Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

The Presence of Two Forms of Succinate Dehydrogenase in Sweet Potato Root Mitochondria

Succinate dehydrogenase was partially purified from sweet potatoroot tissue by solubilization of the enzyme from the submitochondrialparticles, ammonium sulfate fractionation, and DEAE-cellulosecolumn chromatography. Sweet potato succinate dehydrogenaseexisted in two forms; these were separated by disc polyacrylamidegel electrophoresis or by hydroxyapatite column chromatography.There was a difference in the electric charge of the molecule,but not in the molecular weights of the two forms. No differencewas detected between the two forms of succinate dehydrogenasewith respect to their K_m values for succinate, pH-optimums andsubunit compositions. The two subunits that make up the enzymehave molecular weights of about 26,000 and 65,000. ¹ This work was supported in part by Grant-in-Aid 411308 forScientific Research from the Ministry of Education, Scienceand Culture of Japan. (Received November 28, 1981; Accepted February 17, 1982)     

Structural differences in full-length cDNAs for two classes of sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin, which accounts for 80% of the total soluble proteins in sweet potato tuberous roots, consists of two polypeptide classes, A and B. The sporamin cDNA clones can also be classified into sporamin A and B subfamilies based on their sequence homologies, with intra-subfamily homologies being much higher than inter-subfamily homologies. The sequence of an essentially full-length cDNA for sporamin B was compared with that for sporamin A. The coding sequences of two cDNAs share 83% sequence homology. The sequences in the 5- and 3-noncoding regions show many deletions in addition to base substitutions. The endpoints of deletions longer than 4 bp match precisely to the endpoints of short direct repeats present in the other sequence, which suggests that these deletions are generated by slipped mispairing during DNA replication. In the 5- and 3-noncoding region of sporamin B cDNA, there are 5 bp direct repeats with sequences complementary to each other. Since most of these repeats are absent in sporamin A cDNA, these structural features may cause a difference in the secondary structure between A and B mRNAs and affect the translational efficiencies or stabilities of the mRNAs. Precursors for both classes of sporamin carry N-terminal extra-sequences which can be separated into a putative signal peptide segment and a segment enriched with basic amino acids. A two-step processing mechanism for the maturation of sporamin is suggested.     

Temporal and spatial expression pattern of the OSVP1 and OSEM genes during seed development in rice

The spatial and temporal expression patterns of the rice VP1 (OSVP1) gene, as well as the OSEM gene which it controls, were studied during seed development by in situ hybridization and immuno-localization techniques. The expression of OSVP1 could be detected in embryos as early as 2-3 d after pollination (DAP) and thereafter became preferentially localized to shoot, radicle and vascular tissues during the embryo development at both the mRNA and protein levels. In the aleurone layers, OSVP1 mRNA and protein were detected after 6 DAP. OSEM mRNA was detectable after 6 DAP in the embryo and aleurone tissue. The spatial distribution within the embryo of OSEM mRNA and OSVP1 mRNA/protein was very similar after 6 DAP. Transgenic rice carrying a beta-glucuronidase (GUS) gene transcribed from a chimeric promoter consisting of the CaMV 35S minimal promoter (-46) and the 55-bp promoter fragment of OSEM, minimally required for ABA and VP1 regulation, also exhibited a spatial pattern of GUS expression similar to that of OSEM and OSVP1. These results suggest that (OS)VP1 is a major determinant not only of the seed specificity but also of the spatial pattern of OSEM expression in the developing seed.     

Isolation and characterization of two tightly linked catalase genes from castor bean that are differentially regulated

Two catalase genes,cat1 andcat2, have been isolated from the castor bean genome. They were located in the same direction on a chromosome at a distance of 2.4 kb,cat1 being on the downstream side ofcat2. The two genes contained introns at the same positions except that one of the 7 introns incat1 is missing incat2 and the corresponding introns differed in size and sequence between the two genes. The translated regions of the two genes had the same number of nucleotides and exhibited 81.3% nucleotide sequence identity. In addition to introns, the nucleotide sequences of the 5-and 3-flanking regions are highly divergent between the two genes. In etiolated seedlings,cat1 mRNA was present abundantly in endosperms and cotyledons and only in a small amount in roots. Thecat1 mRNA could not be detected in hypocotyls. By contrast,cat2 mRNA is most abundant in hypocotyls and roots, while endosperms and cotyledons contained only low levels ofcat2 mRNA. Although neithercat1 norcat2 mRNA could be detected in dry seeds, both mRNAs showed temporal accumulation in the endosperm in response to germination. These results suggest that expression of two tightly linked catalase genes of castor bean,cat1 andcat2, are differentially regulated during development.