單胞藻 (Chlamydomonas reinhardtii) 可利用 CO2 作為自營 (Sueoka high
salt medium + 5% CO2) 培養或利用 acetate 作為混營 (Tris-acetate-phosphate) 培
養。本研究目的為釐清 HSM + 5% CO2 自營培養及 TAP 混營培養之
methionine sulfoxide reductase (MSR) 基因表現之差異及對高光 (1,000 μE m-2 s-1)
之反應，並了解光合電子傳遞鏈參與高光調控基因表現之角色。高光抑制自營培
養和混營培養單胞藻之 PSII 活性 (Fv/Fm 及 Fv''/Fm'')，抑制程度相同。高光促進
自營培養之 CrMSR isoform 基因表現數目較混營培養多，兩者受高光促進之
isoform 種類不同，高光促進自營培養之 CrMSRA1、CrMSRA2、CrMSRA3、
CrMSRA5 、CrMSRB1.2 和 CrMSRB2.1 基因表現而抑制 CrMSRA4 和
CrMSRB2.2 基因表現， 高光促進混營培養之 CrMSRA3 、CrMSRA5 和
CrMSRB2.1 基因表現而抑制 CrMSRA1、CrMSRA4 和 CrMSRB2.2 基因表現。
利用光合電子傳遞鏈抑制劑證實光合電子傳遞鏈為高光影響自營培養和混營培
養之 CrMSR isoform 基因表現的因子，但是兩者受電子傳遞鏈之不同位置調
控，尤其是 CrMSRA 基因表現。自營培養的 CrMSRA 表現多受 QA 還原態 (-)
調控，混營培養則多由 PQ 還原態 (-) 調控，自營培養 CrMSRB 表現受光合電
子傳遞鏈影響大，主要由 QA.(-) 和 Cytb6f (-) 調控，混營培養 CrMSRB 表現受
光合電子傳遞鏈影響較小，僅 CrMSRB2.2 受 Cytb6f (-) 調控。高光不誘導自營
培養與混營培養的 H2O2 產生，推測 CrMSR isoform 基因表現與 H2O2 無關。
本研究證實自營培養和混營培養 CrMSR 基因表現受高光調控情形不同，並討論
碳源利用方式與 CrMSR 基因表現調控之關係。

Chlamydomonas reinhardtii can utilize CO2 for autotrophic growth (HSM plus
5% CO2) or acetate for mixotrophic growth (TAP). This study was to elucidate the
differential regulation of methionine sulfoxide reductase (MSR) gene expression
between HSM plus 5% CO2 and TAP cultured cells, and also to determine the
difference of gene expression in response to high light (1,000 μE m-2 s-1). The role of
photosynthetic electron transport (PET) in the regulation of MSR gene expression was
also examined by the use of PET inhibitors. High light inhibited PSII activity (Fv/Fm
and Fv''/Fm'') of HSM plus 5% CO2 and TAP cultured cells., while the responses of
CrMSR gene expression in mixotrophically grown cells were different from
autotrophically grown cells, High light increased the expression of CrMSRA1,
CrMSRA2, CrMSRA3, CrMSRA5, CrMSRB1.2, and CrMSRB2.1, but inhibited the
expression of CrMSRA4 and CrMSRB2.2 in autotrophically grown cells. The
expression of CrMSRA3, CrMSRA5, and CrMSRB2.1 in mixotrophically grown cells
was increased by high light but that of CrMSRA1, CrMSRA4, and CrMSRB2.2 was
inhbited. The number of MSR isoform that was up-regulated by high light was greater
in autotrophically grown cell than that in mixotrophically grown cells. Using the PET
inhibitors (3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU) and
2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)), most of the CrMSRA
expression was regulated by reduced QA for autotrophically grown cells while
reduced PQ was the main site for mixotrophically grown cells by high light. The
expression of CrMSRB in autotrophically grown cells was mainy modulated by QA (-)
or Cytb6f (-), while that was not affected by PET, except a role of Cytb6f (-) on the
high light-induced CrMSRB2.2 expression. We fouind that CrMSRB gene expression
in autotrophically grown cells was highly affected by PET but not for micotrophically
grtown cells. The present result that H2O2 did not accumulate in autotrophically and
mixotrophically grown cells suggests that H2O2 may be not involved in the regulation
of high light regulation of CrMSR gene expression. The present study shows that the
mRNA expression of CrMSR isoforms in Chlamydomonas was diffrerentially
regulated between autotrophically and mixttrophically grown cells. The relationship
between the utilization of different C source and CrMSR gene expression will be
discussed.

目錄
論文審定書 ..................................................................................................................... i
誌謝 ............................................................................................................................... ii
中文摘要 .......................................................................................................................iii
英文摘要 ....................................................................................................................... iv
縮寫對照表 ................................................................................................................... vi
目錄 .............................................................................................................................viii
圖目次 ........................................................................................................................... ix
表目次 ........................................................................................................................... xi
附錄目次 ...................................................................................................................... xii
壹、 緒論 ...................................................................................................................... 1
貳、 論文研究目的及實驗架構 ................................................................................ 15
參、 材料與方法 ........................................................................................................ 17
肆、 結果 .................................................................................................................... 27
伍、 討論 .................................................................................................................... 36
陸、 參考文獻 ............................................................................................................ 43
柒、 附錄 .................................................................................................................... 74

許媛婷 (2008)。光合作用電子傳遞鏈訊息調節大型綠藻列石蓴光誘導之
methionine sulfoxide reductase (MSR) 基因表現。國立中山大學海洋生物研
究所碩士論文。台灣，中華民國。
曾宇璐 (2010)。電子傳遞鏈參與光強調控單胞藻 (Chlamydomonas reinhardtii)
methionine sulfoxide reductaseA (MSRA) 核基因之差異表現。國立中山大
學海洋生物研究所碩士論文。台灣，中華民國。
Allen JF, Bennett J (1981) Photosynthetic protein-phosphorylation in intact
chloroplasts-inhibition by DCMU and by the onset of CO2 fixation. FEBS
Letters 123: 67-70
Allen MD, Kropat J, Tottey S, Del Campo JA, Merchant SS (2007) Manganese
deficiency in Chlamydomonas results in loss of photosystem II and MnSOD
function, sensitivity to peroxides, and secondary phosphorus and iron
deficiency. Plant Physiology 143: 263-277
Apel K, Hirt H (2004) Reactive oxygen species: Metabolism, oxidative stress, and
signal transduction. Annual Review of Plant Biology 55: 373-399
Arner ESJ, Holmgren A (2000) Physiological functions of thioredoxin and
thioredoxin reductase. European Journal of Biochemistry 267: 6102-6109
Asada K (1999) The water-water cycle in chloroplasts: Scavenging of active oxygens
and dissipation of excess photons. Annual Review of Plant Physiology and
Plant Molecular Biology 50: 601-639
Asada K (2006) Production and scavenging of reactive oxygen species in chloroplasts
and their functions. Plant Physiology 141: 391-396
Batschauer A, Mosinger E, Kreuz K, Dorr I, Apel K (1986) The implication of a
plastid-derived faction n the transcriptional control of nuclear genes encoding
the light-harvesting chlorophyll a-b protein. European Journal of Biochemistry
154: 625-634
Bechtold U, Murphy DJ, Mullineaux PM (2004) Arabidopsis peptide methionine
sulfoxide reductase 2 prevents cellular oxidative damage in long nights. Plant
Cell 16: 908-919
Bennett ME, Hobbie JE (1972) Uptke of glucose by Chlamydomonas sp. Journal of
44
Phycology 8: 392-398
Boschi-Muller S, Azza S, Sanglier-Cianferani S, Talfournier F, Van Dorsselear A,
Branlant G (2000) A sulfenic acid enzyme intermediate is involved in the
catalytic mechanism of peptide methionine sulfoxide reductase from
Escherichia coli. Journal of Biological Chemistry 275: 35908-35913
Boyle NR, Morgan JA (2009) Flux balance analysis of primary metabolism in
Chlamydomonas reinhardtii. BMC Systems Biology 3: 4
Brot N, Weissbach H (1983) Biochemistry and physiological-role of methionine
sulfoxide residues in proteins. Archives of Biochemistry and Biophysics 223:
271-281
Brot N, Weissbach L, Werth J, Weissbach H (1981) Enzymatic reduction of
protein-bound methionine sulfoxide. Proceedings of the National Academy of
Sciences of the United States of America-Biological Sciences 78: 2155-2158
Brown EC, Somanchi A, Mayfield SP (2001) Interorganellar crosstalk: new
perspectives on signaling from the chloroplast to the nucleus. Genome Biology
2: 1021.1-1021.4
Davies MJ (2005) The oxidative environment and protein damage. Biochimica et
Biophysica Acta-Proteins and Proteomics 1703: 93-109
Delaye L, Becerra A, Orgel L, Lazcano A (2007) Molecular evolution of peptide
methionine sulfoxide reductases (MsrA and MsrB): On the early development
of a mechanism that protects against oxidative damage. Journal of Molecular
Evolution 64: 15-32
Dietz KJ (2003) Redox control, redox signaling, and redox homeostasis in plant cells.
In International Review of Cytology - a Survey of Cell Biology 228 141-193
Durnford DG, Falkowski PG (1997) Chloroplast redox regulation of nuclear gene
transcription during photoacclimation. Photosynthesis Research 53: 229-241
Endo T, Asada K (1996) Dark induction of the non-photochemical quenching of
chlorophyll fluorescence by acetate in Chlamydomonas reinhardtii. Plant and
Cell Physiology 37: 551-555
Escoubas JM, Lomas M, Laroche J, Falkowski PG (1995) Light-intensity
regulation of cab gene-transcription is signaled by the redox state of the
plastoquinone pool. Proceedings of the National Academy of Sciences of the
United States of America 92: 10237-10241
Fey V, Wagner R, Brautigam K, Pfannschmidt T (2005) Photosynthetic redox
45
control of nuclear gene expression. Journal of Experimental Botany 56:
1491-1498
Fischer BB, Eggen RIL, Trebst A, Krieger-Liszkay A (2006) The glutathione
peroxidase homologous gene Gpxh in Chlamydomonas reinhardtii is
upregulated by singlet oxygen produced in photosystem II. Planta 223:
583-590
Fischer BB, Krieger-Liszkay A, Eggen RIL (2004) Photosensitizers neutral red
(Type I) and rose bengal (Type II) cause light-dependent toxicity in
Chlamydomonas reinhardtii and induce the Gpxh gene via increased singlet
oxygen formation. Environmental Science & Technology 38: 6307-6313
Foote CS (1968) Mechanisms of photosensitized oxidation there are several different
types of photosensitized oxidation which may be important in biological
systems. Science 162: 963-970
Foyer CH, Lelandais M, Kunert KJ (1994) Photooxidative stress in plants.
Physiologia Plantarum 92: 696-717
Grimaud R, Ezraty B, Mitchell JK, Lafitte D, Briand C, Derrick PJ, Barras F
(2001) Repair of oxidized proteins - Identification of a new methionine
sulfoxide reductase. Journal of Biological Chemistry 276: 48915-48920
Grossman AR, Harris EE, Hauser C, Lefebvre PA, Martinez D, Rokhsar D,
Shrager J, Silflow CD, Stern D, Vallon O, Zhang ZD (2003)
Chlamydomonas reinhardtii at the crossroads of genomics. Eukaryotic Cell 2:
1137-1150
Grossman AR, Im CS, Moseley J, Eberhard S, Pollock S, Pootakham W (2005)
The use of genomic tools to examine the responses of chlamydomonas
reinhardtii to its environment. Phycologia 44: 94
Harris EH (2001) Chlamydomonas as a model organism. Annual Review of Plant
Physiology and Plant Molecular Biology 52: 363-406
Harris EH (2009) The Chlamydomonas Soursebook, 2nd edition, Volume 1:
Introduction to Chlamydomonas and its Laboratory Use, Elsevier, San Diego,
USA.
Hsu YT, Lee TM (2010) Photosynthetic electron transport mediates the
light-controlled up-regulation of expression of methionine sulfoxide reductase
a and b from marine macroalga Ulva fasciata. Journal of Phycology 46:
112-122
46
Johanningmeier U (1988) Possible control of transcript levels by chlorophyll
precursors in Chlamydomonas. European Journal of Biochemistry 177:
417-424
Johanningmeier U, Howell SH (1984) Regulation of light-harvesting
chlorophyll-binding protein messenger-RNA accumulation in Chlamydomonas
reinhardtii possible involvement of chlorophyll synthesis precursors. Journal
of Biological Chemistry 259: 3541-3549
Karpinski S, Escobar C, Karpinska B, Creissen G, Mullineaux PM (1997)
Photosynthetic electron transport regulates the expression of cytosolic
ascorbate peroxidase genes in Arabidopsis during excess light stress. Plant
Cell 9: 627-640
Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P
(1999) Systemic signaling and acclimation in response to excess excitation
energy in Arabidopsis. Science 284: 654-657
Kimura M, Yoshizumi T, Manabe K, Yamamoto YY, Matsui M (2001)
Arabidopsis transcriptional regulation by light stress via hydrogen
peroxide-dependent and -independent pathways. Genes to Cells 6: 607-617
Kumar RA, Koc A, Cerny RL, Gladyshev VN (2002) Reaction mechanism,
evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide
reductase. Journal of Biological Chemistry 277: 37527-37535
Kwon SJ, Kwon SI, Bae MS, Cho EJ, Park OK (2007) Role of the methionine
sulfoxide reductase MsrB3 in cold acclimation in Arabidopsis. Plant and Cell
Physiology 48: 1713-1723
Leisinger U, Rüfenacht K, Fischer B, Pesaro M, Spengler A, Zehnder AJB,
Eggen RIL (2001) The glutathione peroxidase homologous gene from
Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet
oxygen. Plant Molecular Biology 46: 395-408
Lemaire SD, Guillon B, Le Marechal P, Keryer E, Miginiac-Maslow M,
Decottignies P (2004) New thioredoxin targets in the unicellular
photosynthetic eukaryote Chlamydomonas reinhardtii. Proceedings of the
National Academy of Sciences of the United States of America 101:
7475-7480
Lowther, WT, Brot, N, Weissbach, H, Honek, JF, Matthews, BW (2000)
Thiol-disulfide exchange is involved in the catalytic mechanism of peptide
47
methionine sulfoxide reductase. Proceedings of the National Academy of
Sciences of the United States of America 97: 6463-6468
Lowther WT, Weissbach H, Etienne F, Brot N, Matthews BW (2002) The
mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB.
Nature Structural Biology 9: 348-352
Lu IF, Sung MS, Lee TM (2006) Salinity stress and hydrogen peroxide regulation of
antioxidant defense system in Ulva fasciata. Marine Biology 150: 1-15
Lucksch, I. (1932) Ernährungsphysiologische Untersuchungen an
Chlamydomonadeen. Beih. Bot. Centralbl. 50:64-94.
Mayfield SP, Taylor WC (1984) Carotenoid-deficient maize seedlings fail to
accumulate light-harvesting chlorophyll a/b binding-protein (LHCP)
messenger-RNA. European Journal of Biochemistry 144: 79-84
Mayfield SP, Taylor WC (1987) Chloroplast photooxidation inhibits the expression
of a set of nuclear genes. Molecular and General Genetics 208: 309-314
Mehler AH (1951) Studies on reactions of illuminated chloroplasts : I. Mechanism of
the reduction of oxygen and other hill reagents. Archives of Biochemistry and
Biophysics 33: 65-77
Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB,
Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, Marshall
WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV,
Ren QH, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J,
Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft
MT, Dent R, Dutcher S, Fernandez E, Fukuzawa H, Gonzalez-Ballester D,
Gonzalez-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W,
Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV,
Lohr M, Manuell A, Meir I, Mets L, Mittag M, Mittelmeier T, Moroney
JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV, Paulsen
IT, Pazour G, Purton S, Ral JP, Riano-Pachon DM, Riekhof W,
Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N,
Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K,
Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan
JM, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang PF,
Ball S, Bowler C, Dieckmann CL, Gladyshev VN, Green P, Jorgensen R,
Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P,
48
Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo YG,
Martinez D, Ngau WCA, Otillar B, Poliakov A, Porter A, Szajkowski L,
Werner G, Zhou KM, Grigoriev IV, Rokhsar DS, Grossman AR,
Chlamydomonas Annotation JGIAT (2007) The Chlamydomonas genome
reveals the evolution of key animal and plant functions. Science 318: 245-251
Niyogi KK (1999) Photoprotection revisited: Genetic and molecular approaches.
Annual Review of Plant Physiology and Plant Molecular Biology 50: 333-359
Niyogi KK (2009) Chapter 23. Photoprotection and high light responses. In: Stern DB
ed. The Chlamydomonas Sourcebook, 2nd Eedition, Volume 2 Organeller and
Metabolic Processes. Elsevier, San Diego, USA. pp.847-870
Novoselov SV, Rao M, Onoshko NV, Zhi HJ, Kryukov GV, Xiang YB, Weeks DP,
Hatfield DL, Gladyshev VN (2002) Selenoproteins and selenocysteine
insertion system in the model plant cell system, Chlamydomonas reinhardtii.
EMBO Journal 21: 3681-3693
Oelmuller R, Levitan I, Bergfeld R, Rajasekhar VK, Mohr H (1986) Expression
of nuclear gene as affected by treatments acting on the plastids. Planta 168:
482-492
Oh JE, Hong SW, Lee Y, Koh EJ, Kim K, Seo YW, Chung N, Jeong M, Jang CS,
Lee B, Kim KH, Lee H (2005) Modulation of gene expressions and enzyme
activities of methionine sulfoxide reductases by cold, ABA or high salt
treatments in Arabidopsis. Plant Science 169: 1030-1036
Olry A, Boschi-Muller S, Marraud M, Sanglier-Cianferani S, Van Dorsselear A,
Branlant G (2002) Characterization of the methionine sulfoxide reductase
activities of PILB, a probable virulence factor from Neisseria meningitidis.
Journal of Biological Chemistry 277: 12016-12022
Op den Camp RGL, Przybyla D, Ochsenbein C, Laloi C, Kim CH, Danon A,
Wagner D, Hideg E, Gobel C, Feussner I, Nater M, Apel K (2003) Rapid
induction of distinct stress responses after the release of singlet oxygen in
Arabidopsis. Plant Cell 15: 2320-2332
Pfannschmidt T (2003) Chloroplast redox signals: how photosynthesis controls its
own genes. Trends in Plant Science 8: 33-41
Rodermel S (2001) Pathways of plastid-to-nucleus signaling. Trends in Plant Science
6: 471-478
Romero HM, Berlett BS, Jensen PJ, Pell EJ, Tien M (2004) Investigations into the
49
role of the plastidial peptide methionine sulfoxide reductase in response to
oxidative stress in Arabidopsis. Plant Physiology 136: 3784-3794
Rouhier N, Dos Santos CV, Tarrago L, Rey P (2006) Plant methionine sulfoxide
reductase A and B multigenic families. Photosynthesis Research 89: 247-262
Rouhier N, Kauffmann B, Tete-Favier F, Palladino P, Gans P, Branlant G,
Jacquot JP, Boschi-Muller S (2007) Functional and structural aspects of
poplar cytosolic and plastidial type A methionine sulfoxide reductases. Journal
of Biological Chemistry 282: 3367-3378
Sadanandom A, Poghosyan Z, Fairbairn DJ, Murphy DJ (2000) Differential
regulation of plastidial and cytosolic isoforms of peptide methionine sulfoxide
reductase in Arabidopsis. Plant Physiology 123: 255-263
Sanchez J, Nikolau BJ, Stumpf PK (1983) Reduction of N-acetyl methionine
sulfoxide in plants. Plant Physiology 73: 619-623
Shao N, Krieger-Liszkay A, Schroda M, Beck CF (2007) A reporter system for the
individual detection of hydrogen peroxide and singlet oxygen: its use for the
assay of reactive oxygen species produced in vivo. Plant Journal 50: 475-487
Shiu CT, Lee TM (2005) Ultraviolet-B-induced oxidative stress and responses of the
ascorbate-glutathione cycle in a marine macroalga Ulva fasciata. Journal of
Experimental Botany 56: 2851-2865
Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamysomonas
reinhardtii. Proceedings of the National Academy of Sciences of the United
States of America 46: 83-91
Tarrago L, Laugier E, Rey P (2009) Protein-repairing methionine sulfoxide
reductases in photosynthetic organisms: Gene organization, reduction
mechanisms, and physiological roles. Molecular Plant 2: 202-217
Trebitsh T, Levitan A, Sofer A, Danon A (2000) Translation of chloroplast psbA
mRNA is modulated in the light by counteracting oxidizing and reducing
activities. Molecular and Cellular Biology 20: 1116-1123
Vieira Dos Santos C, Cuine S, Rouhier N, Rey P (2005) The Arabidopsis plastidic
methionine sulfoxide reductase B proteins. Sequence and activity
characteristics, comparison of the expression with plastidic methionine
sulfoxide reductase A, and induction by photooxidative stress. Plant
Physiology 138: 909-922
Vieira Dos Santos C, Rey P (2006) Plant thioredoxins are key actors in the oxidative
50
stress response. Trends in Plant Science 11: 329-334
Wagner D, Przybyla D, Op den Camp R, Kim C, Landgraf F, Lee KP, Wursch
M, Laloi C, Nater M, Hideg E, Apel K (2004) The genetic basis of singlet
oxygen-induced stress responses of Arabidopsis thaliana. Science 306:
1183-1185