Suresh K. Batta

Sucrose metabolism in sugar cane grown under varying climatic conditions: Synthesis and storage of sucrose in relation to the activities of sucrose synthase, sucrose-phosphate synthase and invertase

Abstract The contents of sucrose and hexoses in relation to the activities of sucrose synthase, sucrose-phosphate synthase and soluble invertase in leaf blade and of soluble and wall-bound invertase in stem tissues of sugar cane growing under naturally varying weather conditions were determined. In leaf blades, sucrose synthase, sucrose-phosphate synthase and soluble invertase were most active at tillering. Sucrose synthase was relatively more active than sucrose-phosphate synthase throughout the growth of the cane. In stem tissues the activity of soluble acid invertase was highest at the stem elongation stage and then declined, whereas the activities of soluble neutral invertase and wall-bound acid invertase increased with age. With the ageing of the cane, sucrose accumulation in the stem gradually increased with a concomitant fall in hexose content. A narrow ratio ofsucrose in the basal to top portion of the cane relates to maturity of the cane. On feeding uniformly labelled sucrose and hexoses to storage tissue for 4 hr. ca 85–90 % of the 14C in intracellular sugars was present in sucrose. Synthesis of sucrose from glucose in leaf disks was stimulated by Mg2+ and Mn2+.

Multiple forms of soluble invertases in sugarcane juice: Kinetic and thermodynamic analysis

Soluble invertases were partially purified from storage tissue of an early maturing and high sucrose accumulating genotype Co89003 using ammonium sulphate fractionation and DEAE-cellulose column chromatography. The three different acid invertase isoforms named as A-I, A-II and A-III were identified at late grand growth stage of cane development, whereas only one neutral invertase isoform was detected at maturity stage of cane growth. The final purification achieved for A-I, A-II, A-III and neutral invertase isoform was 37, 45, 7 and 17-fold, respectively. The optimum pH values for A-I, A-II, A-III and neutral isoform were observed to be 5.0, 4.5, 4.5 and 7.0, respectively. Apparent Michaelis constant i.e. Km of different acidic isoforms at its optimum pH and temperature varied from 50.0 to 83.3 mM. For neutral invertase isoform, Km value was 44.0mM at its optimum pH and temperature. The highest Vmax/Km value of A-I isoform indicates the maximum kinetic perfection of this isoform and it may be the major vacuolar enzyme involved in sucrose hydrolysis. From the study of effect of temperature on Km and Vmax values of neutral invertase isoform, the enthalpy change, entropy change and energy of activation were observed to be -21.1 kJ mol-1,-40.5 JK-1 mol-1, and 42.5 kJmol-1, respectively. Among the different metal ions tested, manganese chloride strongly inhibited the activity of all soluble acid invertase isoforms and thus, may be utilized to induce early maturity and controlling sucrose inversion in sugarcane thereby increasing sugar recovery.

Sucrose accumulation in sugarcane: a potential target for crop improvement

Sugarcane is a highly productive crop plant with the capacity of storing large amounts of sucrose. Sucrose accumulation in the stem of sugarcane has been studied extensively. The initial recognition and characterization of the enzymes involved in sucrose synthesis and cleavage led to the widely accepted models of how sucrose accumulation occurs in the storage tissue. New insights were gained into the physiological role of individual enzyme activities in the process of sucrose accumulation in sugarcane. Studies on cell cultures and on isolated cell fragments initially supported and strengthened these models, but more recent research has revealed their weaknesses. A dynamic model of rapid cycling of sucrose and turnover of sucrose between vacuole, metabolic and apoplastic compartments explains much of the data, but the details of how the cycling is regulated needs to be explored. Genomic research into sucrose metabolism has been based on the premise that cataloging genes expressed in association with the stalk development would ultimately lead to the identification of genes controlling the accumulation of sucrose. Considerable progress has been made in understanding and manipulating the sugarcane genome using biotechnological and cell biology approaches. Thus, the greater understanding of physiology of sucrose accumulation and the sugarcane genome will play a significant role in the future sugarcane improvement programs and will offer new opportunities to develop it as a new-generation industrial crop.

Post harvest quality deterioration in sugarcane under different environmental conditions

Post harvest changes in the juice quality parameters in relation to storage time were investigated in three sugarcane genotypes varying in maturity behaviour viz.CoJ83 (early), CoJ88 (mid) and S70/00(late) under different environmental conditions i.e in the month of November, January and March. A gradual decrease in cane weight, % juice extraction, sucrose (% juice), purity (% juice) and pH with simultaneous increase in TSS%, titrable acidity, dextran, reducing sugars and activities of acid and neutral invertases was found in juice during 12 days of cane storage in all genotypes under all environmental conditions. Irrespective of the genotype and environmental condition, the level of neutral invertase was found to be higher as compared to acid invertase except in genotype S70/00, where higher activity of acid invertase was found as compared to neutral invertase during the month of November. The rate of decrease/increase per day of all the quality parameters on staling was highest during the late crushing period i.e. March than during November and January in all genotypes. More deterioration in quality parameters was found in genotype CoJ83 during March and in genotype S70/00 during November, which may be due to their over mature and immature conditions, respectively, at that time. Hence, the variation in the rate of change in quality parameters during staling may be attributed to the difference in maturity level among genotypes during these three months.

Sucrose accumulation and expression of enzyme activities in early and mid-late maturing sugarcane genotypes

Metabolic changes in the contents of sucrose and hexoses in relation to activities of the principal enzymes involved in accumulation of sucrose in internodal storage tissue have been investigated in four sugarcane genotypes varying in maturity behaviour viz. Sel 69/01, Sel 943/98 (both early maturing) and CoS 8436, Sel 17/00 (both mid-late maturing) at different physiological stages of crop growth. In storage tissue, the higher rate of decline in the activity of soluble acid invertase (pH 5.5) with age of cane resulted in faster rate of sucrose accumulation in early maturing sugarcane genotypes and was related to higher sink strength of these genotypes. Unlike soluble acid invertase, the activity of soluble neutral invertase increased with cane maturation in each of the genotypes. The wall-bound acid invertase (pH 5.5) showed maximum activity at stem elongation stage. Mid-late maturing genotypes showed higher activity of this enzyme than early maturing genotypes. No relationship was observed between the activities of sucrose synthase, sucrose phosphate synthase and maturity behaviour of the genotypes.

Sucrose metabolism and expression of key enzyme activities in low and high sucrose storing sugarcane genotypes

Metabolic changes in the levels of sucrose and hexoses in relation to the activities of sucrose synthase, sucrose phosphate synthase and invertase in low (CoS96260) and high (Co89003) sucrose storing genotypes of sugarcane have been studied. Higher activities of sucrose synthesizing enzymes in leaf and stem tissues of Co89003 as compared to CoS96260 were observed. The activities of soluble acid invertase in leaf tissue declined with the age of the crop in both the cultivars. The higher rate of decline in the activity of soluble acid invertase in the stem tissue concomitant with a higher rate of rise in the ratio of sucrose/reducing sugars indicated better sink strength of Co89003 over CoS96260. Unlike soluble acid invertase, the activity of soluble neutral invertase in stem increased with the maturity of the cane. The wall bound acid invertase activity was higher in the immature portion of the stem in both the cultivars. The negative correlation between vacuolar soluble acid invertase activity and sucrose storage, and the promotory effect of apoplastic acid invertase promise a good potential for the manipulation of invertase genes to increase sucrose accumulation in sugarcane

Synthesis and Storage of Sucrose in Relation to Activities of Its Metabolizing Enzymes in Sugarcane Cultivars Differing in Maturity

Metabolic changes in the contents of sucrose and hexoses in relation to the activities of invertase, sucrose synthase and sucrose-phosphate synthase in early (CoJ 64) and late (Co 1148) maturing cultivars of sugarcane have been studied. During early stages of cane growth, lower activities of sucrose synthase and sucrose-phosphate synthase in leaf blade In CoJ 64 over Co 1148 were observed. However, sucrose content in sheath/blade was higher in CoJ 64 than in Co 1148. With the advancing age, the activity of soluble acid invertase (pH 5.4) in stem declined more rapidly in CoJ 64. This resulted in building up of high ratio of sucroselinvert sugars in stem tissue of this cultivar. Feeding uniformly-labelled sucrose and glucose to the cut discs of leaf sheath resulted in higher uptake of 14C in CoJ 64 than in Co 1148. Uptake by stem tissue discs of 14C from sucrose was less than that from hexoses. Based on these results, it is suggested that (i) the rapid fall in the activity of soluble acid invertase in stem concomitant with fast accumulation of sucrose in this tissue is an index of early maturity of the cane, and (ii) high content of sucrose in sheath is a reflection of an efficient translocation of this sugar in early maturing cultivars.

Dextranase production from Paecilomyces lilacinus and its application for dextran removal from sugarcane juice

Dextranase was produced from fungus Paecilomyces lilacinus by submerged fermentation under different cultural conditions viz. pH, incubation temperature, inoculum size and days of incubation for maximum enzyme production. Maximum enzyme production was achieved at pH 6.0 of Mandel media and at temperature 30°C. Inoculum size of 1 × 107 spores/ml was found to be optimum for maximum enzyme production. Enzyme production increased with the increase in days of incubation from 3 to 5 days and then declined thereafter. Dextranase units (from 1 to 15 U/100 ml juice) were exogenously added to sugarcane juice with an aim to optimize dextranase application for removal of dextran from cane juice. Addition of dextranase in juice of cane variety CoS 8436, resulted in relatively less increase in dextran content as compared to control during storage. Whereas, dextran content in untreated juice increased 10 times during 24 h of storage, the increase during this period was only 2.3 times in juice treated with 15 U of dextranase. With the application of 1, 2, 5, 10 and 15 units of dextranase in 100 ml of juice, dextran content was decreased by 15.51, 30.82, 50.20, 68.20 and 73.30 % as compared to the control (with no dextranase added) after 24 h of storage. This study finds application in minimizing the dextran problem in sugar industry.

Internodal soluble invertase isoenzymes in relation to sucrose storage and maturity behaviour in sugarcane cultivars

Changes in activities of internodal soluble invertases in relation to sucrose storage in two sugarcane cultivars viz. Co 89003 (early maturing) and Co 1148 (late maturing) have been studied at different physiological stages of cane development. The higher rate of decline in the activity of soluble acid invertase with ageing of the cane coupled with rapid rise in sucrose/reducing sugars ratio indicated better sink strength and higher sucrose storage in Co 89003. Six isoenzymes of soluble acid invertase in Co 89003 and five isoforms of this enzyme in Co 1148 were identified at internodal elongation stage. However, no neutral invertase isoenzyme was detected in either cultivar at this stage. At maturity stage, three isoenzymes of neutral and four isoenzymes of acid invertase were identified in Co 89003 whereas five isoenzymes of neutral and two isoenzymes of acid invertase were identified in Co 1148. These internodal soluble invertase isoenzymes differed with respect to their catalytic efficiencies for sucrose mobilization in storage tissue. Relative to Co 1148, soluble acid invertase isoenzymes of Co 89003 were less efficient catalytically because of their low Vmax/Km values. Marked differences were also observed in the inhibitory action of Mn++ on various isoenzymes of invertase.

Partial Purification and Characterization of Acid Invertase from the Fresh and Stale Sugarcane Juice

Soluble acid invertases from the fresh and stale cane juice of variety CoJ83 were purified by fractional precipitation with ammonium sulphate and ion exchange chromatography with DEAE- cellulose. Three isoforms of acid invertase were identified in juice of fresh cane and four isoforms of enzyme were found in juice of stale cane. The isoforms obtained were characterized for their kinetic parameters. Optimum pH and optimum temperature range of isoforms from fresh cane juice were 4.5–5.0 and 35–45°C, however isoforms of invertase from juice of stale cane were having optimum pH of 4.0–4.5 and optimum temperature 40–55°C. Isoforms identified from juice of stale canes were kinetically more efficient in comparison to isoforms identified from juice of fresh canes, as they were having higher Vmax/Km values than isoforms from fresh cane juice. MnCl2 inhibited the soluble acid invertase isoforms of fresh cane completely, but it was unable to inhibit completely the enzyme isoforms of juice of stale cane. ZnCl2, NiCl2, KCl, sodium metasilicate, sodium lauryl sulphate and KMnO4 inhibited the activity of different invertase isoforms in both fresh and stale cane juice, but this inhibition percent was relatively less in stale cane enzyme isoforms as compared to enzyme isoforms from juice of fresh cane. This points to the existence of structural and conformational differences among invertase isoforms in juice of fresh and stale cane.