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• W2103796739 abstract "The consequences of mitochondrial dysfunction are not limited to the development of oxidative stress or initiation of apoptosis but can result in the establishment of stress tolerance. Using maize mitochondrial mutants, we show that permanent mitochondrial deficiencies trigger novel Ca2+-independent signaling pathways, leading to constitutive expression of genes for molecular chaperones, heat shock proteins (HSPs) of different classes. The signaling to activate hsp genes appears to originate from a reduced mitochondrial transmembrane potential. Upon depolarization of mitochondrial membranes in transient assays, gene induction for mitochondrial HSPs occurred more rapidly than that for cytosolic HSPs. We also demonstrate that in the nematode Caenorhabditis elegans transcription of hsp genes can be induced by RNA interference of nuclear respiratory genes. In both organisms, activation of hsp genes in response to mitochondrial impairment is distinct from their responses to heat shock and is not associated with oxidative stress. Thus, mitochondria-to-nucleus signaling to express a hsp gene network is apparently a widespread retrograde mechanism to facilitate cell defense and survival. The consequences of mitochondrial dysfunction are not limited to the development of oxidative stress or initiation of apoptosis but can result in the establishment of stress tolerance. Using maize mitochondrial mutants, we show that permanent mitochondrial deficiencies trigger novel Ca2+-independent signaling pathways, leading to constitutive expression of genes for molecular chaperones, heat shock proteins (HSPs) of different classes. The signaling to activate hsp genes appears to originate from a reduced mitochondrial transmembrane potential. Upon depolarization of mitochondrial membranes in transient assays, gene induction for mitochondrial HSPs occurred more rapidly than that for cytosolic HSPs. We also demonstrate that in the nematode Caenorhabditis elegans transcription of hsp genes can be induced by RNA interference of nuclear respiratory genes. In both organisms, activation of hsp genes in response to mitochondrial impairment is distinct from their responses to heat shock and is not associated with oxidative stress. Thus, mitochondria-to-nucleus signaling to express a hsp gene network is apparently a widespread retrograde mechanism to facilitate cell defense and survival. In response to transient stress, impairment of mitochondrial function is a key event (1Jones A. Trends Plant Sci. 2000; 5: 225-230Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) that leads to overproduction of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; mtDNA, mitochondrial DNA; HSP, heat shock protein; sHSP, small HSP; NCS, non-chromosomal stripe; RNAi, RNA interference; AOX, alternative oxidase; ETC, electron transfer chain. 1The abbreviations used are: ROS, reactive oxygen species; mtDNA, mitochondrial DNA; HSP, heat shock protein; sHSP, small HSP; NCS, non-chromosomal stripe; RNAi, RNA interference; AOX, alternative oxidase; ETC, electron transfer chain. and, if oxidative damage is severe, to execution of programmed cell death. In contrast, mitochondrial mutants acclimated to permanent respiratory deficiencies do not develop oxidative stress and have enhanced tolerance to apoptotic stimuli and environmental stresses (2Dey R. Moraes C.T. J. Biol. Chem. 2000; 275: 7087-7094Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar, 4Dutilleul C. Garmier M. Noctor G. Mathieu C. Chetrit P. Foyer C.H. De Paepe R. Plant Cell. 2003; 15: 1212-1226Crossref PubMed Scopus (412) Google Scholar). It has been shown that osteosarcoma ρ0 cell lines devoid of mitochondrial DNA (mtDNA) are more resistant to staurosporine-induced apoptosis than are the parental ρ+ lines (2Dey R. Moraes C.T. J. Biol. Chem. 2000; 275: 7087-7094Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). We found that maize mutants with mtDNA deletions did not undergo oxidative stress despite severe respiratory deficiencies (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar). Moreover, similar mitochondrial mutants in tobacco appeared to develop higher tolerance to biotic (viral infection) and abiotic (ozone) stresses (4Dutilleul C. Garmier M. Noctor G. Mathieu C. Chetrit P. Foyer C.H. De Paepe R. Plant Cell. 2003; 15: 1212-1226Crossref PubMed Scopus (412) Google Scholar). We hypothesized that the enhanced stress resistance of mitochondrial mutants might be contributed by the up-regulation of hsp genes. It was previously demonstrated that cytosolic HSP70 can preserve mitochondrial capacity to maintain membrane potential under oxidative conditions (5Polla B.S. Kantengwa S. Francois D. Salvioli S. Franceschi C. Marsac C. Cossarizza A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6458-6463Crossref PubMed Scopus (393) Google Scholar). Cytosolic HSP70 can also prevent mitochondria from inducing both caspase-dependent and -independent pathways of programmed cell death (6Saleh A. Srinivasula S.M. Balkir L. Robbins P.D. Alnemri E.S. Nature Cell Biol. 2000; 2: 476-483Crossref PubMed Scopus (736) Google Scholar, 7Ravagnan L. Gurbuxani S. Susin S.A. Maisse C. Daugas E. Zamzami N. Mak T. Jaattela M. Penninger J.M. Garrido C. Kroemer G. Nature Cell Biol. 2001; 3: 839-843Crossref PubMed Scopus (749) Google Scholar). One of the α-crystallin type small HSPs (sHSPs) appears to block cytochrome c release from mitochondria (8Paul C. Manero F. Gonin S. Kretz-Remy C. Virot S. Arrigo A.P. Mol. Cell. Biol. 2002; 22: 816-834Crossref PubMed Scopus (373) Google Scholar). Thus, some HSPs exert anti-apoptotic effects in addition to their well documented functions (9Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1878) Google Scholar) in preventing aggregation of non-native proteins (sHSPs) and promoting ATP-dependent renaturation (HSP70). In plants, along with diverse sHSPs (10Waters E.R. Lee G.J. Vierling E. J. Exp. Bot. 1996; 47: 325-338Crossref Scopus (578) Google Scholar), another chaperone, ClpB/HSP101, is crucial for recovery after severe damage to cellular proteins (11Queitsch C. Hong S.-W. Vierling E. Lindquist S. Plant Cell. 2000; 12: 479-492Crossref PubMed Scopus (529) Google Scholar). Its yeast homolog, HSP104, resolves cytosolic aggregates of denatured proteins (12Glover J.R. Lindquist S. Cell. 1998; 94: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar) in a functional complex with SSA1 (HSP70) and Ydj1 (HSP40). To test whether permanent mitochondrial respiratory defects affect expression of hsp genes, we use two experimental models: (i) maize respiratory-deficient NCS (non-chromosomal stripe; reviewed in Ref. 13Newton K.J. Gabay-Laughnan S. Singh K.K. Mitochondrial DNA Mutations in Aging, Disease, and Cancer. Springer-Verlag, Berlin1998: 365-381Crossref Google Scholar) mutants with defined deletions in mtDNA (14Marienfeld J.R. Newton K.J. Genetics. 1994; 138: 855-863Crossref PubMed Google Scholar, 15Hunt M.D. Newton K.J. EMBO J. 1991; 10: 1045-1052Crossref PubMed Scopus (98) Google Scholar, 16Newton K.J. Mariano J.M. Gibson C.M. Kuzmin E.V. Gabay Laughnan S. Dev. Genet. 1996; 19: 277-286Crossref PubMed Scopus (30) Google Scholar, 17Lauer M.K.C. Newton K. Gabay-Laughnan S. Laughnan J. New Biol. 1990; 2: 107-113PubMed Google Scholar, 18Newton K.J. Knudsen C. Gabay-Laughnan S. Laughnan J.R. Plant Cell. 1990; 2: 107-113PubMed Google Scholar), and (ii) the nematode Caenorhabditis elegans with loss of function of different nuclear respiratory genes mediated by RNA interference. In both cases, we show that in response to mitochondrial impairment activation of a number of hsp genes occurs, including heat-inducible, constitutive, and cryptic ones. We present evidence that the primary signaling may originate from a reduction in mitochondrial transmembrane potential and does not involve either Ca2+- or ROS-dependent pathways. Altogether, these features characterize the mitochondria-dependent expression of hsp genes as a novel physiologically important process of retrograde regulation. Plant Material—Maize mutants carrying maternally inherited deletions in mtDNA were analyzed. In NCS2, the nad4 gene is partially deleted and mutant plants are deficient in the NADH ubiquinone oxidoreductase, the respiratory complex I (14Marienfeld J.R. Newton K.J. Genetics. 1994; 138: 855-863Crossref PubMed Google Scholar). The NCS4 deletion removes the 5′-end of the mitochondrial gene encoding the RPS3 ribosomal protein and is associated with reduced levels of mitochondrial translation (15Hunt M.D. Newton K.J. EMBO J. 1991; 10: 1045-1052Crossref PubMed Scopus (98) Google Scholar, 16Newton K.J. Mariano J.M. Gibson C.M. Kuzmin E.V. Gabay Laughnan S. Dev. Genet. 1996; 19: 277-286Crossref PubMed Scopus (30) Google Scholar). Deletion of the 5′-end of the cox2 gene in NCS6 mutants causes a deficiency in cytochrome c oxidase (17Lauer M.K.C. Newton K. Gabay-Laughnan S. Laughnan J. New Biol. 1990; 2: 107-113PubMed Google Scholar, 18Newton K.J. Knudsen C. Gabay-Laughnan S. Laughnan J.R. Plant Cell. 1990; 2: 107-113PubMed Google Scholar), the respiratory complex IV. Mutant plants are heteroplasmic for the mutations, so plants with the most defective phenotypes (with very high levels of mutant mtDNA) (13Newton K.J. Gabay-Laughnan S. Singh K.K. Mitochondrial DNA Mutations in Aging, Disease, and Cancer. Springer-Verlag, Berlin1998: 365-381Crossref Google Scholar) were used for analysis. Such highly mutant individuals are rare, and sufficient amounts of material for analysis could be collected from mature plants only. Control plants with normal phenotype were chosen within the same or closely related NCS families (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar). Heat shock experiments were performed with seedlings, a developmental stage at which the temperature of the whole maize plant can be reliably controlled. 4-day-old maize seedlings (genotypes A619 and B37N) were incubated at 42 °C as described previously (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar), and the roots were used for further analysis. Treatments with inhibitors were performed using 4-day-old normal maize seedlings (genotype B37N) or seedlings with T type cytoplasmic male sterility (B37T) according to Ref. 3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar. Methomyl and chloramphenicol were applied at final concentrations of 7.5 mm and 50 μg/ml, respectively. After 24-h incubations, all seedlings looked normal and retained undamaged mitochondria, because no loss of the outer mitochondrial membrane or mitochondrial matrix content was detected by Western analysis (data not shown). Identification of Maize hsp Coding Sequences—Protein sequences were used to query plant genomic and Expressed Sequence Tag data bases with the tBLASTn algorithm. Arabidopsis homologs of the yeast SSA1-type HSP70 were identified and then used as queries to search the maize EST data bases (Maize Genome Data base, www.zmdb.iastate.edu/cgi-bin/main/ZMDB, and the TIGR Eukaryotic Gene Ortholog (EGO) data base, www.tigr.org/tdb/tgi/ego/orth_search.shtml). The maize orthologs of Arabidopsis mitochondrial HSP70s (19Sung D.Y. Vierling E. Guy C.L. Plant Physiol. 2001; 126: 789-800Crossref PubMed Scopus (379) Google Scholar) were found by homology in the N-terminal protein sequences. The identification of heat-inducible and HS non-responsive maize orthologs of HSP70s was confirmed by gene-specific hybridization of maize total RNA from heat-shocked seedlings. In rice and maize, mitochondrial HSP22s were searched for in the TIGR EGO data base and in the grass genome data base GRAMENE (www.gramene.org/db/searches/blast). The potential mitochondrial targeting of the candidate precursors was evaluated using the Predotar program (www.inra.fr/predotar), and phylogenetic analysis of mitochondrial HSP22s was performed by CLUSTALW alignment (clustalw.genome.ad.jp) of the predicted full-length amino acid sequences. Northern Analysis of Maize Transcripts—Isolation of total RNAs from maize tissues and RNA blot hybridizations was performed as described previously (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar). Gene-specific maize riboprobes were prepared from RT-PCR-amplified total maize (B73N) RNA, using 3′- or 5′-untranslated region-specific primers (Supplemental Table I). To obtain the hsp22B riboprobe, PCR amplification from B73N genomic DNA was used. PCR fragments were cloned into the pGEMT-Easy vector (Promega) and verified by sequencing. To compare the methomyl and chloramphenicol effects, equal RNA amounts were loaded on the same gel, and for each hybridization probe the same x-ray film was used for quantification of hybridization signals (program Image Gauge 3.3; Fuji). Three independent experiments were performed. Protein Analysis—Total protein extracts from unpollinated ear shoots were prepared as described (20Nieto-Sotelo J. Martinez L.M. Ponce G. Cassab G.I. Alagon A. Meeley R.B. Ribaut J.M. Yang R. Plant Cell. 2002; 14: 1621-1633Crossref PubMed Scopus (120) Google Scholar). Cytosolic protein fractions were obtained after trichloroacetic acid precipitation of post-mitochondrial supernatants. Western analyses of total protein extracts (40 μg), cytosolic fractions (100 μg), and mitochondrial protein extracts (50 μg) were performed as described previously (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar). For immunoblot hybridizations, polyclonal antibodies to maize HSP101 (20Nieto-Sotelo J. Martinez L.M. Ponce G. Cassab G.I. Alagon A. Meeley R.B. Ribaut J.M. Yang R. Plant Cell. 2002; 14: 1621-1633Crossref PubMed Scopus (120) Google Scholar), provided by J. Nieto-Sotelo, National Autonomous University of Mexico, Cuernavaca, Mexico, Arabidopsis mitochondrial manganese superoxide dismutase and cytosolic glutathione S-transferase 1 (MnSOD and GST1) (21Kliebenstein D.J. Monde R.A. Last R.L. Plant Physiol. 1998; 118: 637-650Crossref PubMed Scopus (489) Google Scholar), provided by D. Kliebenstein, Cornell University, were used. Monoclonal antibodies to maize mitochondrial proteins, E1α subunit of pyruvate dehydrogenase, HSP70 (isotype 70A), CPN60 (isotype 60B), and HSP22 were supplied by T. E. Elthon (22Lund A.A. Blum P.H. Bhattramakki D. Elthon T.E. Plant Physiol. 1998; 116: 1097-1110Crossref PubMed Scopus (67) Google Scholar). RNAi Silencing in C. elegans—C. elegans (N2) RNA interference (RNAi) by feeding was performed basically as described by Kamath et al. (23Kamath R.S. Martinez-Campos M. Zipperlen P. Fraser A.G. Ahringer J. Genome Biol. 2001; http://genomebiology.com/2000/2/1/research/0002.1PubMed Google Scholar). Escherichia coli strain HT115(DE3) was transformed with L4440-based constructs containing PCR fragments of four genes derived from genomic DNA (primers listed in Supplemental Table II). Adult worms were placed on nematode growth medium plates with recombinant HT115(DE3) clones induced by 0.5 mm isopropyl-1-thio-β-d-galactopyranoside overnight. The worms were allowed to lay eggs and were removed after 12 h. Progeny were grown for 72 h at 23 °C and harvested for RNA extraction by the guanidinium thiocyanate/phenolchloroform method (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 161: 156-159Crossref Scopus (63169) Google Scholar). Developmental stages were scored 10 h before harvesting. Northern hybridizations were performed as described previously (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar). PCR fragments generated from N2 genomic DNA with primers listed in Supplemental Table II were used as hybridization probes. Constitutive Expression of hsp Genes in Maize Mitochondrial Mutants—Northern analysis showed that hsp101 and genes for cytosolic sHSPs, classes I and II (shsp-I and shsp-II), whose products are confirmed to have cytoprotective functions (11Queitsch C. Hong S.-W. Vierling E. Lindquist S. Plant Cell. 2000; 12: 479-492Crossref PubMed Scopus (529) Google Scholar, 25Low D. Brandle K. Nover L. Forreiter C. Planta. 2000; 211: 575-582Crossref PubMed Scopus (97) Google Scholar), are constitutively expressed in all NCS mutants under normal growth conditions (Fig. 1A). Higher levels of all transcripts were found in the translation-impaired NCS4 (rps3) mutant than in the complex I-deficient NCS2 (nad4) and complex IV-deficient NCS6 (cox2) mutants. For expression analysis of genes for cytosolic and mitochondrial HSP70s, we performed a maize Expressed Sequence Tag data base search and then identified the candidate cDNAs (Supplemental Table I) through protein sequence alignments with Arabidopsis orthologs (19Sung D.Y. Vierling E. Guy C.L. Plant Physiol. 2001; 126: 789-800Crossref PubMed Scopus (379) Google Scholar) and response to heat shock (Fig. 1B). Two tested ssa1-homologous genes for cytosolic HSP70s were both highly expressed in all NCS mutants (Fig. 1A), although only one of them was found to be HS-inducible (Fig. 1B). cDNAs for mitochondrial HSP70s were identified via a signature motif in the deduced N-terminal sequences (19Sung D.Y. Vierling E. Guy C.L. Plant Physiol. 2001; 126: 789-800Crossref PubMed Scopus (379) Google Scholar). Both constitutive (mthsc70) and HS-inducible (mthsp70) genes for mitochondrial HSP70s were found to be expressed in NCS mutants at significantly higher levels than in normal plants (Fig. 1, A and B). Transcript levels for CPN60, a different constitutive mitochondrial chaperonin (26Prasad T.K. Stewart C.R. Plant Mol. Biol. 1992; 18: 873-885Crossref PubMed Scopus (50) Google Scholar), were also increased in all NCS mutants (Fig. 1A). In addition to the earlier studied hsp22A gene for the mitochondrial sHSP (22Lund A.A. Blum P.H. Bhattramakki D. Elthon T.E. Plant Physiol. 1998; 116: 1097-1110Crossref PubMed Scopus (67) Google Scholar), we identified another hsp22-like expressed sequence tag (hsp22B; Supplemental Table I) that had orthologs in rice and wheat, but not in Arabidopsis (Fig. 2A). Both genes were dramatically expressed in NCS mutants (Fig. 1A), although hsp22B did not respond to heat shock (Fig. 1B) and its transcripts were not detectable in normal samples of any tissues tested (Figs. 1A and 2B, lanes N; other data not shown). To check expression of an HS-responsive gene that does not belong to the hsp gene families, we used a probe for one of the multiple maize heat shock transcription factor genes, hsfb (Fig. 1B), that had been shown earlier to be HS-inducible (27Gagliardi D. Breton C. Chaboud A. Vergne P. Dumas C. Plant Mol. Biol. 1995; 29: 841-856Crossref PubMed Scopus (64) Google Scholar). None of the transcripts hybridizing to this probe was found at elevated levels in any NCS mutant (Fig. 1A and Table I). Taken together, these results indicate that the expression of hsp genes in NCS mutants differs from a typical heat shock response (e.g. Ref. 28Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1535) Google Scholar).Fig. 2Identification and expression analysis of the maize hsp22B gene. A, phylogenetic tree of plant mitochondrial small HSPs constructed by CLUSTALW alignment on the basis of deduced amino acid sequences of the polypeptide precursors. The data base accession numbers of the predicted polypeptides are as follows: Arabidopsis (At) HSP22A1, GenBank™ CAB79429; At HSP22A2, GenBank™ BAB09755; wheat HSP23.5, GenBank™ AAD03604; wheat HSP23.6, GenBank™ AAD03605; rice HSP22A, GRAMENE GRMP00000073377; rice HSP22B, GRAMENE GRMP00000048381; maize HSP22A, GenBank™ AAC12279. The amino acid sequence of maize HSP22B was translated from cDNA, GenBank™ AY108320. B, transcription of genes for mitochondrial HSPs during maize microsporogenesis. Hybridization of total RNAs isolated from normal fertile (N) and sterile male florets of maize lines with cytoplasmic male sterility, types T, C, and S with gene-specific probes as indicated.View Large Image Figure ViewerDownload (PPT) Role of the Mitochondrial Transmembrane Potential in Activation of hsp Genes—What consequences of mitochondrial mutations could initiate a signaling pathway leading to the transcriptional activation of hsp genes? We considered two effects of the loss of mitochondrial genes: (i) decrease in mitochondrial transmembrane potential Δψm (2Dey R. Moraes C.T. J. Biol. Chem. 2000; 275: 7087-7094Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 29Biswas G. Adebanjo O.A. Freedman B.D. Anandatheerthavarada H.K. Vijayasarathy C. Zaidi M. Kotlikoff M. Avadhani N.G. EMBO J. 1999; 18: 522-533Crossref PubMed Scopus (309) Google Scholar), and (ii) failure to assemble respiratory complexes without mitochondrially encoded components, resulting in accumulation and/or proteolysis of unincorporated subunits (30Karpova O.V. Newton K.J. Plant J. 1999; 17: 511-521Crossref Scopus (61) Google Scholar). These processes can be individually induced in a transient mode by specific inhibitors that cause depolarization of the mitochondrial membrane or arrest of mitochondrial translation. For a membrane discharge that would be highly specific to mitochondria, we chose methomyl treatment of maize seedlings with T-type cytoplasmic male sterility (31Klein R.R. Koeppe D.E. Plant Physiol. 1985; 77: 912-916Crossref PubMed Google Scholar). CMS-T mtDNA encodes an aberrant membrane protein, T-URF13, that can specifically interact with methomyl to form small hydrophilic pores in the inner mitochondrial membrane (32Rhoads D.M. Levings III, C.S. Siedow J.N. J. Bioenerg. Biomembr. 1995; 27: 437-445Crossref PubMed Scopus (43) Google Scholar). Indeed, strong induction of the hsp genes with two types of expression patterns was observed in response to methomyl. Induction of transcripts for mitochondrial HSPs occurred rapidly and was mostly complete within 12 h (Fig. 3A, left). For genes encoding cytosolic HSPs, there was a prolonged lag phase prior to an exponential accumulation of transcripts between 18 and 24 h of treatment (Fig. 3B, left). These results suggest that depolarization of the mitochondrial membrane can trigger divergent signaling pathways for two groups of hsp genes. As we have shown earlier (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar), neither increases in protein oxidation nor elevated levels of marker antioxidant enzymes were detected in maize NCS tissues. Absence of endogenous oxidative stress was further corroborated with the lack of induction of a few highly ROS-responsive genes, including the apx1 for cytosolic ascorbate peroxidase (33Mittler R. Lam E. Shulaev V. Cohen M. Plant Mol. Biol. 1999; 39: 1025-1035Crossref PubMed Scopus (70) Google Scholar). Because no apx1 induction was detected upon methomyl treatment (Fig. 3A, left), the activation of hsp genes was not associated with transient oxidative stress. Chloramphenicol treatment to inhibit mitochondrial translation did not result in any detectable induction of transcripts for cytosolic HSPs (Fig. 3B, right). In addition, chloramphenicol appeared to be a much less potent inducer of genes for mitochondrial HSP22s than methomyl. In contrast, the level of cpn60 induction by chloramphenicol was considerably higher, which was consistent with previous data on cpn60 expression in response to accumulation of a misfolded mitochondrial protein (34Zhao Q. Wang J. Levichkin I.V. Stasinopoulos S. Ryan M.T. Hoogenraad N.J. EMBO J. 2002; 21: 4411-4419Crossref PubMed Scopus (706) Google Scholar). Thus, our results suggest that, except for cpn60, an excess of non-assembled protein subunits within mitochondria is unlikely to generate a primary signal for induction of hsp genes. It is well known that different HSPs are abundantly expressed during pollen development in maize (27Gagliardi D. Breton C. Chaboud A. Vergne P. Dumas C. Plant Mol. Biol. 1995; 29: 841-856Crossref PubMed Scopus (64) Google Scholar, 35Magnard J.-L. Vergne P. Dumas C. Plant Physiol. 1996; 111: 1085-1096Crossref PubMed Scopus (29) Google Scholar, 36Young T.E. Ling J. Geisler-Lee J. Tanguay R.L. Caldwell C. Gallie D.R. Plant Physiol. 2001; 127: 777-791Crossref PubMed Scopus (85) Google Scholar). Indeed, as seen in Fig. 2B, genes for mitochondrial HSC70 and HSP22A are actively transcribed in male florets of fertile and sterile maize lines, including CMS-T with T-URF13-impaired mitochondria and also CMS-C and CMS-S lines that develop unknown mitochondrial dysfunctions during microsporogenesis (13Newton K.J. Gabay-Laughnan S. Singh K.K. Mitochondrial DNA Mutations in Aging, Disease, and Cancer. Springer-Verlag, Berlin1998: 365-381Crossref Google Scholar). However, the hsp22B gene is induced exclusively in CMS-T and the mthsc70 expression is increased in CMS-T, where mitochondrial membrane depolarization was suggested to occur naturally in anthers (37Levings III, C.S. Plant Cell. 1993; 5: 1285-1290Crossref PubMed Google Scholar). Thus, it is likely that the same mechanism of Δψm-dependent hsp activation is turned on in CMS-T maize during microsporogenesis and upon methomyl treatment of vegetative tissues. The differential kinetics of hsp induction in response to methomyl treatment indicates that there is a direct signal from a reduced Δψm to up-regulate the genes for mitochondrial sHSPs. On the other hand, the delayed induction of genes for cytosolic HSPs would suggest another signal generated as a secondary consequence of mitochondrial membrane depolarization. Inhibition of protein import into uncoupled mitochondria leads to accumulation of some mitochondrial precursors in cytosol (38Reid G.A. Schatz G. J. Biol. Chem. 1982; 257: 13056-13061Abstract Full Text PDF PubMed Google Scholar), facilitating their aggregation. In turn, an excess of non-native proteins and their aggregates in cytosol can trigger the induction of hsp genes (9Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1878) Google Scholar). Indeed, in NCS mutants that should have reduced Δψm, we have found that accumulation of the nuclearly encoded E1α subunit of mitochondrial pyruvate dehydrogenase in the cytosol correlates with the significant increase in levels of HSP101 protein (Fig. 4, A and B). Concomitantly in the mitochondria of NCS mutants, high steady-state amounts of HSP22(A), the major HS-responsive maize mitochondrial protein (22Lund A.A. Blum P.H. Bhattramakki D. Elthon T.E. Plant Physiol. 1998; 116: 1097-1110Crossref PubMed Scopus (67) Google Scholar), were detected (Fig. 4C). In addition, analysis of mitochondrial HSP70s with the monoclonal antibodies (22Lund A.A. Blum P.H. Bhattramakki D. Elthon T.E. Plant Physiol. 1998; 116: 1097-1110Crossref PubMed Scopus (67) Google Scholar) revealed the presence of a new immunoreactive protein in addition to the constitutive HSP70. It is likely that the novel HSP70 isoform represents a highly homologous inducible mitochondrial HSP70 (∼50% of the deduced amino acid sequence shows 92% identity to that of constitutive HSP70; data not shown). These changes in protein patterns of inducible mitochondrial HSPs are consistent with high induction of their transcripts in NCS mutants (Fig. 1A), whereas levels of constitutive mitochondrial HSPs, HSP70 and the very abundant CPN60 (Fig. 4C), do not reflect an increase in transcription of the corresponding genes. In mtDNA-depleted mammalian cell cultures, a decrease in Δψm was shown to be associated with a rise in cytosolic Ca2+ levels, and increased expression of some Ca2+-responsive genes was reported (29Biswas G. Adebanjo O.A. Freedman B.D. Anandatheerthavarada H.K. Vijayasarathy C. Zaidi M. Kotlikoff M. Avadhani N.G. EMBO J. 1999; 18: 522-533Crossref PubMed Scopus (309) Google Scholar). We found no difference in transcript levels of the Ca2+-inducible adh1 gene for alcohol dehydrogenase (39Subbaiah C.C. Zhang J. Sachs M.M. Plant Physiol. 1994; 105: 369-376Crossref PubMed Scopus (137) Google Scholar) between NCS mutants and normals in the ear shoot tissues where an overexpression of the hsp genes has been observed (compare Figs. 5A and 1A). To test directly whether genes for mitochondrial and cytosolic HSPs respond to increases in cytosolic Ca2+, we treated B37T maize seedlings with the ionophore A23187, which causes fluxes of intracompartmental Ca2+ into the cytosol. Unlike methomyl, which would also release Ca2+ specifically from mitochondria (40Holden M.J. Sze H. Plant Physiol. 1984; 75: 235-237Crossref PubMed Google Scholar), A23187 does not affect Δψm unless exogenous Ca2+ is supplied (41Curtis M.J. Wolpert T.J. Plant J. 2002; 29: 295-312Crossref PubMed Scopus (87) Google Scholar, 42Kahlert S. Reiser G. FEBS Lett. 2002; 529: 351-355Crossref PubMed Scopus (19) Google Scholar). Adh1 is up-regulated in response to hypoxia, which was shown to be mediated by Ca2+ (43Andrews D.L. Cobb B.G. Johnson J.R. Drew M.C. Plant Physiol. 1993; 101: 407-414Crossref PubMed Scopus (57) Google Scholar). Gradual induction of adh1 is commonly observed in maize seedling roots submerged in water for up to 24 h (Fig. 5B, no additions). Both methomyl and A23187 caused much faster adh1 induction (Fig. 5B), apparently reflecting their effects on Ca2+ efflux into the cytosol. However, neither hsp22A nor shsp-I responded to the A23187 treatment (Fig. 5B). These experiments suggest that a decrease in Δψm itself, rather than changes in cellular Ca2+ fluxes, generates a primary signal to induce expression of hsp genes. RNAi Silencing of Nuclear Respiratory Genes in C. elegans Leads to Induction of hsp Genes—We predicted that signaling from dysfunctional mitochondria to induce hsp genes would exist in organisms other than plants. It may take place not only in mitochondrial mutants but also when the expression of nuclear genes coding for mitochondrial components is impaired. To assess the presence of the mitochondria-mediated signaling to express hsp genes in nematode C. elegans, we used RNAi (44Timmons L. Fire A. Nature. 1998; 395: 854Crossref PubMed Scopus (1425) Google Scholar) of nuclear genes for the components of the ETC. The genes for the following proteins have been chosen as targets: B18 subunit of respiratory complex I, 14-kDa subunit of complex III, cytochrome c1 (complex III), and coxVb subunit of complex IV. All cultures showed developmental delays (mostly L2 and L3 larvae instead of predominant L4 larvae in control culture after 74 h). Like maize mitochondrial mutants (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar), none of the RNAi-treated cultures seemed to experience oxidative stress. No induction of the gst4 and pqm1 transcripts was detected (Fig. 6A), though these genes were shown to be the most ROS-responsive in C. elegans (45Tawe W.N. Eschbach M.L. Walter R.D. Henkle-Duhrsen K. Nucleic Acids Res. 1998; 26: 1621-1627Crossref PubMed Scopus (106) Google Scholar). We have found (Fig. 6A and Supplemental Table II) that RNAi inactivation of respiratory complexes I, III, and IV leads to induction of the major C. elegans heat-inducible gene (46Thakurta D.G. Palomar L. Stormo G.D. Tedesco P. Johnson T.E. Walker D.W. Lithgow G. Kim S. Link C.D. Genome Res. 2002; 12: 701-712Crossref PubMed Scopus (121) Google Scholar) for a cytosolic HSP70, hsp70.6 (but not of the highly homologous hsp70.4; not shown). Moderately increased were transcripts of hsp70A and, more prominently, those of the gene for mitochondrial HSP70 (hsp70F). Among the genes for sHSPs, no induction of hsp12.6 or change in transcript levels of hsp25 was detected (data not shown). The hsp-17 gene, whose expression had not been characterized before, was highly induced in the RNAi-treated cultures, although it did not respond to heat shock (Fig. 6, A and B). Interestingly, the homologous hsp-16.1 and hsp-16.2 genes were induced although no expression of another homologous pair of sHSP genes, hsp-16.48 and hsp-16.41, was detected (Fig. 6A). In contrast, HS induced both types of hsp-16 genes (Fig. 6B). In the C. elegans genome, hsp16s are organized tandemly, hsp-16.1/hsp-16.48 and hsp-16.2/hsp-16.41. In each case, the shared bidirectional promoter regions contain identical heat shock elements, which correlates with co-regulation of tandemly organized hsp16 genes during heat shock by a single heat shock factor, HSF1 (47Jones D. Russnak R.H. Kay R.J. Candido E.P.M. J. Biol. Chem. 1986; 261: 12006-12015Abstract Full Text PDF PubMed Google Scholar, 48Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1124) Google Scholar). Thus, the differential expression of hsp16 genes and activation of hsp17 gene detected in our RNAi experiments suggest that the induction of shsp genes in C. elegans carrying mitochondrial deficiencies is distinct from the HSF1-dependent HS response and may require other transcription factors and cis-recognition sequences. Here we report that acclimation to permanent mitochondrial dysfunction involves steady-state activation of specific sets of hsp genes encoding cytosolic and mitochondrial molecular chaperones. Using maize NCS mutants with defined deletions in mtDNA and cultures of C. elegans with RNAi-inactivated nuclear respiratory genes, we show that mitochondria-dependent hsp gene activation in different organisms has common but distinctive characteristics. (i) Activation of hsp genes depends on inhibition of the electron transfer chain regardless of the type of respiratory defect or its origin (loss of either mitochondrial or nuclear-coded product). In the transient in vivo assays, we have shown that in maize same sets of hsp genes can be induced by depolarization of the mitochondrial membrane. Different kinetics of Δψm-dependent activation suggest different induction pathways for genes encoding mitochondrial and cytosolic HSPs. It is likely that the reduced Δψm, a common result of the ETC inhibition, serves as a primary signal to induce hsp genes coding for mitochondrially targeted proteins. Consequently, inefficient protein import into the de-energized mitochondria might lead to the accumulation of mitochondrial precursors in the cytosol, which was indeed detected in our experiments. This excess of non-native proteins might contribute to the induction of genes for cytosolic HSPs. (ii) In both maize and nematodes, mitochondrial deficiencies differentially activate some HS-inducible hsp genes and also cause high expression of some HS non-responsive hsp genes (including cryptic ones, like hsp22B in maize and hsp-17 in C. elegans). This pattern of hsp expression differs from a typical HS response that involves recognition of heat shock elements by an activated heat shock factor (28Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1535) Google Scholar). It is likely that mitochondria-dependent activation of hsp genes may require additional transcription factors and cis-recognition sequences. (iii) Expression of hsp genes under conditions of permanent respiratory deficiency is not associated with oxidative stress and is not mediated by Ca2+, which clearly distinguishes this type of hsp induction from any others known to date. High expression of some of the tested maize hsp genes at the protein level indicates that activation of hsp genes by impaired mitochondria might be of physiological importance. The remarkable similarities in response to respiratory deficiency in plants and in animals indicate that the constitutive expression of the hsp gene network is a significant part of a program to cope with the disruption of the mitochondrial function. Permanent expression of hsp genes in respiratory-deficient individuals seems to be required for their development and may reflect different changes in genome expression patterns in comparison to the transient responses of normal organisms subjected to temporary biotic/abiotic stresses. Analysis of genome-wide expression in ρ0 mutants compared with wild-type cultures of S. cerevisiae treated with respiratory inhibitors confirmed that gene expression patterns upon mtDNA depletion and after transient inhibition of the ETC are different and overlap only partially (49Epstein C.B. Waddle J.A. Hale IV, W. Dave V. Thornton J. Macatee T.L. Garner H.R. Butow R.A. Mol. Biol. Cell. 2001; 12: 297-308Crossref PubMed Scopus (332) Google Scholar). Our data on maize NCS mutants suggest that mitochondria-dependent hsp expression could be coordinated with another stress defensive mechanism associated with plant-specific mitochondrial alternative oxidase, AOX (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar). As a terminal ubiquinol oxidase, AOX acts as a mild uncoupler of oxidative phosphorylation and as an antioxidant (50Wagner A.M. Moore A.L. Biosci. Rep. 1997; 17: 319-333Crossref PubMed Scopus (149) Google Scholar) and prevents an oxidative stress in NCS mutants (3Karpova O.V. Kuzmin E.V. Elthon T.E. Newton K.J. Plant Cell. 2002; 14: 3271-3284Crossref PubMed Scopus (163) Google Scholar). Thus, in NCS mutants with high constitutive expression of AOX, effective inhibition of ROS generation occurs at the expense of further de-energization of the mitochondrial membrane. In turn, this decrease in Δψm triggers signaling to express cytosolic and mitochondrial HSPs that would have a protective effect on cells and particularly on dysfunctional mitochondria (5Polla B.S. Kantengwa S. Francois D. Salvioli S. Franceschi C. Marsac C. Cossarizza A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6458-6463Crossref PubMed Scopus (393) Google Scholar, 6Saleh A. Srinivasula S.M. Balkir L. Robbins P.D. Alnemri E.S. Nature Cell Biol. 2000; 2: 476-483Crossref PubMed Scopus (736) Google Scholar, 7Ravagnan L. Gurbuxani S. Susin S.A. Maisse C. Daugas E. Zamzami N. Mak T. Jaattela M. Penninger J.M. Garrido C. Kroemer G. Nature Cell Biol. 2001; 3: 839-843Crossref PubMed Scopus (749) Google Scholar, 8Paul C. Manero F. Gonin S. Kretz-Remy C. Virot S. Arrigo A.P. Mol. Cell. Biol. 2002; 22: 816-834Crossref PubMed Scopus (373) Google Scholar, 51Downs C.A. Heckathorn S.A. FEBS Lett. 1998; 430: 246-250Crossref PubMed Scopus (106) Google Scholar). Orchestrated hsp gene expression contributes to a metabolically stable acclimation that prevents oxidative stress and apoptosis in respiratory-deficient mutants (Fig. 7). In experiments with C. elegans, we observed hsp gene induction upon RNAi inactivation of the same nuclear respiratory genes, whose RNAi silencing had been found to give a pronounced increase in lifespan (52Dillin A. Hsu A.-L. Arantes-Oliveira N. Lehrer-Graiwer J. Hsin H. Fraser A.G. Kamath R.S. Ahringer J. Kenyon C. Science. 2002; 298: 2398-2401Crossref PubMed Scopus (790) Google Scholar, 53Lee S.S. Lee R.Y.N. Fraser A.G. Kamath R.S. Ahringer J. Ruvkun G. Nat. Genet. 2003; 33: 40-48Crossref PubMed Scopus (764) Google Scholar). These results suggest that induction of hsp genes through the signaling from impaired mitochondria may represent one of the crucial links between mitochondrial state and longevity (reviewed in Ref. 54Tsang W.Y. Lemire B.D. Biochim. Biophys. Acta. 2003; 1638: 91-105Crossref PubMed Scopus (92) Google Scholar). Thus, we show here that in both plants and animals, there is the mitochondria-dependent hsp gene expression that is different from typical responses to heat shock or oxidative stress and could imply novel signaling pathways and induction mechanisms. Establishing the constitutive expression of hsp genes in the absence of exogenous stress may play a vital role in cell defense and adaptation to permanent respiratory deficiency. We thank J. Nieto-Sotelo and D. Kliebenstein for generous gifts of antisera. We are very grateful to Cathy Gunther, Patrice Albert, and other members of the D. Riddle laboratory (University of Missouri) for helpful advice on Caenorhabditis elegans. Download .pdf (.03 MB) Help with pdf files" @default.
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• W2103796739 title "Mitochondrial Respiratory Deficiencies Signal Up-regulation of Genes for Heat Shock Proteins" @default.
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