Leafsegments were ground with a mortar and pestle either with 80% (v/v) ... exoproteinases, the TCA-soluble supernatant was assayed for. NPCs. An aliquot of ...
Received for publication February 9, 1989 and in revised form July 7, 1989
Plant Physiol. (1989) 91, 1232-1237 0032-0889/89/91/1 232/06/$01 .00/0
Increased Proteolysis of Senescing Rice Leaves in the Presence of NaCI and KCI' Seong-Mo Kang and John S. Titus* Department of Horticulture, Gyeongsang National University, Chinju 660-701, Republic of Korea (S.M.K.); and Department of Horticulture, University of Illinois, Urbana, Illinois 61801 (J.S.T.) However, their activity is not always correlated with changes in proteins in situ (7, 11, 19). Also, the interpretation of results from some studies on proteolytic enzymes has been complicated by variations in assay methods (7, 19) and in experimental systems, such as the detachment of leaves (13, 24, 25) and light/dark conditions (25). Compartmentation is an appealing hypothesis in that the major substrate protein is localized in chloroplasts while the majority of the proteolytic activity is in the vacuoles (21, 23, 24, 26, 28) and, during senescence, there is a change in membrane permeability, resulting in increased leakage of solutes (4, 22). However, chloroplastic (1, 8, 12, 17) and vacuolar membranes (23) remain relatively unchanged until the later stages of senescence. Also, recent evidence indicates that chloroplasts contain a variety of proteolytic enzymes (3, 16, 20, 26). In this report, we present evidence that K+ and Na+ ions promote proteolysis in detached rice leaves, and a significant part of the increased proteolysis can be attributed to the increased efflux of protein hydrolyzates. Increased phloem loading and translocation of carbohydrates in the presence of these ions have previously been reported (14, 15, and the references therein); the same conclusion may now be made for nitrogenous compounds.
ABSTRACT
NaCI and KCI enhanced the degradation of chlorophylls and proteins in detached rice (Oryza sativa) leaves in a concentrationdependent manner. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) accounted for 73 to 80% of the protein lost by day 4 in the light. NaCI at 50 millimolar increased proteolysis by 21% over the control in 4 days, but the addition of cycloheximide reduced the increase to about one-half. Cycloheximide alone had no effect on proteolytic activity during this period. Leaf segments taken from 10-day-old seedlings contained the highest proteolytic activity. Both NaCI and KCI increased the activity of Rubiscodegrading endoproteinases (the amount of ninhydrin-positive compounds measured from HCI-hydrolyzates of trichloroacetic acid-soluble supematant), but decreased the activity of hemoglobin- and Rubisco-degrading exoproteinases (the amount of ninhydrin-positive compounds measured directly from trichloroacetic acid-soluble supematant). Efflux of amino acids from senescing leaf segments into the incubation media increased 7- and 12fold in the presence of KCI and NaCI, respectively. The increased efflux resulted in a negative correlation between salt concentration and amino acid content of leaf segments at the later stage of senescence. It is concluded that, in addition to the induction of new proteinase synthesis, the increased efflux of protein hydrolyzates may play a significant role in increasing proteolysis of salt-treated leaves, especially at the later stages of senescence.
MATERIALS AND METHODS Plant Materials and Treatments Rice seedlings (Oryza sativa L. cv Chilsung) were grown in vermiculite in flats. Seedlings were grown under continuous light (180 ,umol m-2 s-') for 16 ± 2 d (except those shown in Fig. 4) at 28 ± 1°C. Five-cm-long subapical segments from 2 mm below the leaf apex were detached from the second true leaves. Plants were allowed to grow for at least 10 d to obtain segments this long. Five detached segments were placed, base down, into test tubes (1.2 x 10 cm) containing 2-mL test solutions. Five mm sodium phosphate buffer at pH 7.0 served as a control. The buffer solution itself promoted senescence to some extent as measured by Chl decline compared with water controls, but it was useful in maintaining the pH of the incubation media. NaCl, KCI, and CYC were made up in the buffer solution. Leaf segments in the test solutions were incubated for 4 to 8 d under continuous light (180 gmol m-2 s-') at 28 ± 1°C. During the incubation, tube racks were covered with a sheet of saran wrap, and no bacterial growth was observed in test solutions.
Leaf senescence and proteolysis have been studied in cereal Rubisco2 is the most abundant substrate protein degraded during senescence (5, 8, 10, 17, 18, 27). The major regulatory mechanisms involved in proteolysis of senescing leaves include: the synthesis of proteolytic enzymes and the changes in their activity during senescence (2, 5-7, 11, 13, 17, 18, 24, 25, 27) and the physical integrity of cellular organelles and the spatial separation between substrate proteins and enzymes (4, 23-25). The synthesis or existence of endoproteinases with acid pH optima has been reported for several plant species (2, 6). crops.
'Supported in part by the Ministry of Education of the Republic of Korea, the Rockefeller Foundation, and by the Illinois Agricultural Experiment Station. 2Abbreviations: Rubisco, ribulose-1 ,5-bisphosphate carboxylase/ oxygenase; CYC, cycloheximide; Hgb-ase, hemoglobin-degrading enzyme(s); NPC, ninhydrin-positive compound; Leu eq, leucine
equivalent.
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PROTEOLYSIS IN SENESCING RICE LEAVES
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Preparations of Cell-Free Extracts
Leaf segments were ground with a mortar and pestle either with 80% (v/v) acetone or with 5 mm sodium phosphate buffer (pH 7.0) (2-mL extractant/5 leaf segments). The homogenate was centrifuged with a Beckman Microfuge, and the supernatant was directly used to determine either Chl or amino acids and proteins. Enzymes were extracted as above, but the supernatant was passed through a PD-10 column (Pharmacia-LKB) to remove small molecules. Isolation and Determination of Rubisco Protein
Rubisco was purified from 12-d-old seedlings, according to the procedure described by Makino et al. (9). The quantity of Rubisco protein was determined by SDS-PAGE as described (7). After staining and destaining, the gels were scanned with an LKB densitometer. The proportion of Rubisco protein to total soluble proteins was determined by adding the areas under the large and small subunits of Rubisco. Enzyme Assays
