Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation
Debra Rossouw A , Sue Bosch A , Jens Kossmann A , Frederik C. Botha A B and Jan-Hendrik Groenewald A CA 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 Corresponding author. Email: jhgr@sun.ac.za
Functional Plant Biology 34(6) 490-498 https://doi.org/10.1071/FP06214
Submitted: 1 September 2006 Accepted: 14 March 2007 Published: 1 June 2007
Abstract
Suspension cultures were used as a model system to investigate sucrose metabolism in four sugarcane (Saccharum spp. interspecific hybrids) cell lines transformed with antisense neutral invertase (NI) constructs. Throughout a 14-day growth cycle two cell lines in which the antisense sequence was under the control of a tandem CaMV-35S: maize ubiquitin promoter showed a strong reduction in NI activity, as well as reduced hexose and increased sucrose concentrations in comparison to the control line. In lines where the antisense NI sequence was under the control of the weaker CaMV-35S promoter alone, changes in enzyme activity and sugar concentrations were intermediate to those of the more strongly inhibited lines and the control. In comparison to the control line, a higher sucrose to hexose ratio, i.e. increased purity, was obtained in all the lines with reduced NI activity. The in vivo rate of sucrose hydrolysis was reduced in the transgenic lines, suggesting a concomitant reduction in the flux through the ‘futile cycle’ of sucrose breakdown and re-synthesis. Differences between the transgenic cultures and the control were most pronounced during the early stages of the growth cycle and tapered off as the cultures matured. The transgenic cultures displayed impaired growth characteristics suggesting that the growth rate of these cells was retarded because of the reduced availability of hexoses for respiration.
Additional keywords: antisense, sucrose metabolism.
Acknowledgements
The South African Sugarcane Research Institute, the South African Department of Trade and Industry, the Wilhelm Frank Trust and Stellenbosch University funded this research.
Batta SK, Singh R
(1986
) 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. Phytochemistry 25, 2431–2437.
| Crossref | GoogleScholarGoogle Scholar |
Bindon KA, Botha FC
(2001
) Tissue discs as an experimental system for metabolic flux analysis in the sugarcane culm. South African Journal of Botany 67, 244–249.
Bosch S,
Grof CPL, Botha FC
(2004) Expression of neutral invertase in sugarcane. Plant Science 166, 1125–1133.
| Crossref | GoogleScholarGoogle Scholar |
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.
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 | PubMed |
Bugos RC,
Chiang VL,
Zhang EH,
Campbell ER,
Podila GK, Campbell WH
(1995) RNA isolation from plant tissues recalcitrant to extraction in guanidine. BioTechniques 19, 734–737.
| PubMed |
Delaporta SL,
Wood J, Hicks JB
(1983) A plant DNA minipreparation. Plant Molecular Biology Reporter 1, 19–23.
Dickson RE
(1979) Analytical procedures for the sequential extraction of 14C-labeled constituents from leaves, bark and wood of cottonwood plants. Physiologia Plantarum 45, 480–488.
| Crossref | GoogleScholarGoogle Scholar |
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 | PubMed |
Gayler KR, Glasziou KT
(1972) Physiological functions of acid and neutral invertases in growth and sugar storage in sugar cane. Physiologia Plantarum 27, 25–31.
Godt D, Roitsch
(2006) The developmental and organ specific expression of sucrose cleaving enzymes in sugar beet suggests a transition between apoplasmic and symplasmic phloem unloading in tap roots. Plant Physiology and Biochemistry 44, 656–665.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Groenewald J-H,
Hiten NF, Botha FC
(2000) The introduction of an inverted repeat to the 5′ untranslated leader sequence of a transgene strongly inhibits gene expression. Plant Cell Reports 19, 1098–1101.
| Crossref | GoogleScholarGoogle Scholar |
Han Y,
Griffiths A,
Li H, Grierson D
(2004) The effect of endogenous mRNA levels on co-suppression in tomato. FEBS Letters 563, 123–128.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Hatch MD,
Sacher JA, Glasziou KT
(1962) Sugar accumulation cycle in sugar cane. I. Studies on enzymes of the cycle. Plant Physiology 38, 338–343.
Hatch MD, Glasziou KT
(1963) Sugar accumulation cycle in sugar cane. II. Relationship of invertase activity to sugar content, growth rate in storage tissue of plants grown in controlled environments. Plant Physiology 38, 344–348.
| PubMed |
Huber SC, Akazawa TA
(1986) A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells. Plant Physiology 81, 1008–1013.
| PubMed |
Klann EM,
Hall B, Bennett AB
(1996) Antisense acid invertase (T/VT) gene alters soluble sugar composition and size in transgenic tomato fruit. Plant Physiology 112, 1321–l330.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Lindbo JA,
Silva-Rosales L,
Proebsting WM, Dougherty WG
(1993) Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. The Plant Cell 5, 1749–1759.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Lingle SE
(1999) Sugar metabolism during growth and development in sugarcane internodes. Crop Science 39, 480–486.
Lingle SE, Smith RC
(1991) Sucrose metabolism related to growth and ripening in sugarcane internodes. Crop Science 31, 172–177.
Maretzki A, Thom M
(1972) Membrane transport of sugars in cell suspension of sugarcane. I. Evidence of sites and specificity. Plant Physiology 49, 172–182.
| PubMed |
Moore PH
(1995) Temporal and spatial regulation of sucrose accumulation in the sugarcane stem. Australian Journal of Plant Physiology 22, 661–679.
Murashige T, Skoog F
(1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–479.
| Crossref | GoogleScholarGoogle Scholar |
Nonis A,
Ruperti B,
Falchi R,
Casatta E,
Enferadi ST, Vizzotto G
(2007) Differential expression and regulation of a neutral invertase encoding gene from peach (Prunus persica): evidence for a role in fruit development. Physiologia Plantarum 129, 436–446.
| Crossref | GoogleScholarGoogle Scholar |
Que Q,
Wang H-Y,
English JJ, Jorgensen RA
(1997) The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. The Plant Cell 9, 1357–1368.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Rohwer JM, Botha FC
(2001) Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data. Biochemical Journal 358, 437–445.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
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 |
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.
| PubMed |
Schäfer WE,
Rohwer JM, Botha FC
(2004) A kinetic study of sugarcane sucrose synthase. European Journal of Biochemistry 271, 3971–3977.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Schubert D,
Lechtenberg B,
Forsbach A,
Gils M,
Bahadur S, Schmidt R
(2004) Silencing in Arabidopsis T-DNA transformants: the predominant role of a gene-specific RNA sensing mechanism versus position effects. The Plant Cell 16, 2561–2572.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Sergeeva LI,
Keurentjes JJB,
Bentsink L,
Vonk J,
Van der Plas LHW,
Koornneef M, Vreugdenhil D
(2006) Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis. Proceedings of the National Academy of Sciences USA 103, 2994–2999.
| Crossref |
Singh O, Kanwar RS
(1991) Enzymes in ripening of sugar cane at low temperatures. Sugar Cane 4, 2–4.
Thom M,
Maretzki A, Komor E
(1982) Vacuoles from sugarcane suspension cultures. I. Isolation and partial characterization. Plant Physiology 69, 1315–1319.
| PubMed |
Venkataramana S,
Naidu MK, Singh S
(1991) Invertases and growth factors dependent sucrose accumulation in sugarcane. Plant Science 74, 65–72.
| Crossref | GoogleScholarGoogle Scholar |
Venkataramana S, Naidu MK
(1993) Invertase-sucrose relationship in young and mature stem of sugarcane. Phytochemistry 32, 821–822.
| Crossref | GoogleScholarGoogle Scholar |
Vorster DJ, Botha FC
(1999) Sugarcane internodal invertases and tissue maturity. Journal of Plant Physiology 155, 470–476.
Wendler R,
Veith R,
Dancer J,
Stitt M, Komor E
(1990) Sucrose storage in cell suspension cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation. Planta 183, 31–39.
Whittaker A, Botha FC
(1997) Carbon partitioning during sucrose accumulation in sugarcane internodal tissue. Plant Physiology 115, 1651–1659.
| PubMed |
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.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
