The Inhibition of Tomato Fruit Ripening by Silver

The Inhibition of Tomato Fruit Ripening by Silver GRAEME

E. HOBSON, ROYSTON NICHOLS, JACK N. DAVIES and PETER T. ATKEY

Glasshouse Crops Research Institute, Rustington, Littlehampton, West Sussex BN16 3PU, U.K. Received March 5, 1984 . Accepted April 16, 1984

Summary Mature green tomato fruit, infiltrated with STS (up to 10JLmol) while still attached to the plant, ripened unevenly to give extensive green areas on an otherwise red background. Pericarp wall tissue from the two contrasting areas was analysed for various organic constituents. Both the green and, to a cenain extent, the red tissue from treated fruit showed differences from normal in AIS, acidity, and PE activity. PG activity, which usually increases rapidly as tomatoes ripen, was low in the green but not significantly different from normal in the red tissue from ST5-treated fruit. TEM examination revealed that electron-dense panicles were present in the cell walls of phloem elements in vascular bundles of the green tissue, but these deposits were not found in the red tissue from the same fruit. X-ray microanalysis of the panicles suggested that they contained concentrations of silver and sulphur. The results are interpreted as suggesting that silver is affecting those sites in the cell that would subsequently be involved in promoting the synthesis of PG.

Key words: Lycopersicon esculentum, blotc/ry ripening, inhibition by silver, ripening mechanism, systemic infiltration, tomato composition, X-ray microanalysis.

Introduction Several physiological disorders of tomatoes result in an uneven change in colour as the fruit ripen. In one of these, exacerbated by potassium deficiency and known as «blotchy ripening,. (Hobson and Davies 1976), parts of the outer pericarp walls fail to ripen so that green areas of tissue remain on an otherwise red tomato. Compositional analyses have shown that both the red and the green tissue from blotchy fruit are different from normal tissue of comparable colour (Winsor et al. 1962 a). In a survey of the effects of a number of heavy metal ions on the ripening of discs of green tomato wall tissue (Saltveit et al. 1978), silver salts were shown to stop ripening, probably by interfering with the action of ethylene in promoting the normal sequence of developmental changes. While we have confirmed that silver as the nitrate is able to prevent whole green detached tomato fruit from ripening (data not shown), the metal is much more mobile in plant tissue as the anionic thiosulphate complex (Veen 1983). Abbreviations: AlS, alcohol-insoluble solids; PE, pectinesterase; PG, polygalacturonase; STEM, scanning transmission electron microscope; STS, silver thiosulphate; TEM, transmission electron microscope.

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G1lAEME E. HOBSON, ROYSTON NICHOLS, JACK N. DAVIES and PETER T. ATKEY

We have found that if silver as a complex with thiosulphate is infiltrated through the vascular system in part of the peduncle into a fruit truss, then a variable proportion of the outer walls of the tomatoes fails to change colour, reminiscent of blotchy ripening. The composition and pectic enzyme activity in the differently coloured parts of treated fruit have been compared with normal tissue. TEM has located electron-dense particles that are particularly associated with phloem cells in the vascular bundles, and X-ray microanalysis has shown the deposits to be rich in silver and sulphur.

Materials and Methods Tomato [Lycopersicon esculentum (Mill.)] plants, cvs. Kingley Cross and Sonatine, were grown in tarred paper (<
Results

Effects ofsilver infiltration on composition Preliminary experiments were carried out using cv. Kingley Cross. Similar results were later obtained with cv. Sonatine, and it is these data that are presented here. Information on the composition of tomatoes immediately before and after ripening is shown in Table 1. Neither dry matter content nor reducing sugar levels altered signif-

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Table 1: Influence of silver (2 mM STS) infiltration on the composition of locule wall tissue from tomato fruit (LycopersKon esculentum cv. Sonatine). The values in the body of the Table are the means of triplicate determinations. Constituent

Mature green Dry matter (gll00 g fresh wt.) Alcohol-insoluble solids (gll00 g fresh wt.) % glucose + fructose (gl100 g fresh wt.) Titratable acidity (meq/l00 ml expressed sap) Total acidity (meq/100 ml expressed sap) Combined acidity as a percentage of the totala)

Colour of silvertreated tissue

Stage of ripeness of normal tissue Red

Green

Red

Least significant difference at P = O.OSb)

6.19

6.11

7.09

6.66

0.84

3.34

2.20

2.99

2.70

0.50**

3.34

3.77

3.57

3.82

0.73

4.51

6.00

6.10

8.28

1.58**

8.60

9.76

9.80

12.00

1.52**

47.6

38.5

37.6

30.9

a) Combined (or bound) acidity is the difference between total and titratable acidity. b) The symbol ** indicates that the means are significantly different at P <0.01. icantly, although the latter has a tendency to rise with ripeness (Davies and Hobson 1981). AIS usually fall by about a third during this process (Davies and Hobson 1981), and the data for normal tissue in Table 1 are consistent with this. However, the STStreated wall tissue behaved differently and the reduction in AIS was much less than in normal tissue, resembling that observed with blotchy tissue (Winsor et al. 1962 a). It has been consistently found that the titratable acidity of pericarp wall tissue rises sharply from mature green to a maximum following the first appearance of yellow colour (Winsor et al. 1962 b), and then slowly decreases; the data in Table! fit into this pattern, with greater acidity occurring with red tissue than with green. Both the green and red tissue from silver-treated fruit showed a significantly increased titratable acidity, reflected in decreased percentages of combined acidity in the total figures. The change in texture as tomato fruit ripen is accompanied by a loss of pectic material (Hobson 1963) due to the sequential action of two enzymes, PE and PG. Whereas PE activity does not rise much during ripening (Hobson 1963), that of PG increases dramatically (Hobson 1964; Tucker et al. 1980). The softening of tomato fruit through PG activity is almost always accompanied by pigment transformations from green through to red. In blotchy ripening, the near-absence of lycopene and (j-carotene is matched by a severe attenuation of PG synthesis (Hobson 1964). The expected rise in PE activity was shown by normal tissue during ripening (Table 2), but green tissue from silver-treated fruit contained higher activity of this enzyme than is usual for tissue of this colour. PG determinations indicated that although red j. Plant Physiol. Vol. 116. pp. 21-29 (1984)

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GIlAEME E. HOBSON, ROYSTON NICHOLS, JACK N. DAVIES and PETER T. ATKEY

Table 2: Influence of silver (2 mM STS) infUtration on pectic enzyme activity in locule wall tissue from tomato fruit (Lycapersicon esculentum cv. Sonatine). The values in the body of the Table are the means of triplicate determinations. Stage of ripeness of normal tissue

Enzyme activity

PE (units/100 g fresh wt.) PGa) (units/100 g fresh wt.)

Colour of silvertreated tissue

Least significant difference at P = 0.05b)

Mature green

Red

Green

Red

4.01

5.02

4.69

4.86

0.68'"

0

6531 (3.815)

226 (2.355)

4753 (3.677)

(0.32.........)

a) Individual values were statistically analysed after logarithmic transformation. The means and the L.S.D. in logarithmic form are given in parentheses. b) The symbols ... and ......... indicate that the means are significantly different at P < 0.05 and <0.001, respectively.

tissue from fruit that had been exposed to silver exhibited less activity than normal red tissue, the difference was not significant. The walls from green parts of ST5treated fruit displayed less than 5 % of the PG activity of those from red areas. To be effective in preventing ripening, we found that silver treatment must be given prior to the commencement of the autocatalytic phase of ethylene production. Assuming that translocation of silver is quantitative, about 2 #Lmol seems to be the minimum amount that is capable of preventing 100 g tomato wall tissue from ripening. Concentrations of about this magnitude were found in treated tissue, but the particular colorimetric method used suffered from interference from accompanying anions, and c~nsistent results were not obtained.

Evidence from electron microscopy Silver is electron-dense and appears black when unstained biological material is viewed by TEM. However, because sections from tomato fruit had received neither post-fixation in osmium nor heavy metal staining, the contrast was generally of a low order. Nevertheless, dense black particles were found in green tissue from fruit treated with silver, some of which were up to 25 nm in diameter although most were considerably smaller. Such particles were absent from red tissue adjacent to the green, or from untreated control tissue. In sections from fruit exposed to 2 or 10 roM STS, the electron-dense deposits were found in phloem cell walls, particularly associated with the middle lamella (Fig. 1). Additionally, they were observed in the intercellular spaces of the parenchyma of the vascular tissue where the middle lamellae bordered a gap between cells (Fig. 2), suggesting that movement of silver was largely apoplastic. In tissue treated with 20 roM STS, particles were also detected in the walls of xylem vessels.

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Fig. 1: Particles (arrowed), thought to be rich in elemental silver, deposited largely in the middle lamellae and intercellular spaces of phloem in tomato fruit wall tissue infiltrated with 2 mM STS. Bar mark = 1 !Lm; unstained. The centre of the circled portion is enlarged in the inset. Bar mark = 0.25 !Lm.

2 Fig. 2: Enlargement of an intercellular space in phloem showing particles lining the lumen. Bar mark = 0.25 !Lm.

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G1lAEME E. HOBSON, ROYSTON NICHOLS, JACK N. DAVIES and PETEIl T. ATKEY

!', :

3

I

5

~~ 51

CI

I

K

~Cu .

•

keY 4

I . : 1\

rJ:V~ ·Cu

Fig. 3

Si

SCI

~CUl)CU ...

•

keY

..

Fig. 4

Fig. 3 and 4: X-ray energy spectra of sections of tomato fruit tissue. The trace when an area containing electron-dense particles was viewed (Fig. 3), compared with a similar area without such deposits (Fig. 4). Allowing for a greater level of attenuation in Fig. 3, Ag and S are clearly associated with the presence of the particles, but the peaks in Fig. 3 for CI and K are probably not significantly different from background (Fig. 4). j. Plant Physiol. Vol. 116. pp. 21-29 (1984)

Inhibition of tomato fruit ripening

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x-

Typical electron-dense particles from green STS-treated tissue were examined by ray microanalytical techniques. A print-out of the resulting energy spectrum is represented in Fig. 3, and that for an adjacent nonelectron dense area is given in Fig. 4. Characteristic spectral peaks for silver occurred in the first trace (Fig. 3), which were absent in the other (Fig. 4). The sulphur response was also much more prominent in Fig. 3, strongly indicating that the particles contained high concentrations of silver and, to a lesser extent, sulphur. It was concluded that the chlorine and sulphur responses in the control trace derived solely from constituents of the embedding resin, and those for copper from the support grid. Differences in the height of the copper peaks between Figs. 3 and 4 were due to alteration in the attenuation settings of the analyser. Discussion The introduction of silver through only part of the transpiration stream leading to a fruit truss results in a proportion of the outer pericarp wall remaining permanently green while the rest of the wall becomes red, apparently quite normally. This infiltration technique facilitates direct comparison of the two types of tissue from the same fruit. The boundary between the two areas is quite sharp, as with blotchy ripening, and the resulting ripening pattern suggests that there is a direct connection between particular vascular strands in the peduncle and specific parts of the fruit. The response to silver treatment and the onset of blotchy ripening lead to fruit that can be regarded as abnormal (Table 1; Winsor and Massey 1958; Winsor and Massey 1959), while the compositional changes engendered by either condition have some similarities (Tables 1 and 2; Hobson and Davies 1977). Indications are that silver is drawn preferentially to sites in the middle lamellae of selected cells (unless a superabundance of the cation is used), and no evidence of deposits in the cytosol or ground tissue was found. It is clear that the synthesis of PG and the associated colour changes that follow are particularly sensitive to low concentrations of silver, especially when suitably complexed and introduced prior to incipient ripening. Blotchy ripening has been linked with a collapse of the parenchyma round the vascular bundles in the affected regions (Seaton and Gray 1936). Since the trends in composition for silver-treated tissue and that showing blotchy ripening are similar, a failure in autocatalytic ethylene production and PG synthesis from whatever cause leads to tissue the composition of which does not fit into any part of the normal ripening pattern. In an investigation into the fate of silver after STS treatment of cut carnation flowers, Veen et al. (1980) demonstrated that deposits occurred in xylem cells of the receptacle tissue. X-ray analysis showed that the deposits contained both silver and sulphur. Our results suggest that if minimal quantities of STS are used with tomato fruit, particles accumulate preferentially in the walls of phloem and associated parenchyma rather than in xylem. It is evident to us that exogenous ethylene is not able to restore the ability of either j. Plant Physiol. Vol. 116. pp. 21-29 (1984)

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GkAEME E. HOBSON, ROYSTON NICHOLS, JACK N. DAVIES and PETER T. ATKEY

silver-treated or blotchy fruit to ripen uniformly, and we have clear indications that in neither type of unripe tissue is ethylene production likely to be limiting to further development (data not shown). We also have preliminary evidence that silver in amounts sufficient to stop ripening does not affect the capacity of the tissue to synthesise specific proteins such as nitrate reductase. Silver has been shown to be capable of altering the binding characteristics of active sites for ethylene, and to inhibit metabolism of the hormone (see Veen 1983). Either of these two explanations for the action of silver is at present preferable to one involving direct inhibition of PG, or even inactivation of the enzyme following transport to the middle lamella. The quantities of silver involved are thought to be too small to counteract the very considerable amount of the enzyme that is synthesised during normal ripening. The potent effect of silver on the ripening mechanism is being investigated in more detail in attempts to understand the controlling processes more deeply. Acknowledgements

x-

We would like to thank Dr. A. Brown, Jeol (UK) Ltd., for invaluable help and advice with ray microanalysis, Mrs. Carol Frost for cutting the sections, and Mr. J. Pegler for production of the Figures.

References DAVIES, J. N.: Effect of nitrogen, phosphorus and potassium fertilisers on the non-volatile organic acids of tomato fruit. J. Sci. Food Agric. 15,665-673 (1964). DAVIES, J. N. and G. E. HOBSON: The constituents of tomato fruit - the influence of environment, nutrition and genotype. CRC Crit. Rev. Food Sci. & Nutr. 15,205-280 (1981). DAVIES, J. N. and R. J. KEMPTON: Changes in the individual sugars of tomato fruit during ripening. J. Sci. Food Agric. 26, 1103-1110 (1975). HOBSON, G. E.: Pectinesterase in normal and abnormal tomato fruit. Biocheni. J. 86, 358-365 (1963). - Polygalacturonase in normal and abnormal tomato fruit. Biochem. J. 92, 324-332 (1964). - Effect of the introduction of non-ripening mutant genes on the composition and enzyme content of tomato fruit. J. Sci. Food Agric. 31,578-584 (1980). HOBSON, G. E. and J. N. DAVIES: Protein and enzyme changes in tomato fruit in relation to blotchy ripening and potassium nutrition. J. Sci. Food Agric. 27, 15-22 (1976). - - A review of blotchy ripening and allied disorders of the tomato, 1957-1976. Ann. Rep. Glasshouse Crops Res. Inst. for 1976, 139-147 (1977). KARNovSKY, M. K.: A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell BioI. 27, 137A-138A (1965). SALTVEIT, M. E., K. J. BRADFORD, and D. R. DILLEY: Silver ion inhibits ethylene synthesis and action in ripening fruits. J. Amer. Soc. Hort. Sci. 103, 472-475 (1978). SEATON, H. L. and G. F. GRAY: Histological study of tissues from greenhouse tomatoes affected by blotchy ripening. J. Agric. Res. 52, 217-224 (1936). SNELL, F. D. and C. T. SNELL: Colorimetric Methods of Analysis, voI.llA, pp. 25-26. Van Nostrand, Princeton, NJ, 1959. SPURR, A. R.: A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969). TUCKEIl, G. A., N. G. ROBEilTSON, and D. GIlIE1lS0N: Changes in polygalacturonase isoenzymes during the «ripening» ofnormal and mutant tomato fruit. Eur. J. Biochem. 112, 119-124(1980).

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H: Silver thiosulphate: an experimental tool in plant science. Scient. Hort. 20, 211-224 (1983). VEEN, H., S. HENSTRA, and W. C. DE BRUYN: Ultrastructural localisation of silver deposits in the receptacle cells of carnation flowers. Planta 148, 245-250 (1980). WINSOR, G. W. and D. M. MASSEY: The composition of tomato fruit. I. The expressed sap of normal and «blotchy" tomatoes. J. Sci. Food Agric. 9, 493-498 (1958). - - The compositon of tomato fruit. II. Sap expressed from fruit showing colourless areas in the walls. J. Sci. Food Agric. 10, 304-307 (1959). WINSOR, G. W.,J. N. DAVIES, and D. M. MAsSEY: The composition of tomato fruit. IV. Changes in some constituents of the fruit walls during ripening. J. Sci. Food Agric. 13, 141-145 (1962 a). - - - The composition of tomato fruit. V. Comparison of the differently coloured areas of the walls of «blotchy" tomatoes. J. Sci. Food Agric. 13, 145-148 (1962 b). VEEN,

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