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; Jens Kossmann; Frederik C. Botha; Jan-Hendrik Groenewald

doi:10.1071/fp08210

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

Functional Plant Biology ◽

10.1071/fp08210 ◽

2010 ◽

Vol 37 (1) ◽

pp. 22 ◽

Cited By ~ 24

Author(s):

Debra Rossouw ◽

Jens Kossmann ◽

Frederik C. Botha ◽

Jan-Hendrik Groenewald

Keyword(s):

Carbohydrate Metabolism ◽

Cell Cultures ◽

Saccharum Officinarum ◽

Invertase Activity ◽

Growth Cycle ◽

Suspension Cultures ◽

Transgenic Sugarcane ◽

Feeding Experiments ◽

Neutral Invertase ◽

Transgenic Lines

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.

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Effects of Plant Bioregulators on the Production of Iridoid Derived Terpenoids in Valeriana wallichii and Fedia cornucopiae Cell Suspension Cultures

Zeitschrift für Naturforschung C ◽

10.1515/znc-1987-1-207 ◽

1987 ◽

Vol 42 (1-2) ◽

pp. 33-40 ◽

Cited By ~ 6

Author(s):

Wolfram Förster ◽

Hans Becker

Keyword(s):

Cell Suspension ◽

Cell Cultures ◽

Exponential Growth ◽

Cell Suspension Cultures ◽

Growth Cycle ◽

Suspension Cultures ◽

Tissue Cultures ◽

Cell Systems ◽

Optimal Activity ◽

Plant Tissue Cultures

Abstract Four plant bioregulators were tested for their effects on production of valepotriates in Valeriana wallichii and Fedia cornucopiae cell suspension cultures. Concentrations of more than 10 ppm reduced valepotriate yield. At lower concentrations production was increased. For optimal activity, bioregulators had to be applied during early exponential growth, up to day 8 of the growth cycle. At equimolar concentrations dim ethylm orpholinium bromide (4 ppm) and dimethylpiperidinium chloride (3 ppm) significantly im proved total valepotriates in V. wallichii (up to 23%) and in F cornucupiae (up to 50% ) 2-(3,4-dichlorophenoxy ) - triethylamine (6 ppm ) and 2-(3,5-diisopropylphenoxy)-triethylam ine (6.4 ppm) increased valepotriate production in both cell cultures up to 40%. With dimethylpiperidinium chloride and dimethylmorpholinium bromide the ratio of m onoene to diene valepotriates in both cell systems was significantly shifted to the m onoene com pounds. A general use of these bioregulators to increase production of terpenoid secondary m etabolites in plant tissue cultures is indicated.

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Metabolic engineering of invertase activities in different subcellular compartments affects sucrose accumulation in sugarcane cells

Functional Plant Biology ◽

10.1071/pp00029 ◽

2000 ◽

Vol 27 (11) ◽

pp. 1021 ◽

Cited By ~ 5

Author(s):

Hongmei Ma ◽

Henrik H. Albert ◽

Robert Paull ◽

Paul H. Moore

Keyword(s):

Fold Increase ◽

Saccharum Officinarum ◽

Invertase Activity ◽

Acid Invertase ◽

Sucrose Accumulation ◽

Sugar Composition ◽

Transgenic Sugarcane ◽

Soluble Acid Invertase ◽

Invertase Gene ◽

Soluble Acid

Transgenic sugarcane (Saccharum officinarum L.) lines were created to express altered invertase isoform activity to elucidate the role(s) of invertase in the sucrose accumulation process. A sugarcane soluble acid invertase cDNA (SCINVm, AF062734) in the antisense orientation was used to decrease invertase activity. The Saccharomyces cerevisiae invertase gene (SUC2), fused with appropriate targeting elements, was used to increase invertase activity in the apoplast, cytoplasm and vacuole. A callus/liquid culture system was established to evaluate change in invertase activity and sugar concentration in the transgenic lines. Increased invertase activity in the apoplast led to rapid hydrolysis of sucrose and rapid increase of hexose in the medium. The cellular hexose content increased dramatically and the sucrose level decreased. Cells with higher cytoplasmic invertase activity did not show a significant change in the sugar composition in the medium, but did significantly reduce the sucrose content in the cells. Transformation with the sugarcane antisense acid invertase gene produced a cell line with moderate inhibition of soluble acid invertase activity and a 2-fold increase in sucrose accumulation. Overall, intracellular and extracellular sugar composition was very sensitive to the change in invertase activities. Lowering acid invertase activity increased sucrose accumulation.

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TRANSFORMASI GEN SUCROSE PHOSPHATE SYNTHASE (SoSPS1) MENGGUNAKAN Agrobacterium tumefaciens UNTUK MENINGKATKAN SINTESIS SUKROSA PADA TANAMAN TEBU (Saccharum officinarum L.)

Journal of Biological Researches ◽

10.23869/bphjbr.12.2.20077 ◽

2007 ◽

Vol 12 (2) ◽

pp. 137-143

Author(s):

Miswar Miswar ◽

Bambang Sugiharto ◽

Joedoro Soedarsono ◽

Sukarti Moeljapawiro

Keyword(s):

Agrobacterium Tumefaciens ◽

Starch Content ◽

Sucrose Phosphate Synthase ◽

Saccharum Officinarum ◽

Invertase Activity ◽

Pcr Analysis ◽

Sucrose Synthesis ◽

Transgenic Sugarcane ◽

Sucrose Phosphate ◽

Phosphate Synthase

Sucrose phosphate synthase (SPS EC 2.3.1.14) plays an important role in partition of assimilated carbon in most plants. SPS catalyses the penultimate reaction in the pathway of sucrose synthesis, in which sucrose-6-phosphate (Suc6P) is synthesized from UDPglucose (UDPG) and fructose-6-P (Fru6P). To increase the capacity of sugarcane in sucrose synthesis, spindle leaves of sugarcane cv R579 were transformed with cDNA SoSPS1 from sugarcane under the control of constitutive promoter (35S CaMV) that constructed in pBI 121 (pKYS) using Agrobacterium tumefaciens. Based on PCR analysis, we have detected the existence of SPS transgene in some lines of transformed sugarcane, called line 4, 5, 6, and 7. The SPS transgene in transformed sugarcane could be expressed into translation level and increased the amount of leaves SPS protein, so the activity of leaves SPS was higher than wild type sugarcane as control. The transformed sugarcane line 4, 5, 6, and 7 showed 1.4–2.9 fold increases in SPS activity and 1,76–2,2 fold increases in leaves sucrose content. Increasing in SPS activity in transgenic sugarcane was coupled by the increase in invertase activity and ratio between sucrose and starch content.

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Physiological basis for enhanced sucrose accumulation in an engineered sugarcane cell line

Functional Plant Biology ◽

10.1071/fp10055 ◽

2010 ◽

Vol 37 (12) ◽

pp. 1161 ◽

Cited By ~ 15

Author(s):

Luguang Wu ◽

Robert G. Birch

Keyword(s):

Sucrose Synthase ◽

Sucrose Phosphate Synthase ◽

Saccharum Officinarum ◽

Invertase Activity ◽

Negative Regulator ◽

Sucrose Accumulation ◽

Physiological Basis ◽

Transgenic Sugarcane ◽

Extracellular Invertase ◽

Sucrose Phosphate

Transgenic sugarcane (Saccharum officinarum L. interspecific hybrids) line N3.2 engineered to express a vacuole-targeted sucrose isomerase was found to accumulate sucrose to twice the level of the background genotype Q117 in heterotrophic cell cultures, without adverse effects on cell growth. Isomaltulose levels declined over successive subcultures, but the enhanced sucrose accumulation was stable. Detailed physiological characterisation revealed multiple processes altered in line N3.2 in a direction consistent with enhanced sucrose accumulation. Striking differences from the Q117 control included reduced extracellular invertase activity, slower extracellular sucrose depletion, lower activities of symplastic sucrose-cleavage enzymes (particularly sucrose synthase breakage activity), and enhanced levels of symplastic hexose-6-phosphate and trehalose-6-phosphate (T6P) in advance of enhanced sucrose accumulation. Sucrose biosynthesis by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) was substantially faster in assays conducted to reflect the elevation in key allosteric metabolite glucose-6-phosphate (G6P). Sucrose-non-fermenting-1-related protein kinase 1 (SnRK1, which typically activates sucrose synthase breakage activity while downregulating SPS in plants) was significantly lower in line N3.2 during the period of fastest sucrose accumulation. For the first time, T6P is also shown to be a negative regulator of SnRK1 activity from sugarcane sink cells, hinting at a control circuitry for parallel activation of key enzymes for enhanced sucrose accumulation in sugarcane.

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Down-regulation of neutral invertase activity in sugarcane cell suspension cultures leads to increased sucrose accumulation

South African Journal of Botany ◽

10.1016/j.sajb.2007.02.061 ◽

2007 ◽

Vol 73 (2) ◽

pp. 293

Author(s):

D. Joubert ◽

S. Bosch ◽

F.C. Botha ◽

J. Kossmann ◽

J.-H. Groenewald

Keyword(s):

Cell Suspension ◽

Cell Suspension Cultures ◽

Invertase Activity ◽

Suspension Cultures ◽

Sucrose Accumulation ◽

Down Regulation ◽

Neutral Invertase

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Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation

Functional Plant Biology ◽

10.1071/fp06214 ◽

2007 ◽

Vol 34 (6) ◽

pp. 490 ◽

Cited By ~ 29

Author(s):

Debra Rossouw ◽

Sue Bosch ◽

Jens Kossmann ◽

Frederik C. Botha ◽

Jan-Hendrik Groenewald

Keyword(s):

Cell Lines ◽

Growth Cycle ◽

Suspension Cultures ◽

35S Promoter ◽

Control Line ◽

Sucrose Hydrolysis ◽

Futile Cycle ◽

Camv 35S ◽

Neutral Invertase

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.

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744 PB 076 IDENTIFICATION OF BOTH ACID AND NEUTRAL INVERTASE ACTIVITY IN DEVELOPING STRAWBERRY FRUIT

HortScience ◽

10.21273/hortsci.29.5.539f ◽

1994 ◽

Vol 29 (5) ◽

pp. 539f-539

Author(s):

Kirk W. Pomper ◽

Patrick J. Breen

Keyword(s):

Extraction Procedure ◽

Invertase Activity ◽

Assimilate Transport ◽

Strawberry Fruit ◽

Fruit Extracts ◽

Neutral Invertase ◽

Optimum Ph ◽

Cold Acetone ◽

Ripe Stage ◽

Sugar Levels

Invertase (INV) may influence sugar levels and assimilate transport in strawberry fruit. Several groups, including our own, have only detected acid INV (optimum pH 4.6) in strawberry fruit, however, recently Hubbard et al. (Physiol. Plant. 82:191-196, 1991) reported the presence of a neutral INV (pH 7.5). Since dissimilar isolation protocols may have contributed to the different findings, we re-examined our work with developing `Brighton' strawberry using the extraction procedure of Hubbard et al. Neutral INV activity per gFW (pH 7.5-8.0) increased many fold as fruit developed from green to the red ripe stage. Acid INV activity decreased markedly from green-white to the red stage. In addition, when fruit extracts were precipitated with cold acetone, a pellet contained 60% of the acid INV activity, and a surface coagulation of protein contained 60% of the neutral INV activity. This allowed easy separation of these two enzymes. Extraction methodologies affect isolation of neutral INV activity from strawberry fruit.

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Internodal invertases and stalk maturity in sugarcane

The Journal of Agricultural Science ◽

10.1017/s0021859600077637 ◽

1991 ◽

Vol 116 (2) ◽

pp. 239-243 ◽

Cited By ~ 7

Author(s):

H. L. Sehtiya ◽

J. P. S. Dendsay ◽

A. K. Dhawan

Keyword(s):

Enzyme Activity ◽

Cell Expansion ◽

Reducing Sugars ◽

Sucrose Solution ◽

Invertase Activity ◽

Neutral Invertase ◽

High Enzyme ◽

Stem Slices ◽

High Enzyme Activity

SUMMARYAcid and neutral invertase activities in the stem of an early (CoJ 64) and a late cultivar (Col 148) of sugarcane were estimated by incubating stem slices in buffered sucrose solution and measuring the production of reducing sugars. High enzyme activity occurred in young tissue but the activity of both enzymes was considerably lower in the mature internodes. Acid and neutral invertase activity was highest in the midinternode position, corresponding to the region of cell expansion.

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