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.

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K(m) at 38 C of 1.8 x 10(-7)m. The K(m) for casein was 1.1 x 10(-6)m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.

Changes in photosynthesis,rlbulose bisphosphate carboxylase (RuBPCase), and proteolytic activity were followed in the leaves of field-grown soybeans [Giycine max (L.) Merr. cv. Kent] from flowering through senescence. These parameters were followed in relation to changes in leaf resistance, chlorophyll, protein, starch, total N levels, and seed development. In addition, changes in leaf ultrastructure were observed. The initial symptoms of senescence (evident 3 to 4 weeks after flowering) were a decline in photosynthesis, chlorophyll, and total leaf N and an increase in proteolytic activity. Preceding these changes there was a swelling of the chloroplasts and a disorientation of the chloroplast lamellae, possibly resulting from the apparent increase in starch deposition. Also, large numbers of osmiophilic granules appeared within the chloroplasts.These changes were evident prior to the time the seed entered its most rapid period of growth which was 4 to 7 weeks after flowering, The initial decline in photosynthesis did not appear to be due to an increase in leaf resistance or a decline in RuBPCase activity or level. The decline in protein levels began between 5 and 6 weeks after flowering and was paralleled by the decline in carboxylase activity and level. Associated with these changes were an increase in the size of the osmiophilic granules within the chloroplasts, a decrease in the number of chloroplasts with a corresponding increase in the apparent cellular breakdown products, and a dissolution of the vacuoles. No large increase in leaf resistance or change in specific activity of carboxylase was observed until late in senescence.

Changes in ribulose bisphosphate carboxylase (RuBPCase) and proteolytic activity were folowed in the flag leaf and second leaf of field-grown winter wheat (cv. Arthur). These changes were foUlowed in relation to changes in leaf chlorophyll, protein, and photosynthesis, and seed development. Levels of RuBPCase were determined by rocket immunoelectrophoresis as described previously (Wittenbach 1978 RuBPCase in the flag leaf and the leaf immediately below it have been followed from full leaf expansion through senescence. These changes have been followed with respect to changes in leaf Chl, protein, and photosynthesis. In addition, proteolytic activity in these leaves was followed, since these enzymes are responsible for the breakdown of RuBPCase. MATERIALS AND METHODSPlant Material. Samples of flag leaves, second leaves (leaves immediately below the flag leaves), and seed heads were collected from field-grown winter wheat (Triticum aestivum L. cv. Arthur) beginning 1 week prior to anthesis and ending at harvest during the 1977 and 1978 seasons. All collections and photosynthesis measurements were made between 2 and 4 PM. Samples were immediately frozen in liquid N2 and transported to the lab on dry ice. They were then stored in liquid N2 until assayed. Freezing in liquid N2 had no significant effect on RuBPCase activity, and no activity was lost following storage for at least 1 month.Photosynthesis and RuBPCase Assays. Photosynthetic measurements were made using a 14C02-pulsing apparatus similar to that of Naylor and Teare (1 1). All determinations were made on clear days in full sunlight (-1,800 ,uE/m2. s) with the temperature between 22 and 30 C. Leaves were pulsed for 15 s with air (364 ,ul/l CO2) containing 14CO2. Leaf discs were removed with a cork borer, placed in scintillation vials with I ml of tissue solubilizer, and left ovemight on a shaker to digest. Counting solution was then added and the dpm determined by liquid scintillation spectroscopy.RuBPCase activity was determined by following 04C02 incorporation into acid-stable products as described previously (16). Leaf samples of 300 mg were extracted in 5 ml of 25 mm Hepes (pH 7.5) containing 4 mm DTT, 1 mm Na2EDTA, and 1% (w/v) PVP using a Polytron homogenizer. The extracts were centrifuged at 30,000g for 20 min, and the supernatant fractions were used to assay for RuBPCase activity.The levels of RuBPCase were quantitated using the procedure of rocket immunoelectrophoresis (15

In conjunction with a study of the effects of ear removal on the senescence of whole maize (Zea mays L.) plants, visual symptoms and associated changes in constituent contents and activities of a selected leaf (first leaf above the ear) were determined. Leaves were sampled from field-rown eared and earless Pioneer brand 3382, B73 x Mol7, and Farm Services brand 854 maize hybrids at nine times during the grainfilling period.Visual with the attainment of full leaf expansion, appears to predispose the leafto senescence; however, the subsequent rate ofsenescence development can be affected by environment, growth regulators, ammonium nitrate (at least in Nicotiana), and genotype (14). With maize, another metabolic change associated with full leaf expansion was that the entry of nitrate into the leaf was greatly diminished or terminated (9). Although the changes in constituents and metabolic activities (loss of Chl, protein, enzyme activities, and accumulation of carbohydrates) have been intensively studied and associated with senescence, they are judged as symptoms rather than the cause of senescence.The objectives of the current study concerning senescence of a selected leaf of maize during the grain-filling period were to (a) measure changes in selected metabolic parameters previously associated with senescence, (b) compare and contrast these patterns ofchange for eared and earless hybrids that exhibit different patterns of visual senescence, and (c) identify the trait that was most closely associated with effective leaf area duration of the three hybrids. MATERIALS AND METHODSCultural Procedures. Cultural procedures, including ear removal and experimental design, were as described (5). Of the three maize hybrids used, Pioneer brand 3382 and Farm Services brand 854 are classed as 'stay-green' cultivars because their leaves remain green, while leaves of B73 x Mol7 are brownish yellow by the time of grain maturity.Sampling. Plants were sampled between 0900 and 1100 h, nine times during the grain fill period; 4, 11, 19, 25, 30, 37, 44, 54, and 60 DAA.2 Anthesis occurred at approximately July 12. At each sampling time, the selected leaf(leafabove the ear) from each of three representative plants per plot were combined for a sample to provide a total of 30 samples (five replications, six treatments). Leaves were placed on ice for transport to the laboratory. For each sample, the three leaves were stacked and folded once. A cork borer was used to cut discs (a total of 42 discs, 0.785 cm2 disc-') from the midportion ofthe laminae. The disks were transferred to a preweighed vial, reweighed, frozen with liquid N2, and stored at -20°C until extracted for gel electrophoresis. The midribs of the three leaves were then removed and discarded, and the laminae were chopped into 1 x 2 cm sections and thoroughly mixed, and subsamples were taken