Abstract
Cordyceps militaris is a large fungus whose fruiting body is prone to degeneration. In the study of C. militaris FREQUENCY (CmFRQ), the negative regulator of C. militaris circadian clock, it was found that after the high concentration of mannitol was applied to the regeneration culture of conidial protoplast of degenerated C. militaris strain F3411D, the rejuvenated strain CmFRQ-454 with the increased content of CmFRQ was obtained, and its growth vitality of fruiting body was recovered. After six successive asexual subcultures of the rejuvenated C. militaris strain CmFRQ-454, the accumulation of CmFRQ in mycelia gradually decreased, and the fruiting bodies of C. militaris also tended to degenerate from generation to generation. After treatment with proteasome inhibitor MG132 or high concentration of sorbitol, the degenerated asexual reproduction strain CmFRQ-454 to the 7th or 11th generation regained its growth vitality, and the accumulation of CmFRQ in mycelium was increased. Our results demonstrate that after hypertonic solution or MG132 treatment, the key circadian regulator CmFRQ was accumulate in mycelium, which brought the rejuvenation to the degenerated C. militaris.
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Abbreviations
- DNA:
-
Deoxyribonucleic acid
- dsRNA:
-
Double-stranded ribonucleic acid
- ROS:
-
Reactive oxygen species
- FRQ:
-
Clock protein FREQUENCY
- CmFRQ:
-
Cordyceps militaris FREQUENCY
- FRH:
-
FRQ interacting RNA helicase
- MG132:
-
Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal
- WC-1:
-
WHITE COLLAR-1
- WC-2:
-
WHITE COLLAR-2
- WCC:
-
Dimer composed of WC-1 and WC-2
- PAS:
-
PER-ARNT-SIM domain
- ELISA:
-
Enzyme linked immunosorbent assay
- KLH:
-
Keyhole Limpet Hemocyanin
- BSA:
-
Bovine serum albumin
- Sulfo-SMCC:
-
Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate
- PDA:
-
Potato dextrose agar
- PDB:
-
Potato dextrose broth
References
Alexeyev MF, Ledoux SP, Wilson GL (2004) Mitochondrial DNA and aging. Clin Sci 107:355–364. https://doi.org/10.1042/CS20040148
Aronson BD, Johnson KA, Loros JJ, Dunlap JC (1994) Negative feedback defining a circadian clock: autoregulation of the clock gene frequency. Science 263:1578–1584. https://doi.org/10.1126/science.8128244
Baker CL, Loros JJ, Dunlap JC (2012) The circadian clock of Neurospora Crassa. FEMS Microbiol Rev 36(1):95–110. https://doi.org/10.1111/j.1574-6976.2011.00288.x
Barja G, Herrero A (2000) Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals. FASEB J 14:312–318. https://doi.org/10.1096/fasebj.14.2.312
Belden WJ, Loros JJ, Dunlap JC (2007) Execution of the circadian negative feedback loop in Neurospora requires the ATP-dependent chromatin-remodeling enzyme CLOCKSWITCH. Mol Cell 25:587–600. https://doi.org/10.1016/j.molcel.2007.01.010
Bell-Pedersen D (2000) Understanding circadian rhythmicity in Neurospora crassa: from behavior to genes and back again. Fungal Genet Biol 29(1):1–18. https://doi.org/10.1006/fgbi.2000.1185
Butt TM, Wang C, Shah FA, Hall R (2006) Degeneration of entomogenous fungi. In: Eilenberg J, Hokkanen HMT (eds) An ecological and societal approach to biological control, vol 10. Springer, New York, US Chap, pp 213–226
Cha J, Zhou M, Liu Y (2015) Mechanism of the Neurospora Circadian Clock, a FREQUENCY-centric view. Biochemistry 54:150–156. https://doi.org/10.1021/bi5005624
Chen CH, Dunlap JC, Loros JJ (2010) Neurospora illuminates fungal photoreception. Fungal Genet Biol 47(11):922–929. https://doi.org/10.1016/j.fgb.2010.07.005
Cheng P, Yang Y, Gardner KH, Liu Y (2002) PAS domain-mediated WC-1/WC-2 interaction is essential for maintaining the steady-state level of WC-1 and the function of both proteins in circadian clock and light responses of Neurospora. Mol Cell Biol 22:517–524. https://doi.org/10.1128/MCB.22.2.517-524.2002
Cheng P, Yang Y, Wang L, He Q, Liu Y (2003) WHITE COLLAR-1, a multifunctional neurospora protein involved in the circadian feedback loops, light sensing, and transcription repression of wc-2. J Biol Chem 278:3801–3808. https://doi.org/10.1074/jbc.M209592200
Cheng P, He Q, Wang L, Liu Y (2005) Regulation of the Neurospora circadian clock by an RNA helicase. Genes Dev 19:234–241. https://doi.org/10.1101/gad.1266805
Crosthwaite SK, Dunlap JC, Loros JJ (1997) Neurospora wc-1 and wc-2: Transcription, photoresponses, and the origins of circadian rhythmicity. Science 76:763–769. https://doi.org/10.1126/science.276.5313.763
Dufour E, Boulay J, Rincheval V, Sainsard-Chanet A (2000) A causal link between respiration and senescence in Podospora Anserina. Proc Natl Acad Sci USA 97(8):4138–4143. https://doi.org/10.1073/pnas.070501997
Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96(2):271–290. https://doi.org/10.1016/S0092-8674(00)80566-8
Dunlap JC, Loros JJ (2004) The Neurospora circadian system. J Biol Rhythms 19(5):414–424. https://doi.org/10.1177/0748730404269116
Dunlap JC, Borkovich KA, Henn MR, Turner GE, Sachs MS, Glass NL, McCluskey K, Plamann M, Galagan JE, Birren BW, Weiss RL, Townsend JP, Loros JJ, Nelson MA, Lambreghts R, Colot HV, Park G, Collopy P, Ringelberg C, Crew C, Litvinkova L, DeCaprio D, Hood HM, Curilla S, Shi M, Crawford M, Koerhsen M, Montgomery P, Larson L, Pearson M, Kasuga T, Tian C, Baştürkmen M, Altamirano L, Xu J (2007) Enabling a community to dissect an organism: overview of the Neurospora functional genomics project. Adv Genet 57:49–96. https://doi.org/10.1016/S0065-2660(06)57002-6
Feng L, Li XR, Fu MJ, Huang ZY, Wei WQ, Xiao SP (2021) Morphological observation of Cordyceps militaris treated with blue light and proteasome inhibitor MG132. Mycosystema 40(11):2953–2961. https://doi.org/10.13346/j.mycosystema.210160
Feng L, Li XR, Fu MJ, Huang ZY, Wei WQ, Xiao SP (2022) Effects of blue light and proteasome inhibitor MG132 treatments on the transcription of Cmfrq, Cmwc-1 and Cmwc-2 in Cordyceps Militaris. Mycosystema 41(4):587–600. https://doi.org/10.13346/j.mycosystema.210352
Froehlich AC, Loros JJ, Dunlap JC (2003) Rhythmic binding of a WHITE COLLAR-containing complex to the frequency promoter is inhibited by FREQUENCY. Proc Natl Acad Sci USA 100:5914–5919. https://doi.org/10.1073/pnas.1030057100
Görl M, Merrow M, Huttner B, Johnson J, Roenneberg T, Brunner M (2001) A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora Crassa. EMBO J 20:7074–7084. https://doi.org/10.1093/emboj/20.24.7074
Harman D (1973) Free radical theory of aging. Triangle 12:153–158
Harman D (1991) The aging process: major risk factor for disease and death. Proc Natl Acad Sci USA 88:5360–5363. https://doi.org/10.1073/pnas.88.12.5360
He Q, Liu Y (2005) Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin-proteasome pathway. Biochem Soc Trans 33:953–956. https://doi.org/10.1042/BST20050953
He Q, Cheng P, Yang Y, He Q, Yu H, Liu Y (2003) FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation. EMBO J 22:4421–4430. https://doi.org/10.1093/emboj/cdg425
He Q, Cheng P, He Q, Liu Y (2005) The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex. Genes Dev 19:1518–1531. https://doi.org/10.1101/gad.1322205
He Q, Cha J, Lee HC, Yang Y, Liu Y (2006) CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop. Genes Dev 20:2552–2565. https://doi.org/10.1101/gad.1463506
Heintzen C, Liu Y (2007) The Neurospora crassa circadian clock. Adv Genet 58:25–66. https://doi.org/10.1016/S0065-2660(06)58002-2
Liu Y, Loros J, Dunlap JC (2000) Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock. Proc Natl Acad Sci USA 97:234–239. https://doi.org/10.1073/pnas.97.1.234
Lou H, Lin J, Guo L, Wang X, Tian S, Liu C, Zhao Y, Zhao R (2019) Advances in research on cordyceps militaris degeneration. Appl Microbiol Biotechnol 103(19):7835–7841. https://doi.org/10.1007/s00253-019-10074-z
Lou H, Yu Y, Lin J, Guo L, Ye Z, Li Y, Wei T, Yun F (2020) Optimization of the preparation conditions of protoplasts from mononuclear blastospores of Cordyceps Militaris. J Chin Inst Food Sci Technol 20(6):129–136. https://doi.org/10.16429/j.1009-7848.2020.06.016
Pregueiro AM, Liu Q, Baker CL, Dunlap JC, Loros JJ (2006) The Neurospora checkpoint kinase 2: a regulatory link between the circadian and cell cycles. Science 313:644–649. https://doi.org/10.1126/science.1121716
Reppert SM, Weaver DR (2001) Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol 63:647–676. https://doi.org/10.1146/annurev.physiol.63.1.647
Richter C, Park JW, Ames BN (1988) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci USA 85:6465–6467. https://doi.org/10.1073/pnas.85.17.6465
Schafmeier T, Haase A, Káldi K, Scholz J, Fuchs M, Brunner M (2005) Transcriptional feedback of Neurospora circadian clock gene by phosphorylation-dependent inactivation of its transcription factor. Cell 122(2):235–246. https://doi.org/10.1016/j.cell.2005.05.032
Scheckhuber CQ, Mitterbauer R, Osiewacz HD (2009) Molecular basis of and interference into degenerative processes in fungi: potential relevance for improving biotechnological performance of microorganisms. Appl Microbiol Biotechnol 85:27–35. https://doi.org/10.1007/s00253-009-2205-3
Silar P, Lalucque H, Vierny C (2001) Cell degeneration in the model system Podospora anserina. Biogerontology. 2001; 2(1):1–17. https://doi.org/10.1023/a:1010000816277
Soerensen M, Gredilla R, Müller-Ohldach M, Werner A, Bohr VA, Osiewacz HD, Stevnsner T (2009) A potential impact of DNA repair on ageing and lifespan in the ageing model organism Podospora anserina: Decrease in mitochondrial DNA repair activity during ageing. Mech Ageing Dev. 2009; 130(8):487–496. https://doi.org/10.1016/j.mad.2009.05.003
Wang B, Kettenbach AN, Zhou X, Loros JJ, Dunlap JC (2019) The Phospho-Code determining Circadian Feedback Loop Closure and output in Neurospora. Mol Cell 74(4):771–784e3. https://doi.org/10.1016/j.molcel.2019.03.003
Yang Y, Cheng P, Zhi G, Liu Y (2001) Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY. J Biol Chem 276:41064–41072. https://doi.org/10.1074/jbc.M106905200
Yang Y, Cheng P, He Q, Wang L, Liu Y (2003) Phosphorylation of FREQUENCY protein by casein kinase II is necessary for the function of the Neurospora circadian clock. Mol Cell Biol 23:6221–6228. https://doi.org/10.1128/MCB.23.17.6221-6228.2003
Yin J, Xin X, Weng Y, Gui Z (2017) Transcriptome-wide analysis reveals the progress of Cordyceps militaris subculture degeneration. PLoS ONE 12(10):e0186279. https://doi.org/10.1371/journal.pone.0186279
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We are grateful for the financial support from the National Natural Science Foundation of China (Grant Number 31760601).
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Fu, MJ., Zhang, DD., Peng, JM. et al. The level of CmFRQ is associated with the degeneration and rejuvenation of Cordyceps militaris fruiting body. Biologia 79, 1513–1524 (2024). https://doi.org/10.1007/s11756-024-01623-7
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DOI: https://doi.org/10.1007/s11756-024-01623-7