«by zac McDonald Submitted in fulfillment of the academic requirements for the degree of Master of Science in the Department of Biology University of ...»
THE METABOLIC FATE OF SUCROSE IN INTACT
SUGARCANE INTERNODAL TISSUE
Submitted in fulfillment of the academic requirements
for the degree of
Master of Science
Department of Biology
University of Natal
The experimental work described in this thesis was carried out from January
1997 to December 1998 under the supervision of Prof. FC Botha and cosupervision of Dr. Bl Huckett in the Biotechnology Department of the South African Sugar Association Experiment Station (SASEX), Mt Edgecombe, Kwazulu-Natal. The facilities of the Institute of Plant Biotechnology (IPB), Stellenbosch were also made use of during the course of the study These studies represent original work by the author and have not been submitted in any form to another University. Where use was made of the work of others, it has been duly acknowledged in the text.
ACKNOWLEDGEMENTSFirst and foremost I would like to thank my supervisor, Professor FC Botha and my co-supervisor, Dr Bl Huckett for their enthusiastic assistance and guidance during the course of the study. Their contribution of both time and effort ensured that the study successfully attained all of its objectives.
I am also very grateful to my colleagues in the Biotechnology Department of Sasex and to the extended Sasex family for going out of their way to make the study as trouble free as possible.
The financial support provided by Sasex and the FRD was greatly appreciated.
Finally, I would like to thank my family and friends for their prayers and words of encouragement. They kept me focused on my destination and made the journey much less arduous.
ABSTRACTThe study was aimed at determining the metabolic fate of sucrose in intact sugarcane internodal tissue. Three aspects of the fate of sucrose in storage tissue of whole plants formed the main focus of the work. These were the rate of sucrose accumulation in the developing culm, the characterisation of partitioning of carbon into different cellular organic fractions in the developing culm and the occurrence of sucrose turnover in both immature and mature stem tissues.
Specific attention was paid to confirming the occurrence of sucrose turnover in both immature and mature internodal tissue. This sucrose turnover has been described previously in both tissue slices and cell suspension cultures. However, certain results from previous work at the whole plant level have indicated that sucrose turnover does not occur in mature internodal tissue.
Radiolabeled carbon dioxide (14CO2) was fed to leaf 6 of sugarcane culms of a high sucrose storing variety (Saccharum spp. hybrid cv. Nco376). All plants were of similar age (12 months) and were grown under similar conditions. The movement and metabolic fate of radiolabeled sucrose was determined at four time points, (6 hours, 24 hours, 7 days and 6 weeks) during a 6 week period.
The metabolic fate of sucrose was determined in internodes number 3, number 6 and number 9. Internode 3 was found to have a relatively high hexose sugar content of 42 mg glc&fruc fw g'1 and a low sucrose content of 14 mg sue fw g"1.
In contrast the sucrose content of internode 9 was much higher at 157 mg sue fw g"1 and the hexose sugar content much lower at 4.3 mg glc&fruc fw g"1. Based on previous work, the sugar content of internode 3 and internode 9 are characteristic of immature and mature tissues respectively. Internode 6 occupies an intermediary position between internode 3 and 6 with its sucrose content higher than its hexose sugar content, but with the hexose sugar content still
Although the metabolic fate of sucrose within sink tissue was the focal point of the study, the expenmental design also allowed for certain aspects of sucrose production in the source to be investigated. The average photosynthetic rate for leaf 6 in full sunlight was estimated at 48 mg CO2 dm'2 s "1. During photosynthesis, only 30% of the fixed carbon was partitioned into the storage carbohydrate pool while the remaining 70% was partitioned into sucrose for immediate export from the leaf. This high rate of carbon fixation combined with a high rate of carbon export is characteristic of C4 plants such as sugarcane.
On entering the culm, translocation of radiolabeled sucrose was predominantly basipetal with relatively little acropetal translocation. The majority of the radiolabeled carbon was found to be stored in mature internodes. No significant loss of radiolabeled carbon was observed in mature and elongating internodes over the study period. A 22% loss of total radiolabeled carbon was observed in immature internodes over the same period. This can probably be attributed to the higher rates of cellular respiration known to occur in immature tissues.
There appear to be three phases of sucrose accumulation in the developing culm. Initially, the accumulation rate in rapidly growing tissue, as intemode 3 develops into intemode 6, is relatively low. This is followed by a rapid increase in the rate of sucrose accumulation during intemode elongation, as intemode 6 becomes intemode 9. Finally, a decrease in the rate of sucrose accumulation is observed during late maturation, as intemode 9 becomes intemode 12.
Determination of the sucrose content in internodes 3, 6 and 9 revealed that there is a notable increase in sucrose content during intemode maturation. It is proposed that the higher sucrose content of mature tissue is not merely a consequence of the longer growth period of mature tissue, but is due to the increased rate of sucrose accumulation observed during intemode elongation.
Short-term (24 hours) analysis of carbon partitioning revealed that intemodal
maturation was associated with a redirection of carbon from non-sucrose cellular organic fractions to sucrose storage. In immature internodes only 20% of the total radiolabeled carton was present in the sucrose pool 24 hours after feeding.
In elongating intemodes the figure increased to 54% while in mature internodes as much as 77% of the total radiolabeled carbon was retained in the sucrose pool. Concomitant with the increased carbon partitioning into stored sucrose down the developing culm is a decrease in carton partitioning into the hexose sugar pool. In immature tissue, 42 % of the total radiolabel is present in the hexose sugar pool, while in mature tissue the percentage drops to 11%. This decrease is probably indicative of decreased levels of carbon cycling between the sucrose and hexose sugar pool as a result of intemode maturation.
Intemode maturation was also found to be associated with a decrease in the amount of carbon in the water insoluble matter pool and the amino acid/ organic acid/ sugar phosphate pool. Thus, intemode maturation is associated with a redirection of carbon from total respiration to sucrose storage. Long-term (6 weeks) analysis of carbon partitioning confirmed that sucrose storage in mature tissue is greater than that in immature tissue. From the 6 hour time point to the 6 week time point, an 87% reduction in the stored radiolabeled sucrose content was observed in immature intemodes. During the same period only a 25% reduction in the stored radiolabeled sucrose was observed in mature intemodes.
Radiolabel loss from the radiolabeled sucrose pool in both mature and immature intemodes was accounted for by relative radiolabel gains in other cellular organic fractions.
Poovaiah BW and Veluthambi K (1985) Auxin regulated invertase activity in strawberry fruits. J. Am. Soc. Hortic. Sci. 110: 258-261
Preiss J and Sivak L (1996). Starch synthesis in sinks and soyrces pg 63-95. In:
Photoassimilate Distribution in Plants and Crops. Source-Sink relationships Zamski E and Schaffer AA (eds). Marcel Dekker Inc. New York Basel Hong Kong Preisser J and Komor E (1988). Analysis of the reaction products from incubation of sugarcane vacuoles with undine diphosphate-glucose : no evidence for the group translocator. Plant Physiol. 88 259-265 Preisser J, Sprugel H, Komor E (1992). Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole. Planta 186 : 203-211 Pressey R (1968). Invertase inhibitors from red beet, sugar beet and sweet potato roots. Plant Physiol. 43:1430-1434 Quick PW and Schaffer AA (1996). Sucrose metabolism in sources and sinks.
In: Photoassimilate Distribution in Plants and Crops. Source-Sink relationships.
Zamski E and Schaffer AA (eds). Marcel Dekker Inc. New York Basel Hong Kong Ricardo CP (1976). Effects of sugars, giberellic acid and kinetin on acid invertase of the devloping carrot roots. Phytochemistry 15: 615-617 Ricardo CP and apRees T (1970). Invertase activity during the development of carrot roots. Phytochemistry 9: 239-247 Ricardo CP and Sovia D (1974). Development of tuberous roots and sugar
accumulation as related to invertase activity and mineral nutrition. Planta 118:
43-55 Sacher JA, Hatch MD, and Glasziou KT (1963a). Sugar accumulation cycle in sugarcane. III. Physical and metabolic aspects of cycle in immature storage tissue. Plant Physiol. 38: 348-354 Sacher JA, Hatch MD, and Glasziou KT (1963b). Regulation of invertase synthesis in sugarcane by an auxin- and sugar mediated control system.
Physiol. Planta. 16: 836-842 Sacher JA, Hatch MD, and Glasziou KT (1963c). Sugar accumulation cycle in sugarcane. I. Studies on enzymes of the cycle. Plant Physiol. 38: 338-343 Sacher JA (1966). The regulation of sugar uptake and accumulation in bean pod tissue. Plant Physiol 44: 181-189 Saftner RA, Daie J, Wyse RE (1983). Sucrose uptake and compartmentation in sugar beet taproot tissue. Plant Physiol. 72:1-6 Servaites JC, Fondy BR, LI B, Geiger DR (1989). Source of carbon for export from spinach leaves throughout the day. Plant Physiol. 90:1168-1174 Shiroya M, Lister Gr, Nelson CD, Krotkov G (1961). Translocation of 14C in tobacco at different stages of devlopment following assimilation of UCO2 by a single leaf. Can. J. Bot. 39: 855-864 Silvius JE, Snyder FW (1979a). Comparitive enzymic studies of sucrose
metabolism in the taproots and fibrous roots of Beta vulgahs L Plant Physiol 64:
1070-1073 Silvius JE, Snyder FW (1979b). Photosynthate partitioning and enzymes of sucrose metabolism in Sugarbeet Roots. Physiol. Plant. 46 : 169-173 Slack CR (1965). The physiology of sugarcane. VII Diurnal fluctuations in the activity of soluble invertase in elongating internodes. Aust. J. Biol. Sci. 18:781Steingrover E (1981). The relationship between cyanide-resistant root respiration and the storage of sugars in the taproot in Daucus carota L. J. Exp.
Bot. 32:911-919 Steudle E (1992). The biophysics of plant water: compartmentation, coupling
with metabolic processes, and flow of water in plant roots. In 'Water and Life:
Comparitive Analysis of Water Relationships at the Organismic, Cellular and Molecular Levels'. (Eds G.N. Somero, C.B. Osmond and C.L Bolis.) 173-204.
(Springer-Verlag Berlin) Stitt M (1990). Fructose-2-6-bisphosphate as a regulatory molecule in plants.
Annu. Rev. Plant Mol. Biol. 41: 153-181 Stitt M and Heldt HW (1985). Control of photosynthetic sucrose synthesis by Fru-2-6-BP. Planta 164: 179-188 Stitt M and Quick WP (1989). Photosynthetic carbon partitioning: its regulation and possibilities for manipulation. Physiol. Plant 77:633-641 Stommel JR (1992). Enzymic components of sucrose accumulation in wild tomato species Lycopersican peruvianum. Plant Physiol. 99: 324-328 Su LY (1982). Proc Natl.Sci.Counc.Repub. China, Part B 6, 172-180.
Su LY and Preiss J (1978). Purification and properties of sucrose synthase from maize kernels. Plant Physiol. 61: 389-393
accumulation and sucrose metabolizing enzymes in developing grapes. Am. J.
Enol. Vitic. 48: 403-407 Thorn M, Komor E, Maretzki A (1982). Vacuoles from sugarcane suspension cultures I. Isolation and partial characterisation. Plant Physiol. 69: 1315-1319 Thorn M and Komor E (1984). H+-sugar antiport as the mechanism of sugar uptake by sugarcane vacuoles. FEBS Lett. 173, 1-4 Thorn M and Maretzki A (1992). Evidence for the direct uptake of sucrose by sugarcane culm tissue. J. Plant Physiol. 139: 555-559 Thorn M, Komor E, Maretzki A (1982). Vacuoles from sugarcane suspension culture.2. Characterisation of sugar uptake. Plant Physiol.69 1320-1325 Thorn M, Maretzki A (1985). Group translocation as a mechanism for sucrose transfer into vacuoles fr4om sugarcane cells. Proc. Natl. Acad. Sci. USA 82, 4697-4701 Thrower SL (1967). The pattern of translocation during leaf ageing. In: Aspects of the biology of ageing. Pg 483-506, Woolhouse HW (ed) Cambridge University Press.
Tomos AD, Leigh RA, Palta JA and Williams JH (1992). Sucrose and cell water relations. In ' Carbon partitioning within and between organisms'. (Eds Pollock CJ, Farrar JF and Gordon AJ) 71-89. (Bios Scientific Publishers: Oxford Tucker GA, Grlerson D (1987). Fruit ripening-ln: The Biochemistry of Plants. Vol.
12 Davies DD (ed) 265-318. Academic press. New York. NY.ISBN 0-12-675412Van Bel AJE (1993). Strategies of phloem loading. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 44: 253-282 Veith R and Komor E (1993). Regulation of growth, sucrose storage and ion content in sugarcane cells, measured with suspension cells in continuous culture grown under nitrogen, phosphorous or carbon limitation. J. Plant Physiol. 142" 414-424 Vernort L P and Aronoff S (1952). Metabolism of soybean leaves. IV.
Translocate from soybean leaves. Arch. BioCHem. And Biophys. 36:383-398 Vieweg GH (1974). Enzyme des Saccharosestoffwechsels in Wurzeln Planta 116:347-359 Waering PF and Patrick J (1975). Source-sink relations and the partition of ass,milates in the plant- In: Photosynthesis and productivity in dffferent env,ronments. Evans LT (ed) Int. Biol. Progr. 3:481^99, Cambridge University Press.