An Arabidopsis chloroplast-targeted Hsp101 homologue, APG6, has an essential role in chloroplast development as well as heat-stress response.

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.

[1]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[2]  E. Meyerowitz,et al.  Structure and expression of three light-harvesting chlorophyll a/b-binding protein genes in Arabidopsis thaliana. , 1986, Nucleic acids research.

[3]  E. D. Wolf,et al.  High-velocity microprojectiles for delivering nucleic acids into living cells , 1987, Nature.

[4]  R. J. Porra,et al.  Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy , 1989 .

[5]  K. Yamaguchi-Shinozaki,et al.  Characterization of cDNA for a dehydration-inducible gene that encodes a CLP A, B-like protein in Arabidopsis thaliana L. , 1993, Biochemical and biophysical research communications.

[6]  W. Bilger,et al.  Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis , 1994 .

[7]  R. T. Nagao,et al.  A soybean 101-kD heat shock protein complements a yeast HSP104 deletion mutant in acquiring thermotolerance. , 1994, The Plant cell.

[8]  S. Lindquist,et al.  An Arabidopsis heat shock protein complements a thermotolerance defect in yeast. , 1994, The Plant cell.

[9]  J. Shanklin,et al.  The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease. , 1995, The Plant cell.

[10]  Takanori Hirano,et al.  Engineered GFP as a vital reporter in plants , 1996, Current Biology.

[11]  L. Herrera-Estrella,et al.  CLA1, a novel gene required for chloroplast development, is highly conserved in evolution. , 1996, The Plant journal : for cell and molecular biology.

[12]  K. Koch.  CARBOHYDRATE-MODULATED GENE EXPRESSION IN PLANTS. , 1996, Annual review of plant physiology and plant molecular biology.

[13]  J. Khurana,et al.  Nucleotide sequence of the psbP gene encoding precursor of 23-kDa polypeptide of oxygen-evolving complex in Arabidopsis thaliana and its expression in the wild-type and a constitutively photomorphogenic mutant. , 1996, DNA research : an international journal for rapid publication of reports on genes and genomes.

[14]  K. Shinozaki,et al.  A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana. , 1997, The Plant journal : for cell and molecular biology.

[15]  K. Shinozaki,et al.  Two Transcription Factors, DREB1 and DREB2, with an EREBP/AP2 DNA Binding Domain Separate Two Cellular Signal Transduction Pathways in Drought- and Low-Temperature-Responsive Gene Expression, Respectively, in Arabidopsis , 1998, Plant Cell.

[16]  Robert L. Tanguay,et al.  HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status. , 1998, Genes & development.

[17]  Y. Nakamura,et al.  Complete structure of the chloroplast genome of Arabidopsis thaliana. , 1999, DNA research : an international journal for rapid publication of reports on genes and genomes.

[18]  D. Blanchard,et al.  The electronic plant gene register , 1995, Plant physiology.

[19]  L. Martínez,et al.  Characterization of a maize heat-shock protein 101 gene, HSP101, encoding a ClpB/Hsp100 protein homologue. , 1999, Gene.

[20]  Jimin Wang,et al.  New insights into the ATP‐dependent Clp protease: Escherichia coli and beyond , 1999, Molecular microbiology.

[21]  K. Shinozaki,et al.  Identification of clp genes expressed in senescing Arabidopsis leaves. , 1999, Plant & cell physiology.

[22]  Robert L. Tanguay,et al.  Heat Shock Protein HSP101 Binds to the Fed-1 Internal Light Regulatory Element and Mediates Its High Translational Activity , 2000, Plant Cell.

[23]  D. Thureen,et al.  Acquired thermotolerance and expression of the HSP100/ClpB genes of lima bean. , 2000, Plant physiology.

[24]  H. Kawaide,et al.  Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis. , 2000, Plant physiology.

[25]  S. Lindquist,et al.  Heat Shock Protein 101 Plays a Crucial Role in Thermotolerance in Arabidopsis , 2000, Plant Cell.

[26]  W. Sakamoto,et al.  The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis. , 2000, Plant & cell physiology.

[27]  W. Majeran,et al.  Evidence for a Role of ClpP in the Degradation of the Chloroplast Cytochrome b6f Complex , 2000, Plant Cell.

[28]  E. Vierling,et al.  Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Robert L. Tanguay,et al.  Developmental and thermal regulation of the maize heat shock protein, HSP101. , 2001, Plant physiology.

[30]  J. Nieto-Sotelo,et al.  Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum (L.) Moench) inducible by heat, dehydration, and ABA(1). , 2001, Biochimica et biophysica acta.

[31]  P. Westhoff,et al.  Biogenesis and origin of thylakoid membranes. , 2001, Biochimica et biophysica acta.

[32]  K. Shinozaki,et al.  Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature. , 2001, Plant physiology.

[33]  T. Shikanai,et al.  The chloroplast clpP gene, encoding a proteolytic subunit of ATP-dependent protease, is indispensable for chloroplast development in tobacco. , 2001, Plant & cell physiology.

[34]  E. Vierling,et al.  Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress. , 2001, The Plant journal : for cell and molecular biology.

[35]  T. Ito,et al.  An essential role of a TatC homologue of a ΔpH- dependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  K. V. van Wijk,et al.  Identification of a 350-kDa ClpP Protease Complex with 10 Different Clp Isoforms in Chloroplasts of Arabidopsis thaliana * , 2001, The Journal of Biological Chemistry.

[37]  M. Agarwal,et al.  Arabidopsis thaliana Hsp100 proteins: kith and kin , 2001, Cell stress & chaperones.

[38]  G. Ponce,et al.  Maize HSP101 Plays Important Roles in Both Induced and Basal Thermotolerance and Primary Root Growth Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010487. , 2002, The Plant Cell Online.

[39]  J. Glazebrook,et al.  Arabidopsis : a laboratory manual , 2002 .

[40]  M. Agarwal,et al.  Plant Hsp100 proteins: structure, function and regulation , 2002 .

[41]  R. Herrmann,et al.  The gene complement for proteolysis in the cyanobacterium Synechocystis sp. PCC 6803 and Arabidopsis thaliana chloroplasts , 2002, Current Genetics.

[42]  Kazuo Shinozaki,et al.  A New Resource of Locally Transposed DissociationElements for Screening Gene-Knockout Lines in Silico on the Arabidopsis Genome1,212 , 2002, Plant Physiology.

[43]  M. Schmid,et al.  Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana , 2003, Science.

[44]  P. Maliga,et al.  The plastid clpP1 protease gene is essential for plant development , 2003, Nature.

[45]  R. Motohashi,et al.  Functional analysis of the 37 kDa inner envelope membrane polypeptide in chloroplast biogenesis using a Ds-tagged Arabidopsis pale-green mutant. , 2003, The Plant journal : for cell and molecular biology.

[46]  J. Na,et al.  Mechanisms of Glucose Signaling during Germination of Arabidopsis1 , 2003, Plant Physiology.

[47]  A. Pareek,et al.  Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera , 1995, Plant Molecular Biology.

[48]  S. Rodermel,et al.  Mutations in ClpC2/Hsp100 suppress the requirement for FtsH in thylakoid membrane biogenesis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Z. Adam,et al.  Degradation of mistargeted OEE33 in the chloroplast stroma , 1996, Plant Molecular Biology.

[50]  Shenglong Wang,et al.  The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth , 1994, Molecular and General Genetics MGG.

[51]  J. Froehlich,et al.  A Stromal Hsp100 Protein Is Required for Normal Chloroplast Development and Function in Arabidopsis1[w] , 2004, Plant Physiology.

[52]  A. Clarke,et al.  Inactivation of the clpC1 Gene Encoding a Chloroplast Hsp100 Molecular Chaperone Causes Growth Retardation, Leaf Chlorosis, Lower Photosynthetic Activity, and a Specific Reduction in Photosystem Content1 , 2004, Plant Physiology.

[53]  Kazuo Shinozaki,et al.  A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis. , 2004, The Plant journal : for cell and molecular biology.

[54]  M. Agarwal,et al.  Molecular characterization of rice hsp101: complementation of yeast hsp104 mutation by disaggregation of protein granules and differential expression in indica and japonica rice types , 2003, Plant Molecular Biology.

[55]  G. Link,et al.  Identification and characterization of the Arabidopsis thaliana chloroplast DNA region containing the genes psbA, trnH and rps19′ , 1995, Current Genetics.

[56]  Jaroslaw Pillardy,et al.  Clp Protease Complexes from Photosynthetic and Non-photosynthetic Plastids and Mitochondria of Plants, Their Predicted Three-dimensional Structures, and Functional Implications* , 2004, Journal of Biological Chemistry.

[57]  E. Krebbers,et al.  Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana , 1988, Plant Molecular Biology.

[58]  K. Keegstra,et al.  Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue , 1993, Plant Molecular Biology.

[59]  Kazuo Shinozaki,et al.  A resource of 5,814 dissociation transposon-tagged and sequence-indexed lines of Arabidopsis transposed from start loci on chromosome 5. , 2005, Plant & cell physiology.

[60]  A. Pareek,et al.  Plant Hsp100 family with special reference to rice , 1998, Journal of Biosciences.