Genome-wide analysis of rice ClpB/HSP100, ClpC and ClpD genes.
BACKGROUND
ClpB-cyt/HSP100 protein acts as chaperone, mediating disaggregation of denatured proteins. Previous studies have shown that ClpB-cyt/HSP100 gene belongs to the group class I Clp ATPase proteins and ClpB-cyt/HSP100 transcript is regulated by heat stress and developmental cues.


RESULTS
Nine ORFs were noted to constitute rice class I Clp ATPases in the following manner: 3 ClpB proteins (ClpB-cyt, Os05g44340; ClpB-m, Os02g08490; ClpB-c, Os03g31300), 4 ClpC proteins (ClpC1, Os04g32560; ClpC2, Os12g12580; ClpC3, Os11g16590; ClpC4, Os11g16770) and 2 ClpD proteins (ClpD1, Os02g32520; ClpD2, Os04g33210). Using the respective signal sequences cloned upstream to GFP/CFP reporter proteins and transient expression studies with onion epidermal cells, evidence is provided that rice ClpB-m and Clp-c proteins are indeed localized to their respective cell locations mitochondria and chloroplasts, respectively. Associated with their diverse cell locations, domain structures of OsClpB-c, OsClpB-m and OsClpB-cyt proteins are noted to possess a high-level conservation. OsClpB-cyt transcript is shown to be enriched at milk and dough stages of seed development. While expression of OsClpB-m was significantly less as compared to its cytoplasmic and chloroplastic counterparts in different tissues, this transcript showed highest heat-induced expression amongst the 3 ClpB proteins. OsClpC1 and OsClpC2 are predicted to be chloroplast-localized as is the case with all known plant ClpC proteins. However, the fact that OsClpC3 protein appears mitochondrial/chloroplastic with equal probability and OsClpC4 a plasma membrane protein reflects functional diversity of this class. Different class I Clp ATPase transcripts were noted to be cross-induced by a host of different abiotic stress conditions. Complementation assays of Deltahsp104 mutant yeast cells showed that OsClpB-cyt, OsClpB-m, OsClpC1 and OsClpD1 have significantly positive effects. Remarkably, OsClpD1 gene imparted appreciably high level tolerance to the mutant yeast cells.


CONCLUSIONS
Rice class I Clp ATPase gene family is constituted of 9 members. Of these 9, only 3 belonging to ClpB group are heat stress regulated. Distribution of ClpB proteins to different cell organelles indicates that their functioning might be critical in different cell locations. From the complementation assays, OsClpD1 appears to be more effective than OsClpB-cyt protein in rescuing the thermosensitive defect of the yeast ScDeltahsp104 mutant cells.
Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India.
Amanjot
A
Singh
Upasana
U
Singh
Dheeraj
D
Mittal
Anil
A
Grover
D000251:Adenosine Triphosphatases / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005816:Genetic Complementation Test; D018745:Genome, Plant; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D018832:Molecular Chaperones / Q000235:genetics*; D005810:Multigene Family; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D012275:Oryza / Q000235:genetics*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D013312:Stress, Physiological
D018744:DNA, Plant; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010940:Plant Proteins; D000251:Adenosine Triphosphatases