Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells

The ATP content of 7-day-old A vena sativa leaves during senescence in dark and in light, and after treatment with cytokinins and other reagents, has been determined by the luciferin-luciferase method. Special care was taken to avoid decomposition of the ATP, and a detailed procedure is presented for ATP analysis at the picomole level. Preliminary experiments with several inhibitors of photophosphorylation suggest, though not conclusively, that the delaying effect of light on senescence is mediated by photophosphorylation. The ATP values of the leaves senescing in darkness are found to increase in parallel with the large increase in respiratory rate, and kinetin prevents this increase just as completely as it prevents the respiratory rise. It is concluded that the respiratory increase in senescence cannot be simply due to uncoupling. In light the ATP level also rises, though more slowly, and again kinetin prevents this rise. L-Serine, which promotes dark senescence, does not significantly modify the dark ATP level, but both arginine and kinetin, which antagonize the action of serine on senescence, greatly lower the ATP level below that on serine alone. Cycloheximide has a similar effect, and the combination of cycloheximide and kinetin lowers the ATP level drastically. Fusicoccin, which opens stomata in the dark, correspondingly maintains the ATP at a low level. Thus, in general, a low level of ATP is associated with the prevention of dark senescence, ie. probably with ATP utilization, and the ATP level at any time may thus be determined more by the rate of utilization than by the efficiency of respiratory coupling.It is well understood that leaf senescence is dominated by hydrolytic processes. In the darkened oat leaf proteolysis begins within 6 h and polysaccharide breakdown soon follows (25). The increased activity of ribonuclease in leaves of several species was also an early observation (9,28,34) These and other observations could be taken to indicate some relationship between leaf senescence and the products of phosphorylation. It was not clear whether oxidative phosphorylation in the dark and photophosphorylation in the light act in the same way, nor was it at all clear just what the nature of the postulated relationship actually is. As a first step toward clarification of these difficult questions, it was felt essential to determine the levels of ATP during senescence in both darkness and light and to follow the effects on leaf ATP of some of the agents and treatments that modify the senescence process. This paper reports those determinations. A preliminary study has also been made of several inhibitors of photophosphorylation to see whether they modify senescence in light. Two of these inhibitors do in fact interfere with the light effect on senescence and thus the results give general support to the idea that phosphorylation exerts a controlling influence on senescence. MATERIALS AND METHODSA survey of the literature showed that for small samples of plant material the luciferin-luciferase meth...

Section: Discussioncontrasting

confidence: 36%

Metabolism of Oat Leaves during Senescence

Malik¹,

Thimann²

1980

“…The reported preferential alkaline pH for nitrite uptake into chloroplasts (3) also confficted with the characteristic acidic pH optima for anion transport (4,16,22). This anomaly of anion uptake at alkaline pH values has been recognized by other workers (4, 14,27,28) and was clarified by the proposed existence of relatively specific, pH sensitive, anion channels in biological membranes (4,14,27,28).…”

mentioning

confidence: 64%

Nitrite Uptake into Intact Pea Chloroplasts

Brunswick

Cresswell²

1988

ABSTRACIThe relationship between net nitrite uptake and its reduction in intact pea chloroplasts was investigated employing electron transport regulators, uncouplers, and photophosphorylation inhibitors. Observations confirmed the dependence of nitrite uptake on stromal pH and nitrite reduction but also suggested a partial dependance upon PSI phosphorylation. It was also suggested that ammonia stimulates nitrogen assimilation in the dark by association with stromal protons. Inhibition of nitrite uptake by N-ethylmaleimide and dinitrofluorobenzene could not be completely attributed to their inhibition of carbon dioxide fixation. Other protein binding reagents which inhibited photosynthesis showed no effect on nitrite uptake, except for p-chlormercuribenzoate which stimulated nitrite uptake. The results with N-ethylmaleimide and dinitrofluorobenzene tended to support the proposed presence of a protein permeation channel for nitrite uptake in addition to HNO2 penetration. On the basis of a lack of effect by known anion uptake inhibitors, it was concluded that the nitrite uptake mechanism was distinct from that of phosphate and chloride/sulfate transport.

“…4) suggested that transport required an energized membrane. Oligomycin and uncouplers also have been shown to inhibit rapidly the influx of inorganic ions into protoplasts from tobacco suspension cells (29) and to depolarize the membrane potential of oat leaf protoplasts (34). It is not clear whether the mechanism of oligomycin action involved inhibition of the plasmalemma or mitochondrial ATPase.…”

Section: Methodsmentioning

confidence: 99%

“…It is postulated that the plasmalemma ATPase maintains the energized state of the membrane (25,36) and, thus, maintains the sugar transport system in a mobile condition. In vivo, the ATPase may also be involved in uptake and, hence, recycling of K+ (29). The precise stoichiometry of K+ and sugar release is not known.…”

Section: Methodsmentioning

confidence: 99%

Co-transport of Potassium and Sugars across the Plasmalemma of Mesophyll Protoplasts

Huber¹,

Moreland²

1981

Sugars (sucrose + hexoses) produced photosynthetically by isolated mesophyll protoplasts of wheat and tobacco were effluxed across the plasma membrane (3 to 10 micromoles hexose equivalents per milligram chlorophyll per hour). The effiux was sensitive to uncouplers and oligomycin which indicated a requirement for energy. A proton gradient was probably not coupled directly to the transport because changing the proton gradient across the plasma membrane by varying the pH of the medium or by adding sodium acetate had no significant effect on the rate of sugar release.A release of K+ was associated with sugar efflux from the protoplasts. The molar ratio of K' to sugar varied between 1.5 and 2. In most higher plants, sucrose is a major end product of photosynthesis and is also the principal form in which photosynthetically derived carbohydrates are translocated within the plant. When translocation of assimilates from the leaf is reduced, photosynthetic sucrose formation is reduced, whereas starch formation is increased (38). Studies such as these indicate an influence of end-product accumulation (e.g., sucrose) on the partitioning of carbon between starch and sucrose in the leaf cell.Sucrose is also a principal end product of photosynthesis by isolated leaf cells and protoplasts (5,18,35 Previous studies (19,23,33) showed that less that 10% of the '4C02 fixed over 60 min by isolated leaf preparations is released to the medium, which has been taken to suggest that the products of photosynthesis are retained within the protoplast. Recently, we (19) reported an efflux of sucrose and hexoses, measured enzymically, from isolated mesophyll protoplasts at a rate similar to that of photosynthetic sugar formation in vitro. Release of radioactivity to the medium did not quantitatively reflect the sugar released because of dilution with internal pools. The results provided the first evidence for a significant transport of photosynthetic end products across the mesophyll plasma membrane.Characterization of the mechanism involved in transport of sugars across the mesophyll plasma membrane was the subject of the study reported herein. Specific objectives of the study presented here were to determine whether sugar efflux required an energized membrane and, if so, whether it involved co-transport (symport) of H+ or alkali cations. MATERIALS AND METHODSProtoplast Isolation. Protoplasts were isolated from fully expanded leaves of greenhouse-grown 10-to 14-day-old wheat (Triticum aestivum L.) and 4-to 6-week-old tobacco (Nicotiana tabacum L.) plants. Wheat protoplasts were isolated and purified as previously described (6). The purified protoplasts were washed three times in 0.5 M sorbitol and I mm CaCl2 after flotation on sucrose. Tobacco leaf segments (0.5 mm) were incubated in a medium containing 0.5 M sorbitol, I mm CaCl2, 2% (w/v) Cellulysin2 (Calbiochem), 0.4% (w/v) Macerozyme (All Japan Biochemicals), 0.5% (w/v) BSA, and 0.5% (w/v) PVP-40 at pH 5.5 Protoplasts were released after a 1-h incubation at 30 C into a solutio...