Báo cáo lâm nghiệp: "Autophagic response of higher plant cells to a prolonged period of sucrose deprivation"

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Autophagic response of higher plant cells to a prolonged period of sucrose deprivation 1 Dorne 2 Roby 1 R. Douce R. A. C. 1 Bligny Joseph-Fourier, DRFlPCVand , 2 DRFlRMBM 85X, F-38041, Grenoble CEN-G and Université Cedex, France Materials and Methods Introduction cells were One of the most original properties of Sycamore (Acer pseudoplatanus L.) described pre- nutrient medium grown in a as higher plant metabolism lies in the great viously (Bligny, 1977) except Mn was exclud- + 2 flexibility of their adaptation processes ed to prevent excessive broadening of the when faced with variable environmental 31 P orthophosphate resonance in 3! P vacuolar conditions. Thus, sudden temperature nuclear magnetic resonance (NMR) experi- ments. Cells harvested from the culture medium drop, water stress or the decrease of the were rinsed 3 times by successive resuspen- circadian light period diminishes the rates sions in fresh culture medium devoid of sucrose of intracellular carbohydrate biosynthesis. and incubated in flasks containing sucrose-free Consequently, the supply of organic car- culture medium. Every 5 or 10 h, cells were har- vested for perchloric acid (PCA) extraction bon necessary for sustaining cell respira- (Roby et aL, 1987), sucrose and starch determi- tion may be decreased. However, plant nations (Journet et al., 1986) and fatty acid + cells, owing to the presence of intracellular al., measurements et (Dome phospholipid pools of carbohydrate and to their ability to 1987). control an autophagic process can survive P 1 3NMR spectra of sycamore cells were for a period of several days without syn- obtained with a Bruker WM200 spectrometer thesizing or receiving any additional or- operating in the pulsed-Fourier transform mode at 81 MHz. The spectra were obtained with ganic carbon. Some morphological obser- compressed cells (4 cm in height, 3 x 10 cells, 8 vations have shown that, in higher plant 9 g wet weight) placed in a 25 mm tube under cells, portions of the cytoplasm, including constant perfusion as described by Roby et al., cell organelles such as mitochondria, may (1987). The perfusate consisted of culture be engulfed by the tonoplast membrane medium devoid of phosphate, manganese and sucrose and was adjusted to pH 6.5. In vivo (for a review, see Matile and Wiemken, spectra were obtained at 25°C after 3000 accu- 1976). In this study, we try to biochemical- mulations with a repetition time of 0.6 s and a ly characterize the changes occurring in pulse angle of 45°. higher plant cells after a prolonged period 31NMR spectra of PCA extracts stabilized of sucrose deprivation followed by a peri- at pH 7.5 with 40 mM HEPES buffer were mea- od of recovery. sured on a Bruker AM400 spectrometer equip-
ped with a 10 mm multinuclear probe tuned at 162 MHz. The deuterium resonance of D 0 2 was used as a lock signal. Each spectrum represents the accumulation of 2048 free induc- tion decay (FID) broad-bands, proton-decou- pled, recorded with a sweep-width of 6000 Hz, a 60° pulse angle and a repetition time of 4 s. The PCA extract spectra were referenced to the position of the 85% H resonance using a 4 PO 3 sample of 180 mM methylene diphosphonic acid (in 30 mM Tris buffer at pH 8.9) located in a coaxial capillary tube (outer diameter, 1.5 mm). The attributions of the resolvable reso- nance rays were made after running a series of spectra obtained by addition of the authentic compounds to the PCA extracts. Cytochrome oxidase, polar lipids and cardiolipin measure- ments were carried out and according to Bligny Douce (1980). Mitochondria were isolated from sycamore cell protoplasts and purified as described by Nishimura et al., (1982) using discontinuous Percoll gradients. The mitochondria subse- quently concentrated by differential centrifuga- tion were better than 95% intact as judged by their impermeability to cytochrome c (Douce et al., 1972). Sycamore cell respiration was measured at 25°C in their culture medium (Bligny and Douce, 1976). Results cates that the rate of 2 0 consumption when the intracellular started declining Effect of sucrose starvation on the rate of sucrose had been consumed. At that 0 consumption by sycamore cells 2 stage, starch content was decreased to less than 30% of that of normal cells. The fact that the rate of 0 consumption For 24 h the respiration rate of cells de- 2 sucrose starvation was always during of sucrose was constant (Fig. 1It It prived lower than the uncoupled rate (Fig. 1 ) sug- then decreased with time. After 50 h of gested that, during all the experiments, starvation, the rate of 0 consumption was 2 the cell respiration rate was limited by the decreased to less than 50% of that of nor- availability of ADP for either oxidative mal growing cells. Similarly, the uncoupled phosphorylation (Jacobus et al., 1982) or rate of 0 consumption obtained after the 2 glycolysis (ap Rees, 1985) in plastids and addition of 2 pM carbonyl cyanide p-tri- cytosolic phase of sycamore cells. This (FCCP) fluoromethoxyphenylhydrazone also suggested by the analysis of P 31 decreased after ca 24 h in the same ratio was NMR spectra (see also Rebeille et al., as the rate of respiration without uncou- 1985, and Roby . al., 1987). pler. Comparison of Figs. 1 and 2 indi- t 9
Effect of starvation the level sucrose on of P-esters in sycamore cells 3 and 4 illustrate the changes that Figs. in sycamore cells (3! P NMR spec- occur tra) when sucrose was omitted from the nutrient medium. Cells were maintained for up to 80 h in a continuously oxygenat- ed circulating solution (P culture free r medium) at pH 6.5. During the first 10 h, little change occurred. Sucrose efflux from the vacuole was rapid enough to maintain optimum phosphate ester concentra- an (RebeiII6 et al., 1985; Journet et al., tion in the cytosol. After 10 h of sucrose sis 1986). During the period ranging from 10 starvation, the glucose 6-P resonance to 35 h, the P molecules liberated from decreased progressively indicating that i phosphate esters entered the vacuole the efflux from the vacuole be- sucrose where they accumulated (P accumulated limiting factor for cytosolic glycoly- i came a
in higher plant cells, the adenylate energy charge (Pradet and Raymond, 1983) was maintained at a high level. When the sucrose deprivation was prolonged after 35 h, i.e., when almost all intracellular sucrose and starch had been consumed, the amount of NTP in the cell decreased progressively. However, this decrease of NTP was not accompanied by a parallel increase in intracellular NDP and NMP (Fig. 4). The NTP/NDP ratio was maintained at a and it was possible therefore high value that the total amount of NTP per unit vol- ume of cytoplasm might be maintained if the total cytoplasmic volume dropped sharply. Fig. 4 also shows that, after 35-40 h of sucrose starvation, there was a marked increase of glycerylphosphoryl- choline (GPC:), glycerylphosphoryletha- nolamine (GPE) and P-choline. Titration curves ptotting chemical shift versus pH for P-choline in solutions of various com- positions indicated that peak b (Fig. 3) corresponded to P-choline above pH 7.5, indicating that P-choline accumulated in the cytoplasnnic compartment. Finally, since GPE, CiPC and P-choline can be considered as deriving from the most abundant polar lipids (phosphatidyl etha- nolamine, PE, and phosphatidyl choline, PC) of sycamore cell membranes, this suggested that membrane systems were hydrolyzed during sucrose starvation to provide substrates for energy metabolism. Effect of sucrose starvation on cell weight and polar lipids of sycamore cells When sucrose was omitted from the in the cytoplasm). Surprisingly, the nutrient medium, the cell wet weight per slightly ml of culture medium appeared to be decreased glucose 6-P concentration was constant for at least 70 h (Fig. 5), whereas not accompanied by a parallel decrease in the dry weight decreased to 50% of the the concentration of nucleotide triphos- control value within the first 30 h. This phate (NTP) (Fig. 3). This indicated that, decline was attributable to the disappear- during the first 35 h of sucrose deprivation
of sucrose from the vacu and Quantitative determination of cytochrome e d ance starch from the plastids (see Fig. 2). aa and cardiolipin in sycamore cell mito- 3 During this time, the cell fatty acid content chondria remained constant (Fig. 5). However, fatty acid content declined after 40 h of sucrose starvation, when almost all the intracellular The values for cytochrome aa cardio- 3 and carbohydrate pool had disappeared. Anal- lipin contents in sycamore cells and syca- ysis of cell phospholipids indicated that more cell mitochondria are given in Table PC and PE, which represent respectively I. Data indicated that cytochrome aa and 3 40-45°t° and 25-27% of the cell polar cardiolipin contents of mitochondria iso- lipids (Bligny and Douce, 1980), de- lated from sycamore cells were constant creased to 30% of the control value within during sucrose depletion. In contrast, they 70 h of sucrose starvation. Similarly, the declined to less than half of the normal galactolipids and the total protein (includ- value after 50 h of sucrose starvation. It is ing the enzymes of the glycolytic pathway noteworthy that the lag phase observed in cytosol) decreased in the same pro- for cardiolipin or cytochrome aa evolution 3 portion during the starvation (Fig. 6) was comparable to that observed period (Jour- net et al., 1986). for 0 uptake evolution (Fig. 1Further- 2 more, comparison of Figs. 1 and 6 indi- Under these conditions, the decrease in cates that the respiration rates decreased the uncoupled rate of 0 consumption progressively in the same ratio as the 2 during the course of sucrose starvation decrease in intracellular cardiolipin or could be attributable to a progressive dimi- cytochrome aa In addition, it was esta- - 3 nution of the cytoplasmic compartment blished that: 1 ) on a protein basis, the rate and particularly to a diminution of the of 0 uptake in state 3 was about the 2 number of mitochondria per cell. Since we same for normal and sucrose-starved have already demonstrated that, in higher mitochondria (Journet ef al., 1986); 2) the plant cells, cardioplipin and cytochrome mitochondrial structure and size were not aa are exclusively localized in the mito- modified by sucrose starvation (electron 3 chondrial inner membrane (Bligny and microscopy data not shown). In conclu- Douce, 1980), we measured the levels of sion, all these results demonstrate that, these 2 specific mitochondrial markers after a long period of sucrose starvation, during sucrose starvation. the progressive decrease in the uncoupled
Addition of sucrose after 70 h of sucrose starvation resulted in a marked increase in the cell dry weight and total fatty acids (Fig. 5). The increase in the cell dry weight was attributable to a rapid accumulation of sucrose in the vacuolar reservoir and starch in plastids (not shown), whereas the increase in total cell fatty acids was attributable to the synthesis of new cyto- plasmic material, such as mitochondria. Of particular interest was the marked de- crease in the amount of P-choline that was reused for the synthesis of PC. Mean- while, the cell respiration rates increased until the normal value was reached. Inter- estingly, the coupled respiration increased more rapidly than the uncoupled respira- tion during the first hours of recovery (Roby et aL, 1987). This was attributable to the fact that such a rapid synthesis of cell metabolites transiently consumed high levels of ATP. rate of 2 0 consumption by sycamore cells Discussion attributable to a progressive diminu- was tion of the number of mitochondria per cell. These results demonstrate that the trans- fer of sycamore cells into a sucrose-free culture medium triggers the following cas- Effect of sucrose replenishment cade of reactions. 1 ) Initially, sucrose present in the vacuole was consumed. Addition of 50 mM sucrose to the nutrient Then the glucose 6-P level declined pro- medium after 35 h of sucrose starvation gressively, liberating inorganic phosphate (i.e., just before the decline in the uncou- which could stimulate the phosphorolysis pled rate of 0 consumption) resulted in a of starch. 2) When almost all the intracel- 2 very rapid increase of glucose 6-P reso- lular carbohydrate pools had disappeared, the cell fatty acids declined progressively nance accompanied by the disappearance of the cytoplasmic P (Fig. 3). In order to with a parallel increase in polar lipid i increase further the intracellular concen- deacylation products, such as P-choline. tration of glucose 6-P to its original level, it During this stage, the cell respiration rates was necessary to add a small amount of declined as a consequence of the de- P (50 pM) to the circulating medium, since crease in the number of mitochondria per i the efflux of P from the vacuole was not cell. Similarly, the total amount of ATP per i sufficient to sustain rapid phosphorylation cell was reduced in the same proportions as cell lipids and proteins. However, the processes.
ATP/ADP ratio remained considerable, long time after the synthesis or the supply even after 70 h of sucrose starvation. The oforganic carbon has been terminated. absence of relative accumulation of ADP in the cytoplasm explains the fact that the uncoupled/coupled respiration rate ratio remained constant throughout the experi- References ment. Another interesting feature is that P i derived from hydrolyzed P-ester was rapidly accumulated in the vacuole after its ap Rees T. (1985) The organization of glycoly- sis and the oxidative pentose phosphate path- cytoplasmic concentration increased to way in plants. In: Encyclopedia of Plant Phy- ca 40%. 3) During the course of sucrose siology, Vol. 18, (Douce R. & Day D.A., eds.), replenishment, carbohydrate and P-ester Springer-Verlag, Heidelberg, pp. 391-417 7 were resynthesized within 2-4 h and Beevers H. (1961) In: Respiratory Metabolism reached their standard cellular rate if in Plants. Row, Peterson and Company, Evans- external P was added. In fact, P which i , i ton, IL, pp. 119-129 has previously been sequestered in the Bligny R. (1977) Growth of suspension-cultured vacuole during the course of sucrose star- Acer pseudoplatanus L. cells in automatic cul- vation, was not readily returned to the ture units of large volume. Plant Physiol. 59, 502-505 cytoplasm for metabolic processes. Thus, several groups (Rebeil[6 et al., 1983; Bligny R. & Douce R. (1976) Les mitochondries de cellules v6g6tales isolbes. Physiol. V6g. 14, Sivak and Walker, 1986; Martinoia et al., 499-5155 1986) have already suggested that the homeostatic process of P transport across R. & Douce R. (1980) A precise localiza- i Bligny cardiolipin in plant cells. Biochim. Bio- tion of the tonoplast is slow and allows short-term phys. Acta 617, 254-263 changes in the cytosolic P size to be -pool i Dome A.J., Bligny R., RebeiII6 F., Roby C. & used as a means of regulating metabolic Douce R. (1987) Fatty acid disappearance and functions, such as starch and sucrose syn- phosphorylcholine accumulation in higher plant theses. cells after a long period of sucrose deprivation. Plant Physiol. Biochem. 25, 589-595 The results presented here also demonstrate that, during the course of Douce R., Christensen E.L. & Bonner W.D. (1972) Preparation of intact plant mitochondria. sucrose replenishment, P-choline was reu- Biochim. Biophys. Acta 275, 148-160 sed for PC synthesis. It may therefore be Jacobus W.E., Moreadith R.W. & Vandegaer concluded that the presence of excess of K.M. (1982) Mitochondrial respiratory control. P-choline in plant cells should be consid- Evidence against the regulation of respiration ered as a good marker of membrane utili- by extramitochondrial phosphorylation poten- zation after a long period of sucrose star- tials or by ATP/ADP ratios. J. Biol. Chem. 257, vation and sucrose synthesis. 2397-2402 Journet E.P., Bligny R. & Douce R. (1986) Bio- In conclusion, it appears that the plant chemical changes during sucrose deprivation in cell metabolism is extremely flexible. The higher plant cells. J. Biol. Chem. 261, 3193- cytoplasm, in particular, can be utilized as 3199 a carbon source after a long period of Martinoia E., Schramm M.J., Kaiser G., Kaiser sucrose starvation without significantly W.M. & Heber U. (1986) Transport of anions in affecting the survival of the cell. Under isolated barley vacuoles. Plant Physiol. 80, 895- these conditions, higher plant cells, owing 901 to the presence of intracellular pools of Matile P. & Wiemken A. (1976) Interactions be- carbohydrate and to their ability to control cytoplasm and vacuole. In: Encyclope- tween an autophagic process, can survive for a dia of Plant Physiology, Vol. 3 (Stocking C.R. &
Heber U., eds.), Springer-Verlag, Heidelberg, tanus cells. Arch. Biochem. 143- Biophys. 225, 148 pp. 255-287 Nishimura M., Douce R. & Akazawa T. (1982) Rebeille F., Bligny R., Martin J.B. & Douce R. Isolation and characterization of metabolically (1985) Effect of sucrose starvation on sycamore competent mitochondria from spinach leaf pro- (Acer pseudoplatanus) cell carbohydrate and P i toplasts. Plant Physiol. 69, 916-920 status. Biochem. J. 226, 679-684 Roby C., Martin J.B., Bligny R. & Douce R. Pradet A. & Raymond P. (1983) Adenine nucleotide ratios and adenylate energy charge (1987) Biochemical changes during sucrose starvation in higher plant cells. J. Biol. Chem. in energy metabolism. Annu. Rev. Plant Phy- 262, 5000-5007 siol. 34, 199-224 Rebeil]6 F., Bligny R., Martin J.B. & Douce R. Sivak M.N. & Walker D.A. (1986) Phosphosyn- (1983) Relationship between the cytoplasm and thesis in vivo can be limited by phosphate sup- the vacuole phosphate pool in Acer pseudopla- ply. New Phytol. 102, 499-512 2