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Dissecting the individual contribution of conserved cysteines to the redox regulation of RubisCO

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Abstract

Oxidation of the cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) leads to inactivation and promotes structural changes that increase the proteolytic sensitivity and membrane association propensity related to its catabolism. To uncover the individual role of the different cysteines, the sequential order of modification under increasing oxidative conditions was determined using chemical labeling and mass spectrometry. Besides, site-directed RubisCO mutants were obtained in Chlamydomonas reinhardtii replacing single conserved cysteines (Cys84, Cys172, Cys192, Cys247, Cys284, Cys427, Cys459 from the large and sCys41, sCys83 from the small subunit) and the redox properties of the mutant enzymes were determined. All mutants retained significant carboxylase activity and grew photoautotrophically, indicating that these conserved cysteines are not essential for catalysis. Cys84 played a noticeable structural role, its replacement producing a structurally altered enzyme. While Cys247, Cys284, and sCys83 were not affected by the redox environment, all other residues were oxidized using a disulfide/thiol ratio of around two, except for Cys172 whose oxidation was distinctly delayed. Remarkably, Cys192 and Cys427 were apparently protective, their absence leading to a premature oxidation of critical residues (Cys172 and Cys459). These cysteines integrate a regulatory network that modulates RubisCO activity and conformation in response to oxidative conditions.

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Abbreviations

AC:

Acrylamide

CSH:

2-Mercaptoethylamine (cysteamine)

CSSC:

2-Mercaptoethylamine disulfide (cystamine)

DTT:

Dithiothreitol

GuCl:

Guanidinium chloride

IAM:

Iodoacetamide

RubisCO:

Ribulose 1,5-bisphosphate carboxylase/oxygenase

SEM:

Standard error of the mean

VP:

4-Vinylpyridine

References

  • Abat JK, Deswal R (2009) Differential modulation of S-nitrosoproteome of Brassica juncea by low temperature: change in S-nitrosylation of Rubisco is responsible for the inactivation of its carboxylase activity. Proteomics 9:4368–4380

    Article  PubMed  CAS  Google Scholar 

  • Albuquerque JA, Esquível MG, Teixeira AR, Ferreira RB (2001) The catabolism of ribulose bisphosphate carboxylase from higher plants. A hypothesis. Plant Sci 161:55–65

    Article  CAS  Google Scholar 

  • Avila-Ospina L, Moison M, Yoshimoto K, Masclaux-Daubresse C (2014) Autophagy, plant senescence, and nutrient recycling. J Exp Bot 65:3799–3811

    Article  PubMed  Google Scholar 

  • Borkhsenious ON, Mason CB, Moroney JV (1998) The intracellular localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. Plant Physiol 116:1585–1591

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538

    Article  PubMed  CAS  Google Scholar 

  • Bracher A, Whitney SM, Hartl UF, Hayer-Hartl M (2017) Biogenesis and metabolic maintenance of Rubisco. Annu Rev Plant Biol 68:29–60

    Article  PubMed  CAS  Google Scholar 

  • Carrión CA, Martínez DE, Costa ML, Guiamet JJ (2014) Senescence-associated vacuoles, a lytic compartment for degradation of chloroplast proteins? Plants 3:498–512

    Article  PubMed  PubMed Central  Google Scholar 

  • Cavanagh AP, Kubien DS (2014) Can phenotypic plasticity in rubisco performance contribute to photosynthetic acclimation? Photosynth Res 119:203–214

    Article  PubMed  CAS  Google Scholar 

  • Chaurasia SP, Deswal R (2017) Identification and in silico analysis of major redox modulated proteins from Brassica juncea seedlings using 2D redox SDS PAGE (2-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis). Protien J 36:64–76

    Article  CAS  Google Scholar 

  • Chiba A, Ishida H, Nishizawa NK, Makino A, Mae T (2003) Exclusion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing leaves of wheat. Plant Cell Physiol 44:914–921

    Article  PubMed  CAS  Google Scholar 

  • Dean C, Pichersky E, Dunsmuir P (1989) Structure, evolution, and regulation of rbcS genes in higher-plants. Annu Rev Plant Physiol Plant Mol Biol 40:415–439

    Article  CAS  Google Scholar 

  • Eckardt NA, Pell EJ (1995) Oxidative modification of Rubisco from potato foliage in response to ozone. Plant Physiol Biochem 33:273–282

    CAS  Google Scholar 

  • Erb TJ, Zarzycki J (2016) Biochemical and synthetic biology approaches to improve photosynthetic CO2-fixation. Curr Opin Chem Biol 34:72–79

    Article  PubMed  CAS  Google Scholar 

  • Esquivel MG, Pinto TS, Marín-Navarro J, Moreno J (2006) Substitution of tyrosine residues at the aromatic cluster around the βA-βB loop of Rubisco small subunit affects the structural stability of the enzyme and the in vivo degradation under stress conditions. Biochemistry 45:5745–5753

    Article  PubMed  CAS  Google Scholar 

  • Evans JR, Seeman JR (1989) The allocation of protein nitrogen in the photosynthetic apparatus: costs, consequences and control. In: Briggs WR (ed) Photosynthesis. Alan R. Liss, New York, pp 183–205

    Google Scholar 

  • Feller U, Anders I, Mae T (2008) Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated. J Exp Bot 59:1615–1624

    Article  PubMed  CAS  Google Scholar 

  • Ferreira RB, Davies DD (1989) Conversion of ribulose-1,5-bisphosphate carboxylase to an acidic and catalytically inactive form by extracts of osmotically stressed Lemna minor fronds. Planta 179:448–455

    Article  PubMed  CAS  Google Scholar 

  • Ferreira RB, Shaw NM (1989) Effect of osmotic stress on protein turnover in Lemna minor fronds. Planta 179:456–465

    Article  PubMed  CAS  Google Scholar 

  • Ferreira RB, Esquivel MG, Teixeira AR (2000) Catabolism of ribulose bisphosphate carboxylase from higher plants. Curr Top Phytochem 3:129–165

    CAS  Google Scholar 

  • Galmés J, Aranjuelo I, Medrano H, Flexas J (2013) Variations in rubisco content and activity under variable climatic factors. Photosynth Res 117:73–90

    Article  PubMed  CAS  Google Scholar 

  • García-Ferris C, Moreno J (1993) Redox regulation of enzymatic activity and proteolytic and susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis. Photosynth Res 35:55–66

    Article  PubMed  Google Scholar 

  • García-Ferris C, Moreno J (1994) Oxidative modification and breakdown of ribulose 1, 5-bisphosphate carboxylase/oxygenase induced in Euglena gracilis by nitrogen starvation. Planta 193:208–215

    Article  Google Scholar 

  • García-Murria MJ, Karkehabadi S, Marín-Navarro J, Satagopan S, Andersson I, Spreitzer RJ, Moreno J (2008) Structural and functional consequences of the substitution of vicinal residues Cys-172 and Cys-192 in the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii. Biochem J 411:241–247

    Article  PubMed  CAS  Google Scholar 

  • Gutteridge S, Gatenby AA (1995) Rubisco synthesis, assembly, mechanism, and regulation. Plant Cell 7:809–819

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Harris EH (2009) The Chlamydomonas sourcebook: an introduction to Chlamydomonas and its laboratory use, 2nd edn. Academic Press, San Diego

    Google Scholar 

  • Ishida H, Shimizu S, Makino A, Mae T (1998) Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves. Planta 204:305–309

    Article  PubMed  CAS  Google Scholar 

  • Ishida H, Yoshimoto K, Reisen D, Makino A, Ohsumi Y, Hanson M, Mae T (2007) Visualization of Rubisco-containing bodies derived from chloroplasts in living cells of Arabidopsis. Photosynth Res 91:275–276

    Google Scholar 

  • Ishida H, Izumi M, Wada S, Makino A (2014) Roles of autophagy in chloroplast recycling. Biochim Biophys Acta 1837:512–521

    Article  PubMed  CAS  Google Scholar 

  • Junqua M, Biolley JP, Pie S, Kanoun M, Duran R, Goulas P (2000) In vivo ocurrence of carbonyl residues in Phaseolus vulgaris proteins as a direct consequence of a chronic ozone stress. Plant Physiol Biochem 38:853–861

    Article  CAS  Google Scholar 

  • Khanna-Chopra R (2012) Leaf senescence and abiotic stress share reactive oxygen species-mediated chloroplast degradation. Protoplasma 249:469–481

    Article  PubMed  CAS  Google Scholar 

  • Marcus Y, Altman-Gueta H, Finkler A, Gurevitz M (2003) Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation. J Bacteriol 185:1509–1517

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Marín-Navarro J, Moreno J (2003) Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase. Biochemistry 42:14930–14938

    Article  PubMed  CAS  Google Scholar 

  • Marín-Navarro J, Moreno J (2006) Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1,5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress. Plant Cell Environ 29:898–908

    Article  PubMed  CAS  Google Scholar 

  • Mehta RA, Fawcett TW, Porath D, Mattoo AK (1992) Oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase. J Biol Chem 267:2810–2816

    PubMed  CAS  Google Scholar 

  • Michaeli S, Galili G (2014) Degradation of organelles or specific organelle components via selective autophagy in plant cells. Int J Mol Sci 15:7624–7638

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mirzahosseini A, Noszál B (2016) Species-specific standard redox potentials of thiol-disulfide systems: a key parameter to develop agents against oxidative stress. Sci Rep 6:37596

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Moreno J, Spreitzer RJ (1999) C172S substitution in the chloroplast-encoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase⁄oxygenase. J Biol Chem 274:26789–26793

    Article  PubMed  CAS  Google Scholar 

  • Moreno J, García-Murria MJ, Marín-Navarro J (2008) Redox modulation of Rubisco conformation and activity through its cysteine residues. J Exp Bot 59:1605–1614

    Article  PubMed  CAS  Google Scholar 

  • Muthuramalingam M, Matros A, Scheibe R, Mock HP, Dietz KJ (2013) The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo. Front Plant Sci 4(54):1–14

    Google Scholar 

  • Nakano R, Ishida H, Makino A, Mae T (2006) In vivo fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species in an intact leaf of cucumber under chilling-light conditions. Plant Cell Physiol 47:270–276

    Article  PubMed  CAS  Google Scholar 

  • Orr DJ, Alcântara A, Kapralov MV, Andralojc PJ, Carmo-Silva E, Parry MAJ (2016) Surveying Rubisco diversity and temperature response to improve crop photosynthetic efficiency. Plant Physiol 172:707–717

    PubMed  PubMed Central  CAS  Google Scholar 

  • Otegui MS, Noh YS, Martínez DE, Vila Petroff MG, Staehelin MA, Amasino RM, Guiamet JJ (2005) Senescence associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean. Plant J 41:831–844

    Article  PubMed  CAS  Google Scholar 

  • Pickersgill RW (1986) An upper limit to the active site concentration of ribulose bisphosphate carboxylase in chloroplasts. Biochem J 236:311

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Prins A, van Heerden PDR, Olmos E, Kunert KJ, Foyer CH (2008) Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies. J Exp Bot 59:1935–1950

    Article  PubMed  CAS  Google Scholar 

  • Raines CA (2003) The Calvin cycle revisited. Photosynth Res 75:1–10

    Article  PubMed  CAS  Google Scholar 

  • Ranty B, Lorimer G, Gutteridge S (1991) An intra-dimeric crosslink of large subunits of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase is formed by oxidation of cysteine 247. Eur J Biochem 200:353–358

    Article  PubMed  CAS  Google Scholar 

  • Roos G, Foloppe N, Messens J (2013) Understanding the pKa of redox cysteines: the key role of hydrogen bonding. Antioxid Redox Signal 18:94–127

    Article  PubMed  CAS  Google Scholar 

  • Roulin S, Feller U (1998) Dithiothreitol triggers photooxidative stress and fragmentation of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in intact pea chloroplasts. Plant Physiol Biochem 36:849–856

    Article  CAS  Google Scholar 

  • Schloss JV, Stringer CD, Hartman FC (1978) Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate. J Biol Chem 253:5707–5711

    PubMed  CAS  Google Scholar 

  • Sedigheh HG, Mortazavian M, Norouzian D, Atyabi M, Akbarzadeh A, Hasanpoor K, Ghorbani M (2011) Oxidative stress and leaf senescence. BMC Res Notes 4:477

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Shimogawara K, Fujiwara S, Grossman A, Usuda H (1998) High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148:1821–1828

    PubMed  PubMed Central  CAS  Google Scholar 

  • Spreitzer RJ. Mets L (1981) Photosynthesis-deficient mutants of Chlamydomonas reinhardtii with associated light-sensitive phenotypes. Plant Physiol 67:565–569

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sudhani HPK, García-Murria MJ, Moreno J (2013) Reversible inhibition of CO2 fixation by ribulose 1, 5- bisphosphate carboxylase/oxygenase through the synergic effect of arsenite and a monothiol. Plant Cell Environ 36:1160–1170

    Article  PubMed  CAS  Google Scholar 

  • Sudhani HPK, García-Murria MJ, Marín-Navarro J, García-Ferris C, Peñarrubia L, Moreno J (2015a) Purification of Rubisco from Chlamydomonas reinhardtii. Bio-protocol 5(23):e-1673, 1–9

    Google Scholar 

  • Sudhani HPK, García-Murria MJ, Marín-Navarro J, García-Ferris C, Peñarrubia L, Moreno J (2015b) Assay of the carboxylase activity of Rubisco from Chlamydomonas reinhardtii. Bio-protocol 5(23):e-1672, 1–8

    Google Scholar 

  • Taylor TC, Backlund A, Bjorhall K, Spreitzer RJ, Andersson I (2001) First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J Biol Chem 276:48159–48164

    Article  PubMed  CAS  Google Scholar 

  • Torchinsky YM (1981) Sulfur in proteins. Pergamon Press, Oxford

    Google Scholar 

  • Wang S, Blumenwald E (2014) Stress-induced chloroplast degradation in Arabidopsis is regulated via a process independent of autophagy and senescence-associated vesicles. Plant Cell 26:4875–4888

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Xie Q, Michaeli S, Peled-Zehavi H, Galili G (2015) Chloroplast degradation: one organelle, multiple degradation pathways. Trends Plant Sci 20:264–265

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Robert J. Spreitzer (University of Nebraska at Lincoln, USA) for advice and material support to obtain the RubisCO cysteine-to-serine mutants, and Luz Valero (SCSIE, University of Valencia) for processing the samples for mass spectrometry. This work was supported by grants of the Spanish Ministry of Science and Research (BFU2009-11965 from MCeI-DGI) and the University of Valencia (UV-INV-AE14-269247). H.P.K.S. received a fellowship for doctoral studies from the Spanish Ministry of Foreign Affairs and Cooperation (MAEC-AECID).

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  1. Department of Biochemistry and Molecular Biology, University of Valencia, c/ Dr. Moliner 50, Burjassot, 46100, Valencia, Spain

    María Jesús García-Murria, Hemanth P. K. Sudhani, Julia Marín-Navarro, Manuel M. Sánchez del Pino & Joaquín Moreno

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  1. María Jesús García-Murria
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  2. Hemanth P. K. Sudhani
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  3. Julia Marín-Navarro
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Correspondence to Joaquín Moreno.

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García-Murria, M.J., Sudhani, H.P.K., Marín-Navarro, J. et al. Dissecting the individual contribution of conserved cysteines to the redox regulation of RubisCO. Photosynth Res 137, 251–262 (2018). https://doi.org/10.1007/s11120-018-0497-9

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