Relationship between changes in membrane permeability, respiration rate, activities of lipase and phospholipase C and ultrastructure in senescing petals of Dianthus caryophyllus (cv. White Sim)

Linda Burger, G.H. de Swardt and A.H.P. Engelbrecht Department of Botany, Rand Afrikaans University, Johannesburg

Senescence of carnation petals was characterized by a typical climacteric respiration sequence, reaching the preclimacteric minimum and the climacteric maximum on the fifth and sixth day after harvest respectively. Except for a marked decrease prior to the onset of the climacteric rise, the membrane permeability, determined as the percentage apparent free space (% AFS), showed a general increase during senescence. The lipase activity remained at a low and constant level for the greater part of the senescing period , except for a fourfold increase on the eighth day after harvest. An initial decrease in the phospholipase C activity was followed by a continuous increase from the fourth day. The increase in lipase activity showed a similar trend to the increase in the % AFS on the eighth day after harvest, while a close relationship was found between changes in the phospholipase C activity and the % AFS during the climacteric rise and postclimacteric phase. Lipid degrading enzymes may therefore be a contributory cause to changes in the lipid fraction of the lipoprotein membrane structure, causing changes in membrane permeability during senescence. Changes in the respiration rate and the % AFS could be associated with changes in the ultrastructure of senescing cells. S. Afr. J. Bot. 1986, 52: 195-200

Verouderende angelierkroonblare toon 'n kenmerkende klimakteriese respirasiepatroon , met bereiking van die preklimakteriese minimum en die klimakteriese maksimum op die vyfde en sesde dag na pluk respektiewelik. Die membraanpermeabiliteit , bepaal as die persentasie skynbare vrye ruimte (% SVR), neem tydens veroudering toe, behalwe vir 'n beduidende afname net voor die aanvang van die klimakteriese styging. Die lipase-aktiwiteit was deurgaans laag en konstant, behalwe vir 'n viervoudige toename op die agtste dag na pluk. 'n Aanvankl ike vermindering in die aktiwiteit van fosfolipase C is deur 'n onomkeerbare toename vanaf die vierde dag na pluk gevolg. Lipase-aktiwiteit toon slegs op die agtste dag na pluk 'n verwantskap met veranderinge in die % SVR, terwyl daar 'n direkte verwantskap tussen veranderinge in die fosfolipase C-aktiwiteit en die % SVR tydens die klimakteriese styging en die naklimakteriese lase bestaan. Dit blyk dus asof lipied-degraderende ensieme tot veranderinge in die lipoprote·ien-struktuur van membrane aanleiding kan gee. Dit sal noodwendig tot veranderinge in membraanpermeabiliteit lei. Daar bestaan 'n verwantskap tussen veranderinge in die respirasietempo, % SVR en ultrastruktuur van verouderende selle. S.·Afr. Tydskr. Plantk. 1986, 52: 195-200

Keywords: Dianthus caryophyllus, lipase, membrane permeability, phospholipase C, senescence

Linda Burger, G.H. de Swardt* and A.H.P . Engelbrecht Department of Botany, Rand Afrikaans University, P.O. Box 524, Johannesburg, 2000 Republic of South Africa

*To whom correspondence should be addressed

Accepted 14 November 1985

Introduction

Senescence of plant tissues is generally characterized by increased enzyme activities (Hulme 1972), changes in the respiration rate, membrane permeability (Sacher 1962) and ultrastructure (Bain & Mercer 1964).

Many of the enzymes involved in the ageing of flowers show an increase during ageing and usually have a hydrolytic action (Kende & Hanson 1976). It is generally accepted that lysosomes contain lipid degrading enzymes and that these organelles are intimately associated with senescence (Pitt 1975) so that the vacuole and lysosomes can be considered as the lysosomal system or compartment of the cell (Berjak & Villiers 1972). Degradation of the tonoplast, which generally occurs in senescing plant cells (Shaw & Manocha 1965; Platonova eta/. 1974), will lead to mixing of the lysosomal hydrolases with the cytoplasm (Berjak & Villiers 1972). This leads to degradation of the protoplast, which may be responsible for the climacteric rise (Sacher 1962; Bain & Mercer 1964).

Very little is known regarding the primary cause of changes in permeability of membranes during senescence (Sacher 1973). A reduction in the lipid fraction of membranes (Fer­guson & Simon 1973) will modify the lipoprotein structure and can lead to a loss of permeability (De Swardt & Rousseau 1973). An increase in membrane permeability is a common phenomenon in senescing carnations (Mayak et a/. 1977).

This investigation was carried out to determine the relation­ship between changes in the respiration rate and membrane permeability of senescing carnation petals and the role of lipase and phospholipase C in the loss of membrane permeability.

Materials and Methods Commercially harvested carnation flowers were obtained from a local nursery. Stems were shortened to a length of 30 em and the lower leaves were removed. The flowers were placed in 500 ml tap water and held in a growth chamber at 21 oc and 500Jo relative humidity with a continuous 12 h light/ dark cycle. Flowers for individual experiments were selected daily for eight consecutive days after harvest. All experiments were repeated ten times.

Measurement of respiration rate Four petals, one from each of the four outer whorls of the same flower, were selected arbitrarily and the respiration rate was measured at 25 °C over a period of 70 min with a Gilson differential respirometer. No incubation medium was used. The respiration rate was expressed as volume oxygen uptake (cm3

) per kg fresh petal tissue per h .

I96

Determination of percentage apparent free space (% AFS)

A modification of Sacher's method (1966) was used to determine the change in the fJ!o AFS in petal tissue at eight progressive stages of senescence. Five discs of petal tissue per sample, each one from corresponding positions on five similar petals from the outer whorl of the same flower, were taken with a 5 mm cork borer. The volume of the sample was determined by weighing, assuming the specific gravity to be I ,0. The samples from each senescing stage were placed in separate sampling tubes containing 2 cm3 incubation medium, which consisted of 0,05 mol dm - 3 sucrose solution in O,OI mol dm- 3 phosphate buffer, pH 6,5 and 3,7 x I if Bq [UI4C] sucrose. The discs were totally immersed using a glass rod, with due care to prevent bruising. Incubation was carried out for 80 min at 1 o to 2°C. Following the incubation period the discs were removed and rinsed with deionized water . This water was combined with the 2,0 cm3 incubation medium and diluted to 50 cm3 with deionized water. One cm3 of the diluted solution was mixed with 10 cm3 Scintisol Complete in a scintillation vial for radiometric analysis. The rinsed tissue discs were transferred to new scintillation vials with 10 cm3 Scintisol Complete. The vials were left for 16 h to allow complete digestion of the tissue. Counting rates of the various samples from incubation solutions and digested tissue were measured over a period of 10 min with a liquid scintillation spectrometer. The % AFS was determined, using the following formula (Sacher 1966):

h VI % AFS = f;" X V

2 X 100

where I I = counting rate of incubation liquid after incubation h = counting rate of digested petal tissue after incubation VI volume (2 cm3

) of incubation medium v2 = volume of incubated petal tissue discs (cm3

) .

Determination of the incubation period Eight samples of petal tissue, all at the same physiological stage, were selected and incubated for periods of 20, 40, 60, 80, 100, 120, 140 and 160 min respectively at 1 o to 2°C. The % AFS for each sample was determined after radiometric analysis.

Enzyme extraction The enzyme extraction at oo to 2°C was based on a method of Van Loon & Van Kammen (I968). Approximately 5 g petal tissue was added to 40 cm3 0, I mol dm - 3 Tris-HCl buffer (pH 8,0) containing 0, I mol dm - 3 NaCl and 0,1% L- cysteine­He!. Extraction was carried out with a Virtis '23' homogenizer for 5 min at maximum speed, after which the homogenate was stirred for 30 min. The extract was centrifuged for 30 min at 37000 x g and 2°C. The supernatant was then centri­fuged for another 30 min at the same speed. The enzyme­containing supernatant was brought to 95% saturation with solid ammonium sulphate. After standing for I h, the enzymes were collected by centrifuging for IS min at 37000 x g. The precipitate was dissolved in 2,0 cm3 0,06 mol dm- 3 Tris­phosphate buffer (pH 6,9) and dialyzed for 24 h against the same buffer. The dialyzed enzyme solution was subsequently centrifuged for 10 min at 37000 x g and the supernatant was used for the determination of the enzyme activities and protein concentration. The protein concentration was determined by the method of Lowry et at. (195I) and expressed as mg protein per g dry petal tissue.

S.-Afr. T ydskr. Plantk., 1986, 52(3)

Determination of lipase activity Lipase activity was determined by a modified method of Singer & Hofstee (I948). Two cm3 0,75 mol dm - 3 glycerine tri-acetate and 2,0 cm3 0,075 mol dm - 3 sodium carbonate were added to the main compartment of a Gilson flask. The substrate, as well as I cm3 enzyme extract in the side arm, were equilibrated for 10 min at 37°C. The substrate and enzyme were thoroughly mixed and the production of carbon dioxide was measured over a period of 35 min. The specific activity of lipase was determined as follows:

. . llmol C02 per min Enzyme uruts (EU)/ mg protem =

mg enzyme

Determination of phospholipase C activity Phospholipase C activity was measured by a modified method of MacFarlane & Knight (194I). A volume of 0,5 cm3 enzyme extract was mixed with 2 cm3 0, I mol dm - 3 Tris-maleate buffer (pH 7 ,3); 0,5 cm3 0,05 mol dm - 3 calcium chloride; 1,5 cm3 2% ovolecithin and 0,5 cm3 I% albumin. The mixture was incubated for 30 min at 37°C after which 5 cm3 5% trichloracetic acid was added. This mixture was centrifuged for 30 min at 5000 x g. After centrifugation 1,0 cm3 of the supernatant was evaporated with 1 ,0 cm3 2,5 mol dm- 3

sulphuric acid. The residue was heated with I ,0 cm3 2,5 mol dm - 3 nitric acid to fuming point, cooled and diluted to 7,0 cm3 with deionized water and I ,0 cm3 2,5% ammonium molybdate. After addition of I ,6 cm3 deionized water and 0,4 cm3 reducing agent (solution of 100 mg I-amino-2-naphthol- sulphonic acid in 39 cm 3 IS% sodium bisulphite and I cm3 20% sodium sulphite) the reaction mixture was shaken for IS min at 50°C and the absorbance measured at 660 nm after 10 min. The phosphorus content was determined from a standard curve. The specific activity of phospholipase C was determined as follows :

Enzyme units (EU)/ mg protein

_ llmol phosphorus/I,O cm3 x 2 - 30 (min) x mg protein/cm3 reaction mixture

Investigation of ultrastructural changes One intact petal was selected daily from the outer whorl of the same flower. Petal tissue was fixed for 2 h in 4% glutaral­dehyde in 0,05 mol dm - 3 sodium cacodylate buffer at pH 7 ,0, rinsed with fresh buffer for 5 min, immersed for 90 min in 2% osmium tetroxide in buffer and followed by a rinse for 10 min in fresh buffer. Dehydration was done in an ethanol series followed by I ,2-epoxypropane. The tissue was embedded in a low viscosity epoxy/resin mixture (Spurr I969) and left for 12 h at 70°C to allow polymerization of the resin. Sections were stained in uranyl acetate and lead citrate.

Results and Discussion Senescing petals of carnation flowers showed a typical climac­teric respiration pattern (Figure I). The respiration rate was thus used as a non-subjective criterion to determine the stage of senescence of carnation petals and allow selection of samples at the same physiological stage. The expected stimu­lation of the respiration due to the initial loss of compartmen­tation did not occur. Instead there was a gradual decrease in respiration rate and the preclimacteric minimum was reached on the fifth day after harvest. This decrease, which can be attributed to the depletion of respirable substrates (Mayak & Halevy I980), coincided with a thickening of the outer mitochondrial membrane (Figure 5, M), and is

S. Afr. J. Bot., 1986, 52(3)

~ "' " ., 0.

"' "' ~ c "' ~ 0. 0. <(

11

10

9

8

7

6

550

~ 500 ,;;

~450 "' til 400 c. " c-1350 0

"'E 300 s ~ 250

g ~ ·c. "' "' a:

Standard errors

I % AFS I Respiration rate

I Lipase I Phospholipase C

'2 ·~

e 0.

0,30 ~

025 2 . (.)

0,20 ~ :g_

0,15 'g ~

0,10 _g "-

0,30 ~ 0,05 c: 'iii 0 c. ~ 2 " "' 0,10 :g_

t====~==~=!==::==! _ _j0,05

:.J

2 3 4 56 7 8

Days af1er harvest

Figure I Changes in the percentage apparent free space (o--o), lipase specific activity (</J--</J); phospholipase C specific activity (•-e) and the respiration rate (o - - - o) in senescing carnation petals.

comparable to structural changes that have been reported in other plant tissues (Bain & Mercer 1964; Nakamura & Asahi 1976). The subsequent abrupt increase in the respiration rate to reach the climacteric maximum on the following day, coincided with a thinning of the outer mitochondrial mem­brane (Figure 7, M), which probably resulted from the formation of phospholipid-deficient mitochondrial membranes (Nakamura & Asahi 1976). The climacteric rise may also be attributed to a considerably increased mitochondrial activity (Bogin & Erickson 1965; Ku et a/. 1968). A decrease in respirable substrates and enzymes, rather than the degenera­tion of mitochondria, was probably responsible for the final decrease in the respiration rate, as mitochondria with a normal structure could still be observed during the post-climacteric period (Figure 8, M). These findings correspond with the detection of identifiable mitochondria in post-climacteric cells of different types of tissue (Opik 1965; Shaw & Manocha 1965; Matile & Winkenbach 1971; Platonova eta/. 1974).

Senescing carnation petals showed a general increase in the CT/o AFS (Figure 1), comparable to the increased membrane permeability detected by Mayak eta/. (1977). Incubation was carried out for 80 min, which was found to be optimal (Figure 2). Energy is essential for maintenance of membrane integrity (Eilam 1965) and any shortage which might have developed during the preclirnacteric phase, could have contributed to the initial increase in the CT/o AFS. A decrease in membrane lipids (Draper 1969), especially phospholipids (Ferguson & Simon 1973; Beutelmann & Kende 1977; Borochov eta/. 1978; Suttle & Kende 1980), changes in lipoprotein structure of membranes (De Swardt & Rousseau 1973), increased sterol: phospholipid ratio (Thompson et a/. 1981; Lurie & Ben-Arie 1983) and accumulation of peroxidized lipids in membranes (Pauls & Thompson 1984) can contribute to the loss of membrane integrity and ensuing membrane dysfunction. The initial increase in the CT/o AFS during the first three days after harvest can be associated with drastic structural changes in the membrane system. In certain cells the tonoplast showed

197

20

18

~ 16 w (.) <( 14 a. C/)

w 12 w EE

10 !Z ~ 8

B: 6 <(

4

2

40 60 80 100 120 140 160

INCUBA TlON PERIOD (minutes)

Figure 2 Changes in the percentage apparent free space of carnation petals incubated for different periods of time.

discontinuities (Figure 3, arrows) and was apparently replaced by flattened vesiculae (Figure 3, VE), while other cells lacked a tonoplast completely (Figure 4). The cause of the formation of these discontinuities is unknown. The plasmalemma moved away from the cell wall during this phase and appeared relatively thin (Figure 4, PM).

The recovery of membrane permeability on the fourth day after harvest was associated with the reappearance of cells with a normal structure (Figure 6) and intact, but markedly thickened, tonoplasts (Figures 5 & 6, T). Changes in normal protoplasmic compartmentation led to an increased contact between substrates and enzymes, causing temporary rapid anabolic reactions, which may bring about an impression of high metabolic activity and a temporary recovery of mem­brane structure (De Swardt & Rousseau 1973). The thickening of the tonoplast can be ascribed to the deposition of electron­dense material present in the vacuole (Figures 5 & 6, arrows) on the membrane. A reaction between the polyphenolic products of catabolic processes (De Swardt & Rousseau 1973) in the vacuole and the osmium tetroxide (fixative) may be responsible for the formation of the electron-dense material (Platonova et a/. 1974). Similar phenomena were observed in senescing apple tissue (Platonova et a/. 1974) and the abscission zones of leaf tissue (Webster eta/. 1976). The role of this material in the recovery of differential permeability is unknown.

In pericarp tissue of tomatoes, the CT/o AFS increased irreversibly after the temporary recovery in membrane per­meability just prior to the preclirnacteric minimum (De Swardt & Rousseau 1973). However, the CT/o AFS remained relatively constant in carnation petal tissue during this period, until the first day after the climacteric maximum, when the CT/o AFS increased irreversibly on the eighth day after harvest. This fmal abrupt increase in the CT/o AFS corresponded with the total loss of the tonoplast and plasmalemma in most cells (Figure 8). Prolonged deposition of the electron-dense material on the tonoplast may lead to irreversible disintegration and ultimate total loss of compartmentation (Platonova et a/. 1974). Dis­organization of the endomembrane system occurred at this stage and only a few vesiculae and mitochondria (Figure 8, VE & M) could still be observed in the residual cell contents.

The large variations observed in changes of enzyme activities

198 S.-Afr. Tydskr. Plantk. , 1986, 52(3)

,,;r,

3 ·t• • .. \ . .,

~ ' '-......

,~ ~( .. v t ~ . '' ~ · .... \ ,,,

.: ... ~· <I>

., ,. !l \ \ -~ , t

PM • Figures 3- 6 Cells of Dianthus caryophyllus at different stages of senescence. 3. First day after harvest. 4. Third day after harvest. 5 & 6. Fourth day after harvest. Electron dense material in the vacuole and on the tonoplast (arrows); incomplete tonoplast (double arrows); endoplasmic reticulum (ER); microbody (LM); mitochondrium (M); plasmalemma (PM); ribosomes (R); cytoplasmic vacuole (SV); tonoplast (T); vacuole (V); vesiculae (VE). Scale line = 0,5 J.lm.

S. Afr. J . Bot. , 1986, 52(3) 199

•

v •

v •

8 Figures 7-8 Cells of Dianthus caryophyllus at different stages of senescence. 7. Fifth day after harvest. 8. Eighth day after harvest. Endoplasmic reticulum (ER); mitochondrium (M); plasmalemma (PM); ribosomes (R); tonoplast (T); vacuole (V); vesiculae (VE); cell wall (W) . Scale line = 0,5 ~-tm.

in ripening fruit (Sacher 1973) can be ascribed to inactivation or precipitation of enzymes by phenolic compounds (pro­anthocyanidins) during extractions (Loomis & Battaile 1966). As no traces of pro-anthocyanidins could be detected at any stage of senescence in carnation petals, no special precautions needed to be taken to eliminate inactivation by pro-antho­cyanidins during enzyme extractions.

No significant changes in the lipase activity occurred in the first seven days after harvest. The activity remained relatively low compared to that of apple tissue, which showed a con­siderable increase in lipase activity during the climacteric rise, to reach a peak value at the climacteric maximum (Rhodes & Wooltorton 1967). The lipase activity in carnations showed no relationship with changes in the OJo AFS except on the eighth day after harvest when there was a fourfold increase in lipase activity, coinciding with the final abrupt increase in the % AFS. The initial low lipase activity may be ascribed to a lysosomal localization of the enzyme. Lipase may form a structural component of the membranes of lipid bodies (Lin et at. 1983) and lysosomes (Pitt 1975) and therefore, of the tonoplast as part of the lysosomal system (Matile & Wink en­bach 1971; Berjak & Villiers 1972). Degradation of the tono­plast can thus lead to the release or activation of lipase, which can in turn contribute to the degradation of membrane lipids during the final stage of senescence.

A reduction in phospholipase C activity during the first three days after harvest coincided with a simultaneous increase in the% AFS during the same period (Figure 1). It therefore seems unlikely that the loss of differential permeability during this period could be due to degradation of membrane phos­pholipids by phospholipase C. A reduction in the level of

phospholipids was, however, found in a variety of senescing plant tissues (Ferguson & Simon 1973; Beutelmann & Kende 1977; Borochov et at. 1978; Suttle & Kende 1980), caused by increased activities of phospholipases during ageing (Borochov eta!. 1978; Arnihud eta!. 1979), thus enhancing permeability of the plasma membrane (Sacher 1973; Simon 1974). The synthesizing enzyme and the precursor of phospholipase C could possibly be separated from each other by the tonoplast (Kende & Baumgartner 1974). The initial loss of compartmen­tation could, therefore, lead directly to a de novo synthesis of phospholipase C on the fourth day after harvest. This increased phospholipase C activity could cause the termination of the recovery of membrane integrity on the fourth day after harvest, leading to a synthesis of phospholipase C. The synthesis is gradual at first and increases abruptly on the eighth day. It is possible that phospholipase C can be responsible for the loss of differential permeability from the fifth day after harvest onwards, as the increased activity of this enzyme could be closely associated with the increase in the % AFS during this period. The threefold increase in the activity on the eighth day after harvest leads to the abrupt and final loss of dif­ferential permeability.

The primary cause of the changes in membrane perme­ability remains yet unknown. The cytoplasmic membranes determine the intracellular distribution and orientation of enzymes and substrates and the increase in the % AFS is an early indication of senescence (Eilam 1965) in carnation petals. The results obtained suggest that the precursors of lipid degrading enzymes or the enzymes themselves are located in the lysosomal system. Modification of the membrane structure of this system will result in changes in the normal metabolism

200

and finally in the irreversible degradation of membrane lipids. As membranes regulate normal metabolic processes a deterio­ration in their structural integrity will lead to the initiation and acceleration of senescence.

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