Thien, Vun Yee (2017) Transcriptomic and carrageenan analysis under different light wavelenghts with carbon dioxide enrichment. Doctoral thesis, Universiti Malaysia Sabah.
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Abstract
Kappaphycus a/varezii, a marine red algae belonging to the order Gigartinales (Rhodophyta), is one of the most valuable and cultivated Kappaphycus species along with Kappaphycus striatum and Eucheuma denticulatum. K. alvarezii was selected for this study owing to the fact that it has adapted to survive in an ecological niche which is characterised by varying light wavelengths. The process of carbon assimilation in algae is governed by panoply of factors yet much information from the unicellular green alga. Transcriptomes associated with the process of photosynthesis and carbon fixation have offered insights into the mechanism of gene regulation in terrestrial plants; however limited information is available as far as macroalgae/red algae are concerned. The study aims to characterise K. alvarezii transcriptome under specific light wavelengths and CO₂ enrichment. The hypothesis was that K. alvarezii would respond to different wavelength of light at the molecular level and that it would be possible to detect molecular changes in the treated-cultures and gain some insights into the carbon fixation pathway in this red alga. High-throughput paired-end RNA-sequencing was applied to profile the transcriptome of K. alvarezii irradiated with different wavelengths of light (blue 492-455 nm (BL), green 577-492 nm (GL) and red 780- 622 nm (RL)) and those exposed to full light spectrum (WL). In addition to this the carbon dioxide (CO₂) induced transcriptome was analysed to identify differentially expressed transcripts. The sequencing reads were subjected to quality filtering and assembly using Trinity. The growth performance of K. alvarezii for each treatments was determined and a one way analysis of variance was used to evaluate significant differences in daily growth rate of cultures and difference was consider significant at p<0.05. In addition, the chemical structure of K-carrageenan obtained from K. a/varezii specimens subjected to different photosynthetic spectra was assessed using Fourier Transform Infrared (FTIR) spectroscopy in order to determine the effect of light on chemical composition of Kappa-carrageenan. A de novo assembly of the transcriptome of K. alvarezii generated 76,871 qualified transcripts with a mean length of 979bp and a NS0 length of 1,707bp and 55.83% transcripts were annotated on the basis of function. A total of 28,079 annotated transcripts were assigned to 49 functional groups and 3,246 GO terms. KEGG pathway enrichment resulted in 10,460 transcripts being mapped to 273 pathways. The carbon fixation pathway was analysed and key genes encoding enzymes involved in the carbon fixation pathway in K. alvarezii such as PPC, PEPC, PRK, PGK, PPDK, provided that unequivocal molecular evidence that most of the (3 and (4 pathway genes were actively transcribed in K. alvarezii. Setting FDR less than le-10 and log2 fold change of 2 as the cutoff, 315 DEGs were obtained. The most DEGs were detected between WL and WL +CO₂. The DEGs detected under different light qualities (BL, GL, RL, WL) and CO₂ accounted for 3.46% of the identified genes. Based on the gene expression patterns among the four lights, the DEGs were designated to four categories: SL-regulated, GL-regulated, RL-regulated or either BL, GL, RL regulated. The numbers of genes encoding light-harvesting proteins transcripts were significantly down-regulated under different light spectra (BL and RL) and there was no significant difference in gene expression upon CO₂ enrichment. Fucoxanthin chlorophyll a/c-binding and early light-induced proteins were up-regulated under GL. Meanwhile, similar responses of K. alvarezii were found under BL and RL that both fucoxanthin chlorophyll a/c-binding and high light-induced proteins were down-regulated. Different light spectra appeared to induce the same effect on phycobilisome and photosystem proteins in K. alvarezii. Meanwhile, five DEGs encoding photosystem proteins were influenced by the ambient C0₂ concentration. Transcripts encoding phytochrome-like protein were observed expressed only under WL. On the other hand, the growth rate of K. alvarezii was found to be dependent on the wavelengths of light they are exposed to, with longer wavelengths promoting a faster growth rate. Red light (8.1±1.4% day-1) had the most significant impact on the growth rate of K. alvarezii as compared to those treated with blue light (3.5±1.2% day-1). The FTIR fingerprint of the ground seaweed was found to be identical to that of commercial Kappa carrageenan (Sigma). All the samples produced similar FTIR spectral profiles, demonstrated that different wavelengths of light and supplementation with C0₂ have no influence to the chemical structure of Kappa-carrageenan in K. a/varezii. The vast majority of transcripts had similar mRNA levels. GL had more pronounced effect on transcriptome as compared to BL and RL did. GL was found more efficient in triggering the biosynthesis of light-harvesting complex as the upregulation of fucoxanthin and early light-induced proteins. Regulation of gene expression by C0₂ enrichment appears to be more extensive than expected. All the genes necessary to encode the enzymes involved in photosynthetic inorganic carbon fixation were identified in K. alvarezii, especially (4-CCM pathway. Interestingly, most of the key enzymes of (3 were highly expressed under C0₂ enrichment cultures. These results suggested C02 enhancement may alter carbon metabolism and lead to Crtype carbon metabolism in K. alvarezii. The better growth rate in RL as compared to BL is probably due to higher photosynthetic efficiency and quantum yield associated with RL as RL favours the excitation of photosystem II rather than photosystem I. Carrageenan obtained from in vitro K. a/varezii belonged to Kappa-carrageenan. All the samples produced similar FTIR spectral profiles, suggesting that genes related to the carrageenan biosynthesis are not affected by different wavelengths of light or C0₂. Blue, green and red light all have demonstrated roles in modulating light responses, such as changes in gene expression and increase in the growth rate of thalli. A small proportion genes were significantly differentially expressed, suggested that light-regulated gene expression in K. a/varezii is not a unique/single light response. The findings of this project will contribute to our understanding of molecular mechanisms underlying light-induced responses in lower plants as well as facilitate our understanding in inorganic carbon fixation in red algae. The transcriptome of K. a/varezii has large number of hypothetical proteins. The transcriptome studies of the seaweeds are still scarce caused insufficient sequence data available for genome annotation. It is suggested that the proteogenomics could be utilized to improve genome annotations. This study also recommends that seaweed cultivators could utilize red light to enhance the growth rate of K. a/varezii during seedling productions.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Keyword: | Kappaphycus Alvarezii, Algae, Gigartinales |
| Subjects: | Q Science > QK Botany > QK1-989 Botany > QK504-(638) Cryptogams |
| Department: | INSTITUTE > Biotechnology Research Institute (BRI) |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 12 Jan 2024 09:34 |
| Last Modified: | 12 Jan 2024 09:34 |
| URI: | https://eprints.ums.edu.my/id/eprint/37920 |
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