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...

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The pathway of phloem unloading and the metabolism of translocated sucrose were determined in corn (Zea mays) There are several possible pathways for assimilate unloading in sink regions (Fig. 1). One possibility is that sucrose exits the phloem via either a passive, facilitated, or energy-dependent transfer step and enters the apoplast. Sucrose can then either be hydrolyzed by a cell wall invertase to hexoses which are then accumulated by hexose-specific carriers in adjacent consuming cells or sucrose can enter without hydrolysis via a sucrose-specific carrier. Alternatively, assimilates can be unloaded via a symplastic route through plasmadesmata. In the latter scheme, intracellular hydrolysis of sucrose could occur within the cytoplasm or vacuole depending on the intracellular location of invertase activity.

Inoculation of corn (Zea mays) seeds with Azospirillum brasilense strain Cd or Sp 7 significantly enhanced (30 to 50% over controls) the uptake of N03 , K+, and H2PO4 into 3-to 4-dayand 2-week-old root segments. No gross changes in root morphology were observed; altered cell arrangement in the outer four or five layers of the cortex was seen in photomicrographs of cross sections of inoculated corn roots. The surface activity involved in ion uptake probably increased, as shown by the darker staining by methylene blue of the affected area. Shoot dry weight increased 20 to 30% in inoculated plants after 3 weeks, presumably by enhancement of mineral uptake. Corn and sorghum plants grown to maturity on limiting nutrients in the greenhouse showed improved growth from inoculation approaching that of plants grown on normal nutrient concentrations. Enhanced ion uptake may be a significant factor in the crop yield enhancement reported for Azospirillum inoculation.

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MATERIALS AND METHODSThe effects of several amino-reactive disulfonic stilbene deriatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Clr, S042-, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4'-isothiocyano-2,2'-stilbenedisulfonic acid, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, 4,4'-diamino-2,2'-stilbenedisulfonic acid, and NAP-taurine inhibited Cl and S042-but not Pi and K+ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl1 or S04-2 uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl and S042-transport inhibitors.A growing interest has emerged on the nature of anion transport in several biological systems. For example, carrier proteins for phosphate, chloride, and sulfate transport have been recently identified and isolated from bacteria (1, 20), fungi (6), and human red blood cells (see reviews 10, 24). In the red blood cell studies (2-4, 8, 11, 17), several nonpermanent, amino-reactive reagents (e.g., SITS,2 DIDS, DADS, and NAP-taurine), which covalently bind to the plasmalemma, have been used to identify and characterize the band 3 membrane protein as the carrier for CF-and s042-transport.Virtually no information is available on the nature of the carrier proteins responsible for ion transport in plant cells, particularly root cells which are the major sites of entry for ions into the plants. The lack of such information is due, in part, to the lack of suitable technique for isolating functional protoplasts from roots which are, by their nature, more amenable to techniques that have been used to characterize membrane transport. Recently, we developed techniques for the large scale and rapid isolation of protoplasts from corn roots (13) which are capable of transporting several ions (K+, H2PO4 , and H+). In this study, several chemical modifiers which have been used to study anion transport into red blood cells were employed to study anion transport into corn root protoplasts with the intent of providing further insight into anion transport in plant cells. Delaware, 19801. 2Abbreviations: SITS, 4-acetamido4'-isothiocyano-2,2'-stilbenedisulfonic acid; DIDS: 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid; DADS, 4,4'-diamino-2,2'-stilbenedisulfonic acid; NAP-taurine, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate; FCCP, (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone; DIFP, diisopropylfluorophosphate.A modification of our previously reported method (13) was used to isolate protoplasts from young corn roots. As outlined in Figure 1, 0.1% pectolyase Y-23 (16) substituted for pectinase and hemicellulase in the digestion enzyme mixture. Also, the tissue incubation time in the digestion mixture was shortened to 1.5 to 2 h and the precentrifugation steps were omitted. The yield of 106 protoplasts/g root tissue and...

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

The correlations between ATP concentration in corn (Zea mays) root tissue and the rate of phosphate absorption by the tissue have been examined. Experimental variation was secured with 2, 4-dinitrophenol, oligomycin, mersalyl, L-ethionine, 2-deoxyglucose, N2 gassing and inhibition of protein synthesis. It is concluded that ATP could be the energy source for potassium phosphate absorption, but only if the transport mechanism possesses certain properties: oligomycin-sensitivity; creation of a proton gradient susceptible to collapse by uncouplers; phosphate transport via a mersalyl-sensitive Pi--OH-transporter; good activity at energy charge as low as 0. This question is receiving a good deal of attention (9,10,15,16,19,28,37), and it seems to be widely believed that the requirement for aerobic respiration or light can be explained in terms of ATP production, at least for cation uptake. The recent work of Hodges and associates demonstrates that a Mg-requiring, cation-stimulated ATPase is present on plasmalemma from oat roots (20,26,27), and that the correlation between ATPase activity and ion uptake is exceptionally good (12 (2,32) could find no correlation between ATP levels in washed carrot and beet tissue and the rates of K+, Na+ and Cluptake. Experimental variations were obtained with anaerobiosis, uncouplers, inhibitors of ATP formation, and adenosinetrapping agents. In washed beet root tissue with low concentrations of salt, anaerobiosis rapidly depressed salt uptake without affecting ATP concentrations significantly. Conversely, L-ethionine lowered ATP concentrations with little depression of ion uptake. Uncouplers gave inhibition of both ion uptake and ATP concentration, but the inhibition of ion uptake preceded the extensive depletion of ATP. Inhibitors of ATP production, such as oligomycin or Dio-9, had a delayed effect on ATP concentrations and ion uptake. Polya (31) found no evidence directly linking protein synthesis to ion uptake, although indirect effects were suggested. Polya and Atkinson (32) believe their results are consistent with transport mechanisms directly involving the mitochondria and oxygen-terminated electron transport.Cram (8) studied active C1-influx into washed carrot root discs and concluded that uncouplers and oligomycin affected only transport into the vacuole. The Cl-influx pump at the plasmalemma was deduced to be more closely linked to the redox reactions of the respiratory chain than to ATP production, thus supporting Atkinson and Polya.Here we report experiments similar to those of Atkinson and Polya and with the same end in mind, but using washed corn root tissue. The correlation obtained between phosphate absorption rates and ATP concentrations is highly dependent on the treatment used to vary ATP. However, in our opinion, the results are not inconsistent with ATP being the basic energy source for active phosphate transport, provided that the mechanism has certain properties.MATERIALS AND METHODS Two-centimeter segments of primary root of corn seedlings (Zea inays L...