Weed Control, Plant Physiology and Biochemistry

Weed Control, Plant Physiology and Biochemistry SUGARCANE ANTHOCYANINS AS COLOUR PRECURSORS AND PHYTOALEXINS P. Smith and P. M. Hall C.S.R. Research Laboratories, Roseville, N.S.W. Australia ABSTRACT Gel filtration using cross-linked dextran gels can be used to great advantage in research on sugar colour. The technique can be simple and the results directly applicable to applied studies. The method suited for applied work is described in sufficient detail for others to use. Modifications are presented that not only reduce labour and time required per test but also increase sensitivity. Lead acetate precipitation and paper chromatography to expand the meaning of. gel filtration is described. Several examples of the use of the methods are illustrated based on anthocyanins associated with sugarcane. Findings are general, but cane tops, shoots and the like introduce anthocyanins to a great extent. The predominant anthocyanins in one variety (NCo 310) are thought to be luteolinidin derivatives. Red rot infection raises the level of anthocyanlns, The materials polymerise rapidly but much of these will be removed in clarification. However, some residual polymeric anthocyanins pass this process step. Degradation products from anthocyanins respond to iron contamination. Differences in anthocyanin content and composition are sufficiently. great to provide a basis for a possible new method for variety identification and might be useful for screening for disease resistance. INTRODUCTION Methods for colour studies, mentioned in a previous paper (Smith, 1971), have been used to advantage in research and factory studies. Nevertheless, for certain work some finer resolution of colour types is necessary, and one of the methods available IS based on gel filtration using cross-linked dextran gels. The method has been used in many studies on sugar, mainly as a research tool for the isolation of individual molecular species. There has been little practical application (Smith, 1966; McDonald, 1966; Gross, 1969; Farber et al., 1968). Yet this technique is directly applicable in the practical field as evidenced by Somers' resolution of wine anthocyanins (Somers, 1967, 1968). We also have used the technique for refinery and mill studies though not with the aim of fine resolution but rather to give broader separation and identification." Techniques are described which have been stream-lined with regard to the effort required and have been modified to give finer detail, than in normal practice. The use of the methods is illustrated by examples based on the anthocyanins associated with the growing point and red rot stalk tissue of sugarcane. 1139

1140 WEED CONTROL, ETC. EXPERIMENTAL METHODS Conventional Gel Filtration Technique for Separation of Sugar Colour "" Sufficient Sephadex gel (Pharmacia G25 fine grade) is prepared to fill a column 40-45 em high and about It cm diameter (bed volume 80-85 ml). Before packing, the gel is soaked for some hours in 50% aqueous acetone. Sufficient hydrochloric acid (1.5 ml) is first added to' each litre of aqueous acetone to give a solution ph of 2. In one experiment I ml of acidic methanol extract from the growing point of a stalk of variety NCo 310 was loaded on the column and eluted with the same aqueous acetone at 6-8 mlyhour. The effluent was collected in a fraction' collector in 3 ml aliquots. The optical densities of the fractions were measured at 495 nm and the results plotted "against cumulative volume of effluent as shown in Fig. IA, The profile consisted of a polymeric (first peak) and 2 monomeric peaks. 2 A. 2 B. = -, on 05., '0 o 20 40 0 Volume eluted.(ml). 20 40 Fig. 1. Molecular profiles of inverse ph-sensitive pigments present in the growing point of NCo 310 as determined by the conventional technique. A = fresh extract room temperature. B = extract 6 hours at room temperature. A Refined Method for Gel }filtration The conventional method has disadvantages in terms of speed, resolution and labour for many absorbance measurements. Therefore a refined method was developed. The colour detection unit of the system was devised by M. B. Wilson of our Laboratories, wherein, the gel separated effluent is monitored at 493 nm by a simple spectrophotometer, fitted with a special flow cell (1 cm light path). The light intensity emerging from the cell is relayed as a signal (mv) to a chart

P. SMITH, P. M. HALL 114l recorder. The chart speed is proportional to the elution rate. Instrumental details will be reported later elsewhere. Contrary to accepted gel filtration experience, we achieve excellent separation by using a much smaller gel bed (15 ml) and more rapid elution rates (21.5 ml/hr). Furthermore, there is minimal mixing of separated components emerging from the column since the flow cell monitors quantities as small as 0.25 ml. Consequently, the molecular profiles by this method have finer detail than would be obtained by the manual method. This is shown in Fig. 2, where t ml acidic methanol extracts of different NCo 310 samples were resolved. 4,------,--------,r--------,.--------..., 5... E c: E'6..., '"... " g (a) (b) (c) (d) ;;. 8.. 9 1,g 10 '-- ---"'0...1.-'-- "" --'- ---... Elutian valume. (1div=,SoSml) Fig. 2. Molecular profiles of acidic methanolic extracts of different NCo 310 samples as determined by the refined technique. (a) inflorescence, (b) growing point, (c) non-arrowed upper stalk, (d) arrowed upper stalk. Three main peak profiles were obtained for samples (a), (b) and (c) but secondary peaks between peaks 2 and 3 were now observed with the superior resolution of the refined method. Molecular Profiles After Lead Acetate Precipitation Anthocyanins are readily precipitated from acidic methanol extracts with the addition of aqueous lead acetate solution (20% wiv). The precipitate may then be filtered off and redissolved in a smaller volume of stronger acidic methanol (10%.HCl). This provides a convenient method for concentrating anthocyanin mixtures for gel filtration studies. This technique again extends the potential of the gel filtration metho. Traces for gel separated pigments (refined method) precipitated from methanol extracts of a few cane varieties are shown in Fig. 3. Paper Chromatography When further resolution and identification of the resolved components are deired, it is quite easy to apply paper. chromatographic techniques: These

1142 WEED CONTROL, ETC. 2, I I I I I I I I I I I I I I t, E 5. ( b) Ie) E c: ; IS 1i i..... c: 10I I I I I I I I I I C I I "r=-r-1 Elution volume. II div 5'5ml) Fig. 3. Molecular profiles of lead acetate precipitated pigments from different cane varieties. (a) Q 83, upper portion, (b) Q 83, lower portion, and (c) Q 63 whole stalk. are not as sophisticated as some othf techniques but still yield useful information. Fractions, collected in conventional gel filtration,corresponding to profile peaks in Fig. 1 and 4 were chromatographed on Whatman 3MM paper in one dimension using the upper phase of butanohacetic acid:water (4: 1:5) 5... I I I i '4 3 ] E""'2 c: II> d d 1 o1/ I' I I o 2 40 50 Volurn. oluhd.(ml). Fig. 4. Molecular profile of pigments extracted from red rot infected cane plant (lower part-e NCo 310). Conventional technique.

i 'I! P. SMITH, 1'. M.HALL 1143 mixture as the developing solvent. The locations of the main components are shown in Fig. 5. The inverse ph-sensitive colourants (certain monomeric antho- 1'0 GROWING POINT (NCo310) INFECTED CANE A [}:{J 1'0 (vis) B 100.. I II a.j:. I A. 11 Ie \; e la A. 13 A. >- : ", i II Co!! :..! l'!':>... 5 I... 'f'!!'. c_ I :: - II.." c c. e c '".!f- 11 I. invm'h ph-senliitlvl! «. "'- 't: :a! I ai '". c u i-.!!. L5 d:;:o.., e r;; "" II:: (VI') brown(vi.) i t l"5 I c::::::::>.g." rod c:::::::> (vi.) c::::m. d ) : :-oci«vj:!> I I I 0 0 0 Peak 1 peak 2 Peak 3 Peak 1 Peak 2 Peak 3 Fig. 5. Paper chromatograms. cyanins) were visualized by spraying with acidic methanol and found to be confined to peaks 2 and 3; peak I was polymeric. These or similar methods can be applied to colour studies throughout the sugar industry, but.for the sake of illustration the findings of the samples cited will be amplified. The emphasis in this paper is on cane anthocyanins. Anthocyanins RESULTS AND DISCUSSION Before we illustrate the applications of the methods some background information on anthocyanins should be reported. Visible spectra of anthocyanin extracts. Anthocyanins are a class of flavonoid compounds which are responsible for the entire range of red, violet and blue pigmentation occurring in plants. They exist in the dissolved state in the cell sap of flowers, fruits and other plant organs. The aglycones, or sugarfree residues of these compounds, are called anthocyanidins. All of the latter have a common structure termed the flav)dium cation: I '5

1144 WEED CONTROL, ETC. Anthocyanins deepen in colour with decreasing ph. A convenient method for detecting them in cane is to' shred it and then contact with acidic methanol (0.1 %-1% HCl). The liquid becomes deep red through the absorbance of the flavylium nucleus at the low ph. Illustration 1. Anthocyanin Concentration in Different Parts of the Plant. Equal amounts (30 g) of cane sampled from the growing point and top and bottom thirds of the stalk were immersed in 200 ml of acidic methanol for 24 hours at room temperature. The visible spectra Fig. 6 were obtained. loll '9 '8 '7 '6 E'5, 2, d"...., 0'3...':':..I)... 2... "".::".:;:.".::,...:..".",.",."'''"...::...:.:.::;.::,.."...::.::,..'''..:.:...".:.:.'''.:.:..'''''''':':''''''",,,,,, 0'0 1 Ll-------;tO---:-----5to----- 6( 400 450 490 500 550 600 Wavelength, A.nm Fig. 6. Spectra of acidic methanol extracts from different parts of the cane plant (NCo 310). --- growing point, - - - - - top,..... bottom. Clearly, the highest concentration of anthocyanins occurred in the growing point. The growing point extract had an absorbance maximum (A. max) at 495 nm. This maximum is helpful in making a tentative identification of the anthocyanidin derivatives causing it. Table 1 gives the A. max for the various Table 1. Visible spectra of anthocyanidins (Harborne 1958). Pigment Delphinidin Malvidin Cyanidin Pelargonidin Luteolinidin Apigeninidin Absorbance maximum in methanol (HCl) (nm) 546 542 535 520 493 476 anthocyanidins. On the basis of the evidence, luteolinidin derivatives were likely the anthocyanin components in the growing point region. More specific tests for the broad class of anthocyanins are reported in Appendix 1.

p.smith, P. M. HALL Illustration 2. Anthocyanins as Precursors to Polymeric Colour 1145 The methanolic extract from the growing point, on exposure to air darkened.with time. After 6 hours, 1 ml of the darkened extract was separated b; gel filtration as had been done whenfresh. A 3-peakprofile was again obtained but in this case the polymeric peak was reinforced at the expense of the monomeric ones (Fig. 1). Clearly the monomeric anthocyanins were precursors to the polymeric material; probably oxidation had effected the polymerization. This short experiment demonstrated the high instability of these growing point constituents with respect to colour formation. Probably this same colour reaction occurs when cane tops are cut and could explain why they darken soon after cutting. The juice itself would be unstable and, although clarification efficiently removes polymeric colourants, it is possible for this process step to be overloaded, allowing residual polymeric colourants from the anthocyanins to remain after clarification. Illustration 3. Anthocyanins in Infected Cane Yet another example may be cited; Fig. 5 is relevant. Several infected portions of stalk were cut from the lower part of a cane plant (NCo 310). The infection was said to be "red rot." The lower part was deliberately selected because it contained the lowest concentration of anthocyanins when the stalk was healthy. The infected cane was allowed to soak in acidic methanol for 24 hours, and 1 ml of the extract was separated by gel filtration as previously described. A 3-peak profile was again obtained (Fig. 4). The effluent fractions corresponding to each peak were separated by paper chromatography as previously described. The inverse ph-sensitive colourants (monomeric anthocyanins) were located as shown in Fig. 5. Their absence in peak 2 sharply differentiated the infected cane tissue from sound material. The colourants in peak 2 streaked on the chromatogram failed to separate into discrete bands. It is tempting to postulate that the peak-2 components originally contained inverse ph-sensitive colourants (anthocyanins) which were more copiously produced and subsequently altered chemically following the infection of plant tissue. Polyphenol-oxidase enzyme systems are known to oxidise flavonoids to quinones and tannins in response to tissue infection in other plants. These are thought to act as toxins to the fungal enzyme system (Bryde, 1960). The practical importance from these findings is not clear, but as the study continues some directly useful facts may emerge. They may be through variety selection with relevance to the biochemistry and physiology of cane. Illustration 4. Lead Acetate Precipitated Pig;}nents The use of lead acetate precipitation may be illustrated by an experiment to which Fig. 3 is applicable. Sufficient lead acetate solution was added to 600 ml aliquots of acidic methanol (l% HCI) extracts of 325 g samples of cane to precipitate anthocyanins. The samples were from Q 83 (upper portion of stalk), Q 83 (lower portion of stalk) and Q 63 (whole stalk). The precipitates were re-

1146 WEED CONTROL, ETC. dissolved in 25 ml aliquots of acidic methanol (10% H'Cl). Extracts (1 ml) were separated as before. The profiles are shown in Fig. 3. The upper part of the stalk of Q 83 was dark red, while the lower portion was lightly coloured. It is not unexpected then that the profiles of the 2. Q83 samples should also differ. The upper profile was characterised by a dominant polymeric anthocyanin peak and anapparenlabsence of the third peak, while the lower profile consisted.of a small polymer peak and 2 large monomeric peaks. \. The Q63 cane variety did not have a highly coloured stalk. Its profile was characterised by 2 dominant monomeric peaks and a minor polymeric one. Much knowledge has been built up about cane variety pedigrees and disease resistance of different kinds by long painstaking field trials. We are now in a position to attempt some correlation between anthocyanin profiles and inherited disease resistance for particular cane varieties. It is well established that polyphenols (of which anthocyanins are a class) are natural inhibitors to various plant diseases. There seems to be more than a remote chance of finding some characteristic anthocyanin molecular profile for varieties with a common resistance to a given disease. Also the technique paves the way for more rapid surveys of both commercial and experimental cane varieties with respect to their potential colour contribution in milling and refining. 'REVERENCES Bate-Smith, E. C. 1948. Nature, 161:835. Bryde, R. J. W. 1960. Plant Phenolic Symp. Phenolics of plants in health and disease. Pergamon Press, London. p. 95. Farber, L., E. J. McDonald, and F. G. Carpenter. 1968. Proc, Tech. Session Cane Sugar Refining Res. p. 85-104. ' Geissman, T. A. 1962. The Chemistry of Flavonoid Compounds, Pergamon Press, p. 51 and 252. Gross, D. 1967. Int. Sugar J., 69:323-328, 360-365. Harborne, J. B. 1958. Biochem. J., 70:22. Harborne, J. B. 1959. Chromatographic Reviews 1:213. McDonald, E. J., and J. P. Madacsi. 1966. Proc, Tech. Session Cane Sugar Refining Res. p. 136-139.. Shriner, R. L. 1950. Rec. Chern. Prog, 11:121. Smith, P., and P. E. Gregory. Proc, ISSCT, 14. Smith,N. H. lq66. Proc, Tech. Session Cane Sugar Refining Res. p. 84-102. Somers, T. C. 1967. J. Sci. Fd. Agric., 18:193-196. Somers, T. C.196B. Vitis, 7:303-320. APPENDIX I The following chemical tests were carried out to confirm the anthocyanin basis of the red colour of the different acidic methanol extracts of cane samples. References pertaining to. their use in literature are also listed. (a) Addition of 20% aqueous lead acetate solution removed all red colour of the methanol extract with the formation of a blue lead salt precipitate (Geismann, 1962). (b) The pink bands located on one dimensional paper chromatograms after resolution of the methanol extracts intensified in colour after visualizing with acidic methanol sprays (Shriner, 1950). (c) The same pink bands in (b) turned blue after. fuming the chromatograms with ammonia vapour (Bate-Smith, 1948; Harborne, 1959; Ceismann, 1962).