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Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology
RESEARCH ARTICLE

Reduced neutral invertase activity in the culm tissues of transgenic sugarcane plants results in a decrease in respiration and sucrose cycling and an increase in the sucrose to hexose ratio

Debra Rossouw A , Jens Kossmann A , Frederik C. Botha A B C and Jan-Hendrik Groenewald A D E
+ Author Affiliations
- Author Affiliations

A Institute for Plant Biotechnology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.

B South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe 4300, South Africa.

C Present address: BSES Ltd, PO Box 86, Indooroopilly, Qld 4068, Australia.

D Present address: Biosafety South Africa, 105 Wentworth, Somerset Links Office Park, De Beers Avenue, Somerset West 7130, South Africa.

E Corresponding author. Email: hennie@biosafety.org.za

Functional Plant Biology 37(1) 22-31 https://doi.org/10.1071/FP08210
Submitted: 20 July 2008  Accepted: 17 September 2009   Published: 5 January 2010

Abstract

Transgenic sugarcane plants (Saccharum officinarum L. interspecific hybrids) were regenerated from previously described cell lines with reduced neutral invertase (NI) activity. The effects that were observed in the differentiated culm tissues at different stages of maturity paralleled those observed across the growth cycle of the suspension cultures. Reduced NI activity correlated with an increase in sucrose and a decrease in hexose levels. However, the magnitude of the reduction in enzyme activity and the accompanying changes in carbohydrate metabolism were not as pronounced as in the suspension cultures. Feeding experiments with radio-labelled fructose provided evidence that the cycling of sucrose as well as the total respiration rate correlated directly with NI activity. Sucrose synthase activity was upregulated in the transgenic plants, possibly to compensate for the reduction in invertase activity. Despite this partial compensation, the respiratory rates of the transgenic lines were still significantly lower than those of the untransformed control lines. This study clearly demonstrates the importance of NI in directing carbon towards respiratory processes in the sugarcane culm. In addition, this is the first report in which data obtained from genetically modified sugarcane suspension cell cultures and their regenerated, whole-plant counterparts are compared. The observed correlations support the use of cell cultures as a model system for sugarcane internodes, which could significantly accelerate reverse genetic studies on sugarcane carbohydrate metabolism in the future.

Additional keywords: carbohydrate, carbon flux.


Acknowledgements

We thank Fletcher Hiten, Charmaine Stander and Suereta Fortuin for their important technical contribution to this work. The South African Sugarcane Research Institute, the South African Department of Trade and Industry, the Wilhelm Frank Trust and Stellenbosch University funded this research.


References


Albertson PL, Peters KF, Grof CPL (2001) An improved method for the measurement of cell wall invertase activity in sugarcane tissue. Australian Journal of Plant Physiology 28, 323–328.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Alonso AP, Vigeolas H, Raymond P, Rolin D, Dieuaide-Noubhani M (2005) A new substrate cycle in plants. Evidence for a high glucose-phosphate-to-glucose turnover from in vivo steady-state and pulse-labeling experiments with [13C]glucose and [14C]glucose. Plant Physiology 138, 2220–2232.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Barratt DH, Derbyshire P, Findlay K, Pike M, Wellner N, Lunn J, Feil R, Simpson C, Maule AJ, Smith AM (2009) Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase. Proceedings of the National Academy of Sciences of the United States of America 106, 13 124–13 129.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Batta SK, Singh R (1986) Sucrose metabolism in sugarcane grown under varying climatic conditions: synthesis and storage of sucrose in relation to the activities of sucrose synthase, sucrose-phosphate synthase and invertase. Phytochemistry 25(11), 2431–2437.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Bekker JPI (2007) Genetic manipulation of the cell wall composition of sugarcane. PhD Thesis, Stellenbosch University, Stellenbosch, South Africa.

Benhamou N, Genier J, Crispeels MJ (1991) Accumulation of B-fructosidase in the cell walls of tomato roots following infection with a fungal wild pathogen. Plant Physiology 97, 739–750.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Bergmeyer HU , Bernt E (1974) Sucrose. In ‘Methods of enzymatic qnalysis. Vol. 3’. (Ed. HU Bergmeyer) pp. 1176–1179. (Academic Press Inc.: New York)

Bindon KA, Botha FC (2000) Tissue disks as an experimental system for metabolic flux analysis in the sugarcane culm. South African Journal of Botany 66, 260–264.
Crossref | GoogleScholarGoogle Scholar | open url image1

Bindon KA, Botha FC (2002) Carbon allocation to the insoluble fraction. Respiration and triose-phosphate cycling in the sugarcane culm. Physiologia Plantarum 116, 12–19.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Bosch S, Grof CPL, Botha FC (2004) Expression of neutral invertase in sugarcane. Plant Science 166, 1125–1133.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Botha FC, Black KG (2000) Sucrose phosphate synthase and sucrose synthase activity during maturation of internodal tissue in sugarcane. Australian Journal of Plant Physiology 27, 81–85.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Botha FC , Whittaker A , Vorster DJ , Black KG (1996) ‘Sucrose accumulation rate, carbon partitioning and expression of key enzyme activities in sugarcane stem tissue.’ (CSIRO Division of Tropical Crops and Pastures: Brisbane)

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Chourey PS, Nelson OE (1976) The enzymatic deficiency conditioned by the shrunken-1 mutation in maize. Biochemical Genetics 14, 1041–1055.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Cortès S, Gromova M, Evrard A, Roby C, Heyraud A, Rolin DB, Raymond P, Brouquisse RM (2003) In plants, 3-O-methylglucose is phosphorylated by hexokinase but not perceived as a sugar. Plant Physiology 131, 824–837.
PubMed |
open url image1

D’Aoust MA, Yelle S, Nguyen-Quoc B (1999) Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit. The Plant Cell 11, 2407–2418.
CAS | Crossref | PubMed |
open url image1

Dancer J, Hatzfield W, Stitt M (1990) Cytosolic cycles regulate the turnover of sucrose in heterotrophic cell-suspension cultures of Chenopodium rubrum L. Planta 182, 223–231.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Dendsay JPS, Singh P, Dhawan AK, Sehtiya HL (1995) Activities of internodal invertases during maturation of sugarcane stalks. Sugarcane 6, 17–19. open url image1

Dieuaide-Noubani M, Raffard G, Canioni P, Pradet A, Raymond P (1995) Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose. Journal of Biological Chemistry 270, 13 147–13 159.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Ebrahim MK, Zingheim O, El-Shourbagy MN, Moore PH, Komor E (1998) Growth and sugar storage in sugarcane grown at temperatures below and above optimum. Journal of Plant Physiology 153, 593–602.
CAS |
open url image1

Echeverria E (1998) Acid invertase (sucrose hydrolysis) is not required for sucrose mobilization from the vacuole. Physiologia Plantarum 104, 17–21.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Fernie AR, Willmitzer RN, Trethewey RN (2002) Sucrose to starch: a transition in molecular plant physiology. Trends in Plant Science 7, 35–41.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Ferreira SJ, Kossmann J, Lloyd JR, Groenewald J-H (2008) The reduction of starch accumulation in transgenic sugarcane lines. Biotechnology Journal 3, 1398–1406.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Flemetakis E, Efrose RC, Ott T, Stedel C, Aivalakis G, Udvardi MK, Katinakis P (2006) Spatial and temporal organization of sucrose metabolism in Lotus japonicus nitrogen-fixing nodules suggests a role for the elusive alkaline/neutral invertase. Plant Molecular Biology 62, 53–69.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Gayler KR, Glasziou KT (1972) Physiological functions of acid and neutral invertases in growth and sugar storage in sugarcane. Physiologia Plantarum 27, 25–31.
CAS |
open url image1

Geigenberger P, Reimholz R, Geiger M, Merlo L, Canale V, Stitt M (1997) Regulation of sucrose and starch metabolism in potato tubers in response to short-term water deficit. Planta 201, 502–518.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Glasziou KT (1961) Accumulation and transformation of sugars in sugarcane. Origin of glucose and fructose in the inner space. Plant Physiology 36, 175–179.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Gonzalez MC, Cejudo FJ (2007) Gibberellin-regulated expression of neutral and vacuolar invertase genes in petioles of sugar beet plants. Plant Science 172, 839–846.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Groenewald J-H, Botha FC (2008) Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes. Transgenic Research 17, 85–92.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Grof CPL, Campbell JA (2001) Sugarcane sucrose metabolism: scope for molecular manipulation. Australian Journal of Plant Physiology 28, 1–12.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Grof CPL, Knight DP, McNeil SD, Lunn JE, Campbell JA (1998) A modified assay method shows leaf sucrose-phosphate synthase activity is correlated with leaf sucrose content across a range of sugarcane varieties. Australian Journal of Plant Physiology 25, 499–502.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Hatch MD, Glasziou KT (1963) Sugar accumulation cycle in sugarcane. II. Relationship of invertase activity to sugar content and growth rate in storage tissue of plants grown in controlled environments. Plant Physiology 38, 344–348.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Hatch MD, Sacher JA, Glasziou KT (1963) Sugar accumulation cycle in sugarcane. I. Studies on enzymes of the cycle. Plant Physiology 38, 338–343.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Hawker JS (1985) Sucrose. In ‘Biochemistry of storage carbohydrates in green plants’. (Eds PM Dey, RA Dixon) pp. 1–51. (Academic Press: New York)

Hawker JS, Hatch MD (1965) Mechanism of sugar storage by mature stem tissue of sugarcane. Physiologia Plantarum 18, 444–453.
Crossref | GoogleScholarGoogle Scholar | open url image1

Hill SA, ap Rees T (1994) Fluxes of carbohydrate metabolism in ripening bananas. Planta 192, 52–60.
CAS |
open url image1

Huber SC, Akazawa TA (1986) A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells. Plant Physiology 81, 1008–1013.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Iyer LM, Kumpatla SP, Chandrasekharan MB, Hall TC (2000) Transgene silencing in monocots. Plant Molecular Biology 43, 323–346.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Jackson PA (2005) Breeding for improved sugar content in sugarcane. Field Crops Research 92, 277–290.
Crossref | GoogleScholarGoogle Scholar | open url image1

Klann EM, Chetelat RT, Bennett AB (1993) Expression of acid invertase gene controls sugar composition in tomato (Lycopersion) fruit. Plant Physiology 103, 863–870.
CAS | PubMed |
open url image1

Klann EM, Hall B, Bennett AB (1996) Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit. Plant Physiology 112, 1321–1330.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Lee H, Vattuone MA (1996) Purification and characterization of neutral and alkaline invertase from carrot. Plant Physiology 112, 1513–1522.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Lingle SE, Irvine JE (1994) Sucrose synthase and natural ripening in sugarcane. Crop Science 34, 1279–1283.
CAS |
open url image1

Lou Y, Gou JY, Xue HW (2007) PIP5K9, an Arabidopsis phosphatidylinositol monophosphate kinase, interacts with a cytosolic invertase to negatively regulate sugar-mediated root growth. The Plant Cell 19, 163–181.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Ma H, Albert HH, Paull R, Moore PH (2000) Metabolic engineering of invertase activities in different subcellular compartments affects sucrose accumulation in sugarcane cells. Australian Journal of Plant Physiology 27, 1021–1030.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Meyer RF, Boyer JS (1981) Osmoregulation, solute distribution and growth in soybean seedlings having low water potentials. Planta 151, 482–489.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Moore PH (1995) Temporal and spatial regulation of sucrose accumulation in the sugarcane stem. Australian Journal of Plant Physiology 22, 661–679.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Moore PH (2005) Integration of sucrose accumulation processes across hierarchical scales: towards developing an understanding of the gene-to-crop continuum. Field Crops Research 92, 119–135.
Crossref | GoogleScholarGoogle Scholar | open url image1

Perry CA, Leigh RA, Tomos AD, Wyse RE, Hall JL (1987) The regulation of turgor pressure during sucrose mobilization and salt accumulation by excised storage-root tissue of red beet. Planta 170, 353–361.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Qi X, Wu Z, Li J, Mo X, Wu S, Chu J, Wu P (2007) AtCYT-INV1, a neutral invertase, is involved in osmotic stress-induced inhibition on lateral root growth in Arabidopsis. Plant Molecular Biology 64, 575–587.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Rohwer J, Botha FC (2001) Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data. The Biochemical Journal 358, 437.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Rose S, Botha FC (2000) Distribution patterns of neutral invertase and sugar content in sugarcane internodal tissues. Plant Physiology and Biochemistry 38, 819–824.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Rossouw D, Bosch S, Kossmann J, Botha FC, Groenewald J-H (2007) Downregulation of neutral invertase activity in transgenic sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation. Functional Plant Biology 34, 490–498.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Sacher JA, Hatch MD, Glasziou KT (1963) Sugar accumulation cycle in sugar cane. III. Physical and metabolic aspects in immature storage tissues. Plant Physiology 38, 348–354.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Schäfer WE, Rohwer JM, Botha FC (2004) Protein-level expression and localization of sucrose synthase in the sugarcane culm. Physiologia Plantarum 121, 187–195.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Schäfer WE, Rohwer JM, Botha FC (2005) Partial purification and characterisation of sucrose synthase in sugarcane. Journal of Plant Physiology 162, 11–20.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Singh U, Kanwar RS (1991) Enzymes in ripening of sugarcane at low temperatures. Sugarcane 4, 2–4. open url image1

Sonnewald U, Brauer M, von Schaewen A, Stitt M, Willmitzer L (1991) Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or the apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions. The Plant Journal 1, 95–106.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Sonnewald U, Hajirezaei M-R, Kossmann J, Heyer A, Trethewey RN, Willmitzer L (1997) Increased potato tuber size resulting from apoplastic expression of a yeast invertase. Nature Biotechnology 15, 794–797.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Sturm A (1999) Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning. Plant Physiology 121, 1–8.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Sturm A, Crispeels ML (1990) cDNA cloning of carrot extracellular β-fructosidase and its expression in response to wounding and bacterial infection. The Plant Cell 2, 1107–1119.
CAS | Crossref | PubMed |
open url image1

Tang GQ, Sturm A (1999) Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning. Plant Molecular Biology 41, 465–479.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Tang G-Q, Lüscher M, Sturm A (1999) Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning. The Plant Cell 11, 177–189.
CAS | Crossref | PubMed |
open url image1

Van Dillewijn C (1952) Growth: general, grand period and growth formulae. In ‘Botany of sugarcane Vol. 1’. (Ed. C Van Dillewijn) pp. 97–162. (Veenen & Zonen: Wageningen, The Netherlands)

Veith R, Komor E (1993) Regulation of growth, sucrose storage and ion content in sugarcane cells, measured with suspension cells in continuous culture grown under nitrogen, phosphorus or carbon limitation. Journal of Plant Physiology 142, 414–424.
CAS |
open url image1

Vilhar B, Kladnik A, Blejec A, Chourey PS, Dermastia M (2002) Cytometrical evidence that the loss of seed weight in the miniature1 seed mutant of maize is associated with reduced mitotic activity in the developing endosperm. Plant Physiology 129, 23–30.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Vorster DJ, Botha FC (1999) Sugarcane internodal invertases and tissue maturity. Journal of Plant Physiology 155, 470–476.
CAS |
open url image1

Welbaum GE, Meinzer FC (1990) Compartmentation of solutes and water in developing sugarcane stalk tissue. Plant Physiology 93, 1147–1153.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Wendler R, Veith R, Dancer J, Stitt M, Komor E (1990) Sucrose storage in suspension cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation. Planta 183, 31–39. open url image1

Whittaker A, Botha FC (1997) Carbon partitioning during sucrose accumulation in sugarcane internodal tissue. Plant Physiology 115, 1651–1659.
CAS | PubMed |
open url image1

Wyse RE, Zamski E, Tomos AD (1986) Turgor regulation of sucrose transport in sugar beet taproot tissue. Plant Physiology 81, 478–481.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Yao SG, Kodama R, Wang H, Ichii M, Taketa S, Yoshida H (2009) Analysis of the rice SHORT-ROOT5 gene revealed functional diversification of plant neutral/alkaline invertase family. Plant Science 176, 627–634.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Zhu YJ, Komor E, Moore PH (1997) Sucrose accumulation in the sugarcane stem is regulated by the difference between the activities of soluble acid invertase and sucrose phosphate synthase. Plant Physiology 115, 609–616.
CAS | Crossref | PubMed |
open url image1

Zrenner R, Salanoubat M, Willmitzer L, Sonnewald U (1995) Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). The Plant Journal 7, 97–107.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1