Mycosporine-like amino acid and aromatic amino acid transcriptome response to UV and far-red light in the cyanobacterium Chlorogloeopsis fritschii PCC 6912

The “UV sunscreen” compounds, the mycosporine-like amino acids (MAAs) are widely reported in cyanobacteria and are known to be induced under ultra-violet (UV) light. However, the impact of far red (FR) light on MAA biosynthesis has not been studied. We report results from two experiments measuring transcriptional regulation of MAA and aromatic amino acid pathways in the filamentous cyanobacterium Chlorogloeopsis fritschii PCC 6912. The first experiment, comparing UV with white light, shows the expected upregulation of the characteristic MAA mys gene cluster. The second experiment, comparing FR with white light, shows that three genes of the four mys gene cluster encoding up to mycosporine-glycine are also upregulated under FR light. This is a new discovery. We observed corresponding increases in MAAs under FR light using HPLC analysis. The tryptophan pathway was upregulated under UV, with no change under FR. The tyrosine and phenylalanine pathways were unaltered under both conditions. However, nitrate ABC transporter genes were upregulated under UV and FR light indicating increased nitrogen requirement under both light conditions. The discovery that MAAs are upregulated under FR light supports MAAs playing a role in photon dissipation and thermoregulation with a possible role in contributing to Earth surface temperature regulation.


Scientific Reports
| (2020) 10:20638 | https://doi.org/10.1038/s41598-020-77402-6 www.nature.com/scientificreports/ the MAA and AAA pathways. In the second experiment, we explore the effects of exposure to FR light on the same pathways. Both experiments use white light as the control. The experiments were carried out using different sequencing and bioinformatic techniques due to experiments being undertaken at different times and places. Whilst the results from the two experiments are not directly comparable they each provide unique insights into MAA and AAA pathways and the results from the FR light experiment reveals novel and unexpected findings.

Results
The overall change in expression of genes in C. fritschii PCC 6912 exposed to UV-B and FR is shown in Fig. 1; this highlights the significant changes found associated with the MAA and AAA pathways. Associated details on the identified and significantly up and downregulated gene homologs associated with MAAs, AAAs and nitrogen transport are listed in Table 1. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Most strikingly, from across the whole genone was that the MAA mys gene homolog cluster ( Fig. 2) stood out as being significantly upregulated not only in C. fritschii PCC 6912 exposed to UV, but also when exposed to FR (Fig. 1). Other notable features included the gene homologs encoding nitrate ABC transporters, required to transport nitrogen into the cell, which were found to be significantly upregulated under both light conditions, and genes associated with the shikimate pathway to produce tryptophan which were predominately upregulated under UV-B but not affected by FR. Below we report these findings in more detail.

Pathway to 3-dehydroquinate. 3-DHQ is the branch point to producing both MAAs and AAAs.
3-DHQ is formed from phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), which are intermediates in glycolysis and the pentose phosphate pathway respectively (Fig. 3A). The first step is the production of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) using DAHP synthase (AroA: E.C 2.5.1.54; Fig. 3A). In the second step, DAHP is converted to 3-DHQ using 3-dehydroquinate synthase (DHQS) 19,20,23 . We found three annotated DAHP synthase gene homologs in the C. fritschii PCC 6912 genome ( MAA synthesis. We found that the gene homologs representing the mys cluster (  Table 1). However, most remarkably, we also found upregulation of mys A, B and C gene homolog cluster in the FR experiment. Here, however the upregulation was not as high as observed in our UV-B experiment (log2 fold change of between 1.51 and 3.55) (Fig. 3B). In contrast to the pattern seen under UV-B, under FR the fourth gene homolog in the MAA cluster, mysE, which catalyses the conversion of mycosporine-glycine to shinorine was slightly downregulated (WP_016876762; log2 fold change − 1.13) although this was not deemed as significant (Fig. 3B). The induction of MAAs under UV light has previously been established in Gloeocapsa sp and in C. fritschii PCC 6912 14,33 , however this is the first time that is has been observed in cells exposed to FR light.
The DHQS-like gene homolog predicted as DDGS associated with the MAA cluster was upregulated under both FR and UV (WP_016876765; UV/FR log2 fold change 2.51 and 6.49 respectively; Fig. 3B). This DHQSlike (DDGS) gene homolog has been designated as mysA 17 . Two copies of DHQS have also been reported for Anabaena variabilis PCC 7937 and Anabaena sp. PCC 7120 where they were considered to be catalysing different reactions including one for MAA synthesis and the other for AAA synthesis 34 . We now know that one of these DHQS copies was most likely a DDGS 18 . Annotation of these sugar phosphate cyclase family proteins was confounded by DHQS sharing significant sequence similarity to 2-ep-5epi-valionone synthase (EEVS) and making current database annotation unreliable 35 . To confirm the identity of the sugar phosphate cyclase family proteins in C. fritschii PCC 6912, we used BLAST with representative cyanobacterial proteins, alignments and examination of the published discriminatory motifs 36 . From this we confirmed WP_016876765 as DGGS and WP_016876414a as DHQS (Fig. 4). There were no further sugar phosphate cyclase family proteins present.
High performance liquid chromatography (HPLC) analysis of the FR experimental biomass confirmed increases in MAAs under FR with a significant increase in mycosporine-glycine (Table 2; Supplementary Fig. S1). An increase was also observed for shinorine but because of variability across the three experimental replicate samples, this was not deemed to be significant. HPLC analysis of the UV-B samples revealed levels under the white light control to be below detection limit with low levels of shinorine and mycosporine-glycine induced in the samples corresponding to the transcriptome experiment (Table 2). We also observed that in additional samples collected at 4 h in the UV experiment, MAA levels were much higher at this time point, indicating the dynamic nature in acclimation of cells to the stress environment.  www.nature.com/scientificreports/   Table 1). Shikimate is converted to chorismate in a further 3 steps, via shikimate kinase, EPSP synthase and chorismate synthase. We found a bacterial shikimate kinase which was unaltered (WP_016875893.1: AroK). We also found an adenylate kinase family shikimate kinase, similar to those found in Nostoc and Anabaena, which was transcriptionally upregulated under UV (WP_016878669.1: AdK: UV log2 fold change 2.01 (Fig. 5, Table 1). There were no changes in transcriptional regulation under UV or FR for chorismate synthase or for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. Chorismate is the branch point to the AAAs tryptophan, tyrosine and phenylalanine, as well as folate (B9) and to phylloquinone (vitamin K) involving multiple steps for each pathway ( Supplementary Fig. S2). From chorismate, anthranilate synthase produces anthranilite on the pathway to produce tryptophan and indole or chorismate mutase produces prephenate as the branch point to tyrosine www.nature.com/scientificreports/ and phenylalanine. We found an upregulation of gene homologs under UV encoding enzymes for the conversion of chorismate to tryptophan; these were found in a gene cluster (WP_016875298 to WP_016875302; tyrA, B, C and D; UV log2 fold change 1.86-3.17) (Fig. 5). There was no change in expression in this gene cluster under FR (Fig. 5). It was within this cluster that we found DAHP which was, as indicated above, upregulated only under UV (WP_016875297; EC.2.5.1.54, aroA.2.1; UV log2 fold change 3.15) (Fig. 3).  Figs. S2, S3). A reduction in tyrosine and phenylalanine in C. fritschii PCC 6912 exposed to UV-B has previously been observed 39 .

AAA synthesis.
Next we investigated the tryptophan pathways. We first looked for a change in regulation associated with any tryptophan-rich sensory translocator protein (TSPOs). TSPOs are known to be associated with stress adaptation and have recently been found to be upregulated by green light and in response to nutrient defficiency and stress in the cyanobacteria Fremyella diplosiphon 26,40 . We found two similar adjacent TSPOs in C. fritschii with significant homology (> 75%) to TSPOs in the cyanobacteria Fremyella displosiphon. Expression was not changed for the gene similar to Fremyella tspo2, but the tspo3 homolog was moderately downregulated under UV-B and not affected by FR (WP_016876598; UV log2 fold change − 2.10). A third, less related TSPO homologous to proteins found in Nostoc sp. PCC7120 and Anabaena variabilis was downregulated under FR and unchanged under UV (WP_016874465; FR log2 fold change − 1.66). These TSPOs were thus differentially modulated under different light regimens.
Next we investigated if UVR8. Tryptophan has been associated with UVR8 protein serving as the UV-B chromophore that triggers a signalling pathway for UV protection 24 . We found no homolog to UVR8 in C. fritschii. Finally, we were interested to determine if there were changes in the tryptophan related indole acetic acid (IAA). There is some indication that the phytohormone related IAA is modulated by tryptophan and light 41 . One of the pathways to IAA is via indolepyruvate. Conversion of indolepyruvate to indoleacetaldehyde by indolepyruvate decarboxylase (IpdC) is the rate-limiting step in this pathway to IAA synthesis. We found indolepyruvate decarboxylase was upregulated under UV-B but not FR (WP_016877391: E.C 4.1.1.74; IpdC; UV log2 fold change 3.93) ( Table 1; Supplementary Fig. S4). We also found upregulation under UV of a gene homolog involved in the production of indole-3-pyruvate monooxygenase which is also involved in IAA synthesis (WP_016875162: E.C.1.14.13.168; UV log2 fold change 3.24). In contrast, two gene homologs involved in tryptophan metabolism and formation of kynurenine and niacin using tryptophan 2,3 dioxygenase were downregulated under UV (WP_016874813; EC: 1.2.13; UV log2 fold change − 3.94 and WP_016874602; EC 1.13.11.11; UV log2 fold change − 2.75 respectively).
The role of nitrogen. Nitrogen, with its major source being nitrate in cyanobacteria, is essential to the biosynthesis of MAAs and AAAs. We found that in addition to the mys cluster standing out for upregulation under FR, that the nitrate transport genes were also prominent for their upregulation under FR. Nitrate transporters activate transport of nitrate into the cell for glutamine production 42 . For AAAs, nitrogen is introduced with glutamine converting chorismate to anthranilate (Fig. 5). Glutamine, required for anthranilate production, utilises a source of nitrogen which is primarily sourced through nitrate in the growth medium. Glutamine is required to introduce nitrogen into the aromatic chorismate ring structure converting it using anthranilate synthase into anthranilate on the pathway to tryptophan (Fig. 5). For MAAs, nitrogen is added to 4-deoxygadusol using an ATP grasp (Fig. 2). Tyrosine and phenyalanine require additional conversion before nitrogen is added via aminotransferases 23 . Nitrate transporter genes, nrtA and nrtB were found within an nitrogen regulation cluster and were upregulated under both UV and FR (WP_016877346-WP_016877345; log2 fold change range 1.59-1.81). Associated nirA, nrtC, nrtD and nrtP were upregulated only under FR (WP_016877347-WP_016877342 FR log2 fold change 1.53-2.31) ( Table 1). Our results showing upregulation of genes associated with nitrate assimilation indicates that increased nitrogen is required under both UV and FR light conditions.

Conclusion
MAAs and AAAs are two groups of low molecular weight aromatic compounds containing nitrogen that are related in their biosynthetic pathways. MAAs are widely recognised as being important in photoprotection and AAAs are important in the synthesis of proteins and other essential end products. MAAs in addition to their important physiological role and evolutionary and ecological relevance are increasingly sought after for their use in sunscreen and cosmetic products. Likewise, in addition to their important physiological role, AAAs have a wide application in human health and more widely in human medicine and nutrition.
Our results provide new understanding on the regulation of MAA and AAA biosynthetic pathways under low level UV and FR light. Most importantly, we have shown for the first time that the mys gene homolog cluster in C. fritschii PCC 6912 is associated with MAA synthesis up to mycosporine-glycine is upregulated when exposed to UV and to FR light. Increases in MAAs measured using HPLC was found in extracts of the corresponding samples of C. fritschii PCC 6912 exposed to FR.
Our finding that there is upregulation of the MAA pathway under FR suggests that MAAs may have a role in the photon dissipation of light and thermodynamic optimisation. Recently scytonemin, a related UV-A sunscreen compound, was shown to increase soil temperature in cyanobacterial biocrust communities, decreasing soil albedo significantly with the potential for impacting biosphere feedback and affecting the climate 43 . The ability of FR to influence production of MAAs supports the role of these compounds as photon dissipators opening up new possibilites on the importance of these compounds in heat regulation on Earth.
We also found a cluster of genes associated with the pathway to tryptophan upregulated under UV but not under FR. Pathways to tyrosine and phenylalanine biosynthesis were unaltered under both UV and FR. Nitrate transporters were also found to be upregulated under both UV and FR light with some only being upregulated under FR indicating the requirement for additional nitrogen. Our results highlight the complex finely tuned

UV-B light exposure experiment.
Experimental set up for the low level UV-B light exposure was as previously reported 44 . Chlorogloeopsis fritschii (Mitra) PCC 6912 was inoculated at 1:50 dilution from a master culture and cultivated in 5 L Erlenmeyer flasks containing 2 L BG11 media with 10 mM HEPES buffer at pH 7.5. The culture was perfused with 1% CO 2 and was maintained at 38 °C using white light (410-750 nm) with an intensity of 60 µmol photons m −2 s −1 (Grolux fluorescent tubes). At exponential growth phase, cells were harvested and transferred to nine quartz Erlenmeyer 500 mL flasks containing 200 mL of fresh BG11, and 10 mM HEPES at pH 7.5 to give a concentration of 0.44 g L −1 wet weight (approximating to 0.04 g L −1 dry weight). All 9 flasks were exposed to the same white light as for the stock culture for 4 days and 4 h (100 h in total). Three flasks were exposed to white light with no UV-B light acting as the control (white) for 100 h, three flasks were exposed to white light supplemented with UV-B light for the final 4 h of the experiment (samples referred to as; 4 h) and, three flasks were exposed to white light supplemented with 4 h of UV-B light each day for 4 days (samples referred to as; 4h4d). Flasks exposed to UV-B light were placed 10 cm from UV-B tubes (Philips) supplying 3 μmol m −2 s −1 at wavelength range 300-310 nm (Supplementary S1). At the end of the experiment, all 9 flasks were placed on ice and were centrifuged (3000 g) at 4 °C. The pelleted biomass was snap frozen in liquid nitrogen before storage at − 80 °C. Transcriptomics to determine differential gene regulation was undertaken on the white light (control) and 4h4d samples. HPLC analysis to determine MAA content was undertaken on the white, 4 h and 4h4d samples.
RNA preparation and sequencing. RNA was extracted with Trizol followed by terminator exonuclease digestion to enrich for mRNA and subsequently cleaned using a Qiagen RNeasy column. RNA sequencing was conducted at the Centre for Genomic Research, Institute of Integrative Biology at the University of Liverpool, UK, L69 7ZB, using the Life Technologies SOLiD sequencing platform. For each sample, at least 49,034,856 sequences were obtained (50 bp, min average quality 20 as per manufacturer specifications; per sample average sequence number: 57,516,996.44).
Alignment of reads was carried out using the C. fritschii PCC 6912 genome as reference. The sequences obtained for each sample were aligned on to the reference using Bowtie version 0.12.7, using the colour space option. Prior the alignment step, the sequences required the conversion to a pseudo-FASTQ file required as input for Bowtie. For each sequence, only the best alignment was reported by Bowtie, or one was randomly chosen if many were equally best. The average percentage of unambiguously aligned sequences was 47.67%, with a minimum of 35.9% and the maximum equal to 51.9. Considering the known 6968 genes, an average of 83.11% of these were identified as expressed across all the samples (ranging between 73.95% and 87.15%). All the obtained alignment files were processed using HTSeq-count 45 , and reads aligning to the reference genome sequences were counted according to the gene features that they mapped to, as defined in the GTF files.
The differential expression analyses between white and the 4h4d samples were performed using R (version 2.14) and edgeR package. The gene-counts were normalised using "loess smooth" method from the 'limma' package. The "GLM" model was applied to the normalised data (with EdgeR package), and the dispersion related to each gene (genewise dispersion) and the pairwise group comparisons were performed to identify differentially expressed genes for each of the three possible group comparisons. For each contrast, each gene with a p value below 0.05 (after adjusting for multiple testing effect using the False Discovery Rate approach 46 were selected as differentially expressed for that contrast. Significant changes in regulation were defined as log2 fold change ≤ − 1. RNA preparation and sequencing. Samples in centrifuge tubes were immediately cooled on ice then pellets for RNA extraction were prepared by centrifugation for 15 min at 4 °C at 3500 rpm, then further concentrated in a reweighed microfuge tubes at 4 °C at 5000 rpm. After supernatant removal, tubes were weighed on ice to calculate wet weights and the pellets were flash frozen in liquid nitrogen and stored at − 80 °C. Pellet wet weights were between 30 and 50 mg. For extraction, pellets were resuspended in 1 mL cold Trizol reagent (Thermo Fisher Scientific) and homogenised using 0.5 mm glass beads (VK05) in a Precellys 24 homogenizer (Bertin) at 6500 rpm for 2 × 20 s with a 10 s break. After 5 min incubation, the sample was extracted with 0.2 mL chloroform followed by centrifugation at 12,000×g for 15 min at 4 °C. The upper aqueous phase was mixed with an equal volume of 70% ethanol and applied to a PureLink RNA Mini Kit spin cartridge (Thermo Fisher Scientific). The sample was washed and treated on-column with PureLink DNAse (Thermo Fisher Scientific) according to the manufacturer's instructions except for an extra wash step, before drying and elution in 100 µL RNase free H 2 0. RNA concentrations were determined using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). DNA and RNA concentrations were also measured separately using a Qubit 3.0 Fluorometer (Thermo Fisher www.nature.com/scientificreports/ Scientific). Residual DNA contamination was removed by two 30 min treatments at 37 °C using 1.5 µL TURBO DNase (Thermo Fisher Scientific) followed by enzyme removal using the inactivation reagent supplied in a TURBO DNA-free Kit (Thermo Fisher Scientific) according to the instructions. To monitor the presence of DNA, primers were designed to SecA, a protein translocase subunit suitable as a reference gene in the heterocystous cyanobacteria Nostoc sp. PCC 7120 47 and realtime PCR was performed before and after treatment using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) with PerfeCTa SYBR Green FastMix (Quantabio). This confirmed complete removal of amplifiable DNA from all samples. RNA purity and quality was confirmed by running on an Agilent 2100 Bioanalyzer using a RNA 6000 Nano Kit and by cDNA synthesis using qScript cDNA SuperMix (Quantabio) followed by realtime PCR amplification as above. Prior to analyses, adapter sequences were removed from sample reads using the cutadapt tool. Total RNA was used as starting material for the generation of sequence ready libraries. Briefly, bacterial ribosomal RNA was removed from the samples with use of a RiboZero bacteria kit. To preserve strand specificity, rRNA free RNA was subjected to TruSeq stranded mRNA sample preparation. Final libraries were normalised to 4 nM prior to pooling. A final library concentration of 20 pM was used to sequence the libraries on a MiSeq platform at the Swansea University Sequencing Facility, generating a total of 99,448,410 high quality reads between all samples using multiple V3 2×75 bp PE sequencing runs 48 .
Due to an incomplete genome assembly and annotation of the C. fritschii genome, analysis of gene expression was carried out in two phases. Initially transcripts were de novo assembled using Rockhopper 2 49,50 in order to identify novel transcripts absent from the current genome annotation due to the genome's draft status (gene truncation due to contig boundary interruption of coding sequence causing absence of annotations). The de novo gene list was added to the current annotation. Reads for each sample were mapped to the draft genome using the Subread aligner and read summarization carried out by feature Counts using our improved annotation 51,52 .
Un-normalised Read count summarisation was fed into DeSeq2 R package 53 for differential gene expression analysis measuring the effect of the two conditions, white light and red light, on gene expression levels. Log2 fold change along with Wald test p values and adjusted p values were generated from the DeSeq2 normalised dataset. Significant changes in regulation were defined as log2 fold change ≤ − 1.3 or ≥ 1.3 and p adj < 0.05.

MAA extraction and analysis for UV_B and FR samples.
Each pellet of a known weight was resuspended in 100% HPLC grade MeOH (1 mL) and left in the dark at 4 °C overnight (24 h). After centrifugation (5 min at 12,000 rpm), the supernatant was removed and evaporated to dryness using a rotary vacuum concentrator. The dried extract was re-dissolved in 600 µL of deionised water and transferred to autosample vials for HPLC analysis 13 .
For the FR experiment, MAA HPLC analysis was performed using an Agilent 1100 system with a binary pump, an autosampler injector and diode array. The stationary phase was an Alltima Altech C18, 4.6 × 150 mm, 5 µm column thermostated at 35 °C. The re-suspended extracts were injected (100 µL) using an auto-sampler. The mobile phases consisted of; Eluent A: Water (0.01% TFA, v/v) and Eluent B: 70% methanol (0.054% TFA, v/v) with a gradient of; 99% A for 10 min, to 80% A over 5 min, to 1% A over 5 min, held for 3 min and increased to 99% A over 2 min. MAAs were monitored at 320 nm with spectral scanning of HPLC separated peaks from 250 to 400 nm. MAAs were identified based on spectral matching of the two main characterised MAAs (shinorine, λmax = 334 nm; m-gly, λmax = 310 nm) known to be present in C. fritschii 14 . Concentrations expressed as µg g −1 dry weight were estimated from HPLC peak areas and published extinction coefficients using the Beerlambert law using ε = extinction coefficient (shinorine, ε = 44,700 M −1 cm −1 ; m-gly, ε = 28,790 M −1 cm −1 ) 38 , p value significance was determined using a two-tailed t-test.