Skip to main content
SpringerLink
Log in

RNase activity requires formation of disulfide bonds and is regulated by the redox state

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitroby changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as β-ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivoin wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Abel, S. and Glund, K. 1986. Localization of RNA-degrading enzyme activity within vacuoles of cultured tomato cells. Physiol. Plant 66: 79–86.

    Google Scholar 

  • Abel, S. and Glund, K. 1987. Ribonuclease in plant vacuoles: Purification and molecular properties of the enzyme from cultured tomato cells. Planta 172: 71–78.

    Google Scholar 

  • Aoyagi, S., Sugiyama, M. and Fukuda, H. 1998. BEN1 and ZEN1 cDNAs encoding S1-type DNases that are associated with programmed cell death in plants. FEBS Lett. 429: 134–138.

    Google Scholar 

  • Bariola, P.B. and Green, P.J. 1997. Plant Ribonucleases. In: J.F. Riordan, and D'Alessio, G. (Eds), Ribonucleases: Structure and Function, Academic Press, New York pp. 163–190.

    Google Scholar 

  • Bariola, P.A., Howard, C.J., Taylor, C.B., Verburg, M.T., Jaglan, V.D. and Green, P.J. 1994. The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J. 6: 673–685.

    Google Scholar 

  • Baumgartner, B. and Matile, P. 1976. Immunochemical localization of acid ribonuclease in morning glory flower tissue. Biochem. Physiol. Pflanz 170: 279.

    Google Scholar 

  • Blank, A., Sugiyama, H. and Dekker, C.A. 1982. Activity staining of nucleolytic enzymes after sodium dodecyl sulfatepolyacryamide gel electrophoresis: use of aqueous isopropanol to remove detergent from gels. Anal. Biochem. 120: 267–275.

    Google Scholar 

  • Blank, A. and McKeon, T.A. 1989. Single-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated. Proc. Natl. Acad. Sci. USA 86: 3169–3173.

    Google Scholar 

  • Blank, A. and McKeon, T.A. 1991a. Three RNases in senescent and nonsenescent wheat leaves. Plant Physiol. 97: 1402–1408.

    Google Scholar 

  • Blank, A. and McKeon, T.A. 1991b. Expression of three RNase activities during natural and dark-induced senescence of wheat leaves. Plant Physiol. 97: 1409–1413.

    Google Scholar 

  • Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Google Scholar 

  • Brown, P.H. and Ho, T.-H.D. 1986. Barley aleurone layers secrete a nuclease in response to gibberellic acid. Plant Physiol. 82: 801–806.

    Google Scholar 

  • Chang, S.-C. and Gallie, D.R. 1997. RNase activity decreases following a heat shock in wheat leaves and correlates with its posttranscriptional modification. Plant Physiol. 113: 1253–1263.

    Google Scholar 

  • Chen, Z., Young, T.E., Ling, J., Chang, S.C. and Gallie, D.R. 2003. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc. Natl. Acad. Sci. USA 100: 3525–3530.

    Google Scholar 

  • Crafts-Brandner, S.J., Holzer, R. and Feller, U. 1998. Influence of nitrogen deficiency on senescence and the amounts of RNA and proteins in wheat leaves. Physiol. Plant. 102: 192–200.

    Google Scholar 

  • Cuozzo, J.W. and Kaiser, C.A. 1999. Competition between glutathione and protein thiols for disulphide-bond formation. Nat. Cell. Biol. 1: 130–135.

    Google Scholar 

  • Dodds, P.N., Clarke, A.E. and Newbegin, E. 1996. Molecular characterisation of an S-like RNase of Nicotiana alata that is induced by phosphate starvation. Plant Mol. Biol. 31: 227–238.

    Google Scholar 

  • Farkas, G.L. 1982. Ribonucleases and ribonucleic acid breakdown. In: B. Parthier and D. Boulter, (Eds.), Encyclopedia of Plant Physiology, New Series, Berlin: Springer-Verlag, 14B: pp. 224–262.

    Google Scholar 

  • Fath, A., Bethke, P.C. and Jones, R.L. 1999. Barley aleurone cell death is not apoptotic: Characterization of nuclease activities and DNA degradation. Plant J. 20: 305–315.

    Google Scholar 

  • Foyer, C., Rowell, J. and Walker, D. 1983. Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157: 239–244.

    Google Scholar 

  • Gallie, D.R. and Chang, S.-C. 1999. RNase activity is posttranscriptionally controlled during the darkinduced senescence program. In: A.K., Chang, C., Klee, H., Bleecker, A.B., Pech, J.C. and Grierson, D. (Eds.), Biology and Biotechnology of the Plant Hormone Ethylene II. Kanellis, Kluwer Academic Publishers. Dordrecht, The Netherlands, pp. 221–226.

    Google Scholar 

  • Gallie, D.R., Chang, S-C. and Young, T.E. 2002. Induction of RNase and nuclease activity in cultured maize endosperm cells following sucrose starvation. Plant Cell. Tissue. Organ Cult. 68: 163–170.

    Google Scholar 

  • Green, P.J. 1994. The ribonucleases of higher plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 45: 421–445

    Google Scholar 

  • Griffith, O.W. 1980. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106: 207–212.

    Google Scholar 

  • Groover, A. and Jones, A.M. 1999. Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol. 119: 375–384.

    Google Scholar 

  • Hartley, R.W. 1997. Barnase and barstar. In: D'Alessio, G. and Riordan, Ribonucleases; Structure and Function, Academic Press, New York, pp. 51–100.

    Google Scholar 

  • Ito, J. and Fukuda, H. 2002. ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements. Plant Cell 14: 3201–3211.

    Google Scholar 

  • Jost, W., Bak, H., Glund, K., Terpstra, P. and Beintema, J.J. 1991. Amino acid sequence of an extracellular, phosphatestarvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur. J. Biochem. 198: 1–6.

    Google Scholar 

  • Karpinski, S., Escobar, C., Karpinska, B., Creissen, G. and Mullineaux, P.M. 1997. Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress. Plant Cell 9: 627–640.

    Google Scholar 

  • Klink, T.A., Woycechowsky, K.J., Taylor, K.M. and Raines, R.T. 2000. Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A. Eur. J. Biochem. 267: 566–572.

    Google Scholar 

  • Kock, M., Loffler, A., Abel, S. and Glund, K. 1995. cDNA structure and regulatory properties of a family of starvationinduced ribonucleases from tomato. Plant Mol. Biol. 27: 477–485.

    Google Scholar 

  • Kocsy, G., von Ballmoos, P., Suter, M., Ruegsegger, A., Galli, U., Szalai, G., Galiba, G. and Brunold, C. 2000. Inhibition of glutathione synthesis reduces chilling tolerance in maize. Planta 211: 528–536.

    Google Scholar 

  • Kuriyama, H. 1999. Loss of tonoplast integrity programmed in tracheary element differentiation. Plant Physiol. 121: 763–774.

    Google Scholar 

  • Lers, A., Khalchitski, A., Lomaniec, E., Burd, S. and Green, P.J. 1998. Senescence-induced RNases in tomato. Plant Mol. Biol. 36: 439–449.

    Google Scholar 

  • Locker, J.K. and Griffiths, G. 1999. An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins. J. Cell. Biol. 144: 267–279.

    Google Scholar 

  • Loffler, A., Abel, S., Jost, W., Beintema, J.J. and Glund, K. 1992. Phosphate-regulated induction of intracellular ribonucleases in cultured tomato (Lycopersicon esculentum) cells. Plant Physiol. 98: 1472–1478.

    Google Scholar 

  • Loffler, A., Glund, K. and Irie, M. 1993. Amino acid sequence of an intracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur. J. Biochem. 214: 627–633.

    Google Scholar 

  • Lohman, K.N., Gan, S., John, M.C. and Amasino, R.M. 1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol. Plant. 92: 322–328.

    Google Scholar 

  • Mayr, L.M., Willbold, D., Landt, O. and Schmid, F.X. 1994. Role of the Cys 2-Cys 10 disulfide bond for the structure, 95 stability, and folding kinetics of ribonuclease T1. Protein Sci. 3: 227–329.

    Google Scholar 

  • McHale, J.S. and Dove, L.D. 1968. Ribonuclease activity in tomato leaves as related to development and senescence. New Phytol. 67: 505–515.

    Google Scholar 

  • Muramoto, Y., Watanabe, A., Nakamura, T. and Takabe, T. 1999. Enhanced expression of a nuclease gene in leaves of barley plants under salt stress. Gene 234: 315–321.

    Google Scholar 

  • Nakagawa, A., Tanaka, I., Sakai, R., Nakashima, T., Funatsu, G. and Kimura, M. 1999. Crystal structure of a ribonuclease from the seeds of bitter gourd (Momordica charantia) at 1.75 A resolution. Biochim. Biophys. Acta. 1433: 253–260.

    Google Scholar 

  • Nurnberger, T., Abel, S., Jost, W. and Glund, K. 1990. Induction of an extracellular ribonuclease in cultured tomato cells upon phosphate starvation. Plant Physiol. 92: 970–976.

    Google Scholar 

  • Obara, K., Kuriyama, H. and Fukuda, H. 2001. Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia. Plant Physiol. 125: 615–626.

    Google Scholar 

  • Oxley, D. and Bacic, A. 1996. Disulphide bonding in a stylar self-incompatibility ribonuclease of Nicotiana alata. Eur. J. Biochem. 242: 75–80.

    Google Scholar 

  • Pace, C.N., Grimsley, G.R., Thomson, J.A. and Barnett, B.J. 1988. Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. J. Biol. Chem. 263: 11820–11825.

    Google Scholar 

  • Panavas, T., LeVangie, R., Mistler, J., Reid, P.D. and Rubinstein, B. 1999. Identification of senescence associated genes from daylily petals. Plant Mol. Biol. 40: 237–248.

    Google Scholar 

  • Perez-Amador, M.A., Abler, M.L., de Rocher, E.J., Thompson, D.M., van Hoof, A., LeBrasseur, N.D., Lers, A. and Green, P.J. 2000. Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis. Plant Physiol. 122: 169–179.

    Google Scholar 

  • Ruoppolo, M., Vinci, F., Klink, T.A., Raines, R.T. and Marino, G. 2000. Contribution of individual disulfide bonds to the oxidative folding of ribonuclease. Anal. Biochem. 39: 12033–12042.

    Google Scholar 

  • Smirnoff, N. 2000. Ascorbate biosynthesis and function in photoprotection. Phil. Trans. R. Soc. Lond. B Biol. Sci. 355: 1455–1464.

    Google Scholar 

  • Sugiyama, M., Ito, J., Aoyagi, S. and Fukuda, H. 2000. Endonucleases. Plant Mol. Biol. 44: 387–397.

    Google Scholar 

  • Tanaka, N., Arai, J., Inokuchi, N., Koyama, T., Ohgi, K., Irie, M. and Nakamura, K.T. 2000. Crystal structure of a plant ribonuclease, RNase LE. J. Mol. Biol. 298: 859–873.

    Google Scholar 

  • Taylor, C.B., Bariola, P.A., del Cardayre, S.B., Raines, R.T. and Green, P.J. 1993. RNS2: A senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc. Natl. Acad. Sci. USA 90: 5118–5122.

    Google Scholar 

  • Wedemeyer, W.J., Welker, E., Narayan, M. and Scheraga, H.A. 2000. Disulfide bonds and protein folding. Biochemistry 39: 4207–4216.

    Google Scholar 

  • Woycechowsky, K.J. and Raines, R.T. 2000. Native disulfide bond formation in proteins. Curr. Op. Chem. Biol. 4: 533–539.

    Google Scholar 

  • Young, T.E., Gallie, D.R. and DeMason, D.A. 1997. Ethylene mediated programmed cell death during maize endosperm development of Su and sh2 genotypes. Plant Physiol. 115: 737–751.

    Google Scholar 

  • Young, T.E. and Gallie, D.R. 1999. Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals. Plant Mol. Biol. 39: 915–926.

    Google Scholar 

  • Young, T.E. and Gallie, D.R. 2000. Programmed cell death during endosperm development. Plant Mol. Biol. 44: 283–301

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of California, CA, USA

    Zhong Chen, Jun Ling & Daniel Gallie

Authors
  1. Zhong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun Ling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Gallie
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, Z., Ling, J. & Gallie, D. RNase activity requires formation of disulfide bonds and is regulated by the redox state. Plant Mol Biol 55, 83–96 (2004). https://doi.org/10.1007/s11103-004-0438-1

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-004-0438-1

Search

Navigation