A soluble proteinaceous invertase inhibitor was purified from tubers of cv. Provita. N-terminal amino acid sequencing of the purified protein indicated that the invertase inhibitor was a member of a family of small tuber proteins known as Kunitz-type protease inhibitors. The purified protein was completely inhibitory to soluble tuber invertase at quantities that did not inhibit trypsin. Based on amino acid sequence information of invertase inhibitor protein, seven candidate cDNA clones were identified in libraries of Provita and Saturna tubers. The candidate cDNA sequences differed from each other between 2% and 5%, having several non-conservative amino acid substitutions when compared with sequence related protease inhibitors. The results suggest invertase as an alternative target of tuber "protease" inhibitors.

Cold storage of potato tubers at 4 °C is associated with the accumulation of several cold-induced transcripts. By using a previously characterized cDNA (CI7) as probe, we isolated and sequenced the corresponding ci7 gene. The putative promoter of ci7 contains sequence elements that have been shown to mediate expression of stress-responsive genes of Arabidopsis thaliana. CI7 transcripts were differentially induced in response to cold, drought, high salt or exogenous ABA treatment in potato tubers and leaves. Whereas accumulation of CI7 transcript during cold storage occurred within days, induction of CI7 transcript in response to drought, ABA and salt occurred rapidly within few hours. In tubers, accumulation of CI7 protein in response to abiotic stresses and ABA was small when compared to transcript levels. In leaves, the CI7 protein was undetectable after all treatments tested. 3 kb of the 5'-flanking ci7 promoter region were fused to the GUS reporter gene and introduced into S. tuberosum plants. The analysis of tubers of independent transgenic lines did not reveal significant induction of enzymatic GUS activity in response to low temperature. When RNA gel blotting was used to analyze the level of induction of the GUS gene driven by the ci7 promoter, the heterologous GUS fusion was, however, strongly responsive to low temperature. Nuclear run-on transcription studies of the ci7 gene, in comparison with RNA gel blot analyses of the transgenic plants, indicated that most of the temperature-regulated expression of the ci7 gene in tubers may be accounted for by post-transcriptional control mechanisms.

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.

Two nuclear-coded heat-shock proteins (HSP) of pea (Pisum sativum) are synthesized as larger precursors of 26 kDa and 30 kDa in vitro. They are transported post-translationally into isolated, homologous chloroplasts where they are processed to mature proteins of 22 kDa and 25 kDa, respectively. When the chloroplasts used for the transport are isolated from control plants grown at 25 degrees C the 22-kDa and 25-kDa HSPs are located in the stroma of the chloroplasts. However, when chloroplasts are prepared from heat-shocked plants both proteins are found bound to the thylakoid membranes. The transition of the non-binding to the binding status is comparatively sharp and occurs between 36 degrees C and 40 degrees C in the variety 'Rosa Krone'. The transition temperature has been determined at 38 degrees C for 'Rosa Krone' and at 40 degrees C for the variety 'Golf'. At 42 degrees C, 15-min treatment of the plants is sufficient to induce membrane binding, which persists for at least 4-6 h (but not for 24 h) after return to the ambient temperature. Once lost, membrane binding can be reinduced by a second heat-shock treatment in vivo. High light intensities during the heat shock interfere with the binding capacity for heat-shock proteins.