Plant Molecular Biology 44: 487–498, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 487 Identification and characterization of a heat-induced isoform of aldolase in oat chloroplast Regina Michelis and Shimon Gepstein * Faculty of Biology, Technion–Israel Institute of Technology, Haifa, Israel 32000 ( * author for correspondence) Received 19 August 1999; accepted in revised form 21 June 2000 Key words: fructose-bisphophate aldolase, heat shock proteins, heat tolerance, oat leaves Abstract An analysis of protein synthesis at elevated temperatures in oat (Avena sativa) leaves revealed a heat-induced 44 kDa polypeptide. A cDNA library of heat-treated leaves was constructed and screened with specific antibodies raised against this 44 kDa polypeptide. A clone encoding the 44 kDa protein was identified as a form of the chloroplast-localized fructose-bisphosphate aldolase (EC 4.1.2.13). Northern and western blot analyses indicated heat-induced accumulation of the chloroplast aldolase isoform at both the RNA and protein level. Heat inducibility was restricted to the chloroplastic form of the enzyme, and was not observed for the cytoplasmic aldolase. The heat-induced isoform co-purified with thykaloid fractions, as confirmed by immunoassay and activity analyses. However, when thylakoid membranes were treated with proteinase K, the aldolase isoform completely disappeared, suggesting that this enzyme is not embedded but rather tends to adhere to the chloroplast membranes. Immunoblot analysis of other plant species revealed similar heat induction of thykaloid-associated aldolase homologues, suggesting the possible existence of a universal control mechanism for this enzyme’s heat tolerance Abbreviations: HSP, heat-shock protein; 2D-PAGE, two dimensional gel electrophoresis; NEPHGE, non- equilibrium pH gradient electrophoresis; DEPC, diethylpyrocarbonate; LHC II, light-harvesting chlorophyll a/b-binding proteins of photosystem II. Introduction The synthesis of heat-shock proteins (HSPs) in re- sponse to hyperthermic stress is a highly conserved biological phenomenon. The molecular mechanisms of heat-shock gene activation are similar in different eukaryotic cells, and the induction of transcription is mediated by activation of heat-shock factors. The heat- shock response can be elicited through inhibition of synthesis of most unstressed cell proteins and through the induction of mRNA and protein synthesis asso- ciated with gene families coding for HSPs (Vierling, 1991). The heat-shock response has been found to oc- cur in all living organisms observed, including bacte- ria, fungi, algae, higher plants and mammals (Vierling, The nucleotide sequence data reported will appear in the Gen- Bank Nucleotide Sequence Database under the accession number AF216582. 1991). Some of the HSPs share high homology; for example, the HSP70 genes show 75% homology in maize, Drosophila, and man. HSPs have been detected in subcellular organelles, such as chloroplasts and mi- tochondria, though most of the HSPs are encoded in the plant nucleus (Nieto-Sotelo and Ho, 1987; Mar- shall et al., 1990; Drzymalla et al., 1992; Waters, 1995). Of particular interest is the low-molecular- weight chloroplast-localized HSP that protects PSII activity during heat shock (Heckathorn et al., 1998). Although most HSPs have been identified by their intense expression subsequent to heat induction, some are expressed in unstressed cells, or at specific de- velopmental stages, and do not require heat (Koning et al., 1992). The cellular activities of the main HSP families in eukaryotes (HSP70, GroEL homologues and HSP90) were found to be molecular chaperones, necessary for the assembly, unfolding or transport of