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E il naufragare mè dolce in questo mar Ovvero come raddoppiare il contenuto in zucchero.

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Presentation on theme: "E il naufragare mè dolce in questo mar Ovvero come raddoppiare il contenuto in zucchero."— Presentation transcript:

1 E il naufragare mè dolce in questo mar Ovvero come raddoppiare il contenuto in zucchero

2 Barbabietola da zucchero Concentrazione di Saccarosio1802: 5%, 2007: 20% Resa (kg/ha) 1812: : 3500 kg 2005: kg With a root yield 40 t/ha at % sugar content, 6 -7 t sugar/ha. Semente: 1946, 20 kg per Ha, 2005: 1kg (di qualità) Azoto 1953: 240 kg per Ha, 2005: 80 kg Canna da Zucchero At harvest (quindi peso fresco) with best management practices ton/ha in sub-tropical zone tons/ha in tropical zone ( depending on length of growth period ) Per es. in Brasile: 80 t/ha fresh weight (70% di umidità). Sugar content at harvest is usually between 10 and 12 percent of the cane fresh weight

3 Canna da zucchero Whilst improvements in yield have continued to accrue, in Australia there has been no increase in sugar content over the last 40 years (Jackson 2005). 1 ettaro = 2.5 acri 1 ac = 0.4 ha

4 Qualche informazione It is a natural constituent of honey and sugar cane and has a very natural sweet taste. It is particularly suitable as a non-cariogenic sucrose replacement and is favorable in products for diabetics and prediabetic dispositions. Like sucrose, it is fully digested and provides the same caloric value of approximately 4 kcal/g. Unlike sucrose, isomaltulose is toothfriendly and digested much slower leading not only to a low glycemic response but as well to a prolonged glucose supply. Thus, isomaltulose is a slowly released carbohydrate that therefore provides a more sustained energy supply from food and drinks. An optimal energy supply is a topic of increasing importance in research and product development as this may play a role in health (particularly control of obesity) as well as for physical and mental performance. Isomaltulose (trade names Palatinose and NRGylose) a disaccharide that is commercially produced enzymatically from sucrose.

5 * Not metabolized by many microbes (an acariogenic sweetener) * Digested by humans with the same primary products (G & F) * Digested more slowly (lower fluctuation in blood glucose) 6-0-α-D-Glucopyranosyl-D-fructose α-D-glucopyranosyl-(12)-β-D-fructofuranoside Sucrose Isomaltulose ΔG° -12kJ/mol

6 Trehalulose Enzyme from Pantoea dispersa UQ68J (91% IM and 3% TH yield from sucrose at 30–35 C) Enzyme from Pseudomonas mesoacidophila strain MX-45 (91% TH and 8% IM yield from sucrose at 20°C Palatinose

7 Transgenici Tuber-specific expression of an apoplasm- targeted SI in potato. A chimeric sucrose isomerase gene from Erwinia rhapontici under control of a tuber-specific promoter was introduced into potato plants. The enzyme catalyses the conversion of sucrose into palatinose. Expression of the palI gene within the apoplast of transgenic tubers led to a nearly quantitative conversion of sucrose into palatinose. Despite the soluble carbohydrates having been altered within the tubers, growth of palI expressing transgenic potato plants was indistinguishable from wild type plants. Fig. 2. HPLC analysis of the soluble carbohydrates of (A) sugar standards; (B) extract of potato tubers transformed with palI; (C) wt potato. In growing plant tissues, efficient conversion of sucrose into the non-metabolized isomer is lethal or disruptive

8 Riduzione del saccarosio/glucosio Fig. 3. Contents of palatinose, sucrose, glucose and starch of wild type potato tubers and transgenic tubers expressing the chimeric palI gene. Values of wild type (dashed bar) and transgenic lines (black bars) represent the mean of four independent measurements ±S.D. As an additional control transgenic but not expressing lines (n=11) were averaged and the values are given as mean ±S.D. (grey bar).

9 In canna da zucchero High-performance liquid chromatography (HPLC) profiles from the separation of sugar standards (dotted line) and 4000-fold diluted juice from the basal internode (full lines) of Q117 control (A) and transgenic line N3.2H (B). glucose (G, 75.0 μM), fructose (F, 37.5 μM), sucrose (S, 37.5 μM), trehalulose (T, 37.5 μM) and isomaltulose (I, 75.0 μM) (C) Isomaltulose concentrations in juice from the basal internodes of transgenic lines.

10 ControlloTransgenico N3.2 Sugar accumulation profiles of sugarcane cultivar Q117 (A) and transgenic lines N3.2 (B) and N3.2H (C) showing substantial isomaltulose accumulation and enhanced total sugar concentration in juice. TVD, top visual dewlap. Transgenico N3.2H Transgenic lines with the vacuole-targeted NTPP-68J SI expressed from a constitutive ubiquitin (Ubi) promoter, were morphologically similar and equivalent in measured growth parameters to non-transformed controls of the same background genotype. In contrast, expression of a cytosol targeted form of the same SI from the same promoter was very damaging to sugarcane plant development, with low sugar contents and severe stunting in surviving plants.

11 Total sugar, water and fibre contents per unit fresh weight in internodes of sugarcane cultivar Q117 control (squares, full line) and SugarBooster transgenic lines N3.2 (triangles, dotted line) and N3.2H (circles, broken line). Increased photosynthate storage as sugar, rather than altered partitioning between sugar and fibre.

12 Enhanced photosynthesis and sucrose transport in the second fully emerged leaf (TVD2; TVD, top visible dewlap) of SugarBooster transgenic lines N3.2 and N3.2H, relative to sugarcane cultivar Q117 controls. (A) Photosynthetic electron transport rate. (B) CO2 assimilation rate. Leaf senescence was typically delayed by 15–20 days, resulting in an additional one to three leaves functional in photosynthesis per stalk for most of the growth period.

13 Trasporto di Saccarosio (C) Sucrose transport rate into plasma membrane vesicles. Results are means with standard errors from at least three replicates. CCCP, carbonyl cyanide m-chlorophenylhydrazone.

14 Room for improvement? The vacuole stores a correspondingly large proportion of sucrose, which can accumulate to 500 μmol/g FW. Low pH and high protease activity make this a hostile environment to introduced proteins. Even in mature stems with substantial IM accumulation, SI enzyme activity was below the detection limit in cell extracts, consistent with a short half-life of this protein after delivery into the sucrose storage vacuoles.

15 Sucrose concentration ceiling? Mechanisms contributing to the apparent sugar concentration ceiling may include feedback regulation from broad (e.g. osmotic) or specific (e.g. sucrose) sensors, thermodynamic limitations, such as leakage of sucrose through storage compartment membranes, or energetic limitations from the continuous futile cycle of sucrose cleavage and synthesis within the storage pool. Rapid degradation of vacuole targeted SI most likely protects against rapid sucrose depletion in growing tissues. IM accumulates gradually during development, probably because of the following: (i) continuous delivery of SI expressed from the constitutive Ubi promoter; (ii) high catalytic efficiency allowing occasional IM production before SI inactivation and (iii) absence of plant enzymes for IM metabolism. For efficient commercial production of this valued sugar, it will be useful to achieve patterns of developmental expression, compartmentation and enzyme stability yielding high IM content further up the harvested stalk profile.

16 Ingegneria metabolica probabilmente efficace probab. inefficace F Q E (1) P C A B (2) A B (3) P C A B (4) P C A B (5) P C A B (7) P C A B Q TF + SSSSS S (8) P C A B S + (6) P C A B S P ATP Z W Y X (9) P C A B S + + In the storage parenchyma cells of mature sugarcane stems, the sugar storage vacuole occupies about 90% of the symplast and 80% of the total tissue space. The vacuole stores a correspondingly large proportion of sucrose, which can accumulate to 500 μmol/g FW Q

17 Lascia o raddoppia? Doubling the total sugar content in mature internodes of an elite high-sugar cultivar eliminates osmotic limits and osmotic sensing as primary constraints behind the previous concentration ceiling in sugarcane. Our results also challenge the long-standing assumption that high-sugar genotypes require at least 70% moisture content in mature internodes. In the longer term, because sugars ultimately underpin all other biosynthesis in plants, the SugarBooster effect may be a foundation for higher yields of many other biomaterials.

18 Hamerli D, Birch RG (2011) Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field-grown sugarcane. Plant Biotechnology Journal 9: TS transgenic lines yielded up to 350 mM TH without evident reduction in endogenous sugars. Growth and development of the TH-producing lines were not visibly different from UbiKN controls at any stage during callus culture, plant regeneration and growth of the transgenic plants through VG1. No TH was detectable in juice from mature internodes of sugarcane background genotype Q117 or in 35 tested UbiKN control lines harvested after 12 months of growth in the glasshouse. TH was detected at this time, at concentrations up to 542 mM, in 76% of 54 independent transgenic lines that had initially been selected Sugars in juice from mature internodes of UBiTS lines in their second (line 2129) or third vegetative generations in a glasshouse. Each bar represents a single stalk. UniKN control stalks harvested at the same time yielded 420–705 mM sucrose in juice from mature internodes.

19 Sugars in whole-cane juice extracted at maturity from UbiTS lines and UbiKN controls in their first field propagation. Sugar concentration patterns along the sugarcane stem at maturity in their first field propagation of (a) UbiKN control line 2141 and (b) UbiTS line 2130 showing substantial isomer production. Trehalulose concentration in juice increased with internode maturity, reaching about 600 mM, with near-complete conversion of sucrose in the most mature internodes.

20 Field performance of transgenic sugarcane expressing isomaltulose synthase Basnayake S. (2012) Plant Biotechnology Journal 10: Relationship between sucrose, total sugars and isomaltulose (IM) in whole-cane juice at 10 months in FP2 Developmental profiles of sugar accumulation in stalks of low-, moderate- and high-level isomaltulose (IM) producing sugarcane lines.

21 Developmental profiles of sugar accumulation in stalks of field-grown recipient genotype Q117 and transgenic lines N3.2 and N3.2H Developmental profiles of sugar accumulation in stalks of recipient genotype Q117 and transgenic line N3.2 in the field (VG5 at 11 months in May 2007) and on return to a glasshouse (VG6 at 12 months).

22 There was generally a comparable decrease in sucrose concentration, with no overall decrease in total sugars Culture-derived plants were slower to establish and generally gave shorter and thinner stalks at harvest than those grown from field- sourced setts in the initial field generations. However, after several cycles of field propagation, selections were obtained with cane yields similar to the recipient genotypes. Sucrose isomerase activity was low in these transgenic lines, and the results indicate strong potential to develop sugarcane for commercial- scale production of IM if higher activity can be engineered in appropriate developmental patterns.

23 Perchè le stesse linee cresciute in campo non mostrano più il raddoppio dello zucchero? Silenziamento genico? Selection for less efficient expressors? …? I dati nelle diapositive 10 e 21 sono in netto contrasto e non possono essere spiegati in modo semplice. Le stesse linee ricresciute in serra e in campo non mostrano più il fenotipo iniziale e quindi la differenza potrebbe essere dovuta a dei cambiamenti nelle linee.


25 Chong BF, Bonnett GD, Glassop D, O'Shea MG, Brumbley SM (2007) Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink. Plant Biotechnol J. 5: Synthesis of sorbitol in sugarcane using the Malus domestica sorbitol- 6-phosphate dehydrogenase gene (mds6pdh). Rimuovere un prodotto centrale del metabolismo al momento sbagliato rischia di compromettere la crescita (importante la scelta del promotore)

26 The average amounts of sorbitol detected in the most productive line were 120 mg/g dry weight (equivalent to 61% of the soluble sugars) in the leaf lamina and 10 mg/g dry weight in the stalk pith. Sugar concentration (mg/g FW) in mature internodes Sucrose Glucose Fructose ± ± ± 0.36 (Iskandar (2011) BMC Plant Biology 11:12) Cosa dire su Sucrose e Sorbitol???

27 Leaves developed necrosis in a pattern characteristic of early senescence, and the severity was related to the relative quantity of sorbitol accumulated. Section of the last fully expanded leaf from an 8- month-old S-76 plant (bottom leaf) and a control plant (top leaf). Necrosis developed at the S-76 leaf apex and margins Sorbitol-producing sugarcane generated 30%40% less aerial biomass and was 10%30% shorter than control lines. When the Zymomonas mobilis glucokinase (zmglk) gene was co-expressed with mds6pdh … the plants were again smaller, indicating that glucose-6- phosphate deficiency was not responsible for the reduced growth.

28 Le modificazioni della canna da zucchero sembrano promettenti sia in termini di aumento di contenuto globale che in termini di accumulo di composti con maggior valore rispetto al saccarosio. Per una effettiva efficacia dal punto di vista commerciale sarà cruciale modulando lattività del promotore in maniera da far produrre lenzima solo negli internodi più vecchi quando hanno finito di crescere (quando hanno raggiunto leffettiva maturità e non necessitano più saccarosio per crescere). Aggiungi lavoro su derivato benzoico

29 Bibliografia * * Wu L, Birch RG. (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Plant Biotechnol J. 5: * Jackson PJ (2005). Breeding for improved sugar content in sugarcane. Field Crops Research 92:277–290. * * produktion/agriculture/sugar-refining/ (Sugarbeet history) produktion/agriculture/sugar-refining/ Immagini zuccheri:

30 Chong BF, Abeydeera WP, Glassop D, Bonnett GD, O'Shea MG, Brumbley SM (2010) Co-ordinated synthesis of gentiobiitol and sorbitol, evidence of sorbitol glycosylation in transgenic sugarcane. Phytochemistry. 71: See also ppt "sugarcane in the EAA" Biancardi E., Campbell L., Skaracis G., De Biaggi M. (eds.) (2005) Genetics and Breeding of Sugar Beet. Science Publishers Inc., Enfield NH, USA, p.368. Draycott, A. P (2006) Sugar beet. John Wiley & Sons.

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