Subcellular Localization of Proteases in Wheat and Corn Mesophyll Protoplasts

Abstract

proteolytic activity was associated with the chloroplasts of wheat (16). However, the intactness of the chloroplasts in this study was questionable. Much better methods are now available for the isolation of protoplasts (3, 5) and chloroplasts (13) from cereal leaves. Using these methods and a modification of the techniques for isolating plant vacuoles (14, 15), we have studied the localization of RuBPCase2 degrading enzymes in the mesophyll protoplasts of wheat and corn leaves. MATERIALS AND METHODS Plant MateriaL Corn (Zea mays L. B73 x Missouri 17) seedswere disinfected with 20% Clorox prior to planting in pots contaming Jiffy-Mix and sand (3:1, v/v). The pots were maintained in a growth room at 27 C and 75% RH during the day (17 h photoperiod, 1,200 ft-c) and at 20 C and 63% RH overnight.Wheat (Trticwm aestivwm L. var. Chris) seeds were planted in vermiculite and held in a growth cabinet at a constant temperature of 20 C and 70% RH. The photoperiod was 18 h with a light intensity of 1,200 ft-c.Protoplast Isolation. Deveined second and third leaves of 17-to 20-day-old corn plants and primary leaves from 8-to 10-day old wheat plants were cut into 1-to 2-mm wide sections and plasmolyzed with 0.65 M sorbitoL 10 mM CaCl2, 0.5 mm DTT, and 25 mm Mes buffer (pH 6.0) (3). The sections were then transferred to the enzyme mixture (8) consisting of 2% Cellulysin (Calbiochem), 1% hemicellulase (Sigma), 0.5% pectinase (Sigma), 0.6 M mannitol, 0.05% BSA, 0.5 mmt DTT, and 0.2 mm CaCl2 at pH 5.5 (10 ml/g leaves). The sections were infiltrated under a partial vacuum (sufficient for leaf sections to sinkc after the release of the vacuum) for 3 to 5 min and then incubated in a water bath at 30 C for 2 h with constant shaking (40 cycles/min).After enzymic treatment, undigested large pieces were removed by filtering the mixture through a nylon net funnel and crude protoplasts were collected by centrifugation at 300g for 3 min. The protoplasts were purified by a modification of the procedure by Edwards et al. (5). Three ml of 0.5 M sucrose, I mm CaCl2, and 0.5 mm DTT were added to each tube, and the crude protoplast pellet was resuspended by gentle shaking. Then S ml ofthe same sucrose solution were added to each tube and thoroughly but gently mixed by inversion. Six ml of a solution containing 0.4 M sucrose, 0.1 M sorbitoL I mm CaCl2, 0.5 mm DTT, and S mm Mes-KOH buffer (pH 6.0) were then layered onto this, followed by 3 ml of 0.5 M sorbitoL I mm CaCl2, 0.5 mm DTT, and 5 mm Mes-KOH (pH 6.0). After centrifugation at 200g for 5 min, the protoplasts were located in a band between the layers of sorbitol. They were removed, diluted with 20 ml of the top sorbitol solution, and pelleted by centrifugation at 300g for 3 min. Photosynthetic activity of the protoplasts was measured according to the procedure of

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

“…1). A good yield of intact chloroplasts was obtained (peak fractions (10)(11), with only a trace of broken chloroplasts (fractions [17][18]. Although considerable quantities of mitochondria and peroxisomes were occluded within the intact chloroplast band, chloroplast-free zones of peroxisomes (fractions 5-8) and mitochondria (fractions 13-16) were also located in the gradient.…”

“…Characterization of these enzymes (5,15,22,29) has shown that, despite their common role in protein breakdown, they have few similarities with respect to the conditions required for optimal in vitro activity. These differences, particularly differences in optimum assay pH, have led to suggestions of enzyme compartmentalization as a means of regulating protein degradation, since the plant cell contains a number of localized pH zones by virtue of its various organelles.Although acid proteinases are known to be localized in leaf vacuoles (3,11), no other peptide hydrolases have been reported there. Carboxypeptidase activity has been located in protein bodies from the cotyledons of germinating mung beans (7,27) and in vacuoles prepared from the endosperm tissue of 4-d-old castor bean seedlings (18) but has not been reported in vacuoles isolated from leaf tissue.…”

“…Although acid proteinases are known to be localized in leaf vacuoles (3,11), no other peptide hydrolases have been reported there. Carboxypeptidase activity has been located in protein bodies from the cotyledons of germinating mung beans (7,27) and in vacuoles prepared from the endosperm tissue of 4-d-old castor bean seedlings (18) but has not been reported in vacuoles isolated from leaf tissue.…”

Inhibition of Anion Transport in Corn Root Protoplasts

“…2). To avoid possible adverse effects on the surface proteins of the protoplast by proteases from the cell wall digestion enzyme mixture (15), 0.05% BSA and 10 mM DIFP, a specific inhibitor for fungal serine proteases (15), were added to the digestion enzyme mixture.…”

Senescence in wheat leaves: is a cysteine endopeptidase involved in the degradation of the large subunit of Rubisco?

“…Beside this, mRNAs encoding for cysteine proteases were increased or were retained during senescence (Hensel et al 1993;Lohmann et al 1994;Smart et al 1995). It has been shown that Rubisco degrading proteases exist in the vacuole (Lin and Wittenbach 1981;Thayer and Huffaker 1984;Bhalla and Dalling 1986). Yoshida and Minamikawa (1996) suggested the involvement of at least two proteases in the degradation of purified Rubisco by vacuolar lysates.…”