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Published online 30 July 2007
Published in Crop Sci 47:1530-1539 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP PHYSIOLOGY & METABOLISM

Sucrose-Phosphate Synthase, a Biochemical Marker of High Sucrose Accumulation in Sugarcane

Christopher P. L. Grof*, Peter L. Albertson, Johanna Bursle, Jai M. Perroux, Graham D. Bonnett and John M. Manners

CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Rd., St Lucia QLD 4067, Australia

* Corresponding author (Chris.Grof{at}csiro.au).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increasing sucrose content is a major objective of sugarcane breeding programs. One approach employed by breeders is to introgress new genes from genotypes of Saccharum officinarum L. not previously used in breeding. The activity of a suite of key sucrose metabolizing enzymes was measured in progeny of an introgression program to find biochemical markers associated with high sucrose content, measured as commercial cane sugar (CCS). The enzymes sucrose-phosphate synthase (SPS), three isoforms of invertase, and sucrose synthase were measured in four high and four low CCS clones from an initial cross between a S. officinarum and the commercial cultivar Q165. Subsequently, SPS and the two soluble isoforms of invertase were measured in clones derived from a backcross of one of the progeny to another commercial cultivar Mida. Enzyme activities were measured in tissue from internodes taken from four different positions down the stem profile. Of particular significance was the finding that the activity of a key enzyme involved in sucrose synthesis, SPS, was significantly higher in the upper internodes (one to three) of high CCS clones as compared with low CCS clones in both populations, suggesting that this enzyme may have a key role in establishing metabolic and developmental processes necessary for high sugar accumulation during stem growth and maturation.

Abbreviations: BC-H1 to BC-H4, high CCS clones of the backcross population • BC-L1 to BC-L4, low CCS clones of the backcross population • CCS, commercial cane sugar • FW, fresh weight • HPAE-PAD, high performance anion exchange with pulsed amperometric detection • I-H1 to I-H4, high CCS clones of the introgression population • I-L1 to I-L4, low CCS clones of the introgression population • SAI, soluble acid invertase • SPS, sucrose-phosphate synthase • SSf, sucrose synthase forward, functioning to synthesize sucrose • SSr, sucrose synthase reverse, functioning to cleave sucrose


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SUCROSE PLAYS a central role in plant metabolism as it is the primary product of photosynthesis and is the major form of carbohydrate translocated from the leaves and utilized as a C and energy source for growth (ap Rees, 1987). Sugarcane (Saccharum spp.) utilizes the C4 mechanism of CO2 fixation and is grown predominantly in tropical and subtropical regions. Sugarcane produces approximately 70% of the world's sucrose that is used both for food and fermentation generating the biofuel ethanol.

Conventional breeding has made a major contribution to increasing sucrose yields per unit area from sugarcane over the last century. In Australia, varietal improvement has been estimated to have increased sucrose yield by 1 to 1.5% per annum over the last 50 yr while maintaining disease resistance and sugar quality standards (Chapman, 1996). However, this rate of yield increase is well below that achieved in other major field crops such as maize (Zea mays L.), rice (Oryza sativa L.), and wheat (Triticum aestivum L.) (Moore, 1989). Although sucrose yields continue to increase, there has been no increase in sucrose content, at least in Australia, over the last 40 yr (Jackson, 2005). The genome of sugarcane is among the most complex of all crop plants (Grivet and Arruda, 2002). Commercial sugarcane cultivars are derived from initial crosses between Saccharum officinarum L. (2n = 80) and S. spontaneum L. (2n = 60–100) followed by a limited number of backcrosses to S. officinarum. As such, sugarcane varieties are interspecific polyaneuploid hybrids with more than 100 chromosomes per cell. Most sugarcane varieties can be traced back to a very small number of interspecific crosses. Therefore, one significant factor believed to have contributed to the lack of progress in improving stem sucrose content is the narrow gene pool used in current commercial breeding programs (Roach, 1989). Introgression is a breeding strategy to enlarge the available gene pool by incorporation of one or several traits from unadapted exotic germplasm into an elite adapted genetic background. Introgression is likely to require at least two or more backcrosses to an elite commercial parent as exotic germplasm contributes a large proportion of unfavorable genes and only a small number of genes of potential commercial value. A likely constraint to the success of this approach in sugarcane is the difficulty of identifying and tracking the desirable traits from exotic sources through the introgression process.

The complexity of the sugarcane genome, in particular its large ploidy (D'Hont et al., 1998) ensures that DNA marker technology is not as readily applied to genetic improvement as it can be in simple diploids. An alternative approach is the identification of potential biochemical markers that may be associated with a high sucrose phenotype and can be measured at early developmental stages thereby facilitating selection. The strategy described herein targeted key enzymes mediating either sucrose synthesis or cleavage as potential biochemical markers in populations produced to introgress novel germplasm from S. officinarum into elite sugarcane hybrid clones. The enzymes chosen for investigation were the three isoforms of invertase, cell wall, vacuolar acid and neutral invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13), and sucrose-phosphate synthase (SPS; EC 2.4.1.14).

Previously, other investigations have attempted to correlate enzyme activity and sucrose content in diverse sugarcane germplasm. An earlier investigation in sugarcane has examined growth, sugar accumulation and sucrolytic activities of sucrose synthase, soluble acid, and neutral invertases in two cultivars (Lingle, 1997). A further study of seven commercial sugarcane cultivars investigated the possibility of a correlation between sucrose content and the difference between SPS and soluble acid invertase activities (Lingle, 1999). Additionally, the activity of sucrose synthase was examined in relation to the rate of total sucrose accumulation.

Although the internodes of the commercial varieties used in the study exhibited different final sucrose concentrations, these could not be explained by the balance of enzyme activities within the internode.

Biochemical analyses (Zhu et al., 1997) have previously been performed on the hybrid progeny of a cross between high and low sucrose accumulating Saccharum species (female parent Louisiana Purple S. officinarum and male parent S. robustum, Molokai 5829, respectively). The sucrose concentration within the progeny of this cross varied from 26 to 500 µmol g–1 fresh weight (FW) whereas the commercial varieties investigated by Lingle (1999) possessed a much reduced level of variation in sucrose concentration of between 450 and 600 µmol g–1 FW. Zhu et al. (1997) concluded that soluble acid invertase (SAI) activity above a critical threshold level prevented sucrose accumulation in the stem. However, to reach levels of stored sucrose comparable to high sucrose accumulating genotypes, it was also necessary to invoke high activity of SPS.

Mutants in a common genetic background possessing a range of sucrose concentrations are not available in sugarcane and a transgenic approach to produce such plants is difficult because of the pleiotropic effects of the tissue culture and transformation process (Vickers et al., 2005). The production of populations segregating for sucrose is therefore an appropriate alternative means of producing plant material possessing a relatively uniform genetic makeup but differing in sucrose accumulation.

The introgression strategy facilitates the possible identification of novel germplasm associated with high sucrose content. The strategy adopted and described herein was to identify a small number of clones that exhibited either high or low commercial cane sugar (CCS) first in an introgression population and measure the activity of the chosen suite of enzymes. One of the progeny from this population measured to have high CCS and flowering at the appropriate time was subsequently used as a parent in a cross with a different commercial clone. A subset of clones exhibiting either high or low sucrose content were again selected for enzymatic analyses, focused on SPS and the soluble invertases.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
The S. officinarum clone IJ76-514 (2n = 80) was used as the female in a cross with Q165 (2n = 120), an Australian commercial variety. The cross was performed in 1999 and 227 progeny were planted in a replicated field trial in Kalamia, north Queensland (19°32'S, 147°24'E) in 2000. This cross IJ76-514 x Q165, is referred to as the introgression population in the text. A subset of eight clones from the introgression population, representing the extremes of the CCS spectrum were selected for detailed biochemical analysis. Three stalks of each clone were randomly selected in July 2001 from each of three plots, totalling nine stalks per clone. Each stalk was separated into internodal sections and the pith of the internodes 1 to 3, 5 + 6, 9 + 10, and middle (internode midway between internode 10 and the bottom of the stalk) was cored from each section using a cork borer. Internodes were numbered from the top, the tissue was finely chopped and for each clone, internodal material from stalks taken from the same plot was pooled. Two subsamples, one of 5 g for enzyme analyses and a second of 1 g for sugar determinations, were taken from this pool and rapidly cooled in liquid N. The plant material was transferred to dry ice and subsequently stored at –80°C. The final harvest on this trial was performed in August 2001 and agronomic measurements performed.

A subsequent population was produced by crossing an individual (clone KQ99-1410) from the introgression population, that flowered and also was measured to possess high CCS, with a different elite commercial cultivar in a cross designated KQ99-1410 x Mida. For convenience, these progeny are referred to as the backcross population in the text. However, it is recognized that technically this is not a conventional backcross to a recurring parent, but for sugarcane it was necessary to use an alternative elite commercial variety as a parent in this subsequent cross to avoid inbreeding depression. Two hundred eighty-eight progeny from this backcross were sampled as single stools in September 2002 to establish replicated plots of a smaller number of select clones representing the extremes of the spectrum with regard to sucrose content in the overall backcross population. Commercial cane sugar measurements were performed on the sugarcane juice extracted from the stalk using a small mill and subsamples were stored on dry ice and transferred to Brisbane to measure sucrose concentration using the high performance anion exchange with pulsed amperometric detection (HPAE-PAD) (Albertson and Grof, 2007). Twenty high and 20 low CCS clones were selected and planted in replicated plots in a random block design on 24 Oct. 2002. Four high and four low CCS clones were sampled during July 2003 in an identical manner to the introgression population, for detailed biochemical analysis.

Measurement of Commercial Cane Sugar and Sugars
Four mature stalks were sampled from each plot to determine whole stalk CCS for the introgression population in August 2001 and the backcross population in July 2003. Commercial cane sugar was calculated by the application of the CCS formulae described in Albertson and Grof (2004). Commercial cane sugar is the basis of payment to growers in the Australian sugarcane industry and some other parts of the world. It is not a direct measure of sucrose content but rather an estimate of dissolved solids (%) (Brix) adjusted for purity (Pol) and stalk fiber content. The Brix value estimates total soluble sugars whereas Pol estimates sucrose and is determined using a polarimeter.

The sugar composition and concentration in cane juice samples was determined by HPAE-PAD as described by Papageorgiou et al. (1997) and Albertson and Grof (2007). Glucose, fructose, and sucrose calibration curves (R2 ≥ 0.998) were produced from AR grade sugars (Sigma-Aldrich Pty. Ltd., Castle Hill, NSW). The ratio of sucrose to total hexose (sum of glucose and fructose) was also determined.

Enzyme Extraction
Enzymes were extracted from 5 g of tissue in 50 mM Hepes pH 7.5, containing 10 mM MgCl2, 5 mM DTT, 5 mM {alpha}-aminocaproic acid, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM benzamidine, 1 mM benzamide, 2 µM leupeptin, 2 µM antipain, and 0.1% Triton-X 100 as previously described (Albertson et al., 2001). The crude extract was concentrated by a two-step precipitation and resuspension procedure utilizing 15% PEG (polyethylene glycol 8000 MW). A second PEG precipitation was required to remove residual sugars from the extract. The final pellet was resuspended in 2 to 3 mL and desalted on a Sephadex G25 column (50 by 10 mm; Sigma) and stored on ice until required.

Enzyme Assays and Protein Determination
Assays were subsequently conducted with 50 mM Hepes, pH 7.3, except for acid invertase, which was assayed at pH 4.5 in 50 mM McIlvaines buffer (citrate–phosphate buffer). Substrates for the enzyme assays were well above reported Km values thereby facilitating maximum enzyme activity (Vmax).

As sucrose synthase is a bidirectional enzyme capable of both cleaving and synthesizing sucrose, activity was measured in both directions. The reaction catalysed by sucrose synthase operating in the direction to synthesize sucrose is termed SSf whereas the reaction operating in the direction of sucrose cleavage is termed SSr.

The full suite of enzyme activities (SPS, SSf, SSr, SAI, cell wall invertase, neutral invertase) in four internodes down the stem of the high CCS clones, I-H1 and I-H3 and the low CCS clones I-L2 and I-L3 from the introgression population were measured immediately after the plant material was collected. The activity of SPS and neutral and soluble acid invertase in an additional two high (I-H2 and I-H4) and two low (I-L1 and I-L4) CCS clones from the same population were measured at a later date. Aliquots of partially purified extracts were frozen for subsequent protein determinations using the Bradford reagent and bovine serum albumin as the standard (Bio-Rad, Regents Park, NSW).

Sucrose Cleaving Enzymes
Invertase and SSr assays were conducted in the presence of 125 mM sucrose, the SSr assay also included 15 mM UDP.

Sucrose Synthesizing Enzymes
The SSf assay was conducted in the presence of 30 mM UDP-glucose and 15 mM fructose. The SPS assay was conducted in the presence of 30 mM UDP-glucose, 13.8 mM glucose-6-phosphate, and 4 mM fructose-6-phosphate.

All assays were conducted for 30 min at 37°C except for the SPS assay, which was allowed to proceed for 60 min. The reaction rates were linear over these times. The enzyme assays were initiated by adding a 100 µL aliquot of crude extract to 200 µL of 2x assay buffer plus 100 µL of enzyme substrates. Aliquots at time zero (t0) and 30 min (t30) or 60 min (t60) for all enzyme activity assays except for acid invertase, were stopped initially by freezing in liquid N and once all assays were completed were heated to 80°C for 5 min. The acid invertase assay tubes were neutralized with imidazol before heating. All tubes were then stored at –20°C until the reaction products were analyzed by HPAE-PAD. The difference in product present in the two samples (t30 – t~0) or (t60 – t~0) was then used to calculate enzyme activity.

Statistical Treatment
Results were analyzed using analysis of variance and regression protocols in S-Plus 6.1 (Insightful Corporation, Seattle, WA). Experimental blocks, internodes, high and low CCS classifications, and clones were used as sources of variation. Differences between the high and low CCS groups were tested by Fischer's LSD and differences between clones were tested with a Tukey multiple comparison test.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of High and Low Sucrose Accumulating Progeny
The measurements of CCS, Brix, and Pol in four high and four low CCS clones from the introgression and backcross populations are presented in Tables 1 and 2, respectively. The commercial parent used to produce the introgression population Q165 was measured to have a CCS between I-H2 and H3. The S. officinarum parent IJ76-514 was measured to have a CCS value less than I-L4. The parents of the backcross population Mida and KQ99-1410, possessed CCS values between BC-H3 and H4 and BC-L1 and L2, respectively. The groups designated as high and low CCS clones were highly significantly different for Brix, Pol, and CCS in the introgression population but not in the backcross population. Consequently the high and low CCS categories were used to analyze enzyme activities in the introgression population but in the backcross population individual clone values were used. The higher CCS was obtained through both a higher level of dissolved solids, sugars (Brix), and a higher proportion of the sugars being sucrose (Pol).


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  		Table 1. Measurements of commercial cane sugar (CCS), Brix, and Pol in four high and four low CCS clones sampled at final harvest in August 2001 from introgression population IJ76-514 x Q165.

 

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  		Table 2. Measurements of commercial cane sugar (CCS), Brix, and Pol in four high and four low CCS clones sampled at final harvest in July 2003 from backcross population KQ99-1410 x Mida.

 
Characterization of Sucrose Concentration in Selected Progeny Clones
The sucrose concentrations in four different internodes down the stem profile of selected clones from the IJ76-514 x Q165 introgression and KQ99-1410 x Mida backcross populations are shown in Fig. 1 . These samples were taken at the same time as the tissue used for enzyme analysis. The trends in the data are the same for both populations however, the distinction between the high and low clones in the introgression population are greater than the backcross population, reflecting the CCS results.


Figure 1
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  		Figure 1. Sucrose concentrations in selected internodes on a fresh mass basis for (a) four high and four low commercial cane sugar (CCS) clones from the introgression population (IJ76-514 x Q165) and (b) eight clones from the backcross population (KQ99-1410 x Mida). Each point is the mean ± SE of three measurements. Internode middle denotes that position midway between internode 10 and the bottom of the stalk. FW, fresh weight.

 
The sugars expressed as a ratio of sucrose to hexose are shown in Fig. 2 . In the sugarcane stem, sucrose undergoes a constant cycle of synthesis and degradation sometimes referred to as futile cycling (Moore, 1995). The ratio of sucrose to hexose may reflect the balance shifting toward greater overall sucrose synthesis and maturity. Sucrose as a proportion of total sugar was most markedly greater in internodes 9 to 10, where on average it was at least threefold higher in the high CCS clones in both populations. In the lowest internode measured, all four of the low CCS clones in the introgression population had much reduced sucrose/hexose ratios and were significantly less than the high CCS clones (except I-L3 which was not different from I-H4). In the backcross population, the ratio in the lowest internode measured in the high and low CCS clones were similar.


Figure 2
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  		Figure 2. The sucrose/hexose ratio in selected internodes from four high and four low commercial cane sugar (CCS) clones from (a) the introgression population (IJ76-514 x Q165) and from (b) eight clones from the backcross population (KQ99-1410 x Mida). Each point is the mean ± SE of three measurements. Internode middle denotes that position midway between internode 10 and the bottom of the stalk.

 
Sucrose Metabolism Enzyme Activities in Selected Clones
The activities of the key enzymes, SPS and soluble invertases, were measured in four high and four low CCS clones from the introgression cross by the novel application of the HPAE-PAD system (Albertson and Grof, 2007) and are shown in Fig. 3 . These measurements were performed in two series. Two high and two low CCS clones were investigated in each series. Series 1 enzyme activity measurements are shown in Fig. 3a to 3c whereas series 2 are shown in Fig. 3d to 3f. The extraction and measurement of enzyme activities described as series 1 and 2 were performed at different times. The differences in the specific activities of SPS between the series is attributed to the fact that the extracts came from different clones as well as the variability inherent in the measurement of this complex enzyme.


Figure 3
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  		Figure 3. Activity of key enzymes in high (¡) and low (l) commercial cane sugar (CCS) clones from introgression population, IJ76-514 x Q165. Measurements performed in the first series (a–c); measurements performed in the second series (d–f). Panels a and d are sucrose-phosphate synthase; b and e, neutral invertase; and c and f, soluble acid invertase. The units of activity are µg sucrose formed mg –1 protein min–1 in a and d; µg sucrose cleaved mg –1 protein min–1 in b, c, e, and f. Each point is the mean ± SE of six measurements.

 
Additional enzyme activities, namely sucrose synthase operating in both synthesis (SSf) and cleavage (SSr) directions and cell wall invertase were measured as part of series 1 (Fig. 4 ).


Figure 4
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  		Figure 4. Changes in enzyme activities measured at four positions down the stem in two high (¡) and two low (l) commercial cane sugar (CCS) clones selected from the IJ76-514 x Q165 introgression population. Each point is the mean ± SE of six measurements. Activity of sucrose synthase forward (a) is expressed as µg sucrose produced mg protein–1 min–1 whereas sucrose synthase reverse (b) is expressed as sucrose cleaved mg protein–1 min–1. Cell wall invertase activity (c) is expressed as ng sucrose cleaved g FW–1 min–1.

 
As has been previously reported, there is an inverse correlation between increasing sucrose and soluble acid invertase activity in maturing sugarcane (Hatch and Glasziou, 1963). The large error bars (SE of mean) associated with SAI can be attributed to the very low activity of this invertase isoform at the time of harvest. For example, the scale of measurable SAI activity was an order of magnitude lower than the other enzyme activities measured (Fig. 3c, 3f). The other soluble isoform of invertase, neutral invertase, showed little change in activity down the stem and no apparent difference between the high and low CCS clones (Fig. 3b, 3e).

The key enzyme SPS, catalyzing sucrose synthesis, was of particular interest. In the low CCS clones, the activity of this enzyme changed little down the stem profile. In the high CCS clones, the specific activity measured in internodes 1 to 3 was threefold higher than that measured in the low CCS clones. A significant difference in activity persisted in the high CCS clones (I-H1, I-H3) measured in series 1 as compared with the low CCS clones (I-L2, I-L3) down to internodes 9 to 10 (P < 0.001 internodes 1–3 and 5–6 and P < 0.05 for internodes 9–10; Fig. 3a). A significant difference (P < 0.01) in activity was observed in internodes 1 to 3 between the high (I-H2, I-H4) and low (I-L1, I-L4) CCS clones measured in series 2 (Fig. 3d). A difference in activity in the lower internodes between the high and low CCS clones was not evident however.

Sucrose synthase operating in the reverse direction (SSr) to breakdown sucrose, showed no apparent difference in activity between the low and high CCS clones (Fig. 4b). Similarly, the cell wall isoform of invertase (cell wall invertase) showed no difference between the high and low CCS clones (Fig. 4c) although a decrease in activity down the stem was apparent.

The other key enzyme, sucrose synthase operating in its forward direction of sucrose synthesis (SSf), showed significantly higher activity in the upper internodes (1–3) of the high CCS clones I-H1 and I-H3 (P < 0.01), a difference which was no longer apparent further down the stem (Fig. 4a).

The activities of the enzymes SPS and soluble invertases were also measured in eight clones from the backcross population. The KQ99-1410 parent was one of the progeny from the introgression population. A highly significant relationship (P < 0.01) between SPS activity in the uppermost internodes and CCS of the whole stalk was also evident in the backcross population (Fig. 5 ). About 37% of the variation was explained (r2 = 0.371, P < 0.003) which increased to 65% with the removal of the single outlying point from one replicate of BC-H4, and greatly increased the significance of the relationship (r2 = 0.654, P < 0.0001). No difference in neutral or soluble acid invertase was evident between the high and low CCS clones (data not shown). Overall, the specific activity of SPS measured in the selected clones from the backcross population was lower than in the clones from the introgression population, however it is unknown whether this difference is genotypic or environmentally driven.


Figure 5
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  		Figure 5. Sucrose-phosphate synthase (SPS) activity in the uppermost internodes (1 + 2 + 3) of eight clones from the backcross population KQ99-1410 x Mida plotted against whole stalk commercial cane sugar (CCS) measured at final harvest. Individual values are for one replicate of a single clone. The values in open squares are for clone KQ01–3060 for which one CCS value was much lower than the others. The equation for the regression line shown, SPS = 0.1435CCS – 1.1638 (r2 = 0.371, P < 0.003) is for all of the plotted data.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Two main findings have arisen from the investigation of the two populations described herein: (i) an association between elevated SPS activity in the upper internodes and high sucrose accumulating capacity of clones in progeny from two generations; (ii) the sucrose/hexose ratio provides a clear indicator of early maturing clones which on final harvest possess higher CCS and sucrose concentration. These results provide new insights into the physiological and biochemical regulation of development and sucrose accumulation in the sugarcane stem.

The clonal material from the introgression population used for detailed enzymatic analysis was initially selected on the basis of the industry measure, CCS. However as this measure can be negatively affected by hexoses, particularly at concentrations found in immature internodes (Albertson and Grof, 2004), precise measurements of sucrose, glucose, and fructose were performed on the internodes selected for enzyme analysis.

Sucrose, when expressed as a percentage of fresh mass, is clearly different between the high and low CCS clones of the introgression population, particularly in the lowest two sampling positions. A similar trend is evident although less clear cut in the clones selected from the backcross population. However, when the sugars are expressed as a ratio of sucrose to hexose very obvious trends reflecting differences in the rate of maturation are observed. Sucrose as a proportion of total sugar is most markedly greater in internodes 9 to 10, being twofold or more greater in the high CCS clones. These findings suggest that measurement of sucrose/hexose ratios in maturing internodes is worth further investigation as an early predictor of high CCS in developing sugarcane.

The activity of the key enzyme SPS was consistently higher in the uppermost internodes of those sugarcane clones measured to have higher CCS and sucrose content. The robust nature of this biochemical marker is highlighted by the association of SPS and high sucrose accumulation in both the introgression and backcross populations. A direct correlation to sucrose content in the same tissue rather than at the whole stalk level as determined by CCS, however is difficult to make. From Fig. 1a, an increase in sucrose in the two high CCS clones I-H1 and I-H3 is evident by internode 5 to 6 as compared with the low CCS clones. Furthermore it is these two clones which exhibit the highest and significantly greater SPS activity (Fig. 3a) all the way down to internode 9 to 10.

In a previous study to identify the major enzyme determinants of the concentration of sucrose stored, Zhu et al. (1997) performed detailed analysis on the progeny of a cross between high and low sucrose accumulating varieties (female parent S. officinarum, Louisiana Purple, and male parent S. robustum, Molokai 5829, respectively). A negative correlation between stalk mean SAI activity and whole stalk sucrose was reported. No clear relationship was observed between stalk mean SPS activity and whole stalk sucrose. However when activity was expressed as the difference between SPS and SAI activities, a stronger and positive correlation between enzyme activity and sucrose concentration was observed.

However, the application of enzyme activity measurement as a selection tool for commercial cultivars appears to be limited. In a study using two sugarcane cultivars Lingle (1997) demonstrated a significant correlation between sucrose synthase activity and sugar accumulation rate; however, in a later study on seven cultivars this correlation was not corroborated. In the later study, performed over 2 yr, a significant correlation however was noted between neutral invertase as well as SPS and sugar accumulation rate in the second year. Although not highlighted by Lingle (1999) a significant negative correlation was found between SPS activity and elongation rate in both years. A positive correlation was also found between SPS activity and total sucrose content in both years. The elongation phase would approximate the stages of development between internodes 1 and 5 whereas the second phase of dry matter accumulation as defined by Lingle (1999) would roughly equate to internodes 5 to 10 of the study described herein. Taken together these observations suggest that SPS activity in the uppermost part of the stem in these cultivars is an indicator of higher sucrose content. Lingle (1999) was predominantly focused on elucidating a consistent correlation between enzyme activity and rate of sugar accumulation taking place in internodes which had completed elongation, as a selection tool for commercial cultivars. The final sucrose content of cultivars examined by Lingle (1999) varied from 450 to 600 µmol g FW–1 as compared with the study of Zhu et al. (1997) where sucrose varied from 26 to 500 µmol g FW–1. Lingle (1999) attributed the discrepancy of conclusions drawn from the two studies to the lack of variation in sucrose in the commercial cultivars used in the later investigation.

Nevertheless, in three studies performed in different parts of the world and using a broad range of genotypes, the enzyme SPS stands out either as an indicator of, or as a key contributor to, final sucrose content in sugarcane stalks. Although perhaps not useful for further selection of elite commercial cultivars derived from germplasm of very limited diversity, it is likely to have a role in the introgression approach where wild species, possibly with highly variable sucrose content, are being used.

There is a burgeoning amount of data in the literature which demonstrates a correlation between SPS and important agronomic traits such as plant growth and yield. In maize, SPS activity has been shown to be correlated to growth rate in various genotypes (Rocher et al., 1989) and also in young plants (Causse et al., 1995). Furthermore, the SPS activity of maize hybrids was correlated to forage dry matter measured in the field. Independent corroboration of such findings in maize has identified quantitative trait loci linking SPS activity and grain yield (Bertin and Gallais, 2001). In rice, the gene underlying a quantitative trait locus controlling plant height was determined to be SPS (Ishimaru et al., 2004). Plant height has also been reported to be closely correlated to biomass production (Niklas and Enquist, 2000).

Successful overexpression of maize SPS in tomato (Lycopersicon esculentum Mill.) resulting in greater dry weight, more fruit, and higher sucrose concentration was first reported more than 10 yr ago (Worrell et al., 1991; Laporte et al., 1997). Overexpression of SPS has been attempted in a number of crop species with the primary aim of increasing yield, with limited success. However, overexpression of maize SPS in rice resulted in a threefold increase in SPS activity in the transgenic rice plants leading to significantly taller plants identifiable from an early growth stage (Ishimaru et al., 2004).

In sugarcane, the difference in activity between SPS and soluble invertase activity has been postulated to be associated with high sucrose genotypes (Zhu et al., 1997). In the present study a direct correlation between high SPS activity and sucrose accumulation has been observed without the need to invoke a direct role for soluble acid invertase. However, it must be noted that soluble acid invertase was low in all the clones investigated.

Sucrose-phosphate synthase is a key catalytic enzyme in the pathway of sucrose synthesis and is encoded by multiple genes belonging to a small number of families. Phylogenetic analysis of the Arabidopsis genome revealed that SPS was encoded by four genes arranged in three families designated A, B, and C (Längenkamper et al., 2002), whereas in wheat and other grasses five families have been identified (Castleden et al., 2004).

Some preliminary investigation of the genetic basis for the observed differences in SPS and the corresponding phenotype in sugarcane has been performed. Expression of the five families of SPS (Castleden et al., 2004) was measured by quantitative real time PCR in glasshouse-grown plants of the I-H2 and the I-L2 clones (Grof et al., 2006). No difference in expression in either leaves or internodes down the stem profile was observed between the high and low CCS clones, suggesting that SPS activity is posttranscriptionally controlled.

The sugarcane stem is a developmental series of internodes, numbered from the apex. The pattern of stalk development consists of essentially an elongation phase, equating to internodes 1 to 4 (Rae et al., 2006) followed by the sucrose accumulation phase between internodes 5 to 11 (Campbell and Bonnett 1996). Internodes further down the stem profile maintain a relatively constant sucrose concentration. Herein we have described a robust correlation between SPS enzyme activity measured at the apex of the stalk, where the internode is undergoing elongation, and final sucrose content in the sugarcane stem at harvest.

To apply the biochemical marker SPS in a practical sense to sugarcane breeding a number of steps would have to be undertaken. First, SPS would need to be measured in the uppermost internodes of young plants which have grown past the elongation phase (possessing six internodes) to confirm that the SPS activity exhibited the same trend as in the corresponding internodes of mature plants. These measurements would be performed in clones known to be either high or low sucrose accumulators from previous experiments. Developing a robust, high throughput screening process for the measurement of SPS activity would also be essential to implementing SPS as a biochemical marker tool in sugarcane. We are currently optimizing a high throughput system for the measurement of key enzymes including SPS, following the methodology described by Gibon et al. (2004).

Combining these two essential components successfully would markedly reduce the length of time taken to identify high sucrose clones from amongst the large number of progeny produced in sugarcane breeding programs. Identification of clones as high sucrose accumulators when the plants are young would enable these clones to be fast tracked through the selection process as well as enabling a much higher number of clones to be produced and screened if poor performers could be removed earlier.


   ACKNOWLEDGMENTS
 
We wish to thank the Sugar Research and Development Corporation for financial support. Many thanks also to Phillip Jackson, Michael Hewitt, Terry Morgan, and other members of the CSR Technical Field Department for production of the populations and maintenance of the sugarcane field trials. The commercial varieties of sugarcane Q165 and Mida are protected by plant breeder's rights.


   NOTES
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Received for publication December 21, 2006.


   REFERENCES
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