Molecular mechanism of modified clay controlling the brown tide

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Molecular mechanism of modified clay controlling the brown tide organism, Aureococcus anophagefferens revealed by transcriptome analysis Jianan Zhu, zhiming yu, Liyan He, Xihua Cao, Shuya Liu, and Xiuxian Song Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05172 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Environmental Science & Technology

Molecular mechanism of modified clay controlling the brown tide organism Aureococcus anophagefferens revealed by transcriptome analysis Jianan Zhu,a, c Zhiming Yu,a, b, c, * Liyan He,a, b Xihua Cao,a, b Shuya Liu,a, c and Xiuxian Songa, b, c, a

CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology,

Chinese Academy of Sciences, Qingdao 266071, China b

Functional Laboratory of Marine Ecology and Environmental Science, Qingdao National

Laboratory for Marine Science and Technology, Qingdao 266237, China c

University of Chinese Academy of Sciences, Beijing 100049, China

ABSTRACT The data and experiences in mitigating harmful algal blooms (HABs) by modified clay (MC) show that a bloom does not continue after the dispersal of the MC, even though the density of the residual cells in the water is still high, at 20–30% of the initial cell density. This interesting phenomenon indicates that in addition to flocculation, MC has an additional control mechanism. Here, transcriptome sequencing technology was used to study the molecular mechanism of MC in controlling HABs. In residual cells treated with MC, the photosynthetic light reaction was the most affected physiological process. Some genes related to the light harvesting complex, photosystem (PS) I and PS II, were significantly up-regulated (p2 nM) to ensure the quality of the library. Finally, the Illumina HiSeq 4000 (Santiago) was used to carry out double-ended sequencing.

Mapping of Sequence Reads onto Transcripts and Assembly To obtain high-quality reads for further analysis, the raw reads were filtered to remove: a) reads with adaptor sequences, b) low-quality reads (where the number of bases with Qphred ≤20 accounts for more than 50% of the entire read length), and c) reads with high percentages of unknown bases (where unknown bases account for more than 10% of the entire read length). All the processed clean reads for each sample were mapped to the reference genome of A. anophagefferens using the program TopHat2, with 7

ACS Paragon Plus Environment


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no more than two mismatches.34 For the reads that mapped to the reference genome, Cufflinks software was applied to obtain genetic information for the transcripts.36

Transcript Annotation and Analysis of Differentially Expressed Genes The genome information of A. anophagefferens was taken from the National Center for Biotechnology Information (NCBI) database to annotate the assembled transcripts. Gene differential expression analysis was performed using DESeq.37 Firstly, the obtained data were standardized by the DESeq method. And then, the p-value (hypothesis test probability) was calculated using a negative binomial distribution model. Finally, the false discovery rate (FDR) was obtained from the p-value adjusted for multiple testing with the Benjamini-Hochberg procedure.37 The log2 value (sample1/sample2) (log2 fold-change, sample1 is the number of transcripts for the MC treatment sample; sample2 is the number of transcripts for the control sample) was used to determine whether the gene expression is up- or down-regulated. Goseq was used to perform GO (Gene Ontology) enrichment analysis for the differentially expressed genes (DEGs),38 and the GO terms of the gene functional categorization were obtained. When Padj (corrected p-value) 2.46 µmol L-1) and did not reach limiting concentrations during growth. In addition, it has been found that MC changes the pH of algal suspensions and inhibits growth when the values are below 7.5 or above 9.0.9, 33 However, in the present study, the change of pH was almost negligible. Thus, pH was not a factor in inhibiting growth. In summary, changes in water quality brought by MC were not the main factors limiting the growth of algal cells.

Figure 1. The change of cell growth and environmental factors as impacted by MC. A. algal cell growth; B. concentration of total dissolved phosphorus (TDP) and total dissolved nitrogen (TDN); C. pH values. 10


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Effect of MC on the Regulatory Gene Expression of the Residual Cells Sequencing and Transcription Analysis Illumina sequencing data of the 12 tested samples are shown in Table S1 and S2, with 750 million generated raw reads (7.5 Gbps). After removing the residual rRNA and low-quality reads, 720 million clean reads (7.2 Gbps) were obtained for further alignment and analysis. For each sample, the percentage of clean reads mapped to the genome occupied 78.0–81.3% (>70% was available) and the uniquely mapped reads represented 74.4–76.9%. Of the reads that mapped to the genome, 60.3–63.3% mapped to exons (reference transcript), while the others mapped to introns and intergenic regions. These data suggested that the sequencing data were reliable and the results were credible for this study. The transcriptomic sequencing data of fold change for triplicates were provided in the supporting information (Excel file 1).

Differentially Expressed Genes Analysis The transcriptome sequencing data showed that 403 genes were differentially expressed at 3 h after MC addition, of which 294 were up-regulated, and 109 were down-regulated. There were 724 genes that were differentially expressed at 24 h after MC addition, of which 465 were up-regulated, and 259 were down-regulated. From 3 to 24 h, the effect of MC was sustained and enhanced, with the number of differentially expressed genes (DEGs) increasing and the fold-change of some genes expanding. Between 3 and 24 h, there were 196 DEGs in common. The distribution of the DEGs is shown in Fig. S1A and S1B, and the fold-change of the DEGs is displayed in the 11



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supporting information (Excel file 2). Seven genes with distinct expression patterns in response to MC treatment were selected to conduct RT-qPCR to validate the RNA-Seq expression data. The results showed a significant correlation (R2 = 0.738; P < 0.01) between the RNA-Seq and RT-qPCR data, thus indicating that our RNA-Seq data were reliable (Table S3, Fig. S2). Based on the GO database, the DEGs were classified into three functional categories: molecular function, biological process, and cellular component. The results showed that the terms related to photosynthesis, carbon metabolism, cytoskeleton, and transportation were significantly up-regulated. Conversely, the most significant down-regulated term was related to the genetic information processes. The KEGG enrichment results showed that after MC treatment, the up-regulated DEGs of algal cells were enriched in pathways related to the synthesis and metabolism of three major energy-yielding nutrients (carbohydrates, lipids, and proteins) and peroxisomes, while the down-regulated DEGs were enriched for genetic information processes (Fig. 2). The results indicated that the normal physiological processes of non-flocculated cells were disordered. The stress inflicted by MC induced increases in the physiological activities of survival and self-repair, inhibiting normal division and proliferation. Under abiotic stress (e.g., nutrient limitation, high temperature, toxic metal pollution), the up-regulation of genes related to photosynthesis and stress-responsive proteins is universal,13, 20, 42-44 and the overexpression of these genes also emerged in residual cells treated by MC. In addition, the up-regulated wound healing genes 12


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associated with the cytoskeleton represent a specific response to MC treatment. This may be caused by collisions between the clay particles and algal cells, damaging cell membrane and even causing cell lysis (Fig. S3 A, B, C). It has been reported that under circumstances of aluminum stress, genes encoding ABC transporters are up-regulated.45, 46

The modifier used in MC is polyaluminum chloride; however, genes related to ABC

transporters were not up-regulated in the residual cells treated by MC. Thus, it can be inferred that the modifier is non-aluminum-toxicity for the residual cells in the culture suspension.

Figure 2. KEGG classification of assembled transcripts. The top 20 up-regulated pathways are shown on the right, and the top 20 down-regulated pathways are shown on the left. According to the first-level classification, the 20 up-regulated pathways are classified into two categories: A. metabolism and D. cellular process. The secondary-level classification is shown on the right above. From bottom to top, the pathways are biosynthesis of secondary metabolites, fatty acid metabolism, metabolic pathways, ascorbate and aldarate metabolism, citrate cycle (TCA cycle), galactose metabolism, glycolysis/gluconeogenesis, pentose and glucuronate interconversions, 13



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propanoate metabolism, pyruvate metabolism, biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, glycerolipid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, histidine metabolism, beta-alanine metabolism, biotin metabolism, porphyrin and chlorophyll metabolism, terpenoid backbone biosynthesis, and peroxisome. Accordingly, the 20 down-regulated pathways are classified into four categories: A. metabolism, B. genetic information processing, C. environmental information processing, and D. cellular process. The secondary-level classification is shown on the left above. From bottom to top, the pathways are 2−oxocarboxylic acid metabolism, biosynthesis of antibiotics, fatty acid metabolism, metabolic pathways, inositol phosphate metabolism, biosynthesis of unsaturated fatty acids, fatty acid degradation, arginine biosynthesis, histidine metabolism, tyrosine metabolism, glycosaminoglycan degradation, nicotinate and nicotinamide metabolism, protein processing in endoplasmic reticulum, base excision repair, DNA replication, non-homologous end-joining, nucleotide excision repair, ABC transporters, endocytosis, and peroxisome.

Changes in the Biological Process and Cellular Structure Based on DEGs Analysis Analyzing the changes in the transcriptome of algal cells treated by MC, the results showed that DEGs were enriched in the physiological processes and cell structures related to photosynthesis and energy metabolism, stress adaptation and detoxification mechanisms, signal transduction and communication, membrane and cytoskeleton, genetic information processing, and the cell cycle. These data indicated that under the 14


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effects of MC treatment, various physiological activities were changed within the algal cells, threatening their survival and growth.

Photosynthesis and Energy Metabolism Chloroplasts are an important site of energy metabolism for photosynthetic organisms. In residual cells treated with MC, the light reaction process was the most significantly regulated physiological process (p2; + indicates up-regulation. The red arrows indicate increased products. The black dashed arrows indicate the electron transport chain. 1,3-BPG, 1,3-bisphosphoglycerate; 3-PG, 3-phosphoglycerate; A-CoA C, acetyl-CoA carboxylase; A-CoA, acetyl- CoA; Cyt b, cytochrome b; Cyt b6f, cytochrome b6f complex; DLA1, dihydrolipoamide S-acetyltransferase; DLD1, dihydrolipoamide dehydrogenase;

EP,

extrinsic

protein;

FAD,

xanthine

dehydrogenase;

FBA,

fructose-bisphosphate aldolase; FBP, fructose-1,6-diphosphate; Fd, ferredoxin; GAP, glyceraldehyde-3-phosphate; GAPDH,glyceraldehyde-3-phosphate dehydrogenase; LHC, light harvesting complex; M-CoA, malonyl-CoA; Mpv17/PMP22, Mpv17/PMP22 family; OEC, oxygen-evolving complex; Oxa, oxaloacetic acid; PGK, phosphoglycerate kinase; PSI, photosystem I; PSII, photosystem II; PYC, pyruvate carboxylase; RCSIII, photosystem I reaction center subunit III; RCSIV, photosystem I reaction center subunit IV/PsaE;

ROS,

reactive

oxygen

species;

Rubisco,

ribulose-1,5-biphosphate 17



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carboxylase/oxygenase.

Stress Adaptation and Detoxification Mechanisms The MC treatment activated the antioxidant enzyme activity in algal cells.8 In this study, several genes encoding stress-responsive detoxification proteins were significantly up-regulated with MC treatment (p

Molecular mechanism of modified clay controlling the brown tide

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