Abstract
We screened a cDNA library of germinating rice seeds with a cDNA for aleurain (cysteine proteinase from barley) and obtained three distinct types of cDNA clones encoding three species of cysteine proteinases (oryzains alpha, beta, and gamma). The deduced amino acid sequences are distinct in part, but, on the whole, are similar to one another. The three sequences all contain the catalytic triad Cys25-His159-Asn175 (papain numbering). The three oryzains are similar to one another and also to other known cysteine proteinases such as papain and cathepsin H with respect to the sequences around the active site residues and the COOH-terminal Trp-rich region. Amino acid sequence comparison revealed that oryzains alpha and beta are similar not only to each other (70% similarity) but also to actinidin and papain (about 50%), whereas oryzain gamma was rather similar to aleurain (85%) and cathepsin H (60%). Northern blot analysis revealed that the mRNAs for the three oryzains are expressed only in seeds, not in shoots or roots, and show different expression profiles during germination and when the seeds are treated with gibberellic acid. Oryzains alpha and gamma are expressed continuously during germination with a maximum expression 5 days from the start of germination, but are present in neither ripening nor ripened seeds. On the other hand, oryzain beta is expressed not only during germination, but also in ripened seeds before germination. It was noted that the expression of the three oryzain mRNAs is enhanced in different manners by gibberellic acid but is not enhanced by other plant hormones such as auxin. The induction of oryzain beta mRNA is transient, reaching a maximum in 4 h from the addition of giberellic acid and diminishing rapidly thereafter, while the induction of oryzain alpha and gamma mRNAs continues over 5 days. Thus, multiple systems involving cysteine proteinases must be differentially involved in the germination process, probably under hormonal control.
Highlights
We screened a cDNA library of germinating rice already well characterized in animals [14], and refined enzyseeds with a cDNA for aleurain anodbtained three distinct types of cDNA involving cysteine proteinases such as cathepsin H, L, and B, clones encoding three species of cysteine proteinases and cystatins such as stefin A, B, and cystatin 8
We have found a cysteine proteinase in germinating rice seeds whose expression reaches a maximum 5 to 8 days after the startof germination [15]and is enhanced by gibberellic acid (GA,)’ [16]
Isolation and Identification of cDNA Clones for Cysteine Proteinases-Among 1.5 X 10' independent plaques from the XgtlO cDNA library, approximately 20 clones were hybridized with aleurain cDNA
Summary
Applied Biosystems) to give three sequences, Asp-Glu-Arg-Xaa-Asp- ase by partial digestion (see "Experimental Procedures"), tVlTyhyazerle--sdALetqyshnuse,-eAaNnncrHgde-2LLL-eetyeuusr--,mXPPairrnaooa---lGsGGlellqyuuu---eXXXnaaacaaea--o-IAfAlstsehpp-e--ATpsTruprrp-pi-fT-iAheArdrrg-gp-Gr-XolXatuaea-ai-nX-LaLWawyanys-heds,Ai.caoshlbpsot-caoTainrynreread---Prooii-ndfedXnicOtiafZtiieBndlgObt2yh,apetnrooctroeydiznianigcnhoaerymisziatshitnreypc(,y1cs5ot)en.iTtnaheinespnruaocstelieniongtaliesdeoepprseeevnqiuoreeunasdclyesponds to thethird sequence described above. The signal sequences were estimated based on the general characteristic that a signal sequence contains a charged residue within the first five amino acids followedby a core of at least 9 hydrophobic the RNAwas transferredontoa Nylon membrane By this general rule, as well as from the hy-. Amersham) and hybridized with "P-labeled cDNAs a t 65 "C in rapid hybridization solution (Amersham).The filter was washed in 0.1 X SSC containing 0.1% SDS at 65 ' C. dropathy profiles (Fig. 3), we assigned possible signal sequences at Metl-Ala", Met'-Ala", and Met'-Ala24for oryzains a, j3, and y, respectively
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