BACKGROUND
chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm and biotechnology for several decades.
with the first design of a c. reinhardtii microarray  <cit> , transcriptomic analyses in this organism could be conducted.
this first generation microarray contained  <dig> transcript models with  <dig> of them being associated with current transcript models covering about 87% of the predicted genome  <cit>  with nearly  <dig> genes.
however, rapid progress in genome annotation  <cit>  resulted in improved transcriptome data  <cit>  which clearly demanded the design of a new microarray platform for advanced and general transcriptome analyses.
microarrays are relatively cheap and reliable systems to analyze transcript levels on a routine basis and they are perfectly complementary to the recently established rna-seq platforms  <cit> .
the advantages of rna-seq are manifold, e.g.
the higher gene coverage and the increased sensitivity for differential gene expression  <cit> .
the characterization of new gene models and splicing variations are easier to predict, as well as the detection and characterization of mutation sites  <cit> .
however, the results of rna-seq are still critical to examine and high reproducibility is often difficult to achieve.
as a typical consequence, an overestimation of high abundant genes and length dependent amplification has been reported using rna-seq  <cit> .
these internal biases are still under discussion and data analysis and data normalization clearly need to be improved.
additional advantages of microarrays compared to rna-seq are still the significant lower costs  and the good coverage of exon based transcript levels, with around 90%  <cit> , where extremely deep sequencing would be necessary in order to achieve the same transcript coverage with rna-seq.
furthermore, microarray experiments are less time consuming, allow the run of multiple replicates and established analysis platforms for routine transcript level analyses are available.
however, the current microarray platform  <cit> , with  <dig> features, covers just 87% of the predicted genome and many newly annotated genes are missing  <cit> .
based on estimations using the chlamydomonas genome, up to  <dig> transcript models are expected to be present in this green algae  <cit> .
generally, c. reinhardtii adaption to varying stress conditions can be best evaluated by using -omics approaches.
transcriptome studies were performed by different applications, e.g.
microarray or rna-seq, during nutrient starvation  <cit> , anaerobiosis  <cit> , hydrogen production  <cit> , oxidative  <cit>  or light stress  <cit> .
the induction of genes responding to nutrient starvation, e.g.
sulfur and nitrogen starvation is well documented and available data sets are used within this work to test the reliability of our newly designed microarray.
a recent study investigating the effect of sulfur starvation  <cit>  included a comparison of rna-seq data with those acquired from microarray studies.
the data comparison showed a good accordance between both methods.
adaptation to sulfur stress starts with an induction of genes responsible for nutrient transport accompanied by the repression of gene expression related to photosynthetic processes.
in a later step, acclimatization/modulation processes include changes in the amino acid composition  <cit>  of certain target proteins and the synthesis of starch  <cit> .
furthermore, lipid metabolism was shown to be affected under sulfur starvation caused by the physiological shift to anaerobic conditions  <cit> .
in c. reinhardtii it is of particular interest that under sulfur depletion, plastidial hydrogenase activity and consequently hydrogen production is strongly induced  <cit> .
therefore, we also used existing transcriptome data sets of experiments to confirm gene expression pattern under sulfur starvation.
so far, a number of studies employing high-throughput technologies including transcriptomics, proteomics and metabolomics have been carried out to describe the process of hydrogen production in c. reinhardtii  <cit> .
another well documented stress condition is the growth of c. reinhardtii under nitrogen starvation  <cit> .
recent analyses of gene expression variation under nitrogen starvation by rna-seq  <cit>  precisely describe adaptation processes of photosynthesis as well as of anabolic metabolism mechanisms .
the high sensitivity of rna-seq was demonstrated by monitoring differences in expression rates of low abundant genes coding for transcripts involved in regulatory processes.
in detail, it could be concluded that nitrogen starvation results in a decreased photosynthetic gene expression and activity, increased lipid accumulation and induction of gametogenesis.
in this present study, we performed microarray experiments with our newly developed c. reinhardtii full genome microarray to prove its suitability for differential transcript analyses and for comparing time-course global expression profiles of c. reinhardtii under starvation conditions.
additionally, we tested the sensitivity of the array for identifying knockout mutations.
RESULTS
design
microarray design was based on data sets provided by the jgi  <dig>  genome annotation of c. reinhardtii.
this new chlamydomonas reinhardtii microarray platform is now available under the agilent© access number  <dig>  the microarray design is, however, not fixed: a crucial advantage is that newly annotated genes can easily be added to this array.
the adapted new transcriptome consists of  <dig> annotated nucleus-encoded gene models.
we designed  <dig> mer oligonucleotides using two software tools: array-express© and promide©.
the detailed origin for each probe and probe sequence is summarized in additional file  <dig>  table s <dig>  both software tools were used to design temperature and position optimized probes .
as a result, we determined sequence optimized probes for  <dig> gene models, which represent 93% coverage of the transcriptome based on the jgi <dig>  genome annotation.
we were unable to determine specific probes for the remaining transcript models.
in figure  <dig>  the chemical properties of the designed probes are described.
the g/c content of the probes varied between  <dig> and 60%  and melting temperature distribution for all probes was between  <dig> and 90°c, .
additionally, we used  <dig> approved sequences from the first generation c. reinhardtii microarray, adapted to the agilent© platform  <cit>  and added the probes as a third replicate to our microarray platform.
however, according to our blast analysis  are just  <dig> probes specific for one transcript model and we used just proved probes for our new microarray platform.
finally, we compared the new determined probe sequences with the new published annotation  and could confirm in silico by blast analysis http://blast.ncbi.nlm.nih.gov/ the hybridization specificity for 70% of the  <dig> transcript specific sequences.
the remaining probes showed potential cross hybridization properties  or could not be aligned to current transcripts .
it should be noted that the genome annotation of the c. reinhardtii genome is not yet finished and the final number of transcript models is still under discussion.
detailed information about the specificity and potential cross hybridization targets is provided in additional file  <dig>  table s <dig>
testing
we used identical rna samples from cells grown for  <dig> h under sulfur starvation to check cy- <dig> and cy- <dig> labeling.
successful pre-correction was achieved with the feature extraction software .
as a result we could demonstrate that labeling is nearly identical with both dyes.
the cy3/cy <dig> log  <dig> ratio showed a good distribution around  <dig> and therefore an additional dye-specific correction is not necessary .
to ensure that any remaining differences between the two labeling dyes are equalized, all computed log <dig> ratios were normalized.
following the recommendations of  <cit> , a normalization method based on robust local regression  was utilized for this purpose.
normalization and analysis were carried out with the in-house developed emma <dig> software  <cit> .
to test the reproducibility of the data, a  <dig> fold replication with labeled rna samples taken from starvation experiments was performed.
the high similarity of the data sets with a log <dig> ratio variation between  <dig> and 35% for all differential expressed genes derived from three biological and three technical replicas demonstrated the robustness of the system .
the internal c. reinhardtii specific control probes showed a variation smaller than 10%  in all experiments and could be therefore considered as a reliable control parameter for further experiments.
starvation experiments
we performed sulfur and nitrogen starvation experiments with c. reinhardtii wt cc <dig> to analyze the expression of genes responding to nutrient starvation.
as a result  <dig> probes showed a significant fluorescence signal against the background, and were therefore used for data analysis.
with three independent probes per transcript, around  <dig> transcript models could be analyzed in the experiments.
it should be noted that results derived from the newly designed probes showed a good concordance.
however data derived from the probes of the first generation array showed a lower log <dig> ratio in differentially expressed genes and blast analysis showed a high cross hybridization potential for many probes.
we therefore decided not to include these data sets into the analysis.
the data analysis resulted in the identification of a relatively small number of genes responding to the respective nutrient limitation conditions.
we performed a cluster analysis using the software genesis©  for differentially regulated genes, to detect time-dependent gene expression in response to nutrient stress, and could conclude that the majority of the genes showed a fast response to the nutrient stress with constant expression level over the whole time-course.
the late-responding genes mainly belong to transcripts related to secondary effects like phosphorus stress or co <dig> limitation.
nitrogen starvation
the response of gene expression during nitrogen starvation was in general higher and faster than during sulfur starvation conditions.
around  <dig> genes showed twofold increased expression levels whereas approximately  <dig> genes were detected with a twofold reduced expression .
comparison with rna-seq transcriptome data derived from nitrogen starvation experiments  <cit>  revealed a very similar picture with an identical differential gene expression pattern for 60% of the transcripts.
in  <cit>  transcripts were analyzed using illumina solexa© combined with the 454© ultra-fast-sequencing which resulted in the identification of several more transcripts.
differences in the two datasets are most likely a result of differences in time length of starvation  and of differences in the intensity of illumination .
another reason could be the possible bias by overestimation of large transcripts and differences in the range of detection.
within our experiments we were able to confirm the up-regulation of components of the nitrogen transport systems, such as ammonia-, nitrate- and nitrite-transporters .
from  <dig> annotated ammonium transporters , only amt <dig> and amt <dig> were up-regulated.
this result was achieved with all three probes per transcript with a minimal log <dig> ratio of  <dig> and shows high similarities to earlier data provided by  <cit> , however here some of the amt transcripts were most likely miss-annotated.
the nitrate transporter with the highest expression level was encoded by the locus cre <dig> g <dig> t <dig>  as expected, the major nitrate transporter did not response to nutrient stress, since ammonia was provided as the nitrogen source in the growth medium.
furthermore, the up-regulation of one of the four annotated glutamine synthetases  was confirmed, no up-regulation of gln <dig> ; gln <dig>  and gln <dig>  was detectable, similar results were obtained from the rna seq data.
additionally, increased gene expression for one nitrite reductase  and one nitrate reductase  could be confirmed within our experiments.
interestingly, although under these conditions the majority of the photosynthetic gene transcripts were down-regulated, several genes related to photosynthesis showed an up-regulation in transcription rates.
in detail, some genes responsible for ps light harvesting and energy distribution like lhca <dig>  lhcsr <dig> and several photosystem ii subunits like psbx and psbs <dig> were up-regulated during nitrogen starvation.
however, it is noteworthy and somewhat surprisingly that we detected at the same time down-regulation of lhcsr <dig> under nitrogen limitation, a result which is in good accordance to rna-seq data.
it has been reported that lhscr genes are up-regulated under stress conditions and responsible for de-excitation of chlorophyll molecules in ps ii  <cit> .
furthermore in good agreement with  <cit> , none of the ribosomal related genes were up-regulated.
we could further confirm the nit <dig> induction  and the repression of nab <dig>  a nuclear encoded mrna binding factor, which specifically binds and sequesters lhcii mrna and prevents their translation  <cit> .
many carbonic anhydrases showed a down-regulation; however the mitochondrial carbonic anhydrase  was up-regulated.
regarding the tca-cycle the transcript level of the citrate synthase  increased during nitrogen starvation, in contrast to the isocitrate lyase  <dig>  which was not affected in our experiments.
additionally, our data indicated an up-regulation of several genes induced by phosphate starvation, like psr <dig>  as earlier described  <cit> .
psr <dig> however, did not show an increased level of expression within the rna-seq data.
again, the reason for the differences could be due to the longer starvation period leading to secondary effects.
sulfur stress
under sulfur starvation  <dig> genes showed a differential gene expression.
<dig> genes were down-regulated by at least 2-fold whereas around  <dig> genes were at least 2-fold up-regulated.
comparison between first generation and new microarray data derived from sulfur starvation experiments showed high similarities for many genes regarding changes in their differential gene expression pattern .
we confirmed the up-regulation for several sulfate transport systems and induction of several stress response systems .
as shown in figure  <dig> we were able to confirm the increased gene expression of two major aryl sulfatases .
probe specificity for the other ars enzymes could be confirmed but no increased gene expression was detectable.
it should be noted that for ars <dig>  no probes were designed, since this gene has not yet been annotated at the time of the experiment.
the extracellular proteins ecp <dig>  ecp <dig>  slp <dig> , sua  stl <dig>  and sir <dig>  also showed an up-regulation for all determined and tested probes.
similar to the experiments with nitrogen starvation, most photosynthetic related genes were down-regulated however for several proteins involved in light harvesting and light quenching  we could confirm an increase in transcript levels during all  <dig> time points.
overall, we found the majority of the genes  previously described to be induced or repressed by sulfate starvation with the same expression pattern  <cit> .
variations can be explained by the different time scale of sample harvesting and different growth conditions which could also lead to secondary effects like phosphate limitation.
differences in the log <dig> ratios between rna-seq data and microarray data are most likely a result of the higher dynamic range of the rna-seq data, since saturation effects, which can occur for highly expressed genes on microarrays, are not expected.
combing the array data sets derived from the two starvation experiments we identified several genes, which showed an increased or reduced transcript level under both nutrient stress conditions .
most of these genes are either of unknown function or are reported to be involved in transport or metabolism.
lhcsr <dig> knock out detection
to analyze the specificity of the array system we used the knock out mutant npq <dig> , which has been previously shown to be deficient in the lhcsr <dig> gene transcripts  <cit> , to check if we can detect the genotype on the rna level.
it is known that the lhscr <dig>  lhcsr <dig>  and lhscr <dig>  gene expression is enhanced under sulfur starvation and/or during hydrogen production induced by sulfur deprivation  <cit> .
therefore, we analyzed the transcript level for npq <dig> and the parental strain 4a+ under sulfur starvation.
the expression rates under sulfate starvation increased for all three transcripts in the wild type, but no gene expression was detectable in the npq <dig> mutant in the lhcsr <dig>  and lhscr <dig>  isoforms, as expected .
for this reason, we can state a high specificity for our designed probes regarding the lhcsr transcripts and in combination with the analyzed nutrient starvation experiments a good usability of our microarrays.
comparison with wt cc <dig> grown under sulfur deprivation, showed no significant changes regarding the normal response to sulfur starvation.
discussion
in this work we successfully designed and tested a novel microarray platform for chlamydomonas reinhardtii.
we were able to determine unique sequences for most  of the transcript models obtained from the joint genome institute data base version jgi  <dig> .
for a small number of transcript models  we could not identify a specific gene sequence and consequently these genes cannot be analyzed with the current microarray.
we confirmed the specificity for  <dig> probe sets with the current annotation .
we tested our microarray with rna samples from cultures grown under different nutrient stress conditions and compared our data with recent publications.
by doing this, we could confirm with our new system previously published changes in gene expression during nutrient starvation for many genes and hereby proved that this newly designed array is very useful for general transcription analysis.
in addition, rna amplification enabled us to detect several low abundant regulator genes expressed under nutrient starvation with the acquired data being in good accordance with previously published rna-seq and microarray data  <cit> .
microarrays are cheap and reliable tools for monitoring transcript changes, and although rna-seq methods may allow a more detailed view inside the transcriptome, the lower costs, the high reproducibility and the established analysis systems advantages microarrays for routine applications.
therefore, one can now choose the platform, which provides the best conditions for the individual experiment.
in functional tests of the microarrays we were able to show, besides proof of functionality for the analysis of differential gene regulation under nutrient stress conditions, the deletion of the lhcsr <dig>  and lhcsr <dig>  in the knockout strain npq <dig>  <cit>
.
this result let us conclude that the platform is sensitive to investigate knock out or knock down strains.
the successful confirmation of the lack of the corresponding transcript and of induction of expression under sulfur stress for lhcsr <dig> /lhcsr <dig>  clearly proved the suitability of the array for the analysis of gene deletions.
furthermore, as the probes did not show any cross hybridization or increased unspecific binding, the specificity of the designed probes has clearly been demonstrated in this work.
intriguingly, the lhcsr <dig> genes did not show an up-regulation during nitrogen starvation in our work, thus confirming previous experiments  <cit> .
under nutrient starvation the first response of the cells is an up-regulation of transport systems required for the specific nutrient.
we were able to prove gene expression of such transporters for both, nitrogen and sulfate starvation as well as the increased expression for other nitrogen and sulfate specific genes.
both starvation conditions resulted in an increase of transcript levels already after  <dig> hours of nutrient depletion.
these higher levels of transcription rates were consistent throughout the whole time of the experiment.
differences between our and previous studies  <cit>  could be a result of the longer starvation period and use of different chlamydomonas strains cc <dig> and cc <dig> instead of d <dig>  ars <dig>  21gr.
additionally we included into our analysis just those genes with an up-regulation monitored within all time points.
therefore, differences in gene expression level and the number of genes vary.
CONCLUSIONS
in summary, our data proved a high sensitivity of the array as a precondition for further detailed and advanced transcript analyses of mutant vs wt strains in c. reinhardtii.
with probes for  <dig> transcript models  our new array offers a very good coverage of the chlamydomonas genome.
it is noteworthy that current genome annotation predictions estimate around 12000- <dig> gene models, so most likely some transcript models are still missing on the platform, however an updated version of the array can easily be established in the future.

 chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm and biotechnology for several decades. with the first design of a c. reinhardtii microarray, transcriptomic analyses in this organism could be conducted. this first generation microarray contained transcript models with of them being associated with current transcript models covering about 87% of the predicted genome with nearly genes. however, rapid progress in genome annotation resulted in improved transcriptome data which clearly demanded the design of a new microarray platform for advanced and general transcriptome analyses. microarrays are relatively cheap and reliable systems to analyze transcript levels on a routine basis and they are perfectly complementary to the recently established rna-seq platforms. the advantages of rna-seq are manifold, e.g. the higher gene coverage and the increased sensitivity for differential gene expression. the characterization of new gene models and splicing variations are easier to predict, as well as the detection and characterization of mutation sites. however, the results of rna-seq are still critical to examine and high reproducibility is often difficult to achieve. as a typical consequence, an overestimation of high abundant genes and length dependent amplification has been reported using rna-seq. these internal biases are still under discussion and data analysis and data normalization clearly need to be improved. additional advantages of microarrays compared to rna-seq are still the significant lower costs and the good coverage of exon based transcript levels, with around 90%, where extremely deep sequencing would be necessary in order to achieve the same transcript coverage with rna-seq. furthermore, microarray experiments are less time consuming, allow the run of multiple replicates and established analysis platforms for routine transcript level analyses are available. however, the current microarray platform, with features, covers just 87% of the predicted genome and many newly annotated genes are missing. based on estimations using the chlamydomonas genome, up to transcript models are expected to be present in this green algae. generally, c. reinhardtii adaption to varying stress conditions can be best evaluated by using -omics approaches. transcriptome studies were performed by different applications, e.g. microarray or rna-seq, during nutrient starvation, anaerobiosis, hydrogen production, oxidative or light stress. the induction of genes responding to nutrient starvation, e.g. sulfur and nitrogen starvation is well documented and available data sets are used within this work to test the reliability of our newly designed microarray. a recent study investigating the effect of sulfur starvation included a comparison of rna-seq data with those acquired from microarray studies. the data comparison showed a good accordance between both methods. adaptation to sulfur stress starts with an induction of genes responsible for nutrient transport accompanied by the repression of gene expression related to photosynthetic processes. in a later step, acclimatization/modulation processes include changes in the amino acid composition of certain target proteins and the synthesis of starch. furthermore, lipid metabolism was shown to be affected under sulfur starvation caused by the physiological shift to anaerobic conditions. in c. reinhardtii it is of particular interest that under sulfur depletion, plastidial hydrogenase activity and consequently hydrogen production is strongly induced. therefore, we also used existing transcriptome data sets of experiments to confirm gene expression pattern under sulfur starvation. so far, a number of studies employing high-throughput technologies including transcriptomics, proteomics and metabolomics have been carried out to describe the process of hydrogen production in c. reinhardtii. another well documented stress condition is the growth of c. reinhardtii under nitrogen starvation. recent analyses of gene expression variation under nitrogen starvation by rna-seq precisely describe adaptation processes of photosynthesis as well as of anabolic metabolism mechanisms. the high sensitivity of rna-seq was demonstrated by monitoring differences in expression rates of low abundant genes coding for transcripts involved in regulatory processes. in detail, it could be concluded that nitrogen starvation results in a decreased photosynthetic gene expression and activity, increased lipid accumulation and induction of gametogenesis. in this present study, we performed microarray experiments with our newly developed c. reinhardtii full genome microarray to prove its suitability for differential transcript analyses and for comparing time-course global expression profiles of c. reinhardtii under starvation conditions. additionally, we tested the sensitivity of the array for identifying knockout mutations.  design microarray design was based on data sets provided by the jgi genome annotation of c. reinhardtii. this new chlamydomonas reinhardtii microarray platform is now available under the agilent© access number the microarray design is, however, not fixed: a crucial advantage is that newly annotated genes can easily be added to this array. the adapted new transcriptome consists of annotated nucleus-encoded gene models. we designed mer oligonucleotides using two software tools: array-express© and promide©. the detailed origin for each probe and probe sequence is summarized in additional file table s both software tools were used to design temperature and position optimized probes. as a result, we determined sequence optimized probes for gene models, which represent 93% coverage of the transcriptome based on the jgi genome annotation. we were unable to determine specific probes for the remaining transcript models. in figure the chemical properties of the designed probes are described. the g/c content of the probes varied between and 60% and melting temperature distribution for all probes was between and 90°c,. additionally, we used approved sequences from the first generation c. reinhardtii microarray, adapted to the agilent© platform and added the probes as a third replicate to our microarray platform. however, according to our blast analysis are just probes specific for one transcript model and we used just proved probes for our new microarray platform. finally, we compared the new determined probe sequences with the new published annotation and could confirm in silico by blast analysis http://blast.ncbi.nlm.nih.gov/ the hybridization specificity for 70% of the transcript specific sequences. the remaining probes showed potential cross hybridization properties or could not be aligned to current transcripts. it should be noted that the genome annotation of the c. reinhardtii genome is not yet finished and the final number of transcript models is still under discussion. detailed information about the specificity and potential cross hybridization targets is provided in additional file table s testing we used identical rna samples from cells grown for h under sulfur starvation to check cy- and cy- labeling. successful pre-correction was achieved with the feature extraction software. as a result we could demonstrate that labeling is nearly identical with both dyes. the cy3/cy log ratio showed a good distribution around and therefore an additional dye-specific correction is not necessary. to ensure that any remaining differences between the two labeling dyes are equalized, all computed log ratios were normalized. following the recommendations of, a normalization method based on robust local regression was utilized for this purpose. normalization and analysis were carried out with the in-house developed emma software. to test the reproducibility of the data, a fold replication with labeled rna samples taken from starvation experiments was performed. the high similarity of the data sets with a log ratio variation between and 35% for all differential expressed genes derived from three biological and three technical replicas demonstrated the robustness of the system. the internal c. reinhardtii specific control probes showed a variation smaller than 10% in all experiments and could be therefore considered as a reliable control parameter for further experiments. starvation experiments we performed sulfur and nitrogen starvation experiments with c. reinhardtii wt cc to analyze the expression of genes responding to nutrient starvation. as a result probes showed a significant fluorescence signal against the background, and were therefore used for data analysis. with three independent probes per transcript, around transcript models could be analyzed in the experiments. it should be noted that results derived from the newly designed probes showed a good concordance. however data derived from the probes of the first generation array showed a lower log ratio in differentially expressed genes and blast analysis showed a high cross hybridization potential for many probes. we therefore decided not to include these data sets into the analysis. the data analysis resulted in the identification of a relatively small number of genes responding to the respective nutrient limitation conditions. we performed a cluster analysis using the software genesis© for differentially regulated genes, to detect time-dependent gene expression in response to nutrient stress, and could conclude that the majority of the genes showed a fast response to the nutrient stress with constant expression level over the whole time-course. the late-responding genes mainly belong to transcripts related to secondary effects like phosphorus stress or co limitation. nitrogen starvation the response of gene expression during nitrogen starvation was in general higher and faster than during sulfur starvation conditions. around genes showed twofold increased expression levels whereas approximately genes were detected with a twofold reduced expression. comparison with rna-seq transcriptome data derived from nitrogen starvation experiments revealed a very similar picture with an identical differential gene expression pattern for 60% of the transcripts. in transcripts were analyzed using illumina solexa© combined with the 454© ultra-fast-sequencing which resulted in the identification of several more transcripts. differences in the two datasets are most likely a result of differences in time length of starvation and of differences in the intensity of illumination. another reason could be the possible bias by overestimation of large transcripts and differences in the range of detection. within our experiments we were able to confirm the up-regulation of components of the nitrogen transport systems, such as ammonia-, nitrate- and nitrite-transporters. from annotated ammonium transporters, only amt and amt were up-regulated. this result was achieved with all three probes per transcript with a minimal log ratio of and shows high similarities to earlier data provided by, however here some of the amt transcripts were most likely miss-annotated. the nitrate transporter with the highest expression level was encoded by the locus cre g t as expected, the major nitrate transporter did not response to nutrient stress, since ammonia was provided as the nitrogen source in the growth medium. furthermore, the up-regulation of one of the four annotated glutamine synthetases was confirmed, no up-regulation of gln; gln and gln was detectable, similar results were obtained from the rna seq data. additionally, increased gene expression for one nitrite reductase and one nitrate reductase could be confirmed within our experiments. interestingly, although under these conditions the majority of the photosynthetic gene transcripts were down-regulated, several genes related to photosynthesis showed an up-regulation in transcription rates. in detail, some genes responsible for ps light harvesting and energy distribution like lhca lhcsr and several photosystem ii subunits like psbx and psbs were up-regulated during nitrogen starvation. however, it is noteworthy and somewhat surprisingly that we detected at the same time down-regulation of lhcsr under nitrogen limitation, a result which is in good accordance to rna-seq data. it has been reported that lhscr genes are up-regulated under stress conditions and responsible for de-excitation of chlorophyll molecules in ps ii. furthermore in good agreement with, none of the ribosomal related genes were up-regulated. we could further confirm the nit induction and the repression of nab a nuclear encoded mrna binding factor, which specifically binds and sequesters lhcii mrna and prevents their translation. many carbonic anhydrases showed a down-regulation; however the mitochondrial carbonic anhydrase was up-regulated. regarding the tca-cycle the transcript level of the citrate synthase increased during nitrogen starvation, in contrast to the isocitrate lyase which was not affected in our experiments. additionally, our data indicated an up-regulation of several genes induced by phosphate starvation, like psr as earlier described. psr however, did not show an increased level of expression within the rna-seq data. again, the reason for the differences could be due to the longer starvation period leading to secondary effects. sulfur stress under sulfur starvation genes showed a differential gene expression. genes were down-regulated by at least 2-fold whereas around genes were at least 2-fold up-regulated. comparison between first generation and new microarray data derived from sulfur starvation experiments showed high similarities for many genes regarding changes in their differential gene expression pattern. we confirmed the up-regulation for several sulfate transport systems and induction of several stress response systems. as shown in figure we were able to confirm the increased gene expression of two major aryl sulfatases. probe specificity for the other ars enzymes could be confirmed but no increased gene expression was detectable. it should be noted that for ars no probes were designed, since this gene has not yet been annotated at the time of the experiment. the extracellular proteins ecp ecp slp, sua stl and sir also showed an up-regulation for all determined and tested probes. similar to the experiments with nitrogen starvation, most photosynthetic related genes were down-regulated however for several proteins involved in light harvesting and light quenching we could confirm an increase in transcript levels during all time points. overall, we found the majority of the genes previously described to be induced or repressed by sulfate starvation with the same expression pattern. variations can be explained by the different time scale of sample harvesting and different growth conditions which could also lead to secondary effects like phosphate limitation. differences in the log ratios between rna-seq data and microarray data are most likely a result of the higher dynamic range of the rna-seq data, since saturation effects, which can occur for highly expressed genes on microarrays, are not expected. combing the array data sets derived from the two starvation experiments we identified several genes, which showed an increased or reduced transcript level under both nutrient stress conditions. most of these genes are either of unknown function or are reported to be involved in transport or metabolism. lhcsr knock out detection to analyze the specificity of the array system we used the knock out mutant npq, which has been previously shown to be deficient in the lhcsr gene transcripts, to check if we can detect the genotype on the rna level. it is known that the lhscr lhcsr and lhscr gene expression is enhanced under sulfur starvation and/or during hydrogen production induced by sulfur deprivation. therefore, we analyzed the transcript level for npq and the parental strain 4a+ under sulfur starvation. the expression rates under sulfate starvation increased for all three transcripts in the wild type, but no gene expression was detectable in the npq mutant in the lhcsr and lhscr isoforms, as expected. for this reason, we can state a high specificity for our designed probes regarding the lhcsr transcripts and in combination with the analyzed nutrient starvation experiments a good usability of our microarrays. comparison with wt cc grown under sulfur deprivation, showed no significant changes regarding the normal response to sulfur starvation. discussion in this work we successfully designed and tested a novel microarray platform for chlamydomonas reinhardtii. we were able to determine unique sequences for most of the transcript models obtained from the joint genome institute data base version jgi. for a small number of transcript models we could not identify a specific gene sequence and consequently these genes cannot be analyzed with the current microarray. we confirmed the specificity for probe sets with the current annotation. we tested our microarray with rna samples from cultures grown under different nutrient stress conditions and compared our data with recent publications. by doing this, we could confirm with our new system previously published changes in gene expression during nutrient starvation for many genes and hereby proved that this newly designed array is very useful for general transcription analysis. in addition, rna amplification enabled us to detect several low abundant regulator genes expressed under nutrient starvation with the acquired data being in good accordance with previously published rna-seq and microarray data. microarrays are cheap and reliable tools for monitoring transcript changes, and although rna-seq methods may allow a more detailed view inside the transcriptome, the lower costs, the high reproducibility and the established analysis systems advantages microarrays for routine applications. therefore, one can now choose the platform, which provides the best conditions for the individual experiment. in functional tests of the microarrays we were able to show, besides proof of functionality for the analysis of differential gene regulation under nutrient stress conditions, the deletion of the lhcsr and lhcsr in the knockout strain npq. this result let us conclude that the platform is sensitive to investigate knock out or knock down strains. the successful confirmation of the lack of the corresponding transcript and of induction of expression under sulfur stress for lhcsr /lhcsr clearly proved the suitability of the array for the analysis of gene deletions. furthermore, as the probes did not show any cross hybridization or increased unspecific binding, the specificity of the designed probes has clearly been demonstrated in this work. intriguingly, the lhcsr genes did not show an up-regulation during nitrogen starvation in our work, thus confirming previous experiments. under nutrient starvation the first response of the cells is an up-regulation of transport systems required for the specific nutrient. we were able to prove gene expression of such transporters for both, nitrogen and sulfate starvation as well as the increased expression for other nitrogen and sulfate specific genes. both starvation conditions resulted in an increase of transcript levels already after hours of nutrient depletion. these higher levels of transcription rates were consistent throughout the whole time of the experiment. differences between our and previous studies could be a result of the longer starvation period and use of different chlamydomonas strains cc and cc instead of d ars 21gr. additionally we included into our analysis just those genes with an up-regulation monitored within all time points. therefore, differences in gene expression level and the number of genes vary.  in summary, our data proved a high sensitivity of the array as a precondition for further detailed and advanced transcript analyses of mutant vs wt strains in c. reinhardtii. with probes for transcript models our new array offers a very good coverage of the chlamydomonas genome. it is noteworthy that current genome annotation predictions estimate around 12000- gene models, so most likely some transcript models are still missing on the platform, however an updated version of the array can easily be established in the future. 
the complete annotation of the genome allows transcriptomic studies, however a new microarray platform was needed. chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm, cell mobility, phototaxis, and biotechnology. to demonstrate the capabilities of the new microarray, we analyzed transcript levels for cultures grown under nitrogen as well as sulfate limitation, and compared the results with recently published microarray and rna-seq data. we could thereby confirm previous results derived from data on nutrient-starvation induced gene expression of a group of genes related to protein transport and adaptation of the metabolism as well as genes related to efficient light harvesting, light energy distribution and photosynthetic electron transport. based on the completed annotation of chlamydomonas reinhardtii a new microarray on an agilent platform was designed using an extended jgi genome data set which included transcript models. following the estimation of predicted genes in chlamydomonas reinhardtii our new microarray, nevertheless, covers the expected genome by 90-95%. alignment studies with the recently published augustus annotation confirmed transcript models resulting in a very good coverage of 70% of the transcriptome.
 chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm, cell mobility, phototaxis, and biotechnology. the complete annotation of the genome allows transcriptomic studies, however a new microarray platform was needed. based on the completed annotation of chlamydomonas reinhardtii a new microarray on an agilent platform was designed using an extended jgi genome data set which included transcript models.  in total probes were determined covering 93% of the transcriptome. alignment studies with the recently published augustus annotation confirmed transcript models resulting in a very good coverage of 70% of the transcriptome. following the estimation of predicted genes in chlamydomonas reinhardtii our new microarray, nevertheless, covers the expected genome by 90-95%.  to demonstrate the capabilities of the new microarray, we analyzed transcript levels for cultures grown under nitrogen as well as sulfate limitation, and compared the results with recently published microarray and rna-seq data. we could thereby confirm previous results derived from data on nutrient-starvation induced gene expression of a group of genes related to protein transport and adaptation of the metabolism as well as genes related to efficient light harvesting, light energy distribution and photosynthetic electron transport. 