Induction and Development of Increased Ion Absorption in Corn Root Tissue

Evidence is presented that K+ uptake in corn root segments is coupled to an electrogenic H+/K+-exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH-/Pi antiporter. Previous studies with excised corn root segments have shown that washing immediately increased K+ uptake activity (5, 19) and membrane potential (15) with uptake increasing by 3-to 7-fold and the membrane potential increasing to a maximum of 25 mv within 4 h. In contrast to K+, Pi uptake (13, 16) increases only 2-3-fold after a 30-min lag period. Based on these washing response data and the study of the effect of dithioerythritol on membrane potential, resistance, and proton fluxes (17), Hanson and Lin (7,17) suggested that active salt transport and its regulation arise from the functioning of a proton-extruding, cation-carrying ATPase in the membrane, balanced by H+/cation and OH-/anion antiporters. If the main driving forces, A I and ApH generated by electrogenic proton pumps (10) MATERIALS AND METHODSCorn seeds (Zea mays L. B73 x Missouri 17) were germinated and 2-cm (0.5-2.5 cm from the tip) root segments from 3-day-old etiolated seedlings were collected and washed as described by Leonard and Hanson (13). The term "washed tissue" in this paper refers to root tissue washed for 4 h at 30 C in a standard medium of0.2 mM K-phosphate (pH 6.0) plus 0.2 mm CaCl2. Pretreatment with inhibitors was carried out in the same medium for an additional 30 min. Uptake rates were measured by incubating 20 cheesecloth-bound root segments (about 0.3 g of fresh weight) for 30 min (except those in the kinetic studies as noted in figures) in 100 ml of either washing solution labeled with 10,000 cpm/ml of carrier-free 3P in standard medium or wRb in 0.2 mM KCI and CaCl2 solution at pH 6.0. The incubation temperature for uptake was kept at 30 C. Washing, pretreatment, and absorption solutions were held in a water bath with constant shaking (50 cycles/min) and forced aeration. Since tissues that have been washed for 4 h, or more, are "stable" in their physiological activities (7,20), most of the experiments were conducted using washed tissues.In the pH experiments 1 mm Hepes was added to the absorption solution and the pH was adjusted with HCI or NaOH. For proton flux measurements, 80 root segments (about 1.2 g fresh weight) were placed directly in 200 ml of aerated standard medium at 30 C and the pH of the solution was monitored continuously. Net proton flux rate at steady-state was calculated as described by Lin and Hanson (15). Respiration of root tissue was determined with an 02 electrode at 30 C with 10 1-cm root sections in 10 ml of airsaturated standard medium as previously described (17).FC was a generous gift from Dr. E. Marre, and DES, mersalyl, and PCMBS were purchased from Sigma Chemical Co. All other chemicals were ACS reagent grade. RESULTS AND DISCUSSIONAging of plant tissue by washing enhances many physiological activities such as ion uptake, respiration, and cell membrane potential (7,20). In the present study, washing corn ro...

Section: Resultssupporting

confidence: 83%

mentioning

confidence: 96%

Potassium and Phosphate Uptake in Corn Roots

Lin¹

1979

“…16), there is a gradual recovery of these physiological attributes; rapid recovery is produced by fusicoccin. Injury also initiates a rapid net K+ efflux from the tissue, which after 45 to 60 min reverses to a net influx (9,13). Initial leakage of K+ from wounded or shocked plant tissues is a commonly observed phenomenon (15,16).…”

Section: ) With Washing (Or 'Adaptive Aging'mentioning

confidence: 99%

Calcium Influx into Corn Roots as a Result of Cold Shock

Zocchi¹,

Hanson²

1982

Corn roots or washed corn root tissue exposed to cold shock absorb 20 to 24% more "Ca2" into a nonexchangeable phase than control roots.Addition to fusicoccin largely prevents this additional calcium influx. The results are discussed in relation to injury-induced changes in nmebrane permeability of root ceOl membranes.Corn roots respond to a variety of injuries (cutting, rubbing, cold shock, or osmotic shock) by rapid blockage of electrogenic H+ efflux pumping, with resultant decline in cell potential and (8'Rb)K' influx (3, 4, 10, 11). With washing (or 'adaptive aging,' Ref. 16), there is a gradual recovery of these physiological attributes; rapid recovery is produced by fusicoccin. Injury also initiates a rapid net K+ efflux from the tissue, which after 45 to 60 min reverses to a net influx (9, 13). Initial leakage of K+ from wounded or shocked plant tissues is a commonly observed phenomenon (15, 16).There are thus two initial responses to the reception and transmission of an injury signal in corn roots; an endogenous control mechanism is activated to shut down the H+-pumping ATPase, and there is an apparent increase in membrane permeability. These phenomena may be related, but there is no firm evidence on this point.A question can be raised about the reality of the increase in membrane permeability. Under the low-salt conditions of experimentation, the K+ is held in the root cells against an electrochemical gradient, presumably by direct or indirect linkage to the H+ pump (5). Depolarization attendant upon injury-induced inhibition of the H+ pump would increase the potential for K+ efflux and reduce the active influx. Hence, net K+ efflux could be realized without change in the physical conductance of the cell membranes to cations. One way of checking this point is to see if a cation which shows a very large electrochemical gradient for influx, such as Ca 2 (12,14), responds by passive influx subsequent to injury. If it does, there is better reason to speak of injury-induced changes in permeability.We report here an investigation of Ca2+ influx in corn roots due to cold shock. Cold shock is very effective in blocking net H+ efflux (3) and avoids the physical stress associated with cutting, rubbing, and osmotic shrinkage. The experiments show that cold shock increases Ca2+ influx.

“…Seedlings were grown on toweling as described in the accompanying paper (25). Inductive washing was by immersion of sections in well aerated 0.2 mm CaCl2 solution.…”

mentioning

confidence: 99%

Increased Membrane-bound Adenosine Triphosphatase Activity Accompanying Development of Enhanced Solute Uptake in Washed Corn Root Tissue

Leonard

Hanson

1972

Self Cite

Washing of excised corn (Zea mays L., variety WF9xM14) root tissue is accompanied by an increase in (Mg2+ + K+)-stimulated adenosine triphosphatase. This is the adenosine triphosphatase described by Fisher, Hansen, and Hodges as positively correlated with ion accumulation rates. The increase in activity is confined to the microsomal fraction. A close parallel exists between increases in adenosine triphosphatase and phosphate absorption, and they respond similarly to inhibitors of RNA and protein synthesis. However, the amplitude of change is much smaller in adenosine triphosphatase. Possible reasons for this discrepancy are discussed.In the preceding paper (25) we describe the increase in absorption rates of phosphate, and of some other ions and solutes, which accompanies the washing of roots from seedlings grown under highly aerobic conditions. After an inductive lag period of about 30 min, there is a rapid development of phosphate absorption rate without significant change in respiration rate.Inhibitor studies indicate that RNA and protein synthesis are involved. However, no increase in membranes with washing could be found by electron microscopy or phospholipid analysis. Therefore, "transport" enzymes synthesized must be added to existing membranes. Since the apparent Km values for '2P and '6Rb absorption do not change, it would appear that existing carrier systems must be augmented or activated.One enzymatic system which is being explored for a possible role in ion transport is membrane-bound ATPase (1, 4, 5, 7-10, 12, 15, 17, 22, 29, 33 Incorporation and Distribution of "4C-Leucine. Root segments were incubated in 500-mg lots for 15 min in 10 ml of 0.2 mM CaCi2 containing 0.1 ,uc/ml of uniformly labeled 4C-leucine (316 mc/mmole). After being rinsed for 30 sec in ice-cold water and for 20 min in ice-cold unlabeled 1 mM leucine, the tissue was ground with a conical glass homogenizer in 0.2 M sucrose. The homogenate was successively centrifuged at 2,000g for 10 min (cell wall and nuclei), 12,000g for 20 min (mitochondria), and 80,000g for 60 min.The pellets were washed by resuspension and recentrifugation and were suspended in 0.2 M sucrose. Protein was precipitated from the supernatant, first in 50% saturated (NH4),-SO4 and finally in 8% trichloroacetic acid. Each fraction was washed by resuspension and centrifugation and finally was suspended in 0.2 M sucrose. Aliquots of the pellets, precipitates, and final supernatant were assayed for radioactivity.Protein content was determined colorimetrically (27