Microalgae and cyanobacteria are photosynthetic microorganisms that possesses advantageous characteristics to be a potential bio-feedstock for biofuel and biochemical. However, current level of productivity from microalgae does not match up to their potentials due to the growth retardation in different and uncontrollable environmental stresses. Here, this study reports the response of three photosynthetic micro-organisms to different environmental stress such as nitrogen deprivation and temperature fluctuation which ultimately leads to the understanding of how photosynthetic machineries are repressed.
To understand the effect of nitrogen deprivation on the growth of microalgae, Dunaliella tertiolecta LB999 were cultivated under the combination of different light intensity and nitrogen level. Using transcriptome sequencing and transcriptome de novo assembly, genes that respond to both light and nitrogen with an opposite mode of action were investigated. This investigation showed that chlorophyll synthesis, tetrahydrofolate mediated C1 metabolism and GS/GOGAT nitrogen assimilation pathways are repressed in nitrogen deficient condition and upregulated in high-light condition. Glutamate is competitively used in all related pathways, which indicate that the glutamate level in the cells plays a regulatory role.
To understand the effect of temperature changes, Tetraselmis KCTC12432BP, a microalgae strain that show broad temperature tolerance range was cultivated under different temperature of 10°C, 20°C and 30°C. The functional enrichment analysis performed on the differentially expressed genes to different temperatures revealed that upregulated genes in low temperature were enriched in the plastid while the upregulated genes in high temperature were enriched in the mitochondria. In particular, the electron transport chain in the thylakoid and mitochondria showed distinct response which suggests an organelle level response to different temperature stresses. This data indicates that the low energy state
of Tetraselmis under repressed photosynthesis by different temperature stress is compensated by the increased in photosynthetic electron transport chain (PETC) in low temperature, while the even more repressed photosynthesis in high temperature lead to energy salvation by using beta oxidation and mitochondrial oxidative phosphorylation to survive.
To further investigate the change in photosynthetic apparatus to low temperature, cyanobacteria model organism Synechocystis sp. PCC6803 was analyzed using RNA-seq and ribo-seq. Similar to the results from Tetraselmis, the genes composing the PETC were up-regulated, however, the reduced plastoquinone pool remained charged. Investigation of the genome-wide translation level by ribo-seq showed that several subunits of cytochrome b6f complex and ferredoxin complex were repressed at the translation level. Further investigation shows that low temperature globally inhibits translation deficiency. Along with increased ribosome occupancy near the 5’UTR and approximately 50 nt downstream of the start codon the translation initiation factor S1, elongation factor Ts, elongation factor P and RNA helicase crhR were down-regulated either in both transcription and translational level or in translational level.
As a model organism of cyanobacteria, Synechocystis sp. PCC6803’s transcription architecture have been vastly studied for small RNA, anti-sense RNA and transcription start sites. However, the 3’ UTR and terminators of Synechocystis have not been identified. To this end, the 3’ UTR and terminators of Synechocystis were investigated by term-seq which may elucidate translation regulatory mechanisms. A total of 1,017 termination sites were identified, where 755, 104, 68, 79 and 11 were localized at gene 3’ ends, intragenic region, anti-sense direction, 5’UTR and intergenic regions, respectively. Secondary structure prediction and motif searching abled the prediction of the terminator strength of primary terminators. In particular, terminators with C-rich motif found at the intragenic region showed an enrichment of proline-rich amino acids which suggests that the ribosome pausing leads to intragenic transcription termination. Furthermore, due to the proline’s role trans-membrane alpha helix, genes intragenic termination occurrence were strongly enriched in photosynthetic membrane proteins.
미세조류와 사이아노박테리아는 질소결핍이나 온도변화와 같은 환경변화에 의해 성장이 억제된다. 지금까지의 연구는 주로 환경변화에 의해 미세조류와 사이아노박테리아에서 지질 및 탄수화물이 축적되는 것에 초점을 맞추어 왔고, 성장 억제에 대한 연구는 미미한 상황이다. 본 연구에서는 다양한 차세대 시퀀싱 방법을 적용함으로써 환경변화에 의한 성장 억제가 미세조류와 사이아노박테리아의 광합성에 어떤 영향을 주는지 관찰하였다. 질소결핍 조건에서 성장 억제 기작을 살펴보기 위해, 미세조류 Dunaliella tertiolecta LB999의 성장을 억제하는 질소결핍조건과 성장을 촉진하는 고광도 조건에서 전사체 시퀀싱을 진행하고, 각 조건에서 유전자 발현이 어떻게 변화하는지 분석하였다. 그 결과 C1 대사, 엽록소 생성, GS/GOGAT cycle의 억제가 성장에 영향을 미치는 것을 확인하였고, 위 대사경로의 조절 기작이 glutamate level에 의해 영향을 받을 가능성을 제시하였다. 또한 온도변화 조건에서 성장 억제 기작을 연구하기 위해, 10도, 20도, 30도에서 키운 미세조류 Tetraselmis KCTC12432BP에서 각각 전사체 시퀀싱을 진행하였다. 각 조건에서 유전자 발현을 비교했을 때, 저온 조건에서 광합성 전자전달계의 발현이 증가하였으며, 고온 조건에서는 mitochondria의 전자전달계의 발현이 증가하는 것을 관찰할 수 있었다. 이 결과에 의하면 억제되는 광합성을 salvaging하기 위해 저온 조건에서는 광합성을 증가시키며, 고온 조건에서는 beta-oxidation을 이용하여 carbon을 축적하고 oxidative phosphorylation을 이용하여 에너지를 유지하는 것으로 보인다. 이와 같은 저온 조건에서의 광합성 salvaging 패턴이 사이아노박테리아인 Synechocystis에서도 관찰되었다. 그러나 번역체 시퀀싱을 통해 Synechocystis의 유전자 발현을 분석한 결과, 저온 조건에서 광합성 전자전달계의 유전자 발현이 번역 수준에서는 감소하는 것을 확인할 수 있었다. 이로 인한 protein stoichiometry의 불균형에 의해 plastoquinone pool이 reduced(환원?)된 상태로 남아있게 되며, 또한 이와 같이 저온 조건에서 번역 효율이 낮아지는 것은 translation initiation factor S1, elongation factor Ts, P 그리고 RNA helicase 발현이 억제됨에 따른 영향으로 보인다.