The Relevance of Polyamine Levels in Cherimoya (Annona cherimola Mill.) Fruit Ripening

J Plant Physiol. Vol. 143. pp. 207-212 {1994}

The Relevance of Polyamine Levels in Cherimoya (Annona cherimola Mill.) Fruit Ripening MARiA

I. EscRIBANO and CARMEN MERODIO

U.E.I. Refrigeraci6n de Productos Vegetales, Instituto del Frio, C.S.I.S. Ciudad Universitaria s/n, 28040-Madrid, Spain Received May 12, 1993 · Accepted September 29, 1993

Summary

Free polyamine levels were determined in cherimoya (Annona cherimola Mill. cv. ) fruit during ripening at 20 °C. Several parameters including respiration rate, ethylene production and total titratable acidity were analyzed. The polyamine prevailing after harvest was spermidine, although a sharp increase was observed in putrescine content during ripening. The possible relationship between this rise in putrescine level and the ripening process was studied on the basis of the pattern of diamine content in fruit stored over a range of low temperatures (10, 8 and 6 °C). Retardation of the ripening process by cold storage was concurrent with a slower rate of increase in the free putrescine level. Moreover, where ripening was inhibited by storage at the chilling temperature (6 °C), no increase in free putrescine was observed. While no variation was observed in spermidine and spermine content in fruit ripening at 20 °C, levels did undergo change during low temperature storage. The possibility that significant accumulation of the free putrescine titer may be associated with high acidity levels in ripening cherimoya tissues is discussed.

Key words: Cherimoya (Annona cherimola Mill.), chilling temperature, ethylene, acidity, low temperature, polyamines, ripening. Abbreviations: Put =putrescine; Spd = spermidine; Spm = spermine. Introduction

The ripening process, believed to be genetically programmed for fruit in general, has nonetheless been found to vary in different kinds of fruit. The evolution of physiological parameters has been analyzed in Annonaceous fruit (Moreno and De La Plaza, 1983; Bruinsma and Paull, 1984), and the results suggest an unusual ripening pattern. Enhanced ethylene production is preceded by other ripening events such as increased respiration and a sharp decrease in flesh firmess (Palma et al., 1993). Total titratable acidity increased markedly early during ripening, and this increase, a consequence of malic acid accumulation, was matched by a marked decrease in pulp pH (Paull, 1982; Willet al., 1984). In general, the development of ripening capacity in fruit has been associated with the sensitivity of tissues to ethylene. It has been assumed that such sensitivity increases with maturity (Trewavas, 1982), so that the levels of ethylene already © 1994 by Gustav Fischer Verlag, Stuttgart

present in the tissue may be enough to promote changes inherent in ripening. Although ethylene plays an important role in ripening of climacteric fruit, it seems probable that, in cherimoya at least, some other agents may be involved (Kosiyachinda and Young, 1975). Polyamine concentration has been observed to change during development and ripening of fruit (Winer and Apelbaum, 1986; Casas et al., 1990). In general, a correlation has been established between the effects of polyamines and the inhibition of ethylene biosynthesis. This correlation has been explained as a result of metabolic competition for the same precursor, S-adenosylmethionine (Apelbaum et al., 1981; Hyodo and Tanaka, 1986). Also, application of polyamines modified ripening behaviour to a certain extent, depending on the method of application and the tissue that they were applied to (Law et al., 1991; Kramer et al., 1991). Besides, several aspects of the metabolic changes in fruits under stress conditions have been observed to entail poly-

208

MARIA I. EscRIBANO and CAnmN MEllomo

amines and, in general, putrescine accumulation has been found in response to many stress factors (Flores et al., 1985; Slocum et al., 1984). Low temperature storage immediately after harvest is a common technique used to extend the cherimoya ripening period (Wills et al., 1984; Batten, 1990). However, in general, the mechanism by which ripening is delayed at low temperature is blocked at chilling temperature has not yet been elucidated (Wang, 1990). This paper puts forward the possibility that the ripening process of cherimoya could be associated with changes in some specific polyamine titer and with the threshold levels of polyamines. Since free polyamines are suggested to be the active form in the regulation of plant physiological processes (Smith, 1985), this sutdy analyzes the free putrescine (Put), spermidine (Spd) and spermine (Spm) patterns in cherimoya fruit during normal ripening and low temperature storage. Materials and Methods Cherimoya (Annona cherimola, Mill., cv. ) fruit of uniform size and maturity stage (skin becoming yellowish green) were harvested in Almuil.ecar, Granada (Spain). Twenty-four hours after harvest, selected fruit weighing from 180 to 190g per piece was stored in darkness at 20, 10, 8 and 6 °C. Ethylene production and respiration rate measurements were performed on 10 cherimoya fruits enclosed in a glass jar continuously flushed with ethylene- and carbon dioxide-free air at a flow rate of 5 L · h - 1• Ethylene and carbon dioxide content in 1-mL samples of effluent gas were measured with a Varian 3700 gas chromatograph equipped with a Porapak QN (4mx3.2mm) and a molecular sieve (2mx3.2mm) column. After separation, C02 and C2H4 were detected by thermal conductivity and flame ionization detectors, respectively, using helium as carrier. Three fruits were periodically collected, peeled, minced, frozen in liquid nitrogen and stored at - 80 °C until use. Seeds were discarded. Titratable acidity was determined in a homogenate (lOg pulp in 20 mL of distilled water, heated to eliminate volatile acids and cooled to room temperature) by titration with 0.1 N sodium hydroxide to pH = 8.1. Results are expressed in meq. toog- 1 f.w. Three replicates were performed.

Polyamine analysis Extracts for polyamine analysis were prepared by homogenizing 2.0g of frozen tissue in 5% (v/v) ice-cold perchloric acid (400mg tissue·mL- 1 acid). 1,6-hexanediamine (60-125nmol·g- 1 fresh weight) was added as an internal standard before the tissue was ground. The extracts were stored overnight in plastic tubes at 4 °C. After lh on ice the homogenate was centrifuged at 27,000 xg for 30 min at 4 °C. The supernatant was removed and freeze-stored at -20 °C in plastic vials until use for polyamine dansylation. The polyamines were derivatized according to the methods of Flores and Galston (1982). One hundred JlL aliquots of the supernatant were added to 200 J.LL of saturated sodium carbonate and 400 J.LL of dansyl chloride (20 mg · mL - 1 in HPLC grade acetone, preapred fresh) in a 5-mL glass tapered reaction vial. After brief vortexing, the mixture was incubated in the dark at room temperature for 18 h. Excess dansyl reagent was removed by reaction with 100 J,LL of proline ( 100 mg · mL - 1) and incubation for 30 min in the dark. Dansyl polyamines were extracted twice with 750 J.LL of toluene (HPLC grade) with vigorous vortexing for 30 seconds. The poly-

amine-containing organic phase was collected and completely dried under nitrogen, after which the dansyl polyamines were stored in amber glass vials at -20 °C. No variation was observed in the chemical stability of the dansyl polyamines when stored under these conditions for about 1 month. Dansylated extracts were stable for up to 1 month. Dried polyamine residue was redissolved in 1 mL of methanol (HPLC grade) before HPLC analysis.

HPLC Analysis ofDansylated Polyamines Samples were injected into a ftxed 20-J.LL loop for loading onto a reverse phase CIS column (150 x 4.6 mm, 5Jl particle diameter) heated at 40 °C to reduce column back-pressure and solvent viscosity during the run. Samples were eluted from the column with a programmed water: methanol (v/v) solvent gradient progressing from 55% to 95% in 25 min at a flow rate of 0. 9 mL ·min - 1• Dansyl polyamines were detected by a fluorescence spectrophotometer (excitation wavelength, 350 nm; emission wavelength, 495 nm, Kontron Instruments, model SFM 25) and the peaks, areas and retention times were recorded and calculated by an attached computer with a PC Integration Pack Programme (Kontron Instruments). The identity of endogenous polyamines was determined by retention times and coinjection of a known standard. Polyamines in tissues were quantified by using the relative calibration procedure described by Smith and Davies (1985). Known weights (nmol) of individual polyamines and the internal standard, hexanediamine, were dansylated and chromatographed. Weight ratios of individual polyamines to the internal standard were plotted against area ratios. Three curves were generated by this procedure, on each for Put, Spd and Spm. Weight ratios of endogenous polyamines were determined from area ratios and the regression equations for the standard curves. The nmol ratios were then multiplied by the nmol of internal standard added to the extract. This method of quantification eliminates the need for exact measurements of injection quantities and avoids problems encountered due to changes in detector response, since area ratios are constant (McNair and Bonelli, 1969). Multiple variance analysis (ANOVA) was employed to determine the significance of the data for significance level P :S 0.05.

Results

Evolution ofPhysiological Parameters Table 1 shows how the cherimoya ripening parameters varied at different temperatures. The days most representative of the ripening stages are shown. An increase in titratable acidity and an early rise in the respiration rate, previous to ethylene production peak, are characteristic of cherimoya when ripened at 20 °C. Storage at 10 and 8 °C causes a decline in the metabolism of the fruit, concurrent with a lower acidity and respiration rate. Ripening is retarded at these low temperatures, as borne out by the analysis of peak ethylene production, which occurs on the 9th and the 13th day, respectively. The inhibition of ripening when the fruit is stored at 6 °C is associated with very low ethylene production, respiration rate and slight changes in titratable acidity levels.

Changes in polyamine levels during ripening Free polyamine levels in cherimoya during ripening at 20 °C were estimated, finding that after harvest the fruit con-

Table 1: Ethylene production, respiration rate and titratable acidity of cherimoya fruit at different storage temperatures. Titratable Respiration Temperature Day of Ethylene acidity storage production rate (OC) (J!Likg. h) (mg COz/kg ·h) (meq/100 g f.w.) 1.58 ± o.o1b 20 0 1.80" 2.69 ± 0.02 187.82 8.12 1 3.94± 0,01 142.53 26.61 2 3.70 ± 0.06 212.24 12.58 5 2.30 ± 0.06 100.36 10 2 3.57 2.11 ± 0.02 62.05 107.54 6 3.83 ± 0.07 122,02 104.69 9 3.52 ± 0,01 90.81 87.91 13 1.64 ± 0.04 1.85 49.82 8 2 2.95 ± O.Q3 44.54 6 10.70 2.56± 0.05 40.07 13 38.33 2.73 ± 0.04 44.67 39.13 19 2.47 ± 0.05 26.08 6 6 1.13 2.32 ± 0.11 9 1.44 23.51 2.32 ± 0.09 18.45 13 1.56 2.49 ± 0.05 16.63 19 2.52 • Ethylene production and respiration and rate values represent the measurement of ten fruits. bMean ± SD (n = 3).

Titer of polyamines during cherimoya fruit ripening 150

~spermidine

209

U spermine T

Cl

~ 100

E

E

"'

~

~ Q)

·Ec:

5o

.,

>

& 0

0

n

n

2

n n 3

5

lD

Days of storage

Fig. 2: Changes in spermidine and spermine levels in cherimoya fruit pulp during ripening at 20 °C. Vertical bars ± SD.

300

500

100

-250

80 :2

~200

..,~

Cl

i400

....

Cl

~

~

0

E3oo

60

l:

E a;"'

>

-;zoo .Ec ., Q)

>

& 100

I

2

3

4

5

3 c

E

E

'>* 150

0

~

(.)

·~ 100

·.;::;

::l

'tl 0

0. Q)

cQ)

.I

Q)

~::l"'

0..

;;.

£ UJ

0 6

Days of storage

Fig. 1: Changes in putrescine levels (11--8) and ethylene production ( +- - + ) in cherimoya fruit pulp during ripening at 20 °C. In all figures values are the means of three replicates of three cherimoyas each. Vertical bars ± SD.

tains the three basic polyamines: putrescine, spermidine and spermine (Figs. 1 and 2). In this phase, Spd was the prevailing polyamine, accounting for 65.2% of the total free polyamines (Fig. 2). Spd levels were not observed to change significantly during the ripening process, while Spm levels were found to increase approximately 1.5-fold (Fig. 2). However, a sharp rise in Put levels at 20 °C was noted from the first to the third days, remaining steady thereafter. The Put levels at the end of the ripening process were nine times higher than those detected in the fruit after harvest (Fig. 1).

-----~-------------·---QL-~---L--~

0

2

4

6

-------·

__L _ _ J_ __ L_ _J __ _ 8 10 12 14 16

l_~--~

18

20

Days of storage

Fig. 3: Putrescine levels in cherimoya fruit pulp at different storage temperatures: 10 °C (--), 8 °C (· · · · ·} and 6 °C (---- -). Vertical bars± SD.

Changes in polyamine levels during low temperature storage Figure 3 shows Put level patterns at different storage temperatures. A sharp rise in Put levels was also observed during storage at 10 and 8 °C. Nonetheless, as observed in ethylene production and other ripening parameters in cherimoya (shown in Table 1), there is a delay in the increase in this diamine level as compared with ripening at 20 °C. Furthermore, the Put levels reached after 13 days at 10 °C and 19days at 8°C were 265.5 and 281nmolsg- 1 fresh weight, respectively; although the difference between these two levels is not significant, it took 6 days longer at 8 than at 10 °C (Fig. 3). Put levels in fruit after storage at these temperatures are lower than the levels found in fruit ripened at 20 °C (Fig. 1).

210

MARIA I. EscRIBANO and CARMEN MERomo 125,----------------------------------,

~

...:

Cl

~ 100

E

.s

I

t L

A similar situation is found in Spm level patterns during low temperature storage (Fig. 5). After 19 days at 6 °C, the Spm levels reach 84% of the post-harvest content, which is higher than at 8 and 10 °C (60%). Unlike Spd, Spm levels remain steady at 10 °C and even increase slightly at 8 and 6 °C at the beginning of the storage period (first 2 days).

II)

~ ~

Ratio between polyamine pools

CD

c:

:0

.E

75

(j;

c.

(/)

4

6

8

10

12

14

16

18

20

Days of storage

Fig. 4: Spermidine levels in cherimoya fruit pulp at different storage temperatures: 10°C (--), 8°C (·····)and 6°C (-----).Vertical bars± SD.

20r------------------------------------.

~

!:!! 0 E

'',. '

.!!1 10

~

-----·-----------~

~ CD

.Ec: (j;

c.

Discussion

The behaviour of polyamines is not uniform in all cases but seems to depend on both the type of plant species and the metabolic stage of tissues. Examination of cherimoya fruit tissues ripened at 20 °C revealed that Put and polyamine titers did not decline nor was their presence inhibitory to the high production of ethylene during ripening. Moreover, the Put content rose concurrently with increased ethylene production. Thus, peak ethylene production is not related to a significant decrease in polyamine levels in cherimoya fruit, as might be expected if the latter had an inhibitory effect. This pattern is similar to senescence of carnation

15

.s

It was found that the Spd + Spm/Put ratio remained steady or increased during the first days at low temperatures. The ratio did not vary at 6 °C while it gradually declined at 8 and 10 °C until a threshold was reached (Fig. 6) as a result of the rise in Put levels as the fruit ripened. It should be noted that despite the higher total polyamine levels reached during ripening at 20 °C as compared with the levels at 10 and 8 °C, and despite metabolic differences, the polyamine ratio (Fig. 6) reached similar values under all treatments where ripening occurred (0.30 on the 3rd day at 20 °C, 0.33 on the 13th day at 10 °C and 0.34 on the 19th day at 8 °C).

. I

5

(/)

OL-~--~--~---L---L--~--J_--~--~~

0

2

4

6

8

10

12

14

16

18

20

5,--------------------------------,

Days of storage

Fig. 5: Spermine levels in cherimoya fruit pulp at different storage temperatures: 10 °C (--), 8 °C (· · · · ·) and 6 °C (---- -). Vertical bars± SD. At 6 °C, a temperature observed in earlier studies to provoke symptoms of chilling injury in cherimoya (data not shown), inhibition of ripening (Table 1) is concurrent with low Put levels (Fig. 3) throughout the trial period (19 days). Figure 4 shows Spd levels throughout low temperature storage. Contrary to patterns in fruit ripened at 20 °C, during low temperature storage Spd metabolism undergoes significant change. At 6, 8 and 10 °C, Spd levels decline during the first 6 days. Later, Spd patterns depend on storage temperature. By the end of the trial period, the Spd levels in fruit stored at 6 °C reach 94% of the level found in the fruit after harvest, but only 71% and 80% at 10 and 8 °C, respectively (although such differences between the latter two are not significant).

---~-------------1---!

II)

CD

c:

.E

"'

...

1

> 0 c..

0 0

2

4

6

8

10

12

14

··-

16

.....

18

20

Days of storage

Fig. 6: Variations in polyamine ratios in cherimoya fruit pulp at different storage temperatures: 20 °C ( • - - • ), 10 °C (--), 8 °C (·····)and 6 °C (-----).Vertical bars ± SD.

Titer of polyamines during cherimoya fruit ripening flowers where endogenous concentration of Spd and Spm did not change as flowers aged, but Put increased dramatically and paralleled a sharp rise in ethylene {Roberts et al., 1984; Serrano et al., 1991). These results are opposite to those found during avocado and tomato fruit ripening in which the level of polyamines dropped rapidly and an inverse relationship between the level of polyamines and the ethylene production rate was established (Winer and Apelbaum, 1986; Casas et al., 1990). In cherimoya, the sharp rise in Put levels and ethylene production also occurs during low temperature storage {10 and 8 °C), when the fruit begins to ripen. Total titratable acidity also increased markedly, mainly as a consequence of malic acid accumulation {Alique et al., unpublished results). However, at chilling injury temperature, Put levels, ethylene production and other ripening parameters remain constant. This would seem to confirm that in this fruit the significant increase in Put levels is related to ripening and not to chilling injury as other authors have formerly established in other fruits (McDonald and Kushad, 1986; Wang andJi, 1989). The pronounced accumulation of diamine during the early stage of the ripening process could be related to the peculiar elevated levels of organic acid or H+ ions in cherimoya. A similar correlation has been pointed out in plants whose metabolism generates an excess of malic acid in the tissue (Morelet al., 1980). In this way, high levels of putrescine in cheriomoya, thanks to its polycationic nature, could provide a system to maintain cellular structures in these acidic conditions. Although there is no certainty as to whether this abrupt rise in diamine levels is a result of ripening or, whether it plays a key role in this stage, our results could reveal the varying role of the diamine in respect to polyamines. Regarding this, Spd and Spm, whose levels do not vary at 20 °C, do not appear to be related to the cherimoya ripening process, taking place despite the fact that the content of these polyamines declines at low temperatures (10 and 8 °C). At 10 and 8 °C similar levels of Spd and Spm are reached, although it takes longer at 8 °C, due possibly to the fact that the fruit's metabolism is slower during storage at the critical temperature; this pattern differs from polyamine metabolism at 6 °C. Moreover, since treatments preventing the appearance of chilling injury keep Spd and/or Spm levels high or even increase them (Wang and Ji, 1989; Kramer and Wang, 1989), the rise observed in Spm levels during the first days of low temperature storage at 6 °C is probably related to the fruit's initial response to cold stress. The ratio Spd + Spm : Put is observed to decline in those treatments in which the fruit begins to ripen. Further investigations are needed to define whether a biosynthetic relationship among the various polyamines is concurrent with the cherimoya ripening process. Changes in conjugated polyamine content are now under study. Acknowledgements

This study was supported by grants AU 92-1272 from the Comision Interministerial de Ciencias y Tecnolog{a de Alimentos and STD2 0266 ES QR) from the European Communities. The invaluable help of Dr. De La Plaza is gratefully acknowledged.

211

References APELBAUM, A., A. C. BuRGOON, J. D. ANDERSON, M. LIEBEllMAN, R. BEN-AIUE, and A. K. MAtoo: Polyamines inhibit biosynthesis of ethylene in higher plant tissue and protoplasts. Plant Physiol. 68, 453-456 (1981). BATTEN, D. J.: Effect of temperature on ripening and postharvest life of fruit of atemoya (Annona cherimola Mill. x A. squamosa L.) cv. . Scientia Hort. 45, 129-136 (1990). BRUINSMA, J. and R. E. PAULL: Respiration during postharvest development of soursop fruit, Annona muricata L. Plant Physiol. 76, 131-138 (1984). CASAs, J. L., M. AcosTA, J. A. DEL Rm, and F. SABATER: Ethylene evolution during ripening of detached tomato fruit; its relation with polyamine metabolism. Plant Growth Regulat. 9, 89-96 (1990). FtolU!S, H. E. and A. W. GALSTON: Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiol. 69, 701-706 (1982). FLOIU!S, H. E., N.D. YoUNG, and A. W. GALSTON: Polyamine metabolism and plant stress. In: KEY, J. L. and T. KosuGE (eds.): Cellular and Molecular Biology of Plant Stress. Allan R. Liss, New York (1985). HYooo, H. and K. TANAKA: Inhibition of 1-aminocyclopropane-1carboxylic acid synthase activity by polyamines, their related compounds and metabolites of S-adenosylmethionine. Plant Cell Physiol. 27, 391-398 (1986). KoSIYACHINDA, S. and R. E. YOUNG: Ethylene production in relation to the initiation of respiratory climacteric in fruit. Plant Cell Physiol. 16, 595-602 (1975). KRAMER, G. F. and C. Y. WANG: Correlation of reduced chilling injury with increased spermine and spermidine levels in zucchini squash. Physiol. Plant. 76, 479-484 (1989). KRAMER, G. F., C. Y. WANG, and W. S. CoNWAY: Inhibition of softening by polyamine application in apples. J. Amer. Soc. Hort. Sci. 116, 813-817 (1991). LAw, D. M., P. J. DAVIES, and M. A. MuTSCHLilll: Polyamine-induced prolongation of storage in tomato fruits. Plant Growth Regul. 10, 283-290 (1991). McDoNALD, R. E. and M. M. KusHAD: Accumulation of putrescine during chilling injury of fruits. Plant Physiol. 82, 324-326 (1986). McNAIR, H. M. and E. J. BoNELU: Basic gas chromatograph. Consolidated Printer, Berkeley, CA, pp. 150-151 (1%9). MoREL, C., V. R. VII.LANUEvA, and 0. Qumoz: Are polyamines involved in the induction and regulation of the Crassulaceam acid metabolism? Planta 149, 440-444 (1980). MoRENO, J. and J. L. DE LA PLAZA: The respiratory intensity of cherimoya during refrigerated storage: a special case of climacteric fruit? Acta Hortic. 138, 179-186 (1983). PALMA, T., T. M. Amm.ERA, and D. W. STANLEY: A review of postharvest events in cherimoya. Postharvest Bioi. Techno!. 2, 187208 (1993). PAULL, R. E.: Postharvest variation in composition of soursop (An· nona muricata L.) fruit in relation to respiration and ethyele production. J. Am. Soc. Hortic. 107, 582-585 (1982). RoBERTS, D. R., M. A. WALKElt, J. E. THOMPSON, and E. B. DUMBROFF: The effects of inhibitors of polyamine and ethylene biosynthesis on senescence, ethylene production and polyamine levels in cut carnation flowers. Plant Cell Physiol. 25, 315-322 (1984). SElUlANo, M., F. RoMOJARRO, J. L. CASAS, and M. AcosTA: Ethylene and polyamine metabolism in climacteric and nonclimacteric carnation flowers. HortScience 26, 894-896 (1991).

212

MARfA I. EscRIBANO and CARMEN ME~tomo

SLOCUM, R. D., R. KAm-SAWHNEY, and A. W. GALSTON: The physiology and biochemistry of polyamines in plants. Arch. Biochem. Biophys. 235,283-303 (1984). SMITH, T. A.: Polyamines. Annu. Rev. Plant Physiol. 36, 117-143 (1985). SMITH, M.A. and P. J. DAVIES: Separation and quantitation of polyamines in plant tissue by high performance liquid chromatography of their dansyl derivatives. Plant Physiol. 78, 89-91 (1985). TREWAVAS, A. J.: The regulation of development and its relation to growth substances. What's New Plant Physiol. 13, 41-43 (1982).

WANG, C. Y. (ed.): Chilling injury of horticultural crops. CRC Press Inc., Boca Raton, Florida (1990). WANG, C. Y. and Z. L. }1: Effect of low-oxygen storage on chilling injury and polyamines in zucchini squash. Scientia Hort. 39, 1-7 (1989). WILLS, R. B. H., A. Pm, H. GREENFIELD, and C. J. RIGNEY: Postharvest changes in fruit composition of Annona atemoya during ripening and effects of storage temperature on ripening. HortScience 19, 96-97 (1984). WINER., L. and A: Apelbaum: Involvement of polyamines in the development and ripening of avocado fruits. J. Plant Physiol. 126, 223-233 (1986).