低氮对产油尖状栅藻荧光特性及色素蛋白复合物的影响
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摘要
以产油绿藻尖状栅藻(Scenedesmus acuminatus)为实验材料,采用77 K低温荧光、SDS-PAGE圆盘电泳、i TRAQ蛋白定量等方法,研究其在低氮胁迫产油条件下,藻细胞的低温荧光特性及光合色素蛋白复合物的变化。结果表明,在77 K低温条件下尖状栅藻有3个荧光发射峰,分别位于685、695和715 nm处。利用圆盘电泳从藻细胞类囊体膜上分离到6个条带,从上至下依次为CPIa、CPI、CPa1、CPa2、LHCP和FP(游离色素),与对照组(18 mmol·L-1 Na NO3)相比,低氮(3.6 mmol·L-1Na NO3)培养的藻细胞光能耗散增强,分离的类囊体膜色素蛋白复合物代表PSII的条带CPa1和C Pa2模糊,D1蛋白及捕光色素蛋白表达下调。综上低氮限制条件下产油尖状栅藻的PSII核心复合物降解,制约藻细胞吸收和转化光能,进而影响光合效率。
In this study, the photosynthetic apparatus of S. acuminatus was analyzed by purifying the thylakoids and isolating the different pigment-protein complexes upon solubilization. In addition, the effects of N-depleted on photosynthetic apparatus were investigated using SDS-PAGE, 77 K fluorescence and isobaric tags for relative and absolute quantification(i TRAQ). The results showed that, six pigment complexes bands including CPIa, CPI, CPa1, CPa2, LHCP, and FP were isolated from the thylakoid membrane of S. acuminatus. Under N-depleted conditions, the bands CPa1, CPa2 which represented PSII became very vague, meanwhile D1 protein and light-harvesting proteins were down-regulated. The 77 K fluorescence spectra of PSI and PSII were located at 685, 695 and 715 nm. The results from the biochemical and spectroscopic characterization showed that, during the culture period, effects of N-depleted on PSII was greater than PSI, while algal cells decreased light utilization and increased dissipation. So we concluded that, there was difference between microaglal and high plant on photosynthetic traits, otherwise the damage of PSII complexes of S. acuminatus in N-depleted conditions does restrict photosynthetic efficiency.
In this study, the photosynthetic apparatus of S. acuminatus was analyzed by purifying the thylakoids and isolating the different pigment-protein complexes upon solubilization. In addition, the effects of N-depleted on photosynthetic apparatus were investigated using SDS-PAGE, 77 K fluorescence and isobaric tags for relative and absolute quantification(i TRAQ). The results showed that, six pigment complexes bands including CPIa, CPI, CPa1, CPa2, LHCP, and FP were isolated from the thylakoid membrane of S. acuminatus. Under N-depleted conditions, the bands CPa1, CPa2 which represented PSII became very vague, meanwhile D1 protein and light-harvesting proteins were down-regulated. The 77 K fluorescence spectra of PSI and PSII were located at 685, 695 and 715 nm. The results from the biochemical and spectroscopic characterization showed that, during the culture period, effects of N-depleted on PSII was greater than PSI, while algal cells decreased light utilization and increased dissipation. So we concluded that, there was difference between microaglal and high plant on photosynthetic traits, otherwise the damage of PSII complexes of S. acuminatus in N-depleted conditions does restrict photosynthetic efficiency.
引文
陈敏,宫宝安(2003).海洋绿藻77 K低温光谱特性的比较研究.烟台大学学报(自然科学与工程版),16(2):119~123
韩博平,韩志国,付翔(2003).藻类光合作用机理与模型.北京:科学出版社
李爱芬,段舜山,陈敏,宫宝安,周百成(2003).褐藻裙带菜光系统I的能量传递特性.水生生物学报,27(2):214~216
汪亚俊,孙明哲,李爱芬,张成武(2014).不同氮素水平对产油尖状栅藻生长及光合生理的影响.中国生物工程杂志,34(12):51~58
郑锡光,汪河洲,余振新,高兆兰,朱晋昌,蒋丽金(1996).藻胆素的构象变化及其对光吸收的影响.生物化学与生物物理进展,23(5):433~436
Allen JF(1992).Protein phosphorylation in regulation of photosynthesis.Biochim Biophys Acta,1098(3):275~335
Anderson JM,Waldron JC,Thorne SW(1978).Chlorophyll—protein\scomplexes of spinach and barley thylakoids:Spectral characterization of six complexes resolved by an improved electrophoretic\sprocedure.FEBS Lett,92(2):227~233
Basso S,Simionato D,Gerotto C,Segalla A,Giacometti GM,Morosinotto T(2014).Characterization of the photosynthetic apparatus of the Eustigmatophycean Nannochloropsis gaditana:evidence of convergent evolution in the supramolecular organization of photosystem I.Biochim Biophys Acta,1837(2):306~314
Bellafiore S,Barneche F,Peltier G,Rochaix JD(2005).State transitions and light adaptation require chloroplast thylakoid protein\skinase STN7.Nature,433(7028):892~895
Benvenuti G,Bosma R,Cuaresma M,Janssen M,Barbosa MJ,Wijffels RH(2015).Selecting microalgae with high lipid productivity and photosynthetic activity under nitrogen starvation.J Appl\sPhycol,27:1425~1431
Berges A,Charlebois DO,Mauzerall DC,Falkowski PG(1996).Differential effects of nitrogen limitation on photosynthetic efficiency of photosystems I and II in microalgae.Plant Physiol,110(2):689~696
Bulte L,Wollman FA(1992).Evidence for a selective destabilization\sof an integral membrane protein,the cytochrome b6/f complex\sduring gametogenesis in Chlamydomonas reinhardtii.Eur J Bio-chem,204(1):327~336
Cakmak T,Angun P,Demiray YE,Ozkan AD,Elibol Z,Tekinay T(2012).Differential effects of nitrogen and sulfur deprivation on\sgrowth and biodiesel feedstock production of Chlamydomonas\sreinhardtii.Biotechnol Bioeng,109(8):1947~1957
Chukhutsina VU,Büchel C,van Amerongen H(2014).Disentangling\stwo n on-photochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77K.Biochim Biophys Acta,1837(6):899~907
Drop B,Yadav KNS,Boekema EJ,Croce R(2014).Consequences\sof state transitions on the structural and functional organization\sof photosystem I in the green alga Chlamydomonas reinhardtii.Plant J,78(2):181~191
Eisenstadt D,Ohad I,Keren N,Kaplan A(2008).Changes in the photosynthetic reaction centre II in th e diatom Phaeodactylum tricornutum result in non-photochemical fluorescence quenching.Environ Microbiol,10(8):1997~2007
Hankamer B,Lehr F,Rupprecht J,Mussgnug JH,Posten C,Kruse\sO(2007).Photosynthetic biomass and H2 production by green\salgae:from bioengineering to bioreactor scale-up.Physiol Plant,131(1):10~21
Heldt HW,Piechulla B(2010).Plant Biochemistry.Burlington:Elsevier Academic Press
Hu Q,Sommerfeld,M Jarvis E.,Ghirardi M,Posewitz M,Seibert M,Darzins A(2008).Microalgal triacylglycerols as feedstocks for\sbiofuel production:perspectives and advances.Plant J,54(4):621~639
Kuang TY,Argyroudi-Akoyunoglou JH,Nakatani HY,Watson J,Arntzen CJ(1984).The origin of the long-wavelength fluorescence emission band(77 K)from photosystem I.Arch Biochem\sBiophys,235(2):618~627
Niyogi KK(1999).Photoprotection revisited:genetic and molecular\sapproaches.Plant Physiol Biochem,50:333~359
?rd?g V,Stirk WA,Bálint P,Staden JV,Lovász C(2012).Changesin lipid,protein and pigment concentrations in nitrogen-stressed\sChlorella minutissima cultures.J Appl Phycol,24(4):907~914
Pan YY,Wang ST,Chuang LT,Chang YW,Chen CNN(2011).Isolation of thermo-tolerant and high lipid content green microalgae:oil accumulation is predominantly controlled by photosystem\sefficiency during stress treatments in Desmodesmus.Bioresour\sTechnol,102(22):10510~10517
Philipps G,Happe T,Hemschemeier A(2012).Nitrogen deprivation\sresults in photosynthetic hydrogen production in Chlamydomonas reinhardtii.Planta,235(4):729~745
Rochaix JD(2011).Reprint of:regulation of photosynthetic electrontransport.Biochim Biophy s Acta,1807(8):878~886
Salomon E,Bar-Eyal L,Sharon S,Keren N(2013).Balancing photosynthetic electron flow is critical for cyanobacterial acclimation\sto nitrogen limitation.Biochim Biophys Acta,1827(3):340~347
Simionato D,Block MA,La Rocca N,Rocca NL,Jouhet J,Maréchal\sE,Finazzi G,Morosinotto T(2013).The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols,a decrease of chloroplast galactolipids\sand reorganization of the photosynthetic apparatus.Eukaryot\sCell,12(5):665~676
Tüccar G,?z gür T,Ayd?n K(2014).Effect of diesel-microalgae biodiesel-butanul blends on performance and emissions of diesel\sengine.Fuel,132:47~52
Wang GH,Chen LZ,Li GB,Li DH,Hu CX,Chen HF,Liu YD,So ng\sLR(2005).Improving photosynthesis of microalgae by changing\sthe ratio of light-harvesting pigments.Chin Sci Bull,50(15):1622~1626
White S,Anandraj A,Bux F(2011).PAM fluorometry as a tool to assess microalgal nutrient stress and monitor cellular neutral lipids\sBioresour Technol,102(2):1675~1682
Zhang YM,Chen H,He CL,Wang Q(2013).Nitrogen starvation induced oxidative stress in an oil-producing green alga Chlorella\ssorokiniana C3.PLo S ONE,8(7):e69225
韩博平,韩志国,付翔(2003).藻类光合作用机理与模型.北京:科学出版社
李爱芬,段舜山,陈敏,宫宝安,周百成(2003).褐藻裙带菜光系统I的能量传递特性.水生生物学报,27(2):214~216
汪亚俊,孙明哲,李爱芬,张成武(2014).不同氮素水平对产油尖状栅藻生长及光合生理的影响.中国生物工程杂志,34(12):51~58
郑锡光,汪河洲,余振新,高兆兰,朱晋昌,蒋丽金(1996).藻胆素的构象变化及其对光吸收的影响.生物化学与生物物理进展,23(5):433~436
Allen JF(1992).Protein phosphorylation in regulation of photosynthesis.Biochim Biophys Acta,1098(3):275~335
Anderson JM,Waldron JC,Thorne SW(1978).Chlorophyll—protein\scomplexes of spinach and barley thylakoids:Spectral characterization of six complexes resolved by an improved electrophoretic\sprocedure.FEBS Lett,92(2):227~233
Basso S,Simionato D,Gerotto C,Segalla A,Giacometti GM,Morosinotto T(2014).Characterization of the photosynthetic apparatus of the Eustigmatophycean Nannochloropsis gaditana:evidence of convergent evolution in the supramolecular organization of photosystem I.Biochim Biophys Acta,1837(2):306~314
Bellafiore S,Barneche F,Peltier G,Rochaix JD(2005).State transitions and light adaptation require chloroplast thylakoid protein\skinase STN7.Nature,433(7028):892~895
Benvenuti G,Bosma R,Cuaresma M,Janssen M,Barbosa MJ,Wijffels RH(2015).Selecting microalgae with high lipid productivity and photosynthetic activity under nitrogen starvation.J Appl\sPhycol,27:1425~1431
Berges A,Charlebois DO,Mauzerall DC,Falkowski PG(1996).Differential effects of nitrogen limitation on photosynthetic efficiency of photosystems I and II in microalgae.Plant Physiol,110(2):689~696
Bulte L,Wollman FA(1992).Evidence for a selective destabilization\sof an integral membrane protein,the cytochrome b6/f complex\sduring gametogenesis in Chlamydomonas reinhardtii.Eur J Bio-chem,204(1):327~336
Cakmak T,Angun P,Demiray YE,Ozkan AD,Elibol Z,Tekinay T(2012).Differential effects of nitrogen and sulfur deprivation on\sgrowth and biodiesel feedstock production of Chlamydomonas\sreinhardtii.Biotechnol Bioeng,109(8):1947~1957
Chukhutsina VU,Büchel C,van Amerongen H(2014).Disentangling\stwo n on-photochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77K.Biochim Biophys Acta,1837(6):899~907
Drop B,Yadav KNS,Boekema EJ,Croce R(2014).Consequences\sof state transitions on the structural and functional organization\sof photosystem I in the green alga Chlamydomonas reinhardtii.Plant J,78(2):181~191
Eisenstadt D,Ohad I,Keren N,Kaplan A(2008).Changes in the photosynthetic reaction centre II in th e diatom Phaeodactylum tricornutum result in non-photochemical fluorescence quenching.Environ Microbiol,10(8):1997~2007
Hankamer B,Lehr F,Rupprecht J,Mussgnug JH,Posten C,Kruse\sO(2007).Photosynthetic biomass and H2 production by green\salgae:from bioengineering to bioreactor scale-up.Physiol Plant,131(1):10~21
Heldt HW,Piechulla B(2010).Plant Biochemistry.Burlington:Elsevier Academic Press
Hu Q,Sommerfeld,M Jarvis E.,Ghirardi M,Posewitz M,Seibert M,Darzins A(2008).Microalgal triacylglycerols as feedstocks for\sbiofuel production:perspectives and advances.Plant J,54(4):621~639
Kuang TY,Argyroudi-Akoyunoglou JH,Nakatani HY,Watson J,Arntzen CJ(1984).The origin of the long-wavelength fluorescence emission band(77 K)from photosystem I.Arch Biochem\sBiophys,235(2):618~627
Niyogi KK(1999).Photoprotection revisited:genetic and molecular\sapproaches.Plant Physiol Biochem,50:333~359
?rd?g V,Stirk WA,Bálint P,Staden JV,Lovász C(2012).Changesin lipid,protein and pigment concentrations in nitrogen-stressed\sChlorella minutissima cultures.J Appl Phycol,24(4):907~914
Pan YY,Wang ST,Chuang LT,Chang YW,Chen CNN(2011).Isolation of thermo-tolerant and high lipid content green microalgae:oil accumulation is predominantly controlled by photosystem\sefficiency during stress treatments in Desmodesmus.Bioresour\sTechnol,102(22):10510~10517
Philipps G,Happe T,Hemschemeier A(2012).Nitrogen deprivation\sresults in photosynthetic hydrogen production in Chlamydomonas reinhardtii.Planta,235(4):729~745
Rochaix JD(2011).Reprint of:regulation of photosynthetic electrontransport.Biochim Biophy s Acta,1807(8):878~886
Salomon E,Bar-Eyal L,Sharon S,Keren N(2013).Balancing photosynthetic electron flow is critical for cyanobacterial acclimation\sto nitrogen limitation.Biochim Biophys Acta,1827(3):340~347
Simionato D,Block MA,La Rocca N,Rocca NL,Jouhet J,Maréchal\sE,Finazzi G,Morosinotto T(2013).The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols,a decrease of chloroplast galactolipids\sand reorganization of the photosynthetic apparatus.Eukaryot\sCell,12(5):665~676
Tüccar G,?z gür T,Ayd?n K(2014).Effect of diesel-microalgae biodiesel-butanul blends on performance and emissions of diesel\sengine.Fuel,132:47~52
Wang GH,Chen LZ,Li GB,Li DH,Hu CX,Chen HF,Liu YD,So ng\sLR(2005).Improving photosynthesis of microalgae by changing\sthe ratio of light-harvesting pigments.Chin Sci Bull,50(15):1622~1626
White S,Anandraj A,Bux F(2011).PAM fluorometry as a tool to assess microalgal nutrient stress and monitor cellular neutral lipids\sBioresour Technol,102(2):1675~1682
Zhang YM,Chen H,He CL,Wang Q(2013).Nitrogen starvation induced oxidative stress in an oil-producing green alga Chlorella\ssorokiniana C3.PLo S ONE,8(7):e69225