Green Light Suppresses Cell Growth but Enhances Photosynthetic Rate and MIB Biosynthesis in PE-Containing Pseudanabaena

Tengxin Cao1,2,4, Ming Su1,4,✉, Yufan Ai1,4, Ziyi Yang1,4, Jinbo Zhao1,3, and Min Yang1,4,✉

2025-02-25

Green Light Suppresses Cell Growth but Enhances Photosynthetic Rate and MIB Biosynthesis in PE-Containing Pseudanabaena

¹ State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences
² Sino-Danish College, Sino-Danish Center for Education and Research, University of Chinese Academy of Sciences
³ School of Civil Engineering, Chang’an University
⁴ University of Chinese Academy of Sciences

✉ Correspondence: Ming Su <mingsu@rcees.ac.cn>, Min Yang <yangmin@rcees.ac.cn>

Abstract

2-Methylisoborneol (MIB) is a notorious musty odorant in drinking water systems, produced by cyanobacteria during the biosynthesis of photosynthetic pigments. This study investigated the physiological adaptation of Pseudanabaena cinerea, a phycoerythrin (PE)-containing and MIB-producing cyanobacterium, by inducing chromatic acclimation under different light color. Our findings revealed that red light enhanced growth rates by stimulating the tricarboxylic acid (TCA) cycle and associated metabolic processes, while green light significantly increased photosynthetic pigment content and electron transport efficiency. MIB yield correlated nonlinearly with chlorophyll a (Chl a) content, modeled by a logarithmic-linear equation (R² = 0.74, p < 0.01). This was supported by the strong correlation between mic and chlG gene expression at the RNA level (R² = 0.85, p < 0.01). The model showed that <2% of carbon flux is allocated to MIB biosynthesis compared to Chl a production, indicating that MIB biosynthesis is synergistic, not competitive, with photosynthetic pigment production. The red-shift in light spectra due to increased water turbidity observed in the field led to changes in photosynthetic pigments, which decreased MIB levels. This study improves our understanding of MIB-producing cyanobacteria under variable light conditions and offers insights for mitigating MIB occurrences in surface waters.

keywords: 2-methylisoborneol (MIB), mic gene, Chlorophyll a (Chl a), Pseudanabaena cinerea, chromatic acclimation

Introduction

Unpleasant odors in drinking water significantly undermine public trust in water safety and often result in consumer complaints (Suffet et al., 1996; Watson, 2004; WHO, 2011). Among the compounds responsible, 2-methylisoborneol (MIB), a volatile monoterpenoid, is particularly notorious (Devi et al., 2021; Wang et al., 2023). Humans can detect MIB’s musty odor at extremely low concentrations (10 ng L⁻¹, (Suffet et al., 1999)). In addition, MIB can easily pass through most water treatment processes (Srinivasan and Sorial, 2011), making it a persistent and troublesome pollutant in the water industry. It has been implicated in hundreds of odor episodes worldwide (AWWA, 2010; Devi et al., 2021) and detected in over 40% of surface water sources in China (Sun et al., 2014).

MIB production is attributed to actinomycetes (Gerber, 1969), cyanobacteria (Giglio et al., 2011), fungi, and myxobacteria (Yamada et al., 2014), with filamentous cyanobacteria—including Pseudanabaena, Phormidium, Oscillatoria, and Planktothricoides—identified as the primary producers in aquatic environments (Giglio et al., 2011; Izaguirre et al., 1999; Jia et al., 2019; Lee et al., 2017; Lu et al., 2022; Su et al., 2015; Wang et al., 2011; Zhang et al., 2016). The molecular and biochemical mechanisms of MIB biosynthesis in cyanobacteria were elucidated following studies on actinomycetes (Komatsu et al., 2008; Wang and Cane, 2008). This process involves the shared precursor geranyl diphosphate (GPP), also essential for synthesizing photosynthetic pigments like xanthophylls and chlorophylls (Giglio et al., 2011; Su et al., 2023; Zimba et al., 1999). The biosynthesis requires two key enzymatic steps: GPP methylation and subsequent cyclization to MIB, mediated by SAM-dependent methyltransferase and monoterpene cyclase, respectively. Both genes involved in MIB synthesis exhibit light-dependent expression in both culture experiments (Wang et al., 2011; Zhang et al., 2016) and field study (Cao et al., 2023).

As ancient photosynthetic organisms, cyanobacteria rely heavily on light—both intensity and wavelength—for survival and metabolism (Grébert et al., 2018; Jia et al., 2019; Reynolds, 2006; Su et al., 2023). Variations in underwater light spectra, driven by organic matter and suspended particles, promote niche differentiation and chromatic acclimation (Stomp et al., 2007). Cyanobacteria optimize light capture through phytochromes and phycobilisomes (PBS), large antenna complexes that harvest wavelengths (300–750 nm) beyond chlorophyll’s absorption range (Ho et al., 2017; Wiltbank and Kehoe, 2019). For example, phycoerythrin (PE) absorbs green light (495–570 nm), while phycocyanin (PC) and allophycocyanin (APC) absorb green-yellow (550–630 nm) and orange-red (650–670 nm) wavelengths, respectively (Graham et al., 2017; OCarra et al., 1980; Zheng et al., 2021). Cyanobacteria also modify PBS composition through chromatic acclimation (CA), enabling adaptation to changing light environments (Kehoe, 2010). Six types of CA have been identified, involving various adjustments in pigment synthesis to optimize light absorption (Gutu and Kehoe, 2012; Sanfilippo et al., 2019a; Wang and Chen, 2022). These modifications further affected downstream pathways, including photosynthesis, growth, and metabolism at the transcriptional level (Gutu and Kehoe, 2012; Wiltbank and Kehoe, 2019).

Photosynthesis and respiration are two essential physiological processes for photoautotroph (Dey et al., 2022). As a secondary metabolite of photosynthetic pigments, MIB biosynthesis is hypothesised to be a co-regulated process with photosynthesic process. Our previous research has revealed a PE-containing strain Pseudanabaena performs type III CA by accumulating PC and PE under green light, and demonstrated a positive correlation between cellular MIB and chlorophyll a content (Su et al., 2023). The gene expression related to MIB biosynthesis has also been proved to be different under different light colors (Dayarathne et al., 2024). However, the photophysiological responses under different light color and the metabolic interplay among photosynthetic pigment synthesis, MIB production and cell growth remain insufficiently understood.

Advances in transcriptomics have deepened our understanding of metabolic functions and gene expression in cellular processes (Thompson et al., 2011). Porphyrin and chlorophyll metabolism are fundamental to photosynthesis (Simkin et al., 2022), while genes associated with the citric acid (TCA) cycle underscore ATP production and cellular growth (Steinhauser et al., 2012). Moreover, terpenoid backbone biosynthesis plays a key role in MIB production (Giglio et al., 2011). In this study, we induced chromatic acclimation using a phycoerythrin (PE)-containing and MIB-producing cyanobacterium (Pseudanabaena cinerea), so that to investigate the gene regulation of these processes under varying light color exposures. Specifically: 1) mic gene (encoding MIB synthase) expression and MIB production responses to different light color conditions were examined using qPCR at both DNA and RNA levels. 2) Phytophysiological responses under red and green light exposures were compared, focusing on MIB biosynthesis, photosynthesis, and cell growth, as revealed by transcriptomics analysis. 3) A carbon flux allocation model was developed to describe MIB biosynthesis during photosynthetic pigment formation under different light colors. 4) In-situ evidence on the relationship between the MIB variation and light spectra dynamics. Our findings advance the understanding of photophysiological regulation of MIB biosynthesis in cyanobacteria. This knowledge enhances ecological insights into MIB-producing cyanobacteria and supports nature-based solutions for mitigating recurring MIB episodes in surface water sources.

Methods

Culture experiment for Pseudanabaena under different light color

Pseudanabaena cinerea FACHB 1277, obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, was utilized to investigate the impact of light color on cell growth, MIB production, and photophysiological characteristics over a 35-day culture period. Cells in the logarithmic growth phase were centrifuged (200 × g, 2 min) and washed three times with BG11 medium to eliminate extracellular odorous substances. Subsequent culture experiments were conducted at a cell density of approximately 2 × 10⁶ cells L⁻¹. The optimum growth condition of Pseudanabaena cinerea is 25 °C and 30 μmol photons m⁻² s⁻¹ (Cao et al., 2023), and in this study, they were cultured in triplicate at different temperature (10 °C, 25 °C, and 35 °C) and 30 μmol photons m⁻² s⁻¹ under a 12 h/12 h light/dark cycle in 500 mL BG11 medium. Considering the light spectra absorbed by these PBS and Chl a, as well as the relationship between the MIB and photosynthetic pigments, different light colors were generated using four types of LED lamps with red (620 nm), green (520 nm), blue (455 nm), and white fluorescent lamps (Philips, Netherland). Cultures were sampled every 3 or 4 d during a 35 d culture period for quantifying cell density, MIB concentration, and mic gene expression level. Samples in the stationary phase were further analyzed for photosynthetic pigment composition, photophysiological parameters (including effective photochemical quantum yield of PS II (Y (II)) and maximum electron transport rate (ETRmax)), as well as transcriptomic analysis.

Chemical analysis

To determine the concentration of phycobiliproteins, a 50 mL culture was centrifuged and the resulting pellet was suspended to 6 mL of sodium-phosphate buffer (pH 7). The cells were disrupted by sonication for 1 min. Phycobiliproteins were extracted using up to 5 freeze–thaw cycles (freezing in liquid nitrogen and thawing at room temperature), followed by a 10 min centrifugation at 10,000 × g at 4 °C. The absorption spectrum of the supernatant was measured using a UV–Vis spectrophotometer across wavelengths ranging from 250 to 700 nm. The concentration of phycobiliproteins was calculated using the following equations (Bennett and Bogorad, 1973): PC=A615−0.474A6525.34PC=\frac{A_{615}-0.474A_{652}}{5.34}, APC=A652−0.208A6155.09APC=\frac{A_{652}-0.208A_{615}}{5.09} and PE=A562−2.41PC−0.849APC9.62PE=\frac{A_{562} - 2.41PC - 0.849APC}{9.62}.

The Chl a was extracted using 80% acetone and incubated overnight in the dark at 4 °C, followed by a 10-minute centrifugation at 7,000 × g at 4 °C. The concentration of Chl a was determined based on the absorption spectrum at 663 nm (Glazer, 1989).

MIB concentrations were determined using solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC–MS), with the detection limit of 2 ng L⁻¹ (Su et al., 2015). Subsamples (100 mL) added with 5% Lugol’s iodine for phytoplankton cell enumeration were settled for 48 h, then concentrated to 10 mL and kept in the dark until cell counting. The phytoplankton was identified according to (Komarek et al., 2014), and cell density was estimated using a counting chamber (S52, 1 mL, Sedgewick-Rafter) under a microscope (OLYMPUS BX51, Olympus Optical, Tokyo, Japan) following (Su et al., 2015). The Y (II) and ETRmax of cultures were determined by PHYTO-PAM-II (PPAB0145, Germany). The turbidity were measured using a multiple-probe instrument (EXO2, Xylem, USA) in-situ.

Gene expression quantification, transcriptomic sequencing and metagenomic sequencing

The 500 mL subsamples were filtered through 1.2 μm Isopore™ Membrane Filters to collect phytoplankton, and the membrane filters were stored at -80 °C in 1.5 mL centrifuge tubes until DNA and RNA extraction. The DNA of environmental samples was extracted directly from the membrane using the Fast DNA spin kit for soil (MP Biomedicals, USA) following the manufacturer’s instructions. The RNA was extracted using E.Z.N. A.™ Soil RNA Kit (OMEGA, USA). Subsequently, PrimeScript™ RT Master Mix (TaKaRa, Japan) was employed for reverse transcription of RNA to cDNA, with the reaction carried out at 37 °C for 15 min followed by 85 °C for 5 s. The concentration and purity of DNA, RNA and cDNA were identified by microspectrophotometry (NanoDropND-2000, NanoDrop Technologies, Willmington, DE). DNA and cDNA samples were stored at -80 °C until mic gene quantification. The mic genes of water samples were amplified using primers MIBQSF (5’-GACAGCTTCTACACCTCCATGA-3’) and MIBQSR (5’-CAATCTGTAGCACCATGTTGAC-3’) (Suruzzaman et al., 2022). The mic gene abundances were quantified using LightCycler 96 (Roche, USA) with 25 µL volume mixture including 12.5 µL TB GreenTM Premix Ex TaqTM (TaKaRa, Japan), 0.8 µL for each primer (MIBQSF and MIBQSR), 8.9 µL deionized water, and 2 µL template DNA. The quantitative PCR was conducted as follows: pre-incubation at 95 °C for 10 min; 50 cycles at 95 °C for 20 s, 50 °C for 20 s, and 72 °C for 20 s; and DNA melting from 65 °C to 97 °C. The standard curve for quantitative PCR was obtained: cq=−3.4537lg(cmic)+40.13(R2=0.999,p<0.0001)c_q = -3.4537\text{lg}(c_{mic}) + 40.13 (R^2 = 0.999, p < 0.0001) with the efficiency of 95%.

Transcriptomic sequencing was conducted to profile the photophysiology response to different light color. Before RNA library construction, the RNA sample were treated with DNAse to remove possible DNA contamination, and then the rRNA was removed from the total genomic RNA using RiboMinus Transcriptome Isolation Kits for bacteria (ThermoFisher Scientific, USA). The cDNA library was constructed with a TruSeq™ Stranded Total RNA Library Prep Kit (Illumina, USA). Paired-end sequencing was performed on an Illumina Hiseq 2500 platform.

To investigate the succession among different photosynthetic pigments-containing phytoplankton under different light spectra. Metagenomic sequencing was conducted to profile the microbial functional structure. Genomic DNA was fragmented to about 400 bp using Covaris M220 (Gene Company Limited, China), then constructed the paired-end library using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Paired-end sequencing was performed on Illumina Hiseq Xten (Illumina Inc., San Diego, CA, USA) using HiSeq X Reagent Kits.

Data analysis

The raw sequencing reads underwent a cleaning process, which involved trimming adapter contamination reads and low quality reads (quality value < 15) (Bolger et al., 2014). Subsequently, the SortMeRNA software was utilized to eliminate the rRNA sequences (Kopylova et al., 2012). The resulting clean reads were then assembled into Unigene using Trinity (Grabherr et al., 2011), and gene function was annotated by BLASTx against the KEGG database (Kanehisa et al., 2007). Gene expression of assembled unigenes was normalized using fragments per kb per million fragments (FPKM), and significantly differentially expressed genes (DEGs) under different light color treatments were identified based on absolute values of |log2Fold Change|≥2|\log_2\text{Fold Change}|\geq 2, p-value < 0.01, and FDR ≤ 0.001, calculated by DESeq2 R package (Anders and Huber, 2010). Furthermore, pathway enrichment analysis was conducted to determine the differentially expressed gene clusters according to the KEGG database using clusterProfilter package in R version 4.3.1 (Yu et al., 2012). The rich factor means the ratio of the number of differentially expressed genes (DEGs) enriched in a pathway to the total number of annotated genes in that pathway. The raw sequencing data of Metagenomic were trimmed of adaptors, and low-quality reads (length<50 bp or with a quality value <20 or having N bases) were removed by fastp. The clean sequencing data were assembled using MEGAHIT. Contigs with a length over 300 bp were selected as the final assembling result and for further gene annotation. Representative sequences of non-redundant gene catalog were aligned to KEGG database with e-value cutoff of 10⁻⁵ using Diamond for functional annotations. The figures were visualized using the ggplot2 package in R version 4.3.1.

The carbon flux was determined by the MIB and Chl a concentration, along with the relative molecular mass and the number of carbon atoms from the same sample in the stationary phase, and calculated by (Eq. 1) as follows:

carbon flux(MIB)=cMIB×NCmib×mChlacMIB×NCMIB×mChla+cChla×NCChla×mMIB(1) \text{carbon flux}(\text{MIB}) = \frac {c_{\text{MIB}}\times\text{NC}_{\text{mib}}\times m_{\text{Chl} a}}{c_{\text{MIB}}\times\text{NC}_{\text{MIB}}\times m_{\text{Chl} a} + c_{\text{Chl} a}\times\text{NC}_{\text{Chl} a}\times m_{\text{MIB}}} \qquad(1)

Where cMIBc_{\text{MIB}} and cChlac_{\text{Chl} a} are the concentrations of MIB and Chl a, respectively. mMIBm_{\text{MIB}} and mChlam_{\text{Chl} a} represent the relative molecular masses of MIB and Chl a, being 168.3 g mol⁻¹ and 893.5 g mol⁻¹, respectively. NCMIB\text{NC}_{\text{MIB}} and NCChla\text{NC}_{\text{Chl} a} are the number of carbon atoms, with 55 for Chl a and 11 for MIB.

Results

Cellular MIB yield and mic gene expression level of Pseudanabaena cinerea in response to light color

At different temperatures (10 °C, 25 °C, and 35 °C), the Pseudanabaena exhibited distinct responses to light color, and the most remarkable variation in MIB concentration and cell color was witnessed under the optimum temperature (25 °C) (Fig. 1C and D). Subsequently, 25 °C was chosen as the optimal control temperature to explore the mechanisms of the effects of light color on MIB-producing Pseudanabaena. Our previous study highlighted the differences in growth and MIB production characteristics of Pseudanabaena at 25 °C under various light colors (Su et al., 2023). It showed that red light led to the highest cell density and growth rate, followed by white and green light, while Pseudanabaena could not survive under blue light (Fig. S1). In contrast to the growth trends, cellular MIB yield was significantly higher under green light than under red or white light (Fig. S2). To investigate the mechanisms underlying MIB production, we quantified mic gene expression (which encodes the enzyme responsible for MIB biosynthesis) throughout the culture period. The mic gene expression levels (RNA abundances normalized to DNA abundances) ranged from 0.006 to 0.538 under different light conditions, with notably higher levels under green light (0.37 ± 0.08) compared to red (0.028 ± 0.018, p < 0.01) and white light (0.049 ± 0.026, p < 0.01, Fig. 1E). These gene expression patterns mirrored the trends in cellular MIB yield across light conditions, with no significant differences in MIB yield or mic expression between red and white light. Additionally, mic gene expression was significantly positively correlated with cellular MIB yield (R² = 0.822, p < 0.01, Fig. 1F).

Fig. 1: Culture experiment for Pseudanabaena cinerea under 25 °C, 30 μmol photons m⁻² s⁻¹, different light color: cultures were sampled every 3 or 4 d during a 35 d culture period for quantifying cell density, MIB concentration, and mic gene expression level. Samples in the stationary phase were further analyzed for photosynthetic pigment composition, photophysiological parameters and transcriptomic analysis. Effects of light color on MIB production under different temperature was conducted under 10 °C, 25 °C, and 35 °C, and the samples in day 10 to day 15 collected for quantifying cell density, MIB concentration, and mic gene expression level (A). The cell color collected at day 15 under different temperature different light colors (B). The cellular MIB yield (C) and mic gene expression level (D) of Pseudanabaena cinerea under different light color and culture temperature. The mic gene expression level of MIB-producing Pseudanabaena cinerea under different light color at 25 °C (E). Correlation between mic gene expression level and MIB concentration (F).

Photophysiology and photosynthetic pigments regulation in response to light color

The quantity and composition of photosynthetic pigments in Pseudanabaena during the stationary phase varied according to light color (Fig. 2A and 2B). Phycoerythrin (PE), which absorbs green light most effectively, was highest under green light (3986 fg cell⁻¹, 19%) and was also present under white light (2469 fg cell⁻¹, 16%), but was undetectable under red light. In contrast, phycocyanin (PC), which optimally absorbs red light, was most abundant under red light (7039 fg cell⁻¹, 47%) compared to green (5300 fg cell⁻¹, 26%) and white light (4530 fg cell⁻¹, 29%). These pigment variations altered cell color, causing cells to appear red under green light and green under red light. Chlorophyll a (Chl a) production was also highest under green light (11346 fg cell⁻¹, 55%), followed by red (8092 fg cell⁻¹, 53%) and white light (8769 fg cell⁻¹, 55%).

Fig. 2: The cell color of Pseudanabaena cinerea collected at the end of the culture experiment (day 35) under different light colors (A). The photosynthetic pigment contents (B), effective photochemical quantum yield of PS II (Y (II)) (C), and maximum electron transport rate (ETRmax) (D) under different light colors. The expression level (FPKM) of genes encoding phycobilisomes (PE, PC, and APC), Chl a, photosystem II, photosystem I, photoelectron transfer pigments, and carbon fixation (E).

Gene expression patterns aligned with these pigment trends, particularly under green light, where genes related to PE (cpeD, cpeU, and cpeY) and chlorophyll (chlG, chlH, and chlN) were significantly up-regulated compared to red light. Additionally, green light exposure led to increased expression of genes related to photosystem II (PsbC), photosystem I (PsaB and PsaF), and photoelectron transfer (PetD) (Fig. S3). This up-regulation extended to genes involved in carbon fixation (gap2, tktA, ALDO, xfp, and rbcL), further supporting the link between pigment synthesis and carbon assimilation (Fig. 2E, Fig. S4).

Rapid light-response curves showed clear photophysiological differences across light wavelengths. Under green light, Pseudanabaena exhibited significantly higher effective photochemical quantum yield of PS II (Y(II): 0.248 ± 0.068) and relative maximum electron transport rate (ETRmax: 17.778 ± 1.289) than under red (Y(II): 0.098 ± 0.039; ETRmax: 9.433 ± 3.629) or white light (Y(II): 0.208 ± 0.028; ETRmax: 13.667 ± 3.076) (Fig. 2C and 2D).

Photophysiological response to light color

The regulation of photosynthetic pigments influenced downstream pathways that control various cellular processes. Transcriptomic analysis was further conducted to elucidate the physiological responses in response to varying light color. This analysis revealed 539 significantly up-regulated and 719 significantly down-regulated genes (|log2(Fold Change)≥2|\log_2(\text{Fold Change}) \geq 2|, p < 0.01) under green light compared to red light (Fig. 3A).

Fig. 3: Differentially expressed genes of Pseudanabaena cinerea under green light compared to red light (A). The significantly up-regulated (B) and down-regulated genes (C), along with their associated pathways.

KEGG enrichment analysis of these differentially expressed genes (DEGs) categorized them into several key biological pathways, including carbon fixation (8 genes, enrichment score: 0.53), photosynthesis−antenna proteins (15 genes, 0.52), glycolysis/gluconeogenesis (10 genes, 0.38), the pentose phosphate pathway (6 genes, 0.35), glyoxylate and dicarboxylate metabolism (6 genes, 0.33), the citrate cycle (TCA cycle) (4 genes, 0.33), and photosynthesis (17 genes, 0.3) (Fig. 4A, Table S1). This enrichment underscores the interconnectedness between light conditions and pathways essential for energy metabolism and cellular function in Pseudanabaena.

Fig. 4: KEGG enrichment analysis of categories of differentially expressed genes of Pseudanabaena in response to light color (green, red, and white) (A). The up-regulated and down-regulated pathways under green light compared to red light at pathway levels 3 (B). Differentially expressed metabolic pathways (C) of Pseudanabaena cinerea responding to light color. Pink and blue symbols represented the up-regulated and down-regulated genes or pathways by green light compared to red light.

The fold change (FC) of gene expression level under green light relative to red light highlighted several up-regulated pathways primarily linked to photosynthesis proteins (0.122 log10\log_{10}FC), carbon fixation (0.122 log10\log_{10}FC), two−component system (0.097 log10\log_{10}FC), photosynthesis (0.086 log10\log_{10}FC), terpenoid backbone biosynthesis (0.085 log10\log_{10}FC), and porphyrin and chlorophyll metabolism (0.071 log10\log_{10}FC). These pathways emphasize the enhanced metabolic activities supporting photosynthetic and carbon-assimilation functions under green light. Conversely, the down-regulated pathways were mainly associated with nitrogen metabolism (-0.201 log10\log_{10}FC), sulfur metabolism (-0.074 log10\log_{10}FC), the TCA cycle (-0.008 log10\log_{10}FC), and cell growth processes (-0.004 log10\log_{10}FC), indicating a shift away from nitrogen and sulfur utilization and cellular proliferation activities (Fig. 4B and 4C, Fig. S5, Fig. S6).

Relationship between biosynthesis of MIB and photosynthetic pigments

MIB is recognized as a secondary metabolite that shares a precursor with chlorophyll a (Chl a) and is synthesized through the terpenoid backbone biosynthesis pathway (Fig. 5A). Under green light, both Chl a biosynthesis genes (gcpB, ispH, GGPS, FDPS, chlP, and chlG) and MIB biosynthesis genes (mtf and mic) were notably up-regulated, suggesting a synchronized enhancement in the production of these compounds. Furthermore, the mic gene displayed significantly higher expression under green light compared to red and white light, with peak expression observed at the light midpoint (green: 0.52 ± 0.095; red: 0.055 ± 0.007; white: 0.090 ± 0.006), which decreased at the dark midpoint (green: 0.31 ± 0.075; red: 0.043 ± 0.005; white: 0.049 ± 0.002) regardless of the light color (Fig. 5B).

Fig. 5: The detailed biosynthesis pathway of MIB and photosynthetic pigments (A). Red symbols represented the up-regulated genes under green light compared to red light. The expression level of the mic gene at the light and dark midpoints of 12 h/12 h light/dark cycles under various light colors (B). The relationship between the gene expression levels of mic and chlG (C), as well as the association between cellular MIB yield and Chl a yield (D) in response to different light colors. The shading is 95% confidence interval. The carbon flux in the two branches responds to light colors (E).

Quantitative analyses of the expression levels of chlG (a gene encoding an enzyme critical for Chl a biosynthesis) and mic revealed a strong positive logarithmic correlation between the two (R² = 0.85, p < 0.01), indicating a close metabolic link between Chl a and MIB production (Fig. 5C). Similarly, there was a consistent positive logarithmic correlation between cellular MIB yield and Chl a yield across different light colors (R² = 0.74, p < 0.01, Fig. 5D), underscoring the interconnected biosynthetic pathways of these metabolites.

Under green light, Pseudanabaena cinerea exhibited significantly higher cellular yields of MIB (112.11 ± 4.93 fg cell⁻¹) and chlorophyll a (Chl a) (9881.33 ± 1299.78 fg cell⁻¹) compared to red light (MIB yield: 81.85 ± 4.11 fg cell⁻¹; Chl a yield: 4649.84 ± 418.49 fg cell⁻¹) and white light (MIB yield: 94.76 ± 4.61 fg cell⁻¹; Chl a yield: 6800.35 ± 973.44 fg cell⁻¹, Fig. S7). Despite the increased yields, it was noted that only a small fraction of carbon (2%) was allocated to MIB biosynthesis alongside Chl a biosynthesis.

The increase in Chl a yield was associated with a decrease in the carbon allocation for MIB biosynthesis. This is evident from the reduced ratio of carbon to MIB biosynthesis under green light, which was (1.28 ± 0.17)% C, compared to red light ((1.82 ± 0.38)% C) and white light ((1.61 ± 0.02)% C) (p < 0.01, Fig. 5E). This suggests that while higher light availability enhances the overall productivity of both pigments, it may also lead to a more efficient allocation of resources, prioritizing the synthesis of Chl a over MIB under optimal light conditions.

Relationship between the MIB variation and light spectra dynamics in field investigation

In actual waters, the underwater light spectrum is significantly affected by variations in water turbidity (Fig. 6A). As turbidity increased from 0.6 NTU to 25.2 NTU, particles can cause a redshift (from 560 nm to 630 nm) in the underwater light spectrum. Under field conditions, we discovered a succession of phytoplankton as revealed by metagenomic analysis, where the relative abundance of PE decreased ((67.7 ± 16.2) to (31.6 ± 8.7) FPKM) and that of PC increased ((187.2 ± 124.2) to (399.2 ± 109.4) FPKM) with turbidity increasing from 10 NTU to 20 NTU (Fig. 6B). Further, this variation of light spectra led to the variation of MIB concentration under different water turbidity, with a significant decrease (p < 0.01) from (101.2 ± 28.4) ng L⁻¹ in 10 NTU to (34.2 ± 8.2) ng L⁻¹ in 20 NTU (Fig. 6C).

Fig. 6: The relationship between underwater light spectra and water turbidity (A). The variation of the relative abundance of genes encoding different photosynthetic pigments (B) and MIB concentration (C) under different water turbidity.

Discussion

2-methylisoborneol (MIB), produced by a group of cyanobacteria, has led to significant odor problems in aquatic environments. Its biosynthesis has been confirmed as a volatile secondary metabolite intertwined with the production of photosynthetic pigments (Giglio et al., 2011; Komatsu et al., 2008). Given that both MIB and Chl a are synthesized from a shared precursor, GPP, it becomes evident that the synthesis of MIB is closely dependent on light conditions and is affected by the biosynthesis of various photosynthetic pigments (Su et al., 2023). This interdependence underscores the importance of light quality and intensity in regulating both pigment production and the associated metabolic pathways, including those leading to MIB biosynthesis. Our previous study has revealed that higher light intensity induced higher MIB biosynthesis by up-regulating the expression level of the mic gene, and partially explained the variance in MIB concentration in aquatic environments (Cao et al., 2023). In addition, light spectrum has been demonstrated as an essential selective factor for the competition, composition, and evolution of phytoplankton (Hauschild et al., 1991). The distinct light spectrum niches led to diverse cyanobacterial pigment components in marine, freshwater, and terrestrial environments (Chen et al., 2020).

In our experiment, a higher growth rate of Pseudanabaena cinerea was observed under red light. Cyanobacteria have evolved multiple photoreceptor-regulated mechanisms specifically within their photosynthetic processes, enabling them to perceive and adapt to variations in environmental light conditions. The phytochrome superfamily has been extensively identified in cyanobacteria, allowing them to detect changes in ambient light color and intensity (Ikeuchi and Ishizuka, 2008; Quail et al., 1995). This superfamily is responsible for detecting and transmitting light signals to downstream pathways that govern cellular processes such as growth, phototaxis, cell aggregation, and photosynthesis (Bezy et al., 2011; Kehoe, 2010; Nagae et al., 2024). The enrichment analysis revealed top rich factor pathways were carbon fixation and photosynthesis, indicating that the light color has a direct impact on photosynthetic processes, and the variations of these pathways subsequently exert an effect on the downstream pathways regulating cellular processes. Our study observed that two types of cyanobacteriochrome (CBCR), namely iflA and Cph2, were up-regulated under green light conditions, which is consistent with previous studies indicating that iflA and Cph2 are regulated by red and green light during CA3 at both the RNA and protein levels, thereby promoting growth under conditions with a high red-to-far-red light ratio (Bussell and Kehoe, 2013). Furthermore, the two-component system pathway was also up-regulated in green light, which is because the photoreceptors regulate chromatic acclimation via two-component signal transduction pathways (Yeh et al., 1997). The growth enhancement in red light mediated by CBCR may be subject to post-transcriptional regulation, with increasing the expression levels of nitrogen metabolism, sulfur metabolism, and TCA cycle.

The composition of photosynthetic pigments varied under different light colors. Photosynthetic organisms rely on photosynthetic pigments to capture light energy, which is then transferred to the photoreaction center for various metabolic processes. In cyanobacteria, the absorption spectrum of chlorophyll (Chl) features a green gap that is effectively compensated by phycobiliproteins (PBS) (Kehoe, 2010; Marsac, 1977; Sanfilippo et al., 2019b; Wiltbank and Kehoe, 2019). The variation of light color can also induce chromatic acclimation (CA) mechanisms in certain cyanobacteria, influencing the expressions of genes encoding PE and PC, and adjusting the composition of phycobiliproteins (PBS), thereby enabling them to adapt to diverse light conditions (Kehoe, 2010). Laboratory experiments have demonstrated that CA provides Pseudanabaena with competitive advantages in fluctuating light environments (Acinas et al., 2009; Stomp et al., 2008). Furthermore, chromatic acclimators in Synechococcus have been identified as the predominant pigment types in the Tara Oceans dataset, particularly at greater depths and higher latitudes (Grébert et al., 2018). In our experiments, we observed enhanced synthesis of PE under green light compared to red light, while red light led to greater production of PC than green light. This variation in pigment composition aligns with the principles of CA3, and similar regulatory patterns in PBS were noted in several strains of Pseudanabaena (Khan et al., 2019; Mishra et al., 2012).

Furthermore, we observed an elevated effective photochemical quantum yield (Y(II)) and the relative maximum electron transport rate (ETRmax) under green light, which was also attested by the higher expression level of genes encoding photosynthesis, carbon fixation, porphyrin and chlorophyll metabolism. This is consistent with the previous evidence that the increase in phycobilin levels is likely to contribute to an expanded PSII antenna, enhancing the absorption and transfer of energy to Chl a, thereby optimizing the electron transport efficiency (Park and Dinh, 2019). In contrast to the enhanced growth rates observed under red light, elevated chlorophyll a (Chl a) content was achieved under green light. Emerging evidence suggests that green light plays a crucial role in CO₂ assimilation and acts as a pivotal signal for both long-term development and short-term dynamic environmental adaptation (Smith et al., 2017). Similar increases in Chl a yield under green light have been documented in Arthrospira (Park and Dinh, 2019), Phormidium (Hotos and Antoniadis, 2022), and Pseudanabaena (Dayarathne et al., 2024). Chlorophyll a (Chl a) directly harnesses light energy under red and white light, whereas under green light, the majority of light energy is absorbed by PE before being transferred to Chl a (Wehrmeyer, 2003). This reduced efficiency in light absorption under green light may lead to increased production of photosynthetic pigments (Dayarathne et al., 2024), thereby supporting balanced cellular growth.

The influence of light color on Chl a synthesis further extends to the production of MIB. Our findings indicate that green light results in a higher yield of cellular MIB by up-regulating the expression levels of the mic gene. We observed a positive correlation between cellular MIB yield and Chl a production (R² = 0.74, p < 0.01), as evidenced by the co-upregulation of these two biosynthesis pathways under green light, despite their reliance on the same precursor. The variation of MIB yield and mic gene expression level under different water temperatures further clarified the relationship between MIB production and photosynthetic pigments. The significantly higher MIB yield and mic gene expression level under green light were only observed under the optimum temperature (25 °C) along with chromatic acclimation. However, under 10 °C and 35 °C, there was no significant variation in MIB yield in response to light color as the poor growth rate makes it unable to adequately adjust the pigment composition. Furthermore, the higher expression level of the mic gene at the light midpoint compared to that under the dark midpoint regardless of the light color also demonstrated that the biosynthesis of MIB was concomitant with Chl a, which are light-dependent processes. Notably, these findings may be influenced by species differences, variations in physiological age, and the regulation of chromatic adaptation. As previously reported for the light color response of Pseudanabaena foetida var. intermedia, since it does not contain PE, it thus cannot utilize green light and has a lower MIB yield under green light. As noted, in our experiment, only 2% of carbon was allocated to MIB biosynthesis compared to Chl a, suggesting that MIB synthesis does not significantly compete for precursors and has a minimal impact on overall carbon utilization. This aligns with previous studies indicating that MIB accumulation is not constrained by carbon availability and does not function as an “overflow” product for dissipating excess carbon (Zimba et al., 1999). Furthermore, our findings indicated that an elevated Chl a yield under green light led to a slight decrease in the carbon ratio for MIB biosynthesis, potentially attributable to more effective resource allocation strategies. The concurrent biosynthesis of MIB and Chl a implies that environmental factors influencing Chl a production also simultaneously affect MIB synthesis, enhancing our understanding of the occurrence and variability of MIB in aquatic environments.

In natural waters, the underwater light spectrum is dependent on the absorption of light by pure water, along with organic matter and suspended particles. Generally, red light dominates near the surface, while green and blue wavelengths penetrate into greater depths (Kirk, 1994). The scattering and absorption of particles also influenced the distribution of underwater light spectra. It was discovered in field investigation that the light spectrum shifts towards red with the increase of water turbidity (Fig. 6A). Significantly, this alteration led to the succession among different photosynthetic pigments-containing phytoplankton, manifested as less PE and more PC under a more red light spectrum resulting from higher water turbidity. These succession was also attested by other field studies (Stomp et al., 2007) and culture experiments (Stomp et al., 2004), the light spectrum has been illustrated as the drivers for the competition between red and green cyanobacteria. Since the MIB producer has been identified as PE-containing Pseudanabaena cinerea in this reservoir, the relative abundance of PE decreased along with lower MIB yield under a more red light spectrum, and thus, the MIB concentration was observed to decrease significantly under higher water turbidity. Therefore, both the culture experiment and the field investigation indicated that increasing water turbidity is a potential strategy to control the MIB production in actual water. Considering the relationship between the biosynthesis of MIB and photosynthetic pigments, the environmental factors that affect cyanobacterial growth, such as light intensity and nutrients, might affect the MIB production response to light color. In future studies, a comprehensive consideration of the interspecies interaction and environmental factors in shaping MIB concentration should be elucidated in both culture experiments and at the field scale.

Conclusions

In this study, we utilized transcriptomics to elucidate the biosynthesis of 2-methylisoborneol (MIB) in Pseudanabaena cinerea as regulated by photophysiological responses to different light colors. Our findings revealed that red light promotes cell growth, while green light enhances photosynthesis and the production of photosynthetic pigments. The increased synthesis of chlorophyll a (Chl a) under green light was associated with a corresponding rise in MIB production, supported by a positive correlation between MIB and Chl a biosynthesis. Only 2% of carbon was allocated to MIB biosynthesis during Chl a production, indicating minimal competition for precursors and a limited impact on overall carbon availability. This simultaneous biosynthesis of MIB and Chl a provides valuable insights into how environmental factors influence MIB production, thereby contributing to our understanding of its variability in aquatic ecosystems.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (52030002, 51878649).

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