Author/Editor		Kern, Nadja
Title		Vloga proteaz in proteaznih inhibitorjev v listih fižola Phaseolus vulgaris (cv. Češnjevec) v odgovoru rastline na stres
Translated title		Role of proteases and protease inhibitors in leaves of bean Phaseolus vulgaris (cv. Češnjevec) in plant response to stress
Type		monografija
Place		Ljubljana
Publisher		Univerza v Ljubljani, Medicinska fakulteta
Publication year		2000
Volume		str. 81
Language		slo
Abstract		With the aim of elucidating involvement of proteolytic enzymes in plant response to water deficit, activities of proteinases in control and drought stressed leaves of Phaseolus vulgaris, cv. Češnjevec, were compared. The physiological state of plants was determined by measuring water potential in the first trifoliar leaves. Activities of individual groups of proteinases in extracts were measured using synthetic substrates. Enzymes were partially purified by precipitation in 50 % acetone, fractionated by gel filtration and further characterized using specific inhibitors. A serine proteinase FLSP with molecular mass of about 55 kDa increased four times in drought in respect to control. A cysteine proteinase FLCP with molecular mass of 25-30 kDa increased less significantely in drought in respect to control. FLSP and FLCP were further fractionated by gradient native electrophoresis and stained for activities. N-terminal aminoacid sequence APENVLE*VKQDNS, determined in FLCP active bend, showed sequence similarity to other cysteine proteinases with proline in the second position. Based on the N-terminal aminoacid sequence of phytocystatin FLCPI-2 from leaves of Phaseolus vulgaris and the sequence QVV(AS)GT, conserved in related leguminous phytocystatins, degenerate primers were constructed and used in RT-PCR reaction to synthesise a 138 bp cDNA fragment. The deduced aminoacid sequence of inhibitor FLCPI-2 was obtained from the cDNA sequence. The sequence contained the LARFAVEEHN motiv, which is specific for phytocystatins, and showed 91 % identity with soyabean phytocystatin (CPI Gmal) and 89 % identity with phytocystatin from leguminous species Vigna unguiculata (CPI_Vun). It is concluded that FLCPI-2 is a novel phytocystatin from leaves of Phaseolus vulgaris. The DNA fragment with known sequence was used as a probe to study transcriptional levels of phytocystatin FLCPI-2 under drought stress by hybridization. (Abstract truncated at 2000 characters).
Descriptors		LEGUMES CYSTEINE PROTEINASES SERINE PROTEINASES CYSTEINE PROTEINASE INHIBITORS SERINE PROTEINASE INHIBITORS PLANT LEAVES WATER GERMINATION ELECTROPHORESIS, AGAR GEL POLYMERASE CHAIN REACTION AMINO ACID SEQUENCE BASE SEQUENCE TRANSCRIPTION, GENETIC