Removing the pods from soybean (Glycine max [L.] Meff. cv Wye) plants induces a change in leaf function which is characterized by a change in the leaf soluble protein pattern. The synthesis of at least four polypeptides (-27, 29, 54, and 80 also demonstrated a marked increase in the accumulation of protein within the vacuoles of these cells following pod removal. Therefore, the polypeptides that accumulate following depodding may be localized in these cells.The purpose of this study was to further characterize these polypeptides and to purify one or more to obtain antiserum for quantitation and cellular localization work. This paper describes the purification and antiserum production to a glycoprotein apparently composed ofa 27 and 29 kD polypeptide. In addition, quantitation of this protein with development and further characterization of its accumulation are presented. MATERIALS AND METHODS Plant Material. Soybean (Gycine max [L.] Merr. cv Wye[determinate]) plants were grown as previously described (12). Depodding was initiated 1 week after flowering and was repeated at weekly intervals.Protein Purification. Trifoliate leaves were harvested from the main stem of plants which had been depodded for 4 to 5 weeks. The leaves were extracted in 20 mM Tris-HCI buffer (pH 7.6) containing 4 mM DTT and 1 mM EDTA (4 ml: 1 g of tissue) at 4°C using a Waring Blendor. The homogenate was centrifuged at 30,000g for 20 min, and the supernatant fractions were decanted and combined. Saturated (NH4)2SO4 was added to the supematant fraction to bring it to a concentration of 2.0 M and, after standing for 30 min on ice, the solution was centrifuged at 12,000g for 10 min. The precipitate was discarded and the supernatant solution was brought to 2.8 M (NH4)2SO4. After centrifugation, the precipitate was dissolved in 100 mm acetate buffer (pH 5.6) containing 0.9% NaCl. This solution was applied to a Con A-Sepharose column (1 x 5 cm) equilibrated with the resuspension buffer. The column was washed with buffer until no more protein was eluted; then 50 mM l-O-methyl-a-D glucopyranoside in the same buffer was added to elute the bound protein. The fractions containing the bound protein were then applied to a Sephacryl S-200 column (5 x 105 cm) equilibrated with 0.9% NaCl in 20 mm Tris-HCl buffer (pH 7.6). One major peak (A280) was obtained, and the fractions from the center of this peak were combined and concentrated for antiserum production and standardization of the quantitative radial immunodiffusion assay.Production of Antiserum. The purified protein was emulsified in complete Freund's adjuvant. Mice (C57/BL6) were immunized initially by footpad injection with 50 ,ug protein/mouse. Subsequent boosts after 2 and 3 weeks were done intraperitoneally using 10 and 5 gg protein, respectively, plus 1 mg of Alhydrogel/mouse. The mice were then bled 1 week later by retroorbital puncture to obtain antiserum. Thereafter, the mice were given boosts of 1 to 1.5 ,ug protein every 2 to 3 weeks and were bled 1 week after each boost for col...
MesophyH protoplasts isolated from primary leaves of wheat seedlings were used to folow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at al stages of senescence. Al the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.Leaf senescence has often been referred to as a well-organized event (2, 4) because of the sequential loss of organelles with their corresponding function during senescence. Yet, evidence is lacking on how these changes might be controlled. If MATERIALS AND METHODSPlant Material. Wheat (Triticum aestivum L. var. Arthur) seeds were planted in vermiculite in 6-cm pots and held in a growth cabinet at 20°C and 75% RH. The photoperiod was 16 h, with a quantum flux density of 250 ,uE m s'. After 10 days, the second leaf had just emerged and the first leaf was fully expanded. The seedlings were then transferred to a dark room at 22°C to induce senescence. At different times, sections were taken from the primary leaves (8-cm sections cut 2 cm from the tip) and used for protoplast isolation.Protoplast, Chloroplast, and Vacuole Isolation. Protoplasts were isolated as previously described (6), except that 0.1% pectolyase Y-23 was substituted for prectinase and hemicellulase in the digestion enzyme mixture (5), which shortened the incubation time to 1 to 1.5 h. Chloroplasts were isolated according to the procedure of Robinson and Walker (10), as outlined previously (6).Vacuoles were isolated from protoplasts according to a modification of the method of Wagner (11). Twenty-eight ml of 0.5 mm DTT in 170 mm phosphate buffer (pH 8.0) were added to the centrifuge tube containing the protoplast pellet. The mixture was gently stirred for 5 min, poured through three layers of glasswool, and centrifuged at 200g for 3 min to spin down the majority of the protoplasts. The supernatant was transferred to a beaker, and 12 ml of 60o sucrose (w/v) were added to form an 18% sucrose mixture. The mixture was then divided into two centrifuge tubes, and 6 ml o...
Well nodulated, field-grown soybeans (Glycine max [L.] Meff. var Williams) were depodded just prior to seed development and near mid pod-fill. Both treatments caused a considerable increase in leaf dry weight, suggesting continued photosynthate production following pod removal. Moreover, depodding had a marked effect on leaf soluble protein without affecting total proteolytic activity. Early depodding caused a 50% increase in leaf protein, and both early and late depodding caused the retention of protein for several weeks following the decline in control leaves. But despite this retention of protein, leaves of depodded plants showed no difference in the onset of the irreversible decline in photosynthesis. Therefore, although depodding delayed the loss of leaf chlorophyll and protein, it did not delay the onset of functional leaf senescence and in fact, actually appeared to enhance the rate of decline in photosynthesis. There was a good correlation between the irreversible decline in ribulose bisphosphate carboxylase (activity and amount) and that of photosynthesis. In contrast, the correlation did not seem as good between stomatal closure and the onset of the irreversible decline in photosynthesis. The reason total soluble protein remained high following depodding while carboxylase, which normally comprised 40% of the soluble protein, declined was because several polypeptides increased in amounts sufficient to offset the loss of carboxylase. This change in leaf protein composition indicates a change in leaf function; this is discussed in terms of other recent findings.Senescence of soybean leaves is normally characterized by a decline in photosynthesis and the loss of leaf protein and Chl (12), leading to death of the leaf. However, recently it was shown that following pod removal, the leaves lose the ability for photosynthesis but retain high levels of Chl and protein (7, 10), indicating a separation of functional senescence from death of the leaf. Therefore, removing the pods from soybeans apparently does not delay functional senescence of the leaves as has been claimed (5). Instead, depodding causes a change in the soluble protein pattern of the leaf suggesting a change in leaf function (10). The leaf appears to change from a photosynthesizing source organ to a sink organ.In the previous growth room study (10), Wye soybeans, a determinate variety, were grown with applied N which resulted in poor nodulation. Under these conditions, the plants had essentially only one sink, the pods, following flowering. Removing this sink caused a rapid decline of photosynthesis and a 'Contribution No. 3184
Senescence of soybean leaves is characterized by a decline in photosynthesis and the loss of leaf protein and Chl (11,22). Clearly, the most dramatic visual symptom is leaf yellowing, and because of this, it is widely used as an index of plant and leaf senescence. From our field studies with winter wheat (21) and soybeans (22), loss of Chl appears to be a good initial index of leaf senescence. Yet caution must be taken in using this as the only indicator of senescence since Chl loss is not always an inevitable event in senescence (18,19).Over 50 years ago, Molisch (10) recorded the observation that plants delayed in the reproductive stages showed delayed senescence. Leopold et al (7), following up on this report, were able to demonstrate a marked delay in soybean leaf and plant senescence following the continuous removal of either the flowers or pods. In recent years, this response in soybeans has been investigated further in an attempt to understand the 'senescence signal.' Lindoo and Nooden (9) were able to duplicate the pod removal effect by only removing the seeds from the pods, indicating that the senescence signal was associated with the developing seeds. The same laboratory (13) later provided evidence which separated seed dry matter accumulation from the senescence response, thereby contradicting the theory that seeds caused senescence by diverting or withdrawing needed nutrients from the leaves.Although these studies on pod and seed removal imply a maintenance of normal leaf function, this has never been conclusively demonstrated. In fact, Mondal et al. (1 1) found that depodding soybeans partially inhibited and caused photosynthesis to decline earlier than in control, podded plants.
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