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Functional Plant Biology, 2010, 37, 22–31

www.publish.csiro.au/journals/fpb

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 A

Institute for Plant Biotechnology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa. 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: [email protected] B

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.

Introduction Sugarcane (Saccharum officinarum L. interspecific hybrids) is an important commercial crop plant that is capable of accumulating up to 25% (w/w) of its FW as sucrose. Historically, increases in sucrose yield have been accomplished through conventional breeding programs; however, these increases were attained mainly through improvements in cane yield, not in sucrose content (Jackson 2005). In addition, there are several indications that commercial sugarcane is approaching a yield plateau, most probably because the natural genetic potential for sucrose production has been exhausted (e.g. Grof and Campbell 2001; Moore 2005). Genetic engineering could represent an opportunity to add to this potential through the direct manipulation of key enzymes in sucrose metabolism with the aim of increasing the capacity of sugarcane plants to accumulate sucrose. In many heterotrophic plant tissues metabolite cycling is a key feature that allows the plant organ to adapt to environmental challenges. Sucrose accumulation in sugarcane internodes (Glasziou 1961; Sacher et al. 1963) and suspension cells  CSIRO 2010

(Wendler et al. 1990) is regulated by a continuous cycle of synthesis and degradation. It is a carefully controlled process that balances the supply of carbon substrates from the apoplast with the demand of competing pathways for carbon skeletons. Control is exerted on this process through the metabolic regulation of the various enzymes in the synthesis and degradation pathways (see Moore 1995 for a review). Although the apparent ‘futile cycling’ of sucrose is an energetically demanding process (Hill and ap Rees 1994; Dieuaide-Noubani et al. 1995), it could have major physiological functions such as the control of respiration (Dancer et al. 1990), the maintenance of osmotic potential (Geigenberger et al. 1997), the control of sugar accumulation (Rohwer and Botha 2001), sugar signalling (Cortès et al. 2003) and the control of carbon allocation to biosynthetic processes (Fernie et al. 2002). Sucrose cycling is, therefore, neither futile nor wasteful. Various studies in sugarcane have shown that significant effects are exerted on the sucrose cycle by the enzymes sucrose phosphate synthase (SPS; EC 2.4.1.14; Grof et al.

10.1071/FP08210

1445-4408/10/010022

Neutral invertase downregulation in sugarcane

1998; Botha and Black 2000), the invertases (EC 3.2.1.26; Zhu et al. 1997; Echeverria 1998; Vorster and Botha 1999; Rose and Botha 2000; Rohwer and Botha 2001; Bosch et al. 2004) and sucrose synthase (SuSy; EC 2.4.1.13; Lingle and Irvine 1994; Schäfer et al. 2004, 2005), which are collectively responsible for the synthesis and breakdown of sucrose in the various cellular compartments. Although sucrose can be synthesised only in the cytosol by SPS or SuSy activity, it is distributed to various degrees between the apoplast, the cytosol and the vacuole of the storage parenchyma (Hawker 1985; Welbaum and Meinzer 1990). The hydrolytic and/or cleavage activities of the invertases and SuSy in all these subcellular compartments could, therefore, exert an influence on sucrose metabolism, translocation and storage (Lee and Vattuone 1996). The invertases hydrolyse sucrose to glucose and fructose and have been suggested to play a crucial role in the control of metabolic fluxes, sucrose partitioning, and ultimately, plant development and crop productivity (Sonnewald et al. 1991; Klann et al. 1996; Sturm 1999; Tang et al. 1999). The degree to which this statement is true for each of the three invertase isoforms (see below) remains an unresolved issue. Other possible functions of the invertases include the regulation of cell turgor for cell expansion, (Meyer and Boyer 1981; Wyse et al. 1986; Perry et al. 1987), and the control of sugar composition in storage organs (Klann et al. 1993). Furthermore, some of the invertases seem to be involved in the responses of plants to environmental factors, such as wounding and infection (Sturm and Crispeels 1990; Benhamou et al. 1991). In higher plants various isozymes of invertase with different pH optima and subcellular localisations exist (Sturm 1999). There are two kinds of acid invertase, both exhibiting optimum activity between pH 5.0 and 5.5, which are located in two separate cellular compartments, i.e. the vacuole (soluble acid invertase, SAI) and the apoplast (cell wall invertase, CWI). In sugarcane internodes CWI probably controls the flow of sucrose from the conducting tissue to the young growing parenchyma cells (Hatch and Glasziou 1963), whereas SAI may play a role in the remobilisation of stored sucrose from the vacuole (Sacher et al. 1963) and is believed to be important in the regulation of hexose levels in certain tissues (Singh and Kanwar 1991). Mature sucrose-storing internodes of sugarcane contain negligible SAI levels (Hatch and Glasziou 1963), but do, however, exhibit significant SuSy and neutral invertase (NI) activity (Hawker and Hatch 1965). NI is located in the cytosolic compartment, where it functions optimally at pH 7.0. Early studies found that NI activity (expressed on a FW basis) increases with internode maturity and, therefore, correlates positively with sucrose concentrations in internodal tissues (Hatch et al. 1963; Gayler and Glasziou 1972; Batta and Singh 1986; Singh and Kanwar 1991). However, conflicting data have also been reported where NI activity (also on a FW basis) decreases with internode maturation (Dendsay et al. 1995). The most recent studies report that NI activity (on a per mg protein basis) increases up to the fifth internode before declining as the internodes mature further (Ebrahim et al. 1998; Vorster and Botha 1999). In addition, NI is reported to be the only sucrolytic enzyme that shows any correlation to sugar concentrations in mature sugarcane stem tissue, where its activity levels are positively correlated with hexose levels (Gayler and Glasziou

Functional Plant Biology

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1972; Bosch et al. 2004) and negatively correlated with sucrose levels (Rose and Botha 2000). It was initially reported that NI activity expressed on a FW basis showed no particular distribution pattern related to varietal differences (Hatch and Glasziou 1963). However, subsequent research showed that NI activity was low or absent in mature storage tissues of many high-sugar storing, early-maturing varieties of sugarcane (Dendsay et al. 1995). These differential expression patterns of NI suggest that it might be an important determinant of the sucrose accumulation characteristics of sugarcane. As NI is a cytosolic enzyme, it is the only invertase that functions in the compartment where sucrose is actively synthesised and could, therefore, play a crucial role in the process of sucrose accumulation in sugarcane. Of particular importance is the role that NI might play in controlling the cycle of sucrose breakdown and resynthesis. Moreover, a kinetic model for sucrose metabolism in sugarcane identified NI as the key enzyme in determining the flux through this cycle (Rohwer and Botha 2001). Although only theoretical and speculative in nature, this in silico model suggests that NI is a suitable target for a reverse genetic approach aimed at increasing sucrose contents in sugarcane internodes. In a recent study we have described the establishment and characterisation of transgenic sugarcane suspension cultures with reduced NI activity (Rossouw et al. 2007). These suspension cultures showed reduced sucrose cycling, impaired growth characteristic, increased sucrose concentrations and reduced hexose levels. Here we describe the characterisation of transgenic plants that were regenerated from these cell lines. Existing studies on plants where invertase activity has been genetically modulated in the cytosol of a higher plant described the effects of overexpressing a yeast invertase in the cytosolic compartment of transgenic potato and tobacco plants (Sonnewald et al. 1991, 1997; Ma et al. 2000). In these cases the upregulation of cytosolic invertase activity resulted in an increase of respiration rates. The findings in our study are in agreement with those observations, and we present evidence that NI in sugarcane is of crucial importance for providing carbon backbones for respiration and sucrose cycling. In addition, we demonstrate that sugarcane suspension cells represent a valuable experimental model system for metabolic engineering in sugarcane. Materials and methods Biochemicals Chemicals were purchased from Sigma-Aldrich (Johannesburg, South Africa). All coupling enzymes and cofactors used in the sugar and enzyme assays were obtained from Roche Applied Science (Indianapolis, IN, USA). The [U-14C]fructose was from Amersham International (Claremont, South Africa). All other solvents and biochemicals were of analytical grade. Plant material Callus of sugarcane (Saccharum officinarum L.) variety NCo310 was transformed as described earlier (Rossouw et al. 2007) with an antisense NI construct (pASNI510) in which gene expression is regulated by a strong, constitutive tandem promoter, consisting of the maize ubiquitin and CaMV 35S

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Functional Plant Biology

promoters. After regeneration the plantlets were hardened off and grown in a greenhouse until suitable nodes were available for vegetative propagation. Transgenic sugarcane lines destined for metabolic analyses were routinely generated through this ‘two tiered’ system because (i) plants regenerated directly from tissue culture often have highly variable growth characteristics and (ii) it affords an opportunity to generate several independent clones of each line. These vegetatively propagated transgenic plants were grown with appropriate untransformed controls in a greenhouse in Stellenbosch, South Africa for 12 months before harvesting and characterisation. All plants were grown in 3-L pots that were randomly arranged on greenhouse benches in an approximate 3  30 matrix. The two transgenic lines, U1 and U2, as well as control NCo310 plants were harvested between 0900 and 1000 hours towards the end of summer (February). The leaf with the youngest visible dewlap, the node it was attached to and internode just above it was defined as leaf, node and internode number one, respectively, according to the system of Kuijper (Van Dillewijn 1952). Internodes 3 and 4 were grouped together to represent immature tissues and internodes 7 and 8 were grouped as representative of maturing internodal tissues. Independent replicates refer to samples generated from individual plants, i.e. stalks harvested from plants in separate pots. Samples of these tissues were taken for protein and enzyme analyses and 1 mm tissue disc slices were sectioned with a hand microtome from core internodal tissue 6 mm in diameter. The tissue discs were immediately placed in 50 mL ice-cold buffer containing 25 mM K-MES (pH 5.7), 250 mM mannitol and 1 mM CaCl2 for at least 15 min (Whittaker and Botha 1997). Protein extractions Samples were ground in liquid nitrogen and 150 mg tissue extracted in 500 uL extraction buffer (100 mM sodium phosphate, pH 7.5) containing 1 mM EDTA, 10 mM DTT, 10% (v/v) glycerol, 2% polyvinylpolypyrrolidone (PVPP) (w/v) and 0.0016 g mL–1 Complete protease inhibitor (Roche Applied Science). Insoluble residue was pelleted by centrifugation at 10 000g for 15 min. The supernatant was passed through Sephadex G25 size-exclusion minispin columns (Biopharmacia Ltd, Perth, WA) by centrifugation at 2500g for 1 min, and the eluates used as the desalted protein samples. Protein concentrations in the crude and desalted samples were determined according to Bradford (1976) using the Bio-Rad (Hercules, CA, USA) microassay, with BSA as a standard. The pellet was washed three times in extraction buffer (without PVPP) to remove any residual soluble invertase activity. Enzyme assays Cell-wall bound invertase activity was measured in the insoluble pellets according to the method by Albertson et al. (2001). Acid invertase, neutral invertase and SuSy activity was determined in the desalted enzyme extracts: SuSy activity was determined in the breakdown direction as described by Schäfer et al. (2004). Acid invertase activity was assayed by incubation of extracts for 3 h in 125 mM sucrose and 50 mM citrate-phosphate buffer (pH 5.5). Reactions were stopped at 1-h intervals by a 2 min incubation at 90C. Neutral invertase activity was similarly assayed by a 2-h

D. Rossouw et al.

incubation in 50 mM Hepes (pH 7.5) and 125 mM sucrose. At 0, 1 and 2 h, aliquots were transferred to a second microtiter plate (kept at 4C), and the reaction terminated by the addition of 5 mL stop solution (2 M Tris, 22 mM ZnSO4). For both the acid and neutral invertase assasy the reducing sugars were measured using a system coupled to NAD+ (Huber and Akazawa 1986) with a Beckman DU 7500 spectrophotometer (Beckman Coulter, Brea, CA, USA). Radiolabelling experiments For 14C metabolic studies, the tissue discs were labelled as described by Bindon and Botha (2002). Tissue discs were incubated on a rotary shaker at 102 rpm for 4 h in 650 mL buffer containing 5 mM glucose, fructose and sucrose and [U-14C]fructose at a specific activity of 37 Bq nmol–1. The 14 CO2 released during the incubation period was collected in 500 mL 12% (w/v) KOH contained in central wells of the 250 mL Erlenmeyer flasks. The radioactivity present in the KOH vials was determined by a Beckman scintillation counter (Beckman Coulter) following the addition of 4 mL Ultima Flo scintillation cocktail (Perkin Elmer, Waltham, MA, USA). After the labelling period, unincorporated label was removed from the tissue discs by two consecutive washes in 50 mL ice-cold wash buffer (50 mM MES, pH6.5). The discs were then blotted dry and ground in liquid nitrogen. The ground tissues were weighed before being transferred to 2 mL Eppendorf tubes containing 1 mL 80% EtOH and 30 mM Hepes (pH 7.5). The samples were incubated for 2 h at 80C, then centrifuged at 17 000g for 20 min at 4C. The supernatants were removed and the extraction repeated with another 1 mL of 80% EtOH. The supernatant from this step was added to that of the first extraction. Radioactivity in the total soluble fraction was determined by counting 10 mL in 4 mL scintillation cocktail in a Beckman LS 1801 scintillation counter (Beckman Coulter). The remaining pellet was washed five times in 80% EtOH and dried completely in a vacuum centrifuge, after which 2 mL of the organic solvent Soluene-350 was added to dissolve the insoluble residue. Radioactivity in 200-mL aliquots of the cell pellet suspension was also determined with a scintillation counter as described for the KOH solutions. Fractionation of cellular constituents For fractionation of cellular constituents, 1 mL of the total solubles were passed through strong cation and anion exchangers (Supelco Supelclean columns; Supelco, Bellefonte, PA, USA) in tandem to bind all acidic and basic compounds and render an extract containing only the neutral fraction in a volume of 10 mL ddH2O. This neutral fraction was completely reduced in a vacuum centrifuge and resuspended in 10% (v/v) isopropanol. The acid and basic fractions were eluted from the columns with 2 mL of 4 M NH4OH and 4 M formate, respectively. Label recovery in the three fractions was again determined with scintillation counting. The remaining soluble extracts were dried under vacuum and resuspended in 10% isopropanol. Samples were prepared for HPLC analysis by passing through 0.45 mm filters (Millipore, Billerica, MA, USA).

Neutral invertase downregulation in sugarcane

Functional Plant Biology

Enzymatic assay of total sugar content Glucose, fructose and sucrose were assayed according to the method by Bergmeyer and Bernt (1974).

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using other constructs. In addition, the resulting transgenic plants displayed clear signs of reduced vigour, i.e. transgenic sets germinated at only 50% the rate of control NCo310 sets and the resulting plants were ~30% smaller than their untransformed counterparts after 2 months.

Analysis of sugars by HPLC Sugars were fractionated by HPLC (Shimadzu SCL-10AVP system; Shimadzu, Kyoto Japan) using a Hamilton RCX-10 column (Hamilton, Bonaduz, Switzerland). A 30 mM NaOH solution was used as the mobile phase, at a flow rate of 1 mL min–1, and 14C in glucose, fructose and sucrose was determined by inline liquid scintillation spectroscopy (Radiomatic A-500; Perkin Elmer).

A reduction in NI activity leads to an increase in sucrolytic SuSy activity For the initial characterisation of the transgenic lines all enzyme activities that contribute to the breakdown of sucrose were determined in young and maturing internodes. All sucrosehydrolysing activities decreased from young to maturing internodal tissues (Table 1). NI activity was reduced by more than 35% in the U1 and U2 lines in comparison with the control lines. The opposite pattern was evident in the case of SuSy, as activity in the breakdown direction increased in the transgenic lines relative to the control. No significant differences were observed in the activities of the SAI and CWI isoforms.

Statistical analyses All analyses were performed in four independent replicate samples from four individual plants. Analysis of variance (ANOVA) was performed with the data analysis software, Sigma Plot 2000 (V6.1, SPSS, Chicago, IL, USA). P  0.05 was considered as significant unless stated otherwise. Results

A reduction in NI activity leads to an increase in the purity of the sugar samples

The transgenic plant lines in this study were transformed with an antisense NI sequence under the control of a tandem 35Subiquitin promoter, which induces strong, constitutive transgene expression (Rossouw et al. 2007). Only two independently transformed lines, i.e. U1 and U2 that were shown to have reduced NI activity in the corresponding suspension cultures, could be regenerated although several calli lines were stably transformed and grew on selection media. The transgenic calli exhibited high levels of recalcitrance during regeneration and transgenic antisense NI plants were regenerated at less than 5% of the average efficiency of the transformation system when

Clear alterations were found in the sugar compositions of the transgenic tissues in comparison to the control. On average glucose and fructose concentrations were lower, but the average sucrose concentrations were consistently higher in both the young and maturing internodes of both the transgenic lines (Table 2). This consistent reduction in hexose concentrations and concurrent increase in sucrose concentrations resulted in a significant increase in the purity (the percentage ratio of the sucrose v. the total soluble sugar, i.e. sucrose plus + glucose + fructose, molar concentrations) of the transgenic samples. In comparison to the control samples, the purity of the transgenic lines increased

Table 1. Activity of the sucrolytic enzymes in young (internodes 3–4) and maturing (internodes 7–8) internodes of the control (NCo310) and two transgenic sugarcane lines (U1 and U2) Enzyme activity is expressed as nmol min–1 mg–1 protein. Values are the average of four replicates  s.e. Values that differ significantly from the control are indicated: *, P < 0.05 Young internodes U1

NCo310 Neutral invertase Sucrose synthase Acid invertase Cell wall invertase

30.6 ± 2.4 50.6 ± 5.8 32.6 ± 2.2 20.8 ± 3.2

19.2 ± 2.3* 76.8 ± 1.8* 32.1 ± 4.2 23.3 ± 3.6

U2

NCo310

Maturing internodes U1

U2

20.5 ± 2.9* 65.8 ± 11.2* 30.4 ± 5.1 22.4 ± 2.2

22.5 ± 3.6 24.3 ± 4.8 15.6 ± 3.2 11.8 ± 1.6

13.8 ± 0.9* 45.3 ± 3.9* 14.2 ± 1.3 12.0 ± 2.4

14.6 ± 2.7* 36.5 ± 19.8 15.6 ± 2.1 12.1 ± 1.5

Table 2. Concentrations of glucose, fructose and sucrose in extracts from young and mature tissues of the control and two transgenic sugarcane lines (U1 and U2) Sugar concentrations are expressed as mmol mg–1 protein. Values are the average of four replicates  s.e. Values that differ significantly from the control are indicated: *, P < 0.05

Glucose Fructose Sucrose % Purity

NCo310

Young internodes U1

U2

NCo310

Maturing internodes U1

U2

23.9 ± 7.0 19.9 ± 8.2 57.3 ± 15.9 56.8 ± 6.7

17.7 ± 4.4 13.1 ± 2.1 72.1 ± 10.8 70.1 ± 3.9*

17.6 ± 5.4 15.9 ± 7.3 77.3 ± 16.8 70.2 ± 3.9*

22.5 ± 3.2 23.7 ± 4.3 173.6 ± 28.8 79.2 ± 3.0

17.4 ± 1.4* 17.0 ± 2.3 198.1 ± 13.7 85.1 ± 1.2*

19.3 ± 2.6 17.9 ± 7.0 199.4 ± 22.1 84.4 ± 1.6*

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D. Rossouw et al.

by 13 and 6%, respectively, in the young (to 70%) and maturing (to 85%) tissues. A reduction in NI activity leads to an increase in the flux into sucrose To investigate potential changes in the in vivo partitioning of carbon to various key metabolite pools, radio-labelled fructose was fed to excised internodal tissue discs. In general, the labelled fructose was taken up with greater efficiency by the transgenic tissue discs (Table 3). Less than 7% of the label in young internodes and 10% in the maturing internodes were present in fructose after 4 h of labelling with 14C-fructose. This implies that fructose is rapidly metabolised after uptake. This is similar to the previous observations that more than 90% of the glucose taken up by sugarcane tissue is rapidly metabolised (Bindon and Botha 2002). Label allocation to the insoluble fraction decreased considerably as the internodes mature but no differences were apparent between the transformed and untransformed tissues. Significant amounts of label were present in both the sucrose and glucose pools following uptake of labelled fructose (Table 3). Furthermore, the amount of label in sucrose exceeded that in both the hexoses despite the fact that the label was supplied as fructose. The appearance of label in glucose following the uptake of labelled fructose only indicated recycling of carbon from sucrose to the hexoses through invertase activity. In the control plants similar amounts of label were present in glucose

and fructose pointing to the rapid cycling of carbon through invertase. In three of the four transgenic tissues tested the amount of label in fructose significantly (P < 0.05) exceeded that in glucose, suggesting reduced cycling of carbon from sucrose to glucose. Similarly, in comparison with to the control, label incorporation into sucrose was also significantly (P < 0.05) greater in most of the transgenic tissues. The opposite was true for glucose in the U2 transgenic line in which the incorporated radio-label was significantly reduced relative to the control. Although the allocation of label to the amino and organic acid pools was not influenced significantly the released of labelled CO2 was reduced significantly in most (three of four) of the transgenic tissues. The label in sucrose as a percentage of the total label was greater in the transgenic lines than in the control (Fig. 1). This higher proportion of label in sucrose in the transgenic lines is at the expense of label present in glucose, the organic and amino acids, and released CO2. The specific activity of the sugars was determined without any provision for compartmentation of the sugars or the label (Table 4). The internal specific activity of fructose was significantly lower than that of the fructose in the incubation medium. In this specific experimental system, the isotopic steadystate between external and internal metabolic active hexoses is reached after 3 h of incubation (Bindon and Botha 2000). If isotopic equilibrium between the external fructose in the medium and the internal hexose phosphate pool from which sucrose is

Table 3. Distribution of 14C in internodal tissue discs of sugarcane supplied with [U-14C]fructose for 4 h The total activity of each component is expressed as kBq mg–1 protein. Each value is the average  s.d. of four separate samples. Values that differ significantly from the control are indicated: *, P < 0.05 Line Young NCo310 internodes U1 U2 Maturing NCo310 internodes U1 U2

Total respiration

0.96 ± 0.40

0.41 ± 0.08

4.32 ± 0.89

2.79 ± 0.13 2.99 ± 0.93 2.36 ± 0.67

0.69 ± 0.10 0.73 ± 0.07 1.06 ± 0.33

0.22 ± 0.03* 3.69 ± 0.17 0.23 ± 0.07* 3.94 ± 0.97 0.20 ± 0.07 3.62 ± 0.94

2.20 ± 0.63 2.07 ± 0.57

1.08 ± 0.05 0.94 ± 0.17

0.09 ± 0.03* 3.37 ± 0.65 0.15 ± 0.03 3.16 ± 0.70

Insoluble fraction

Sucrose

Glucose

Fructose

20.85 ± 3.88

7.21 ± 0.70

6.73 ± 2.02

1.83 ± 0.66

1.49 ± 0.63

2.95 ± 0.56

30.80 ± 4.56* 7.48 ± 1.11 11.58 ± 2.96* 1.00 ± 0.31 2.08 ± 0.52 26.06 ± 6.96 6.85 ± 1.89 9.89 ± 2.30* 0.76 ± 0.05* 1.30 ± 0.51 21.88 ± 8.83 2.67 ± 1.80 8.94 ± 1.89 1.12 ± 0.32 1.64 ± 0.77 29.47 ± 2.96 24.62 ± 2.62

2.52 ± 1.21 21.39 ± 2.91* 0.63 ± 0.18 2.59 ± 0.37 2.50 ± 1.11 13.26 ± 3.38 0.61 ± 0.18* 2.13 ± 0.50

(a)

Percntage (%)

CO2

Total uptake

Organic acids Amino acids

(b) 100

100

80

80

60

60

40

40

20

20

0

0 NCo310

U1

U2

NCo310

U1

U2

Fig. 1. Percentage allocation of 14C to the various metabolic pools after a 4 h labelling period with [U-14C]fructose in (a) young and (b) maturing internodes of sugarcane. The fractions represented from bottom to top are as follows: fructose ( ), glucose ( ), sucrose ( ), organic acids ( ), amino acids ( ), insoluble ( ) and CO2 ( ).

Neutral invertase downregulation in sugarcane

Functional Plant Biology

Table 4. Specific activities of glucose, fructose and sucrose in the tissue disc extracts from young and mature tissues of the control and two transgenic sugarcane lines Specific activities are expressed as Bq mmol–1 sugar. Values are the averages of four replicates  s.d. Values that differ significantly from the control are indicated: *, P < 0.05

Young internodes

Maturing internodes

Line

Fructose

Glucose

Sucrose

NCo310

60.8 ± 11.2

77.6 ± 24.8

117.7 ± 12.7

U1 U2 NCo310

124.9 ± 23.0* 84.4 ± 11.3* 71.6 ± 9.7

57.7 ± 6.6 46.4 ± 13.5 57.8 ± 15.5

153.1 ± 24.3* 128.1 ± 11.9 53.4 ± 10.4

U1 U2

159.2 ± 31.6* 129.7 ± 42.3*

40.0 ± 12.3 31.3 ± 6.4*

90.0 ± 33.8 69.8 ± 20.0

underestimation of the flux due to the rapid mobilisation of the cytosolic hexoses into metabolism. The ratio between the specific activities of the glucose and fructose pools when label is fed into the system through fructose will be indicative of the cycling of carbon from fructose into sucrose and back to glucose. The latter step is probably largely a reflection of invertase activity. Rapid mobilisation of fructose and return of carbon from sucrose to glucose should result in a ratio close to unity and the slower the return the lower this ratio would be. The calculated ratios demonstrate that the control plants have a more rapid flow of carbon from sucrose to glucose and this process was highest in the immature internodes (Fig. 4). Evidently the reduction in invertase activity in the transgenic lines significantly influenced the rate at which the glucose and fructose pool reach isotopic equilibrium. Discussion Transgenic sugarcane lines with reduced NI activity were regenerated at a very low efficiency when using a strong, constitutive transgene expression system. Reduced NI activity, therefore, seems to induce a phenotype that significantly reduced (a)

0.30

Flux into glucose (nmol min–1 mg–1 protein)

derived is assumed, the flux into sucrose can be calculated (Fig. 2). This is obviously an underestimation of the flux into sucrose as no provision is made for flux of label out of the sucrose pool. In comparison to the control the net flux into sucrose was significantly (P < 0.1) greater in the tissues of the U1 line (Fig. 2). In order to calculate the flux into glucose we used two different methods. First, we assumed isotopic equilibrium between the external fructose, internal hexose phosphate pool and the sucrose pool from which the glucose is derived. Second, we assumed that only 10% of the total sucrose is in the cytosol and that all the label in sucrose is present in the cytosolic sucrose pool (Fig. 3a, b). The calculated flux values for these two systems are similar. Moreover, the calculations suggest that the flux from sucrose to glucose was higher in the control plants in both the young and maturing internodes than in the two transgenic lines, although the converse was true for the flux into glucose (Fig. 3). In the young internodes, the flux into glucose was reduced by ~60% in the transgenic lines, and in the maturing internodes the reduction was between 50 and 75%. The calculated values will be an

0.25 0.20 0.15 * *

0.10

*

0.00

(b)

Flux into sucrose (nmol min–1 mg–1 protein)

2.00 **

1.50

1.00

Flux into glucose (nmol min–1 mg–1 protein)

3.00

2.50

*

0.05

Young internodes

*

27

Maturing internodes

0.70 0.60 0.50 0.40 0.30 * 0.20

*

*

*

0.10 0.00

0.50

Young internodes

0.00 Young internodes

Maturing internodes

Fig. 2. Net flux of [U-14C]fructose into sucrose after a 4 h labelling period in sugarcane. Values are the average of four replicates  s.d. The three bars in each group represent from left to right NCo310 ( ), U1 ( ) and U2 ( ). Values that differ significantly from the control are indicated: *, P < 0.05; **, P < 0.1.

Maturing internodes

Fig. 3. Calculated net flux of label into glucose after a 4-h labelling period in sugarcane assuming isotopic equilibrium between the external fructose, (a) the internal hexose phosphate pool and the sucrose pool from which the glucose is derived or (b) that only 10% of sucrose is in the cytosol and all the label in sucrose is in the cytosol. Values are the average of four replicates  s.d. The three bars in each group represent from left to right NCo310 ( ), U1 ( ) and U2 ( ). Values that differ significantly from the control are indicated: *, P < 0.05.

Ratio of specific activity in glucose/fructose

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D. Rossouw et al.

1.40 1.20 1.00 0.80 0.60

*

*

0.40 *

*

0.20 0.00 Young internodes

Maturing internodes

Fig. 4. The ratio between the specific activities of the glucose and fructose pools after a 4-h labelling period in sugarcane. The three bars in each group represent from left to right NCo310 ( ), U1 ( ) and U2 ( ). Values that differ significantly from the control are indicated: *, P < 0.05.

the viability of in vitro cell lines and potential plants that could be regenerated from these. In addition, the only two transgenic lines that were regenerated still contained significant amounts (~60%) of NI activity. In similar studies in sugarcane with pyrophosphate dependent phosphofructokinase (Groenewald and Botha 2008), UDP-glucose dehydrogenase (Bekker 2007) and ADP-glucose pyrophosphorylase (Ferreira et al. 2008) similar silencing systems reduced the respective catalytic activities much more efficiently. This suggests that the transgenic lines that survived and were able to regenerate in this case were lines in which NI activity was reduced with low efficiency because of, for example, their specific integration characteristics. These observations suggest that a certain minimum amount of NI activity should still be available for the transgenic cells to be viable. The low germination rate (50%) of the transgenic sugarcane sets during vegetative propagation and reduced growth rate of the resulting plants further underlines the impact of reduced NI activity on the vigour of the transgenic plants. In similar experiments conducted in tomato (Solanum lycopersicum L.) plants, transgenic lines with reduced acid invertase levels in ripe fruits had increased sucrose concentrations and were ~30% smaller than the control fruits (Klann et al. 1996). In addition, Vilhar et al. (2002) demonstrated that a deficiency in cell wall invertase activity in maize (Zea mays L.) endosperm results in reduced mitotic activity, cell divisions and cell size, probably because of the reduced availability of the hexoses. Recently Arabidopsis mutants with reduced NI activity displayed severely reduced growth rates (Barratt et al. 2009). Correlations between reduced sucrolytic rates and growth inhibition as reported here have, therefore, been demonstrated previously. Transgenic sugarcane plants that contained ~60% of the NI activity found in control plants, both in immature and maturing internodes, were regenerated and characterised. Glucose, fructose and sucrose concentrations, percentage purity and the respective specific enzyme activities in the control samples were similar to the reported values in other studies on sugarcane internodes (Whittaker and Botha 1997; Vorster and Botha 1999; Bindon

and Botha 2002). Sucrose concentration and the percentage purity of the transgenic lines were consistently higher, and the hexose (glucose and fructose) concentrations lower, than that of the control lines in all the tissues sampled (Table 2). The sucrose concentrations in the immature and maturing internodes of the transgenic plants were up to 35 and 15% higher, respectively, than control levels. The percentage purity of the transgenic samples was ~13 and 6% higher than in the control in the young and maturing internodes, respectively. In general, purity increases in the maturing internodes as the demand for glycolytic intermediates decreases (Botha et al. 1996). Therefore, it appears that the downregulation of NI activity has a smaller influence in the more mature internodes where glycolytic flux is smaller, i.e. the remaining activity and complementary activity of SuSy can cope more effectively with the demand for hexoses in these tissues. This explains the relatively lower increase in the percentage purity of the transgenic samples in the older internodes compared with the increase measured in the immature transgenic tissues. In terms of the allocation of label to the various carbon pools in the tissue discs, some notable differences were evident in the transgenic lines. We noted that the transgenic tissues took up more fructose from the incubation buffer during the labelling period than the controls (Tables 3, 4). A possible explanation is that the lower glucose and fructose concentrations of the transgenic cells resulted in a greater concentration gradient between the medium and the intracellular hexoses. It is also possible that the change in metabolite concentrations in the transgenic tissues altered some aspects of the hexose uptake system in such a way to increase the uptake of hexoses from the incubation buffer. The amount of label in the insoluble pool decreased by ~60% from young to maturing internodes in all the plants sampled, which is in agreement with trends as reported by Bindon and Botha (2002). No differences between the control and transgenic lines were evident for the total incorporation of label in the insoluble pool, suggesting that this branch of carbon metabolism remained relatively unaffected by the changes in the sucrolytic activities in the transgenic tissues. The increase in label allocation to sucrose between the young and maturing internodes can be seen in terms of both the amount of label per mg protein and as a percentage of the total label taken up (Table 3; Fig. 1). In the control plants, the percentage allocation of label to sucrose increased from ~32 to 50% between the immature and maturing internodes, respectively, whereas in the transgenic plants the label allocation increased from 43 to 64%, respectively (Fig. 1). The control values for label allocation to sucrose as well as the other fractions are similar to those reported by others (Whittaker and Botha 1997; Bindon and Botha 2002). Both label recovery in glucose (Table 3) and the flux of label into glucose (Fig. 3) were significantly reduced in both the young and maturing internodes of the transgenic lines, suggesting a clear reduction in the in vivo rate of invertase-mediated sucrose hydrolysis. Traditionally, the phosphorylation, incorporation into sucrose (with the associated randomisation of label in the hexose moieties) and subsequent hydrolysis by invertase is considered to be the only pathway through which label could be incorporated into glucose after feeding labelled fructose. More recently though, Alonso et al. (2005) suggested the presence of a glucose-6-phosphatase activity in maize root tips, which could

Neutral invertase downregulation in sugarcane

be responsible for the direct flux of label from the hexose phosphate pool to glucose. Although the experiments described here were not designed to distinguish between these possible pathways, there are two reasons why our data suggest that the sucrose cycle through NI represents a much more significant flux in sugarcane internodal tissues. First, in both the young and maturing internodes of the control and transgenic lines there is a strong correlation between the amount of NI activity and the flux of label into glucose (Table 1; Fig. 3). Second, in the plants with reduced NI activity the rate in establishing isotopic equilibrium is slowed down while the phosphatase mediated reaction would not be influenced by reduced invertase activity. The major impact of the reduced NI activity on carbon flux from sucrose to glucose is consistent with previous work showing a flux control coefficient of higher than 0.7 for NI in sucrose accumulation in sugarcane (Rohwer and Botha 2001; Bosch et al. 2004). This is also consistent with earlier references to NI’s important role in sucrose cycling in sugarcane tissues (Zhu et al. 1997; Echeverria 1998; Vorster and Botha 1999; Rose and Botha 2000). The apparent increase in the net flux into sucrose in the transgenic lines (Fig. 2) is, therefore, probably due to the decreased hydrolysis rates, which would result in an accumulation of label in the sucrose pool, as opposed to a distribution of label to other pathways and competing carbon pools. In combination then, the data suggests that the decrease in sucrose hydrolysis did not lead to a corresponding decrease in the rate of sucrose synthesis. One pathway that is clearly affected by the altered metabolism of the transgenic cells is respiration. In nearly all the transgenic tissues sampled the CO2 released (the end product of oxidative respiration) contained significantly less labelled carbon than the control samples (Table 3). This suggests a decrease in the respiration rate of these tissues, which could explain the increased recalcitrance in regeneration and the slow-growing phenotype of the transgenic plants. The same trends with regards to the respiration and growth rates were also reported for the suspension cells (Rossouw et al. 2007). The low regeneration efficiency of transgenic calli and relative small reduction (40%) in NI activity in regenerated plants further suggests that the cells become unviable if NI activity is reduced below a certain threshold value as discussed before. Although numerous studies using mutants or transgenic plants with reduced SuSy activity have demonstrated that SuSy is the main cytosolic sucrose hydrolysing activity directing sucrose to other carbon pools (Chourey and Nelson 1976; Zrenner et al. 1995; D’Aoust et al. 1999; Tang and Sturm 1999), a recent study indicate that in at least some tissues this role is primarily fulfilled by NI (Barratt et al. 2009). Arabidopsis mutant plants deficient in two closely related isoforms of neutral invertase activities in roots have severely decreased growth rates. The observed phenotype in these mutant plants is consistent with general carbon starvation due to decreased capacity for sucrose catabolism. The reverse genetic approach that we used in this study now also directly establishes a crucial role for NI in sugarcane tissues, where cytosolic sucrose catabolism specifically seems critical to sustain energy metabolism for normal development and growth. This is supportive of a growing volume of literature that indicates that neutral invertase plays a critical role in growth

Functional Plant Biology

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and development (Flemetakis et al. 2006; Gonzalez and Cejudo 2007; Lou et al. 2007; Qi et al. 2007; Barratt et al. 2009; Yao et al. 2009). Expressed as a percentage of the control, the decrease in NI activity in the internodes is not as substantial as was found in the suspension culture experiments conducted with the same transgenic lines (Rossouw et al. 2007). Although only 33% of the NI activity remained in the transgenic suspension cultures, the internodal tissues contained on average 60% of the NI activity of control tissues. This could be due to possible differences in transgene expression and silencing efficiencies (Iyer et al. 2000) or differences in sink-source relationships, metabolic regulation and cell architecture, e.g. vacuolisation and transport structures (Veith and Komor 1993; Botha et al. 1996). However, the apparent impact of reduced NI activity on the closely associated metabolite levels and fluxes in primary carbon metabolism are similar in both systems – in terms of identity (same metabolites show same changes) and relative magnitude (extent of impact correlates positively with the extent to which NI activity was reduced). An increase in the sucrose to hexose ratio and a decrease in respiratory flux were also apparent in the suspension cultures of the same transgenic lines (Rossouw et al. 2007). In light of these findings, suspension cultures are proposed as a viable model system for the initial characterisation of transgenic interventions in primary carbon metabolism in sugarcane. In conclusion, our data suggest that NI plays an important role in controlling the rate of sucrose hydrolysis and therefore in the provision of hexose substrates to the subsequent cellular processes, particularly in immature internodes. Moreover, sucrose accumulation is not influenced by a decrease in NI activity. Similar to Arabidopsis mutants with reduced NI activity, suppression of NI activity in sugarcane impacts negatively upon respiration resulting in the reduced growth of the transgenic plants. This is most likely due to reduced availability of hexoses as a consequence of the lower NI activity. This also points to the importance of carbohydrate cycles to provide plasticity to metabolism to optimise carbohydrate allocation for both, storage and growth. 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.

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Manuscript received 20 July 2008, accepted 17 September 2009

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