold higher tolerance against paromomycin than the wild-type strains.
genomic integration of chimeric gene constructs
pcr was used to verify the stable integration of the chimeric genes into the genome of v. carteri transformants.
to this end, genomic dna was isolated from transformants that were able to grow for three weeks under selective pressure by exposure to paromomycin.
using aphviii-specific primers and the isolated genomic dna as a template, pcr fragments of the expected size were obtained from seventeen transformants.
the transformants were also checked for the presence of the g-luc reporter gene using g-luc specific primers.
fifteen of the seventeen transformants were shown to contain both the aphviii gene and the g-luc gene.
obviously, two transformants contained only the intact aphviii gene but no intact g-luc gene or no g-luc gene at all.
therefore, both clones were excluded from further investigations.
the parent wild-type strain did not yield any aphviii or g-luc specific products, as expected.
the transformation efficiency was calculated to be at about  <dig>  · 10− <dig>  which is quite similar to previously reported transformation rates of about  <dig>  · 10− <dig>  <cit>  and about 10− <dig>  <cit> .
the co-transformation rate was quite high, but the total number of transformants was too low to allow for a significant statistical analysis.
however, the total number of obtained transformant clones was almost the same, regardless of whether the selectable marker gene and the reporter gene were on two different plasmids or both genes were on the same plasmid.
expression of the g-luc reporter gene in transformants
all transformants that contained both intact aphviii and g-luc genes were assayed for functional expression of luciferase.
luciferase activity was assayed using the bioluminescent substrate coelenterazine and a minilumat luminometer.
pcr-positive transformants clearly showed luciferase activity in contrast to the parent wild-type strain.
however, the expression rate varied between the different transformants.
the expression of the luciferase was analyzed in detail after a long-term incubation of the transformants without selective pressure .
long term stability of dna integration and gene expression
for any biotechnological approach with transgenic organisms, the stability and unimpaired expression of the transgenes for a long period of time is of fundamental importance.
therefore, the genomic integration and expression of the aphviii and g-luc genes was re-examined after more than one year of cultivation of the v. carteri transformants or, in other words, after more than  <dig> generations.
cultivation was without selective pressure during this period.
if the dna of the chimeric aphviii gene  was present in the genome, a 324 bp chimeric fragment, containing 107 bp of the rbcs <dig> promoter region and 217 bp of the aphviii coding region, was expected to be amplified by pcr using the corresponding specific primers .
the results are shown in figure 4a.
transformants were also re-investigated for the presence of the g-luc reporter gene .
if the dna of the former plasmids pnitlucx or pnitluca was present in the genome, a fragment of 119 bp was expected to be amplified with the corresponding specific primers .
in both experiments  the amplified fragments were cloned and sequenced .
the sequences were just as anticipated.
in both cases, the parent wild-type strain did not yield any product, as expected .
as a further control, a 208 bp fragment of an unaffected volvox gene  was amplified both from all transformants and from wild-type strains, just as expected .
thus, it can be concluded that the genomic integrations of both the chimeric aphviii and g-luc genes are still present even more than  <dig> generations after transformation.
therefore, the genomic integrations can be referred to as stable.figure  <dig>
proof of genomic integration of both
aph
viii and
g-luc
genes in transformants.
paromomycin resistant transformants and the parent wild-type strain were analyzed for presence of the aphviii gene in the genome by genomic pcr.
the expected size of the pcr fragment produced in transformants was 324 bp .
genomic pcr analysis of paromomycin resistant transformants and the parent wild-type strain for the presence of the g-luc gene in the genome.
the expected size of the pcr fragment produced in transformants was 119 bp .
as a control, a 208 bp fragment of an unaffected volvox gene  was amplified both from all transformants and from wild-type strains just as expected.
m-lanes refer to the molecular weight marker.
pcr fragments were cloned and sequenced.
the obtained sequences are given right of each gel image.
the positions of the primers and the start codon  are indicated.
this pcr assay was carried out more than  <dig> generations after transformation of the strains.
we also re-examined the functional expression of the chimeric aphviii and g-luc genes by antibiotic resistance and luciferase assays, respectively.
all transformants exhibited about the same antibiotic resistance and luciferase activity compared to the values determined right after transformation.
thus, not only the genomic integration but also the expression of the chimeric aphviii and g-luc genes is stable in v. carteri.
inducibility of the nita promoter-driven g-luc reporter gene in transformants
all transformants were assayed for inducible expression of luciferase.
for it, transformants and wild-type cultures were grown in standard volvox medium with ammonium.
after a media shift to volvox medium with nitrate, incubation was continued for 1 h under standard conditions.
cell extracts were obtained from all strains before and after incubation in nitrate medium.
the cell extracts were checked for luciferase activity using a luminometer .
to allow for quantitative comparison of activity measurement results, all samples were normalized with the respective chlorophyll content.
the results clearly show that luciferase expression is inducible by the nh4+ to no3− switch.figure  <dig>
quantification and inducibility of luciferase activity in transformants.
transformants expressing the g-luc reporter gene under control of the nita promoter and the parent wild-type strain were investigated for luciferase activity.
the diagram shows the measured enzyme activity in untreated specimen  and specimen induced by a switch from nh4
+ to no3
− .
the columns represent the mean of three independent experiments.
the standard deviation is indicated.
the factor of induction is given underneath the columns.
the luciferase activity of each sample was normalized with its chlorophyll concentration.
this luciferase activity assay was carried out more than  <dig> generations after transformation of the strains.
in transformants, the expression increased by a factor of  <dig> to  <dig> after the switch to nitrate.
the average induction factor was  <dig>  +/−  <dig> .
except for the different number of obtained transformants, we couldn’t see any principal differences between transformants generated with pnitlucx and ppmr <dig> or with pnitluca alone.
for later large scale applications, we also visualized the expression and inducibility of luciferase in a more direct way.
although transformants show a wild type phenotype under the light microscope , a glow is visible even to the naked eye in the darkroom when the coelenterazine substrate is added to cell lysates of no3−-induced transformants .
exposure to a light-sensitive film also allows for a simple, semi-quantitative determination of chemiluminescence of many transformants in parallel .figure  <dig>
visualization of nitrate-inducible luciferase activity.
parental wild-type spheroids and transformants expressing the g-luc reporter gene under control of the nita promoter  were investigated for light emission as a result of luciferase activity both under standard conditions  and after induction by a switch from nh4
+ to no3
− .
upper row: standard photo showing the assay setup.
middle row: photo  without any extraneous light after addition of the coelenterazine substrate.
lower row: exposure to a light-sensitive film for 10 min after addition of the coelenterazine substrate.
mrna and protein expression kinetics after induction of the nita promoter-driven chimeric reporter gene
the characteristics of the nita promoter were analyzed after induction of the nita promoter-driven chimeric g-luc reporter gene with a switch from nh4+ to no3−.
taking transformant nitluc-a <dig>  as an example, both the g-luc mrna and the luciferase activity were investigated at several time points from  <dig> to 240 min after the switch to no3− .
for it, total rna was isolated and the g-luc mrna expression level was analyzed using quantitative real-time rt-pcr.
the luciferase enzyme activity was quantified in parallel using the chlorophyll content as a reference.figure  <dig>
mrna and protein expression kinetics after nitrate-induction of the
nita
promoter.
transformant nitluc-a <dig> expressing the g-luc reporter gene under control of the nita promoter was induced by a switch from nh4
+ to no3
− medium at 0 min.
both the g-luc mrna and the luciferase activity were investigated at several time points after induction.
the relative g-luc mrna level was calculated in comparison to the reference gene tbpa using the 2-Δct method for analysis of the real-time rt-pcr results.
the protein expression was determined with luciferase activity assays using the chlorophyll concentration of each sample for normalization.
after the switch to no3−, the g-luc mrna expression increases quickly and peaks at about 30 min.
in the following 2 hours, the expression level decreases slowly and reaches a plateau at about 30% of the maximal expression.
the luciferase enzyme activity shows a similar profile but the maximum is reached about 1 hour after the switch, which corresponds to a delay of 30 min in relation to the mrna peak.
the luciferase activity reaches about  <dig> million relative light units  at the peak .
the luciferase expression level decreases even slower than the mrna expression level and reaches a plateau at about  <dig> million rlu or about 70% of the maximal expression, which might reflect that the luciferase protein turnover is much lower than the turnover of its mrna.
discussion
the codon-adapted luciferase gene of g. princeps has proved to be a suitable reporter gene in v. carteri, even if the codon-optimization has been done for chlamydomonas  <cit> .
although volvox diverged from unicellular ancestors like chlamydomonas at least  <dig> million years ago and codon usage became different from each other  <cit> , the relationship obviously is still close enough to allow for good expression in volvox.
the assay for luciferase activity through detection of emitted light of the chemiluminescent reaction proved to be very effective in volvox just as in several other organisms before  <cit> .
the assay is quick and easy, enzyme activity can be quantified reliably and the measurement is both robust and well reproducible.
the assay also exhibits high sensitivity because even a small amount of enzyme can catalytically process many substrate molecules to produce a detectable signal.
accurate quantification is done with a luminometer.
in large scale analyses, light detection on light-sensitive films or with a camera allows for qualitative assessments.
since gaussia luciferase reaction is quite fast  <cit>  it is important to measure the emitted light immediately after coelenterazine injection.
previous work with the gaussia luciferase showed that this enzyme is very stable even in body fluids such as blood and urine  <cit> , in cell culture medium  <cit>  and in cell extracts  <cit> .
in our standard assay, we measured the enzyme activity in crude cell extracts obtained by ultrasonic treatment.
no further purification of the enzyme was necessary.
in preliminary experiments we even measured the luciferase activity in volvox cultures containing intact spheroids .
the activity values obtained with intact spheroids were almost as high as the activity values obtained with cell extracts.
therefore, in high-throughput screenings, direct measurement of cell cultures without ultrasonic treatment might be an even easier alternative, but reproducibility of the results needs be checked before.
the functionality of the assay in cell cultures without ultrasonic treatment is attributed to the high cell-membrane permeability of coelenterazine.
altogether, the codon-adapted luciferase reporter gene outshines previous reporter genes used in volvox in terms of its sensitivity, robustness and ease of use.
in addition to the g-luc reporter gene, the promoter of the nitrate reductase gene  <cit>  turned out to be a powerful molecular tool for genetic engineering in v. carteri.
two of our vectors  allow for nita-driven expression of any target gene after a single cloning step.
the nita promoter drives expression of the gene of interest by simply changing the nitrogen source from ammonium to nitrate  <cit> , all the other components of the volvox medium remain unchanged throughout.
the change of media is easily managed with volvox because the spheroids can be quickly collected on a nylon screen and transferred to the new medium.
changing the nitrogen source disturbs growth and development, if at all, only to a minimum in contrast to other methods for promoter induction using chemicals  <cit> , hormones  <cit>  or increased temperatures   <cit> .
the switching time of the nita promoter is very short: after induction by changing the nitrogen source, mrna expression increases immediately and reaches its maximum about 30 min after induction , which is similar to results in earlier work with the complete unmodified endogenous nita gene  <cit> .
the expression and induction characteristics caused by our nita fragment demonstrate, that the nita promoter is fully working and all required cis-elements are included.
therefore, our constructs also might be interesting for those who want to analyze this promoter and its regulation in more detail.
a further advantage of using the nita promoter for genetic engineering is that the expression rate of the target gene is not only influenced by nitrate but also by ammonium.
as mentioned, ammonium prevents the uptake of nitrate  <cit>  and nitrate is required for the induction of the nita promoter.
thus, submicromolar amounts of ammonium added together with the normal amounts of nitrate might allow for adjustment of the expression rate of the target gene in a given transformant.
it also might be possible to switch off expression by adding larger amounts of ammonium.
CONCLUSIONS
two new molecular tools are now available for genetic engineering of the green alga volvox carteri.
the nita promoter of v. carteri allows for inducible expression of target genes and induction occurs immediately after changing the nitrogen source from ammonium to nitrate.
the change of the nitrogen source is easily managed and it disturbs neither growth nor development.
the codon-adapted luciferase gene of g. princeps turned out to be a powerful reporter gene in volvox.
the assay for luciferase activity is easy, robust, reproducible and sensible; moreover, it works in crude cell extracts.

 the green alga volvox carteri represents one of the simplest multicellular organisms: volvox is composed of only two cell types, somatic and reproductive. despite its basic organizational concept, volvox algae share many features that characterize the life cycles and developmental histories of complex multicellular organisms. therefore, various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ecm biogenesis have been studied in volvox and its close relatives in the group of the volvocine algae. comparisons among volvocine algae open the possibility to discover universal rules that characterize the transition from unicellular organisms, such as chlamydomonas, to complex, differentiated multicellular organisms like volvox.figure the phenotype of v. carteri: wild-type and transformant. v. carteri consists of ~ small, biflagellate, terminally differentiated, somatic cells at the surface and ~ large reproductive cells in the interior of a spheroid. the reproductive cells develop inside the mother spheroid to embryos and later to juveniles. up to 99% of the volume of such a spheroid consists of a complex, transparent, glycoprotein-rich extracellular matrix that holds all of the cells in place. wild-type spheroid of volvox carteri f. nagariensis short before release of ~ juvenile spheroids. spheroid of transformant nitluc-a functionally expressing the g. princeps luciferase gene under control of the v. carteri nita promoter. transformant nitluc-a shows the same phenotype as its wild-type parent. however, the technical feasibility of genetically manipulating a species of interest is a key factor for any detailed molecular analysis of gene or protein functions in order to reveal key aspects in development and evolution. a limited number of molecular tools and techniques are available in volvox that provide a good basis for molecular manipulations: stable nuclear transformation is possible, unselectable markers can be efficiently co-transformed together with selectable markers, some reporter genes and promoters are available, expression of chimeric genes works, foreign genes can be expressed, gene replacement by homologous recombination is feasible, plasmids or transposons can be used for gene tagging, and the genome of v. carteri has been sequenced. even if several promoters are available for genetic manipulations in v. carteri, there is a need for a strong, inducible promoter that can be switched on easily without affecting further development of the organism. the hsp70a promoter is able to increase expression in response to heat; however, due to its permanent expression and the heat-inducibility of sexual development, it is unsuitable for use as a molecular switch. the arylsulfatase promoter is inducible by sulfur deprivation, but sulfur deprivation disturbs further development or even prevents it. further inducible volvox promoters have not been investigated so far. we considered that the promoter of the endogenous nitrate reductase might be suitable as a novel inducible promoter for genetic engineering in v. carteri. in plants, algae and fungi, nitrate assimilation genes including the nitrate reductase genes are expressed as a response to micromolar concentrations of nitrate and induction occurs within minutes. even if nitrate is the most commonly used nitrogen source because of its major abundance, the energy cost required for ammonium assimilation is lower than that of nitrate. therefore, ammonium provides a negative signal for nitrate assimilation, which leads to repression of nitrate transporters and enzymes of the pathway. also in v. carteri, accumulation of the nitrate reductase transcript has been shown to be both induced by nitrate and repressed by ammonium. aside from inducible promoters, reporter genes are required for genetic engineering. so far, the endogenous genes of arylsulfatase and the sex inducer as well as the heterologous genes of the codon-optimized green fluorescent protein and the hexose/h+ symporter hup have been presented as reporter genes in volvox. only the ars gene allows for easy quantification of the expression product and, therefore, is suitable for continuous use. however, several suitable reporter genes are required in a model organism, e.g., for conducting multiplex assays. a good candidate for a new reporter gene in v. carteri seemed to be the luciferase gene of the deep sea copepod gaussia princeps, which was previously adapted to the nuclear codon usage of c. reinhardtii. the suitability of g-luc genes that were codon optimized for expression in the respective hosts has been shown in different organisms like human, the alga chlamydomonas reinhardtii, the fungus pathogen candida albicans and in bacteria. the g-luc protein does not require any cofactor for activity and it catalyzes the oxidation of the substrate coelenterazine into the excited form of coelenteramide that releases a photon with a wavelength of 480 nm upon electron transition to the ground state. g-luc is the smallest atp-independent luciferase cloned to date and it was shown to exhibit an activity up to -fold higher than renilla reniformis luciferase, firefly luciferase and bacterial luciferases. here, we show that the codon-adapted luciferase gene of g. princeps is a suitable reporter gene in v. carteri. emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer; the chemiluminescence can also be detected on light-sensitive film, with a standard camera or even with the naked eye in the darkroom. we also demonstrate that the promoter of the v. carteri nitrate reductase gene is a powerful molecular switch for induction of transgene expression. strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. in addition, we provide proof of the long-term stability of inducible expression of the chimeric nita/g-luc transgenes after stable nuclear transformation.  chimeric selectable marker and reporter genes for transformation for all transformation experiments, the streptomyces rimosus aphviii gene that confers resistance to paromomycin was used as a selectable marker. the aphviii gene was driven by strong volvox promoters, i.e., either a tandem promoter made of both the hsp70a and the rbcs promoter or the promoter of rbcs alone. for some experiments, this chimeric selectable marker gene was on a separate plasmid and it was co-transformed with the reporter gene-carrying plasmid.figure schematic diagram of the utilized selectable marker and reporter gene plasmids. plasmid ppmr carries the selectable s. rimosus aphviii gene driven by an artificial tandem promoter from the hsp70a and rbcs genes of v. carteri and a 3′-flanking sequence derived from the rbcs gene of v. carteri. plasmid pnitaph contains the chimeric aphviii selectable marker gene derived from plasmid ppmr3; however, the aphviii gene is only driven by the v. carteri rbcs promoter; it also contains the 3′-flanking sequence of the v. carteri rbcs gene. in addition, pnitaph contains the v. carteri nita promoter region and also the 3′ flanking sequence of the nita gene. an artificial multiple cloning site was introduced between the 5′ and 3′ control elements of nita to allow for integration of a protein coding sequence of interest. plasmid pnitlucx contains the same nita cassette as pnitaph; in addition, the g. princeps g-luc reporter gene was cloned into the multiple cloning site between the 5′ and 3′ control elements of nita. plasmid pnitluca is a derivative of pnitaph; the g. princeps g-luc reporter gene was inserted into the multiple cloning site. amplified fragments of rbcs3/aphviii and g-luc are indicated. v.c., volvox carteri; s.r., streptomyces rimosus; g.p., gaussia princeps; g-luc, luciferase gene. to allow for expression of a gene of interest under the control of the nitrate reductase promoter of v. carteri, plasmid pnitx was constructed. this plasmid contains about  kb 5′ flanking sequence of the nita gene and about  kb of its 3′ flanking sequence. the latter sequence includes the terminator sequence of nita. an artificial multiple cloning site with unique restriction sites was introduced between the 5′ and 3′ control elements to allow for integration of the protein coding sequence of interest. plasmid pnitaph was derived from plasmid pnitx; it also carries the chimeric aphviii selectable marker derived from plasmid ppmr. plasmids pnitaph and pnitx are universal starting plasmids for the construction of expression vectors for volvox. for analysis of the expression characteristics of the nita promoter, the coding sequence of the luciferase from gaussia princeps was used as a reporter and, therefore, introduced into plasmids pnitx and pnitaph, respectively. the resulting plasmids pnitlucx and pnitluca contain the g-luc coding sequence, which was previously adapted to the nuclear codon usage of c. reinhardtii, under control of the 5′ and 3′ control elements of the nita gene of v. carteri. the kasi sites within the multiple cloning site and at the end of the g-luc coding sequence allow for dna integrations to produce fusion proteins between the luciferase reporter and a certain protein of interest. plasmid pnitlucx needs to be co-transformed with a selectable marker plasmid, whereas pnitluca already contains the selectable marker aphviii. generation of transgenic volvox strains using the aphviii selectable marker to allow for selection of transformants with even weak transgene-mediated resistance, the lowest concentration of the antibiotic paromomycin that kills all wild-type volvox cells was determined. to achieve this, a series of aliquots with the same number of wild-type v. carteri cells was exposed to increasing concentrations of paromomycin, incubated for six days, and screened for living or dead cells. the wild-type volvox algae tolerated concentrations of up to  μg/ml paromomycin. a concentration of  μg paromomycin/ml or higher led to 100% cell death. to allow for an objective and rapid discrimination between aliquots containing living cells and aliquots containing dead cells even in large-scale screenings of culture plates, red-shifted false-color images were created from standard photographs of the plates.figure analysis of paromomycin resistance in wild-type and transgenic v. carteri strains. for detailed analysis of resistance, identical quantities of v. carteri spheroids were exposed to increasing concentrations of paromomycin and incubated for six days. numbers refer to the concentration of paromomycin utilized. natural color and red-shifted, false-color images are shown. the wild-type v. carteri strain. transgenic v. carteri strain nitluc-x that was co-transformed with ppmr the selectable marker plasmid, and plasmid pnitlucx, which carries the g-luc reporter gene driven by the nita promoter. transgenic v. carteri strain nitluc-a that was transformed only with plasmid pnitluca, which carries both the selectable marker gene and the g-luc reporter gene driven by the nita promoter. for transformation, a logarithmically growing culture of v. carteri was harvested by filtration and subjected to particle bombardment using dna-coated gold microprojectiles as described before. the unselectable plasmid pnitlucx was co-transformed with plasmid ppmr, while the selectable plasmid pnitluca was transformed alone. after the transformation procedure, the algae were incubated in liquid medium and allowed to recover for three days before paromomycin was added. after 10– days of incubation, green and living spheroids were separated under microscopical control. the antibiotic resistance of separated spheroids was justified and concretized by incubation for six days in 2 ml volvox medium with different concentrations of paromomycin ranging from to 500 μg/ml. transformants with the most robust resistance tolerated concentrations up to 50 μg/ml paromomycin. thus, volvox transformants exhibited an up to 125-fold higher tolerance against paromomycin than the wild-type strains. genomic integration of chimeric gene constructs pcr was used to verify the stable integration of the chimeric genes into the genome of v. carteri transformants. to this end, genomic dna was isolated from transformants that were able to grow for three weeks under selective pressure by exposure to paromomycin. using aphviii-specific primers and the isolated genomic dna as a template, pcr fragments of the expected size were obtained from seventeen transformants. the transformants were also checked for the presence of the g-luc reporter gene using g-luc specific primers. fifteen of the seventeen transformants were shown to contain both the aphviii gene and the g-luc gene. obviously, two transformants contained only the intact aphviii gene but no intact g-luc gene or no g-luc gene at all. therefore, both clones were excluded from further investigations. the parent wild-type strain did not yield any aphviii or g-luc specific products, as expected. the transformation efficiency was calculated to be at about  · 10− which is quite similar to previously reported transformation rates of about  · 10− and about 10−. the co-transformation rate was quite high, but the total number of transformants was too low to allow for a significant statistical analysis. however, the total number of obtained transformant clones was almost the same, regardless of whether the selectable marker gene and the reporter gene were on two different plasmids or both genes were on the same plasmid. expression of the g-luc reporter gene in transformants all transformants that contained both intact aphviii and g-luc genes were assayed for functional expression of luciferase. luciferase activity was assayed using the bioluminescent substrate coelenterazine and a minilumat luminometer. pcr-positive transformants clearly showed luciferase activity in contrast to the parent wild-type strain. however, the expression rate varied between the different transformants. the expression of the luciferase was analyzed in detail after a long-term incubation of the transformants without selective pressure. long term stability of dna integration and gene expression for any biotechnological approach with transgenic organisms, the stability and unimpaired expression of the transgenes for a long period of time is of fundamental importance. therefore, the genomic integration and expression of the aphviii and g-luc genes was re-examined after more than one year of cultivation of the v. carteri transformants or, in other words, after more than generations. cultivation was without selective pressure during this period. if the dna of the chimeric aphviii gene was present in the genome, a 324 bp chimeric fragment, containing 107 bp of the rbcs promoter region and 217 bp of the aphviii coding region, was expected to be amplified by pcr using the corresponding specific primers. the results are shown in figure 4a. transformants were also re-investigated for the presence of the g-luc reporter gene. if the dna of the former plasmids pnitlucx or pnitluca was present in the genome, a fragment of 119 bp was expected to be amplified with the corresponding specific primers. in both experiments the amplified fragments were cloned and sequenced. the sequences were just as anticipated. in both cases, the parent wild-type strain did not yield any product, as expected. as a further control, a 208 bp fragment of an unaffected volvox gene was amplified both from all transformants and from wild-type strains, just as expected. thus, it can be concluded that the genomic integrations of both the chimeric aphviii and g-luc genes are still present even more than generations after transformation. therefore, the genomic integrations can be referred to as stable.figure proof of genomic integration of both aph viii and g-luc genes in transformants. paromomycin resistant transformants and the parent wild-type strain were analyzed for presence of the aphviii gene in the genome by genomic pcr. the expected size of the pcr fragment produced in transformants was 324 bp. genomic pcr analysis of paromomycin resistant transformants and the parent wild-type strain for the presence of the g-luc gene in the genome. the expected size of the pcr fragment produced in transformants was 119 bp. as a control, a 208 bp fragment of an unaffected volvox gene was amplified both from all transformants and from wild-type strains just as expected. m-lanes refer to the molecular weight marker. pcr fragments were cloned and sequenced. the obtained sequences are given right of each gel image. the positions of the primers and the start codon are indicated. this pcr assay was carried out more than generations after transformation of the strains. we also re-examined the functional expression of the chimeric aphviii and g-luc genes by antibiotic resistance and luciferase assays, respectively. all transformants exhibited about the same antibiotic resistance and luciferase activity compared to the values determined right after transformation. thus, not only the genomic integration but also the expression of the chimeric aphviii and g-luc genes is stable in v. carteri. inducibility of the nita promoter-driven g-luc reporter gene in transformants all transformants were assayed for inducible expression of luciferase. for it, transformants and wild-type cultures were grown in standard volvox medium with ammonium. after a media shift to volvox medium with nitrate, incubation was continued for 1 h under standard conditions. cell extracts were obtained from all strains before and after incubation in nitrate medium. the cell extracts were checked for luciferase activity using a luminometer. to allow for quantitative comparison of activity measurement results, all samples were normalized with the respective chlorophyll content. the results clearly show that luciferase expression is inducible by the nh4+ to no3− switch.figure quantification and inducibility of luciferase activity in transformants. transformants expressing the g-luc reporter gene under control of the nita promoter and the parent wild-type strain were investigated for luciferase activity. the diagram shows the measured enzyme activity in untreated specimen and specimen induced by a switch from nh4 + to no3 −. the columns represent the mean of three independent experiments. the standard deviation is indicated. the factor of induction is given underneath the columns. the luciferase activity of each sample was normalized with its chlorophyll concentration. this luciferase activity assay was carried out more than generations after transformation of the strains. in transformants, the expression increased by a factor of to after the switch to nitrate. the average induction factor was +/−. except for the different number of obtained transformants, we couldn’t see any principal differences between transformants generated with pnitlucx and ppmr or with pnitluca alone. for later large scale applications, we also visualized the expression and inducibility of luciferase in a more direct way. although transformants show a wild type phenotype under the light microscope, a glow is visible even to the naked eye in the darkroom when the coelenterazine substrate is added to cell lysates of no3−-induced transformants. exposure to a light-sensitive film also allows for a simple, semi-quantitative determination of chemiluminescence of many transformants in parallel.figure visualization of nitrate-inducible luciferase activity. parental wild-type spheroids and transformants expressing the g-luc reporter gene under control of the nita promoter were investigated for light emission as a result of luciferase activity both under standard conditions and after induction by a switch from nh4 + to no3 −. upper row: standard photo showing the assay setup. middle row: photo without any extraneous light after addition of the coelenterazine substrate. lower row: exposure to a light-sensitive film for 10 min after addition of the coelenterazine substrate. mrna and protein expression kinetics after induction of the nita promoter-driven chimeric reporter gene the characteristics of the nita promoter were analyzed after induction of the nita promoter-driven chimeric g-luc reporter gene with a switch from nh4+ to no3−. taking transformant nitluc-a as an example, both the g-luc mrna and the luciferase activity were investigated at several time points from to 240 min after the switch to no3−. for it, total rna was isolated and the g-luc mrna expression level was analyzed using quantitative real-time rt-pcr. the luciferase enzyme activity was quantified in parallel using the chlorophyll content as a reference.figure mrna and protein expression kinetics after nitrate-induction of the nita promoter. transformant nitluc-a expressing the g-luc reporter gene under control of the nita promoter was induced by a switch from nh4 + to no3 − medium at 0 min. both the g-luc mrna and the luciferase activity were investigated at several time points after induction. the relative g-luc mrna level was calculated in comparison to the reference gene tbpa using the 2-Δct method for analysis of the real-time rt-pcr results. the protein expression was determined with luciferase activity assays using the chlorophyll concentration of each sample for normalization. after the switch to no3−, the g-luc mrna expression increases quickly and peaks at about 30 min. in the following 2 hours, the expression level decreases slowly and reaches a plateau at about 30% of the maximal expression. the luciferase enzyme activity shows a similar profile but the maximum is reached about 1 hour after the switch, which corresponds to a delay of 30 min in relation to the mrna peak. the luciferase activity reaches about million relative light units at the peak. the luciferase expression level decreases even slower than the mrna expression level and reaches a plateau at about million rlu or about 70% of the maximal expression, which might reflect that the luciferase protein turnover is much lower than the turnover of its mrna. discussion the codon-adapted luciferase gene of g. princeps has proved to be a suitable reporter gene in v. carteri, even if the codon-optimization has been done for chlamydomonas. although volvox diverged from unicellular ancestors like chlamydomonas at least million years ago and codon usage became different from each other, the relationship obviously is still close enough to allow for good expression in volvox. the assay for luciferase activity through detection of emitted light of the chemiluminescent reaction proved to be very effective in volvox just as in several other organisms before. the assay is quick and easy, enzyme activity can be quantified reliably and the measurement is both robust and well reproducible. the assay also exhibits high sensitivity because even a small amount of enzyme can catalytically process many substrate molecules to produce a detectable signal. accurate quantification is done with a luminometer. in large scale analyses, light detection on light-sensitive films or with a camera allows for qualitative assessments. since gaussia luciferase reaction is quite fast it is important to measure the emitted light immediately after coelenterazine injection. previous work with the gaussia luciferase showed that this enzyme is very stable even in body fluids such as blood and urine, in cell culture medium and in cell extracts. in our standard assay, we measured the enzyme activity in crude cell extracts obtained by ultrasonic treatment. no further purification of the enzyme was necessary. in preliminary experiments we even measured the luciferase activity in volvox cultures containing intact spheroids. the activity values obtained with intact spheroids were almost as high as the activity values obtained with cell extracts. therefore, in high-throughput screenings, direct measurement of cell cultures without ultrasonic treatment might be an even easier alternative, but reproducibility of the results needs be checked before. the functionality of the assay in cell cultures without ultrasonic treatment is attributed to the high cell-membrane permeability of coelenterazine. altogether, the codon-adapted luciferase reporter gene outshines previous reporter genes used in volvox in terms of its sensitivity, robustness and ease of use. in addition to the g-luc reporter gene, the promoter of the nitrate reductase gene turned out to be a powerful molecular tool for genetic engineering in v. carteri. two of our vectors allow for nita-driven expression of any target gene after a single cloning step. the nita promoter drives expression of the gene of interest by simply changing the nitrogen source from ammonium to nitrate, all the other components of the volvox medium remain unchanged throughout. the change of media is easily managed with volvox because the spheroids can be quickly collected on a nylon screen and transferred to the new medium. changing the nitrogen source disturbs growth and development, if at all, only to a minimum in contrast to other methods for promoter induction using chemicals, hormones or increased temperatures. the switching time of the nita promoter is very short: after induction by changing the nitrogen source, mrna expression increases immediately and reaches its maximum about 30 min after induction, which is similar to results in earlier work with the complete unmodified endogenous nita gene. the expression and induction characteristics caused by our nita fragment demonstrate, that the nita promoter is fully working and all required cis-elements are included. therefore, our constructs also might be interesting for those who want to analyze this promoter and its regulation in more detail. a further advantage of using the nita promoter for genetic engineering is that the expression rate of the target gene is not only influenced by nitrate but also by ammonium. as mentioned, ammonium prevents the uptake of nitrate and nitrate is required for the induction of the nita promoter. thus, submicromolar amounts of ammonium added together with the normal amounts of nitrate might allow for adjustment of the expression rate of the target gene in a given transformant. it also might be possible to switch off expression by adding larger amounts of ammonium.  two new molecular tools are now available for genetic engineering of the green alga volvox carteri. the nita promoter of v. carteri allows for inducible expression of target genes and induction occurs immediately after changing the nitrogen source from ammonium to nitrate. the change of the nitrogen source is easily managed and it disturbs neither growth nor development. the codon-adapted luciferase gene of g. princeps turned out to be a powerful reporter gene in volvox. the assay for luciferase activity is easy, robust, reproducible and sensible; moreover, it works in crude cell extracts. 
however, functional and molecular analyses of such processes require a wide array of molecular tools for genetic engineering. so far there are only a limited number of molecular tools available in volvox. two novel molecular tools for genetic engineering of volvox are now available: the nitrate-inducible nita promoter of v. carteri and the codon-adapted luciferase reporter gene of g. princeps. the multicellular green alga volvox carteri represents an attractive model system to study various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ecm biogenesis. long-term stability of inducible expression of the chimeric nita/g-luc transgenes after stable nuclear transformation was demonstrated by transcription analysis and bioluminescence assays. ammoniumco-transformationgaussia princeps luciferase genegenetic engineeringgreen algaeheterologous expressioninducible promoternitrate reductasenitrogen sourcesreporter genesstreptomyces rimosus aphviii genevolvocine algaeissue-copyright-statement© the author 2015 strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. we also show that the luciferase gene from the marine copepod gaussia princeps, which previously was engineered to match the codon usage of the unicellular alga chlamydomonas reinhardtii, is a suitable reporter gene in v. carteri. here, we show that the promoter of the v. carteri nitrate reductase gene is a powerful molecular switch for induction of transgene expression. emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer. these novel tools will be useful for future molecular research in v. carteri.
 the multicellular green alga volvox carteri represents an attractive model system to study various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ecm biogenesis. however, functional and molecular analyses of such processes require a wide array of molecular tools for genetic engineering. so far there are only a limited number of molecular tools available in volvox.  here, we show that the promoter of the v. carteri nitrate reductase gene is a powerful molecular switch for induction of transgene expression. strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. we also show that the luciferase gene from the marine copepod gaussia princeps, which previously was engineered to match the codon usage of the unicellular alga chlamydomonas reinhardtii, is a suitable reporter gene in v. carteri. emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer. long-term stability of inducible expression of the chimeric nita/g-luc transgenes after stable nuclear transformation was demonstrated by transcription analysis and bioluminescence assays.  two novel molecular tools for genetic engineering of volvox are now available: the nitrate-inducible nita promoter of v. carteri and the codon-adapted luciferase reporter gene of g. princeps. these novel tools will be useful for future molecular research in v. carteri. keywords ammoniumco-transformationgaussia princeps luciferase genegenetic engineeringgreen algaeheterologous expressioninducible promoternitrate reductasenitrogen sourcesreporter genesstreptomyces rimosus aphviii genevolvocine algaeissue-copyright-statement© the author 2015 