Peptidohydrolases of Soybean Root Nodules

Abstract

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminoptidase, and carboxypeptidase actiities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-LLeu-p8-naphthylamide, while the second hydrolyzed n-benzoyl-L-Arg-/8-, and azocasein. The third endopeptidase hydrolyzed Bz-L-Arg-8NA, Bz-L-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8.AU of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-p-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-pi-naphthylamides. The major activity of two of the aminopeptidases was directed toward L-Leu-and LMet-p-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-.-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classiffed as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.Senescence of soybean root nodules results in a decreased capacity to reduce atmospheric nitrogen, thus potentially limiting the supply of amino nitrogen to the plant. After the decline in nitrogenase activity, total soluble protein per unit fresh weight of nodule decreases rapidly (22), although the fresh weight per nodule shows little change. In addition, the total number of nodules per plant decreases sharply. Thus, nodule senescence can be characterized by a sharp decline in nitrogenase activity coupled with a degeneration of the nodule leading to loss of soluble protein and eventual abscission of the nodule from the root. The loss of soluble protein is a characteristic feature of all types of plant senescence, and is generally attributed to increased activity of proteolytic enzymes. The role of proteolytic enzymes in nodule senescence is not as well established as in leaf senescence (7, 8, 19, '

The Legume Root Nodule: From Symbiotic Nitrogen Fixation to Senescence

“…The acquisition of vacuolar identity by symbiosomes upon senescence likely allows the delivery of newly formed proteases to facilitate nutrient remobilization and a sink-to-source transition. Indeed, nodule senescence is accompanied by increased plant proteolytic activities that might cause large-scale protein degradation in soybean (Malik et al, 1981), French bean (Pladys et al, 1991) and alfalfa (Pladys & Vance, 1993).…”

“…Various proteases, including those of the acid, serine, aspartic and cysteine types, have been isolated from senescing nodule tissue of soybean, alfalfa, French bean, and pea (Kardailsky & Brewin, 1996;Malik et al, 1981;Pfeiffer et al, 1983;Pladys & Vance, 1993;Pladys & Rigaud, 1985). The induction of cysteine protease genes during nodule senescence has been shown in soybean (Alesandrini et al, 2003), Chinese milk vetch (Naito et al, 2000), pea (Kardailsky & Brewin, 1996) and M. truncatula (Fedorova et al, 2002).…”

Insight into the control of nodule immunity and senescence during Medicago truncatula symbiosis

“…These CP genes are specifically expressed in senescent nodules (Fedorova et al, 2002) and the corresponding proteins are involved in proteolytic activities (Malik et al, 1981;Pladys and Vance, 1993). CPs can be inhibited by PHYTOCYSTATINS, which are proteins involved in control of the cellular proteolytic activities during various developmental processes (Martínez et al, 2012;Díaz-Mendoza et al, 2014).…”

Senescence in French‐bean nodules: Occurrence of different proteolytic activities

“…The involvement of proteases in leaf senescence is well-known (Peterson andHuffaker 1975, Ragster andChrispeels 1979), and the occurrence of protease activity in Medicago nodules was first reported by Vance et al (1979). Several endopeptidases have been characterized by disc-gel electrophoresis in functional soybean nodules (Malik et al 1981) and during dark-induced senescence (Pfeiffer et al 1983a). We have purified ao acid protease from functional Phaseolus nodules by ammonium sulfate fractionation, gel filtration and ion exchange chromatography, and suggested that it might use leghemoglobin isolated from these nodules as substrate (Pladys and Rigaud 1982).…”