Cystatins, cysteine proteinase inhibitors, deserve note because of their regulatory and protective functions in plant tissues. We isolated both genomic DNA and cDNA clones from soybean that encode a cystatin consisting of 245 amino acid residues (soyacystatin). It is, while basically similar in sequence to known cystatins that are generally in the range of 12-15 kDa, characterized by having extremely large extension sequences in both its amino and carboxyl termini. The genomic DNA encoding soyacystatin is also unique in that it consists of four exons with three introns in its coding regions. The mRNA for soyacystatin is distinctly expressed in soybean seeds 2 weeks after flowering. Soyacystatin purified from mature soybean seeds had a molecular mass of about 26 kDa on SDS/PAGE which suggests that it contains the extension sequences. Papain-inhibition experiments demonstrate that this endogenous soyacystatin has almost the same inhibitory activity as that of its deletion mutant (102 amino acid residues) recombinantly produced by truncation of the amino and carboxyl terminal extensions, indicating that the occurrence of the extensions does not affect the cystatin activity. Immunohistochemical experiments reveal that soyacystatin is expressed nearly uniformly in the cotyledons. These results also suggest the possible occurrence of a cysteine proteinase as the target enzyme of soyacystatin.Keywords: cystatin ; cysteine proteinase inhibitor; soybean ; Glycine max.Cystatins refer to proteins that specifically inhibit cysteine proteinases. A number of cystatins of animal origin have been analyzed in detail and classified into three families according to their amino acid sequences [l]. However, cystatins of plant origin have long been unidentified. Oryzacystatins I and I1 from rice seeds are now accepted as the first to have their genomic DNA and cDNA structures characterized [2-51. According to their deduced amino acid sequences, oryzacystatin I and I1 have no disulfide bonds and, therefore, are considered to resemble family 2 cystatins of animal origin [6]. The gene structures of oryzacystatin I and I1 differ from those of animal cystatins, particularly in their intron positions [3, 51. Subsequently, cDNA clones for cystatins were isolated from maize [7] and cowpea [8] and were found to have a structural resemblance to the cDNAs for oryzacystatin I and 11. Recently, a potato multicystatin containing eight inhibitory domains has been characterized [9]. These reports suggest that there are various cystatins to be included in the plant cystatin family.Cystatins have their inhibitory effects on both endogenous and exogenous cysteine proteinases [lo-121. This means that in plants cystatins could play roles in protection against crop Correspondence to T. Misaka, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan 1'13Abbreviations. Bz-Arg-NH-Nap, N-benzoyl-DL-arginine-2-naphthylamide; nt, nucleotide(s); 1 XNaCl/Cit, 0.15...

Corn cystatin I was expressed in Escherichia coli as a mature protein. It was purified by gel filtration on Sephadex G-50, ion-exchange HPLC, and reversed-phase HPLC. The purified protein showed strong inhibitory activities against papain (Ki: 3.7 x 10(-8) M), and cathepsins H (Ki: 5.7 x 10(-9) M) and L (Ki: 1.7 x 10(-8) M), whereas it inhibited cathepsin B to a lesser extent (Ki: 2.9 x 10(-7) M). Western blot analysis using antibody raised against corn cystatin I revealed that in the corn kernel, the protein occurs with a molecular mass of approximately 13 kDa. Localization in the aleurone layer and embryo of the corn kernel was shown by immunostaining microscopy.

Previous studies have identified many cDNA species that encode a variety of G protein alpha-subunits occurring in taste buds. These include the cDNA encoding a taste-bud-specific Galpha, gustducin (G(gust)). Here we carried out comprehensive analyses of Galpha species that occur in the taste buds of rat circumvallate papillae and also in their single cells isolated from the taste buds. Reverse transcriptase-polymerase chain reaction showed the presence of 10 kinds of Galpha cDNAs, including a splice variant of Galphas, among which G(gust), Galphas, Galphai2 and Galphai3 cDNAs were shown to be major species. In situ hybridization and immunohistochemistry showed that Galphai2, as well as G(gust), expressed in a subset of taste bud cells, and the frequency of Galphai2-expression appears to be higher than that of G(gust). Southern analyses of the amplified cDNA from single cells showed that each taste bud cell expresses multiple Galpha mRNA species. For example, some Galphai2-positive cells also express one or more other Galpha species, including Galphas, Galphai3 and G(gust), and there is no apparent correlation in expression among the three Galpha species.

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.