Fertilization, Soils and Cultural Practices

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Fertilization, Soils and Cultural Practices THE EFFECT OF SILICON ON ENZYME ACTIVITY IN VITRO AND SUCROSE PRODUCTION IN SUGARCANE LEAVES Y. Wong You Cheong, A. Heitz and J.

1 Fertilization, Soils and Cultural Practices THE EFFECT OF SILICON ON ENZYME ACTIVITY IN VITRO AND SUCROSE PRODUCTION IN SUGARCANE LEAVES Y. Wong You Cheong, A. Heitz and J. Deville Mauritius Sugar Industry Research Institute Reduit, Mauritius ABSTRACT The efficiency of sugar production in leaf tissue collected from canes of 2 varieties growing in nutrient solution containing different levels of Si was studied as well as the effect of Si in vitro on the activity of cane and yeast invertase and cane tyrosinase. Si deficiency decreased the rate of photosynthesis in symptom-free leaf tissue of cane whose older leaves were showing Sideficiency symptoms. This effect was also obtained in completely symptomless Si-deficient cane growing under a Perspex roof. It would appear, therefore, that the lower efficiency of photosynthesis of Si-deficient leaves was not connected with the appearance of symptoms, i.e., degradative changes in leaf tissue. It was probably related to some other unknown function of Si in plant metabolism. The inhibition of invertase activity by Si in vitro was shown to be uniquely due to ph changes in the incubation medium brought about by the addition of sodium silicate. Si had no effect on cane and yeast invertase or cane tyrosinase activity in vitro. INTRODUCTION It has never been clearly established that Si is an essential element for the optimum growth of plant species. However, there is now increasing evidence that addition of Si to certain plants very low in that element promotes growth. Si in the rice plant has been shown to increase photosynthetic surface (Anon., 1966), oxidising power of roots (Okuda et al., 1964), protection from pests and diseases (Ota et al., 1957), and to reduce transpiration (Yoshida et al., 1959). Beneficial effects of Si on sugarcane growth in the field have been attributed to a reduction in Mn toxicity (Halais and Parish, 1963), but Wong You Cheong and Halais (1970) showed that sugarcane tolerated high levels of Mn and concluded that sugarcane had a basic need for Si. If Si plays an essential role in plant growth, it is biochemical, either as Si-organic complexes or as a catalyst. However, infra-red studies (Yoshida et al., 1959) have shown that the bulk of Si wds present as. SiO, gel in the rice plant. Alexander (1968) reported that Si inhibited the action of invertase although no evidence was obtained on its action on enzymes promoting the production of sucrose. Wong You Cheong et al. (1968) obtained an increase in phosphorylation in both roots and leaves of sugarcane plants deficient in Si when they were supplied with that element. The greater concentrations of sucrose precursors thus obtained should result in an increased sucrose production in the leaves. It was, therefore, the object of this study to verify the effect of Si on sucrose production and on invertase and tyrosinase activity in vitro.

778 FERTILIZATION, ETC. EXPERIMENTAL PROCEDURE The effect of Si on enzyme activity was studied by using commercial yeast invertase in 2 ways, polarimetry and colorimetry.

2 778 FERTILIZATION, ETC. EXPERIMENTAL PROCEDURE The effect of Si on enzyme activity was studied by using commercial yeast invertase in 2 ways, polarimetry and colorimetry. In the polarimetry studies, inversion was carried out at room temperature in an acetate buffer medium (ph = 5.5) adjusted to 2 ionic strengths (I = 0.1 and 0.4). The inversion mixture consisted of 1 ml invertase and 4 g of sucrose in a final volume of 25 ml. Polarimeter readings were taken at regular intervals. For the colorimetry studies, inversion was followed by measuring reducing sugars by the arseno-molybdate method (Snell and Snell, 1953). The incubation mixture consisted of 1 ml sucrose in acetate buffer, giving 30 moles sucrose/nll digest, 0.25 ml water or Si solution, and 0.25 ml diluted invertase preparation. Incubation was carried out at 30 C for 20 min and was stopped by placing the reaction tubes in boiling water for 10 min. Here also, the two ionic strengths of buffer were used. Si was added as sodium metasilicate or silicic acid at 0, 2.8 and 8.0 moles Si/ml of digest. Silicic acid was obtained by passing metasilicate through Amberlite IR-120 in the H form. Phosphate buffer (ph 5.5) was also used at 2 ionic strengths (I = 0.1 and 0.4). The effect of Si on cane invertase and tyrosinase was examined by extracting these enzymes from the meristem of stalks deficient in Si. The meristem was ground with quartzsand in the ratio 3:1, and the juice was expressed through nylon cloth. The residue was again crushed in 5 ml ice-cold distilled water and expressed. It was then shaken in 15 ml ice-cold distilled water: for 5 min and again filtered through nylon cloth. Tyrosinase assays were carried out on the extract as such, but invertase was assayed after dialysis, of the extract. The effect of the addition of Si on cane invertase activity was studied by adding silicic acid and sodium silicate to the extract to give final concentrations of 0, 3 and 6 p moles Si/ml of digest. Concentrations of 0, 2, 4 and 8 p moles Si/ml of digest were used for tryrosinase studies. Tyrosinase activity was afterward assayed by the method described by Alexander (1966). Protein content was determined by the method of Sutherland et al. (1949). The influence of Si on sucrose production was investigated with one-eye cuttings of sugarcane of var M 93/48 and M , taken from plants very low in Si, germinated in sawdust and then transferred to 13-liter polythene buckets containing a modified Arnon and Hoagland solution which was continually aerated. Three levels of Si, as silicic acid, were added, giving 4 treatments as follows: 1) control (nutrient solution only) ; 2) nutrient solution + 50 ppm Si; 3) nutrient solution ppm Si; 4) nutrient solution ppm Si. Leaf samples were taken from the control and Si treatments before sunrise. Strips 10 X 2 cm were removed from the middle portion of the leaf, and the ends were placed between sheets of blotting paper which were always kept moist. These strips were exposed to light for different periods of time. Replicates of the 2 varieties (M 93/48 and M ) were used in a randomized block design. Field samples of leaves taken from canes of var M 93/48 which had undergone 2 treatments (no Si and 15 tons calcium silicatelha) were also exposed to light. For the determination of sugars, the strips were blended in hot water, and sugars were extracted by placing the tubes in a boiling water-bath for 30

I y. WONG YOU CHEONG, A. HEITZ, J. DEVILLE 779 rnin. The extract was then clarified with lead acetate, and reducing sugars were determined by the method of Tanimoto and Burr (1964).

3 I y. WONG YOU CHEONG, A. HEITZ, J. DEVILLE 779 rnin. The extract was then clarified with lead acetate, and reducing sugars were determined by the method of Tanimoto and Burr (1964). Sucrose was determined by subtracting reducing sugars from total reducing sugars obtained after inversion with HCl. RESULTS AND DISCUSSION ) Eflect of Si on Enzyme Activity Invertase. The effect of different levels of Si as sodium silicate on yeast invertase activity is shown in Fig. 1 and 2. Increasing concentrations of sodium Final ph corrected = 0 prnole Si 5.2 x : 2 prnole = 3 prnole si 5.2 = 8 prnole si 5.2 L 1 I I 1 I I I TIME OF INCUBATION (mins) Fig. 1. Effect of Si on invertase activity (ph corrected) silicate raised the ph of the buffer medium (ionic strength = 0.1). When the ph of the medium was brought back to 5.2 with the addition of acetic acid, there was no inhibitory effect of Si on invertase activity (Fig. l), but, when the increase in ph due to the addition of sodium silicate was not corrected, invertase activity was sharply inhibited at the 8 p mole level (Fig. 2). As the increase in ph was also very marked at that level, it would appear that the effect on invertase activity was due to ph, and not t~ Si increases. This was confirmed in an experiment when only silicic acid was used at the 8 p mole level. The results, which followed the same pattern as Fig. 1, showed that silicic acid did not change the ph of the buffer medium and had no inhibitory effect on invertase activity (Table 1). When P buffer (ph = 5.5, I = 0.1) was used, addition of Si as sodium

780 FERTILIZATION, ETC. Final ph uncorrected = 0 prnole Si 5.5 X = 2 prnole Si 5.7 0 = 3 pmole Si 5.9 n= 8prnoleSi 9.3-1 30-0 Y 20-8 10- Q?

4 780 FERTILIZATION, ETC. Final ph uncorrected = 0 prnole Si 5.5 X = 2 prnole Si = 3 pmole Si 5.9 n= 8prnoleSi Y Q? 0 I I I I I I TIME OF INCUBATION (rnins) Fig. 2. Effect of Si on invertase activity (ph not corrected). Table 1. The effect of Si as silicic acid on yeast invertase activity in acetate buffer. I Pol readings for Si treatments Inversion time (min) Controll Siz No Si added to digest p moles Si/ml of digest. - silicate at the 8 p mole level completely inhibited the action of invertase (Table 2), but the ph also sharply changed to 9.8. However, when the ionic strength of the buffer was increased to 0.4, the changes in ph as well as the effect on inverlase activity due to the addition of sodium silicate were only slight (Table 2). When invertase activity was measured by assaying sugars formed after incubation with cane invertase, the same trend was observed. These results show that Si had no effect on both yeast and cane invertase activity in vitro and that the inhibition of invertase activity reported by Alex- I

y. WONG YOU CHEONG, A. HEITZ, J. DEVILLB 78 1 Table 2. The effect of 2 concentrations of Si in P buffer at 2 ionic strengths on yeast invertase activity as measured by changes in Pol.

5 y. WONG YOU CHEONG, A. HEITZ, J. DEVILLB 78 1 Table 2. The effect of 2 concentrations of Si in P buffer at 2 ionic strengths on yeast invertase activity as measured by changes in Pol. Pol readings for following treatments Time of Controll 2.8 p moles Si 8.0 p moles Si incubation (min) I=O.12 I = 0.4 I=O.1 I = 0.4 I = 0.1 I= O Final ph No Si added to digest. 2 Ionic strength. ander (1968) to be due to Si was, in fact, due to changes in ph caused by the addition of sodium silicate to the buffer medium. Tyrosinase. The effect of the addition of Si as both sodium silicate and silicic acid on the activity of tyrosinase extracted from cane meristem deficient in Si was investigated at 6 concentrations of Si. There was no effect of either source of Si on cane tyrosinase. Sucrose Production The rates of production 'of sucrose and total sugars in leaf samples of var M and M 93/48 taken from cane pre-treated with different levels of Si in nutrient solution are given in Tables 3 and 4, respectively. The leaf samples used (3rd leaf) did not exhibit any visible symptoms although such symptoms were apparent in the older leaves of Si-deficient cane. (Comment of General Editor: Tables 3 and 4 contain examples in which the "sucrose production" of a treatment exceeded the "total sugar production," such as the Fero Si treatment under 1'1, hr artificial light. In reply to an inquiry concerning this relationship, the senior author offered the following explanation: "Production means the diflerence between the amount present at a certain interval and that present at zero hour. Total sugars = sucrose + reducing sugars. In some cases, especially tin the experiments with artificial light, the reducing sugar production is negative, i.e. reducing sugars present at zero time having been converted to sucrose; hence sucrose will necessarily be greater than total sugars in these cases.") Table 3 shows that there was a significant increase, due to Si pre-treatment, in the rates of production of both sucrose and total sugars in the leaves of var M exposed to sunlight but not when exposed to artificial light; different levels of Si pre-treatment did not cause significant differences in sugar production, although the rates of sugar production were usually higher at the higher levels of Si. Sunlight was much more efficient than the artificial light used in the production of sugars.

782 FERTILIZATION, KI'C. Table 3. Sugar production in leaf strips of sugarcane var M 147144 following pre-treatment with 4 levels of Si in nutrient solution and exposure to light.

6 782 FERTILIZATION, KI'C. Table 3. Sugar production in leaf strips of sugarcane var M following pre-treatment with 4 levels of Si in nutrient solution and exposure to light. Type of light Si pretreatment of cane (ppm) and time of exposure Artificial light 13 hr 3 hr 4fr hr Mean Sunlight 4fr hr Artificial light 16 hr 3 hr 49 hr Mean Sunlight 4fr hr Sucrose production1 Total sugar production1 1 pg/cmz of leaf surface LSD (.05P) : between means of Si treatments in sucrose production, artificial light = 38 between Si treatments in sucrose production, sunlight = ' 99 between means of Si treatments in total sugar production, artificial light = 41 between Si treatments in total sugar production, sunlight = 149. As for var M 93/48 (Table 4), a significant increase in sucrose production due to Si pre-treatment was obtained on exposure to artificial light, but the difference was not significant for the leaves exposed to sunlight. There was a large varietal difference in rate of sugar production; M produced several times more sugar than did M 93/48. When leaf samples were taken from field plots of cane which had received 0 and 15 tons of calcium silicatelha, the Si-rich leaves were significantly more efficient in producing sugars (Table 5). However, the effect could also have been due to the addition of Ca; but the results in Tables 3 and 4, where cane had been kept in pure nutrient solution, indicate that a deficiency of Si did interfere with the efficiency of photosynthesis. The control plants from which the leaves were sampled in Tables 3-5 all showed Si-deficiency symptoms in the older leaves although the leaves actually used for the production of sugars were free of such symptoms. When Si-deficient cane was kept under a Perspex roof, no Si-deficiency symptoms ever appeared even on the older leaves. In a separate experiment, leaf samples were taken

Y. WONG YOU CHEONG, A. HEITZ, J. DEVILLE 783 Table 4. Sugar production in leaf strips of sugarcane var M 93/48 following pre-treatment with 3 levels of Si in nutrient solution and exposure to light.

7 Y. WONG YOU CHEONG, A. HEITZ, J. DEVILLE 783 Table 4. Sugar production in leaf strips of sugarcane var M 93/48 following pre-treatment with 3 levels of Si in nutrient solution and exposure to light. Type of light and time of exposure Si pre-treatment of cane (ppm) Artificial light 4 hr 8 hr 12 hr Mean Sucrose production1 Sunlight 13 hr hr hr G Mean Total sugar production1 Artificial light 4 hr hr hr Mean Sunlight 14 hr hr hr Mean pg/cm2 of leaf surface LSD (.05P)., : between means of Si treatments in sucrose production, artificial light = 23.7 between means of Si treatments in sucrose production, sunlight = 26.3 between means of Si treatments in total sugar produclion, artificial light = 20.6 between means of S*i treatments in total sugar production, sunlight = from Si-deficient and Si-rich cane growing in pure nutrient solution under Perspex. The rates.of sugar production of these leaves are given in Table 6. Table 6 shows that, even for Si-deficient plants free of deficiency symptoms, the rate of sugar production was significantly lower than for Si-rich. cane. The lower efficiency of sugar production in Si-deficient leaf tissue must, therefore, not be linked with the eventual appearance of deficiency symptoms and would appear to be related to some other unknown function of Si in plant metabolism. These results are, therefore, in accordance with the increased phosphorylation obtained by Wong You Cheong et al. (1968) in the roots and leaves of sugarcane treated with Si.

i 784 FERTILIZATION, ETC. Table 5. Sugar production in leaf discs, exposed to artificial light, of var M 93/48 sampled from field plots receiving 0 and 15 tons of calicium silicatelha.

8 i 784 FERTILIZATION, ETC. Table 5. Sugar production in leaf discs, exposed to artificial light, of var M 93/48 sampled from field plots receiving 0 and 15 tons of calicium silicatelha. I 'I -Total- Sucrose production sugar production Time of exposure CaSi03 CaSiOs of leaf (hr) 0 Si added 0 Si added Mean LSD (.05P) : between means for sucrose production-34 between means for total sugar production Table 6. Sugar production in leaf strips, exposed to artificial light, sampled from Si-deficient and Si-rich cane growing in pure nutrient solution under Perspex. Sucrose production Total sugar production Si pre-treatmen t Si pretreatment Time of of cane (ppm) of cane (ppm) exposure -- of leaves (hr) I I Mean LSD (.05P) : between means for sucrose production-14 between means for total sugar production-16. REFERENCES Alexander, A. G The oxidising enxymes of sugarcane: tyrosinase (polyphenol oxidase). J. Agric. Univ. P. R., 50 (2) : In vitro effects of silicon on hydrolitic and oxidative enzymes of sugarcane. Proc. ISSCT, 13:532. Anon Ann. Rep. International Rice Research Institute (Los Banos) :30. Halais, P., and D. H. Parish Rep. Maurit. Sug. Ind. Res. Inst., 10:74. Hewitt, E. J Sand and water culture methods used in the study of plant nutrition. Commonwealth Agricultural Bureaux. Parnham Royal. Bucks. England. p Okuda, A., and E. Takahashi Le role du silicium. Agron. Trop. (Nogent), 19:534. Ota, M., H. Kobayashi, and Y. Kawaguchi Soil Sci. Plant Nutr. (Tokyo), 3:104. Snell, P. D., and C. T. Snell Colorimetric Methods of Analysis. Vol. 111, 3rd ed. Van Nostrand. p Sutherland, E. W., C. F. Corry, R. Haynes, and N. S. Olsen Purification of the hyperglycemic glycogenolytic factor from insulin and from gastric mucosa. J. Biol. Chem., 180:825.

9 y. WONG YOU CHEONG, A. HEITZ, J. DEVILLE 785 Tanimoto, T., and G. 0. Burr Sugar microdetermination by direct colorimetry of ferricyanide. Hawaiian Planters' Record, 57 (2) : 151. Wong You Cheong, Y., and P. Y. Chan Ann. Rep. Maurit. Sugar Ind. Res. Insti., 15:77., and P. Halajs Needs of sugarcane for silicon when growing in highly weathered latosols. Expl. Agric., 6: Yoshida, S., Y. Ohnishi, and K. Kitagishi Role of silicon in rice nutrition. Soil and Plant Food, 5:

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