在利用遠紅光的藍綠菌Synechococcus sp. PCC 7335中研究光系統II反應中心次單元PsbD3之點突變對於光系統II功能影響

Abstract

某些藍綠菌生長在遠紅光（λ=700~800 nm）時，會進行遠紅光轉換並表達在FaRLiP基因簇中光系統的同源基因，重組在白光（λ=400~700 nm）生長時表現的光系統。 PsbA3、PsbC2和PsbD3是在遠紅光轉換過程中重組光系統 II的三個蛋白質次單元。這三個蛋白質次單元具有保守的殘基，並與吸收遠紅光的葉綠素d及葉綠素f專一地結合。先前的研究中，蛋白質結構和光譜資料說明了這些葉綠素參與了遠紅光下光合作用中的電子傳遞及能量傳遞。然而，這些研究缺乏活體證據。此外，目前仍缺乏能在行遠紅光轉換的藍綠菌中產生點突變的系統，因此難以研究這些殘基點位。 在本研究中，我們嘗試利用 CRISPR-Cpf1 系統在能進行遠紅光轉換的藍綠菌Synechococcus sp. PCC 7335的PsbA3、PsbC2以及PsbD3中產生點突變。在三個次單元中，我們成功的在PsbD3次單元產生了Y191W點突變。這個保守位點的酪氨酸殘基和電子傳遞鏈中的葉綠素a產生建結。並且，相對於白光下表達光系統二的殘基，該酪氨酸被認為能改變此葉綠素a的能量。此外，由於由於引子設計錯誤，造成Y191W外的另一個點突變N220D也被產生了。生長實驗中，突變株與野生型相比在遠紅光下生長得較慢。對突變株的光譜分析顯示，遠紅光下生長的突變株相對於野生型，具有更多白光下表達的光系統。我們在突變株細胞的氧活性測試中，發現突變株的光系統二失去了利用遠紅光的能力。此外，高效液相層析沒有在遠紅光下生長的突變株細胞中檢測到葉綠素d。 我們利用蔗糖梯度進行的光系統分離。與野生型相比，適應遠紅光的突變株分離較多的光系統分數。突變株的光系統二發出與野生型不同的螢光訊號；而在另一個分層中則同時發出來自白光表達光系統二及遠紅光表達光系統一的訊號。最後，我們對突變株進行蛋白質質譜分析，檢測到了遠紅光下表達光系統二中所有次單元的訊號。這些結果表明在突變株中，遠紅光下表達的光系統二的組裝可能因點突變而受到影響，並且葉綠素d的存在可能取決於組裝的完整性。 此研究中，我們利用CRISPR-Cpf1在Synechococcus sp. PCC 7335中產生點突變，而結果顯示該系統仍有待改進。對突變株的分析則顯示，特定殘基可能參與了的光系統二的組裝。這些結果提供了在藍綠菌中基因編輯改進的方向，並增加了我們對利用遠紅光光合作用的理解。

When exposed in far-red light (FRL, λ=700~800 nm), some cyanobacteria perform far-red light photoacclimation (FaRLiP) and express a set of homologue genes of photosystem in the FaRLiP gene cluster that remodel photosystem (PS) expressed in white light (WL, λ=400~700 nm). PsbA3, PsbC2 and PsbD3 are three of the protein subunits that remodel the photosystem II (PSII) during FaRLiP. The three subunits contain conserved residues that provides specific bindings to chlorophylls, including FRL-absorbing chlorophyll (Chl) d and Chl f. Structure and spectroscopic information suggests these Chls contribute to FRL-using photosynthesis and function in the electron transfer chain (ETC) or energy transfer process. However, in vivo evidences for such suggestion is lacking. Furthermore, there is lacking an established mutagenesis method to generate point mutation in FaRLiP cyanobacteria to study these residues. In this study, we attempted to utilize CRISPR-Cpf1 system to generate point mutations in the three PSII subunits of a FaRLiP cyanobacteria, Synechococcus sp. PCC 7335. Point mutations was generated in PsbD3, which introduced an Y191W mutation to an ETC-related Chl a binding tyrosine residue. The residue was suspected to alter the energy of binding Chl a compared to residues in WL-expressed PSII. Additionally, an N220D mutations was accidently generated by a primer design error. We found that the mutant grew slower under FRL compared to wild type. Spectroscopic analysis of mutant grown under FRL revealed higher amount of WL-expressed photosystem in mutant compared to WT, while oxygen evolution showed that the PSII of mutant cells lost FRL-using ability. Moreover, pigment analysis by high performance liquid chromatography detected no Chl d in cells grown under FRL. Photosystem isolation by sucrose gradient showed increased fraction layer compared to WT. The PSII fraction in the mutant emitted distinct signals from WT, while a fraction emitted signals from both WL-expressed PSII and FRL-expressed PSI. Finally, protein mass spectroscopy analysis of the fractions in mutant detected all subunits in the FRL-expressed PSII. These results suggest that the assembly of FRL-expressed PSII is affected due to point mutations, and the integrity of such PSII may be required for the existence of Chl d. Overall, we utilized the CRISPR-Cpf1 to generate point mutations in Synechococcus sp. PCC 7335, showing the system still to be improved, and suggested the assembly-related function of specific residues in photosystem II. These findings pave the way for developing the gene editing system in FaRLiP cyanobacteria and expand the knowledge of FRL-utilizing photosynthesis.