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a Dep. of Biological and Physical Sciences, Univ. of Southern Queensland, Toowoomba, Qld 4350, Australia
b CSIRO Plant Industry, Queensland Biosciences Precinct, 306 Carmody Road, St Lucia, Qld 4067, Australia
c CSIRO Plant Industry, Davies Lab., PMB, PO Aitkenvale, Qld 4814, Australia
d Langwatlstr. 23, CH-8125 Zollikorberg, Switzerland
e CSIRO Plant Industry, PO Box 350, Glen Osmond, SA 5064, Australia
* Corresponding author (graham.bonnett{at}csiro.au).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AS, antisense • COM, commercial • NAS, null antisense • PPO, polyphenol oxidase • S, sense • SPS, sucrose phosphate synthase • TC, tissue culture
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been demonstrated that inhibition of PPO activity in juice by chemical inhibitors, heat, or elevated pH significantly reduces the development of color in juice (Smith, 1976; Tu, 1977; Gross and Coombs, 1976; Coombs and Baldry, 1978; Goodacre et al., 1980; Bucheli and Robinson, 1994). Inhibition of PPO activity by alkaline pH (Tu, 1977) or heat (Smith, 1976) also reduced color intensity in sugar crystals. These observations suggest that a reduction in endogenous PPO activity in planta could lead to a reduction in browning of sugarcane juice during processing. To test this hypothesis, transgenic sugarcane with altered levels of PPO activity were produced. Plants were transformed with either sense or antisense gene constructs carrying the endogenous sugarcane PPO gene. This approach has been used previously in potatoes (Solanum tubersosum L.) to inhibit browning of tubers following injury (Bachem et al., 1994; Coetzer et al., 2001).
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR analysis demonstrated that for Set 1 and 2, all plants called sense and antisense plants had the respective transgene present. For PCR, DNA was extracted from leaves with a commercially available kit (Plant DNA Isolation Kit, Boehringer Mannheim, Germany). PCR was performed in 25-µL reactions with 700 ng to 1 µg genomic DNA, 0.3 µM of each primer, 200 µM dNTPs, 2.6 U DNA polymerase (Expand High Fidelity, Roche Molecular Biochemicals, Germany), and proprietary buffer containing 1.5 mM MgCl2. Reactions were performed at 95°C for 3 min, followed by 35 cycles of annealing at 55°C for 2 min, extension at 72°C for 2 min, and denaturation at 94°C for 1 min. A final annealing period at 55°C for 2 min was followed by extension at 72°C for 5 min. The upstream primer (5'-TTA GCC CTG CCT TCA TAC GC-3') was in the ubiquitin promoter for all four plasmids, and the downstream primer (5'-GTG TTC CCG CTC GCT CTG-3') in the AS PPO gene for both AS plasmids. The downstream primers for pSPPO1 (5'-CTC ACA ATT CCA CAC AAC ATA C-3') and for pSPPO2 (5'-GGC TTC GCA TCC CTG TGG-3') were in the fragment of cloning vector between the promoter and the PPO sense gene, and in the sense PPO gene, respectively. PCR products were 815 bp for both AS plasmids and 1147 and 1008 bp for pSPPO1 and pSPPO2, respectively.
Some plants were also present in Exp. 1 and 2 that had been shot with antisense PPO constructs but did not contain the transgene (NAS, "null antisense" clones). A summary of the plant material in each experiment is presented in Table 2. In Set 2, only the most vigorous plant was saved from each transgenic callus piece, so each numbered clone represented an individual transformation event. For Set 1 more than one plant was taken from most of the transgenic callus pieces. Therefore a minimum of 22 transgenic events was represented in Set 1 (8 S, 9 AS, and 5 NAS). As transgenic callus pieces occasionally contain cells derived from more than one transformation event (Bower et al., 1996), it is likely that the actual number of transgenic events was greater than 22.
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Juice Color
For analysis of color intensity, juice was centrifuged at 17968 g for 10 min at 4°C, the supernatant diluted 10-fold with phosphate buffer (KH2PO4, 25 mM and Na2HPO4, 25 mM, pH 7.0), and filtered through a 0.45-µm filter (Millex-HV Low Protein Binding, Millipore). Absorbance was measured at 420 and 720 nm (an estimate of turbidity) in duplicate. Color intensity was calculated as [1000 x (mean absorbance at 420 nm – mean absorbance at 720 nm) x 10]/[cell size (cm) x sucrose concentration of juice (g/mL)]. A sucrose equivalent was estimated from brix measurement (Table XI, BSES, 1991). This method was modified from those of the Sugar Research Institute (SRI), Mackay, Queensland (K. Miller, personal communication) and that of Bucheli and Robinson, (1994).
Polyphenol Oxidase Activity
PPO activity was measured as the maximum rate of oxygen uptake per milliliter of juice with an oxygen electrode (Hansatech, Kings Lynn, UK) at 25°C (Dry and Robinson, 1994). Voltage output from the electrode control box was recorded on a chart recorder (Yew 3057 or Sekonic SS-250F), or acquired and analyzed electronically (DI-190 Data Acquisition Serial Port Module and Advanced Codas data analysis package, DATAQ Instruments, Akron, Ohio). The reaction mixture (1-mL total volume) consisted of 20 µL of juice in 50 mM NaH2PO4 (pH 6.0), to which chlorogenic acid substrate (final concentration 2 mM) was added to start the reaction. All measurements were performed in duplicate. One unit (U) of PPO activity is 1 µmol of oxygen evolved min–1 mL–1.
Crystal Color Tests
Two clones, one high and the other low in PPO activity and color from Exp. 1, were chosen for crystal color tests. The high and low PPO activity clones were a PPO sense transformed clone (S23), and Q124, a commercial variety, respectively. On 1 Nov. 1998, three samples of stalks of Q124 and S23 were taken from Exp. 1 and transported to the Sugar Research Institute (SRI), Mackay.
Samples were milled and syrup made at SRI from 2–9 Nov. 1998. The cane was crushed multiple times in an experimental mill, thereby extracting most of the juice. The remaining fiber was then macerated with boiling water and recrushed to extract further juice. The juice was strained to remove fiber particles and heated to 78°C for 20 min to simulate the milling process. Lime saccharate (a mixture of CaO at 15 brix and sugar syrup in a ratio of 1:1) was added to the juice with constant stirring to increase pH to 7.8. The temperature was then rapidly increased to boiling point (100°C+) for 2 min to remove entrained gases before settling. Dilute flocculant (A2125, Mitsui-Cytec Ltd Tokyo, Japan) was then added via a syringe below the surface of the juice while stirring by hand with a spatula, to a concentration of 3 µg mL–1. The "mud" was allowed to settle from the juice for 30 min. The clarified juice was then siphoned through a course filter, concentrated to 25% brix by boiling at atmospheric pressure and then to 60% brix under vacuum in a 15-L pilot scale vacuum pan. The syrup was then cooled and stored at –20°C before crystallization.
For crystallization, an aliquot of syrup was drawn into a rotary film evaporator (RFE), operating under vacuum control and boiled at 65°C until enough water had been removed to allow seeding. For Q124, 100 g of seed sugar (commercially available caster sugar that was retained by a 210 µM square aperture sieve) was added to give a crystal content of 25% (w/v). Boiling was resumed and the syrup slowly fed into the RFE to maintain a crystal content of about 30% by mass until the flask was full. The vacuum was then released and a fraction of the total "massecuite" was centrifuged to yield the first crystal sample (mean size 0.5–0.6 mm). Boiling was resumed and the remainder of the syrup was fed on to the retained massecuite as previously. When all of the syrup had been introduced, the contents were allowed to crystallize to give a crystal content of between 40 and 45% (w/v). The heavy massecuite was then centrifuged to give a final crystal size of 0.7- to 0.8-mm size. The sugar was gently dried in a warm air stream with constant mixing then stored for transport.
For S23 samples, only 50 g of seed sugar was used to reflect the lower amount of juice available. Where there was insufficient syrup to grow crystals to 0.8 mm as described above, all of the syrup was fed into the flask before removal of the first crystal. About 75% of the massecuite was then removed and diluted with water to redissolve the crystal giving a solution of about 60% brix. This solution was then fed back to the flask to give a final massecuite as above.
Color of mixed juice before clarification, and the syrup prepared from the clarified juice (liquor) were measured at the time of crystallization. Samples were mixed thoroughly and Celite was added to 0.1 g mL–1. The slurry was filtered under vacuum through a filter paper (Whatman No. 1). The refractive index of the filtrate was measured at 20°C. Juice was then diluted volumetrically, filtered through a prefilter and finally a 0.45-µm filter under pressure. 30 mL aliquots were adjusted to pH 7 ± 0.1 using 0.1 M HCl or 0.1 M NaOH. Absorbance was read immediately using filtered distilled water as a reference. Color was calculated by the following equation: (1000 x absorbance at 420 nm)/(cell size x concentration g/mL). Purity was measured and color/impurity ratio of the crystallized sugar was calculated. Purity was measured as the percentage ratio of sucrose to total soluble solids and the color to impurity ratio of the crystallized sugar was calculated.
Northern Analysis
From selected clones, two stalks from the ratoon crop of the Ayr experiment were harvested on 24 Sep. 2001. The internode sheathed by the leaf five below the youngest fully expanded leaf (last visible dewlap) was removed from each stalk. Internodes were placed in a 50-mL tube and frozen in liquid nitrogen, transported on dry ice, and then stored at –80°C.
Total RNA was isolated from internodal sugarcane tissue. As PPO expression has been shown to be strong in the meristem, weak in the internode below the meristem, and absent from more mature internodes (Bucheli et al., 1996) an internode several below the meristem was chosen as it would have no PPO expression and constitutive overexpression in sense transformed plants could be seen. The actual internode used was the internode behind the fifth leaf below the last fully expanded leaf. The tissue was frozen in liquid nitrogen and ground to a fine powder in a chilled mortar and pestle. Total RNA was then isolated (Ausubel et al., 1994). For northern analysis, total RNA was fractionated on 1% (w/v) formaldehyde-agarose gels, transferred to Hybond N+ nylon membrane (Amersham Biosciences, Uppsala, Sweden), and hybridized as previously described (Peters et al., 1996). The sugppo1 probe, a 2.2-kb clone, encoding the full-length cDNA, described by Bucheli et al. (1996), was labeled with the Strip-EZ DNA kit (Ambion, Austin, TX).
Statistical Analysis
Analysis of variance (ANOVA) and other statistical analyses were performed by S-PLUS (Insightful Corporation, Seattle, WA). Differences between means of traits for different clone type were tested by applying ANOVA using blocks and clone types as sources of variation. Significant differences between types were determined using the multiple comparison option and the Sidak method.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Relationship between Juice Color and Raw Sugar Color
Polyphenol oxidase activity was higher and color darker for clone S23 than for Q124 at both May and September sampling times in Exp. 1. Clone S23 showed one of the highest PPO activities and darkest color, in both the May and the September data sets, whereas Q124 showed one of the lowest PPO activities and lightest colored juice. Polyphenol oxidase activity was also measured in juice samples before crystallization. The mean juice PPO activity of clone S23 was significantly higher than that of Q124 (p 
0.01) (Table 4). PPO activities in these samples were lower than those measured on the same clones in September. This difference was due to dilution of the first expressed juice by the boiling water used for extraction of bagasse, and probably also because of the elevated temperature of the water when added to the juice.
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Polyphenol Oxidase Activity Does Not Correlate with Higher Color at Higher Polyphenol Oxidase Activities
The sense-transformed clones in the second set of experiment showed a much greater range of PPO activities than the other types of clone without any continued trend of increase in color (Fig. 2). When the sense clones are removed from the correlation analysis then the correlation coefficients were –0.01 (ns), 0.61 (p = 0.001), and 0.58 (p = 0.01) for the three experiments, respectively. For the latter two experiments, 4 and 5, the coefficients are very similar to the first set of experiments, 1 and 2. Why the experiment at Samford should be so different is not clear, but in both sets of experiments, the PPO activity was higher at Samford than elsewhere, possibly because of the younger age of the crop when sampled. The improvement of the correlation with the removal of the sense clones, in the last two experiments, shows that the relationship between PPO and juice color is strong in both sets of experiments and that at higher levels of PPO activity, the relationship ceases to be linear.
Overexpression of Polyphenol Oxidase in Sense Plants
Although the production of transgenic plants often appears to result in elevated PPO activity and darker juice color, PPO activity can also be altered by specific effects of transformation. The Set 2 sense clones had significantly higher PPO activity than the other plants in all experiments in which they were grown (Exp. 3–5, Table 3). The PPO activities of the individual sense clones grown in Exp. 4 are shown with Q117 as a comparison in Fig. 3
. Three of the sense plants (478, 498, and 574) had RNA extracted from the fifth internode (from the top). The RNA was subjected to northern blot analysis and probed with the PPO gene. The result (Fig. 4)
clearly shows the expected 2.2-kb band expressed in the sense transformed plants which is absent from the commercial varieties and the tissue culture control. As a full-length cDNA encoding a native PPO enzyme was used in the transformation, an equivalent sized band mRNA would have been expected in the commercial varieties and tissue-cultured control had the native PPO gene been expressed.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is not clear why PPO activity is elevated in the juice of transgenic plants. Either more PPO activity is released into the juice from the transgenic plants or the transgenic plants have a higher level of activity than nontransformed controls. Roles have been proposed for PPO activity such as defense against pathogens (Mayer and Harel, 1979) or in regulating oxygen evolution in photosynthesis (Vaughn et al., 1988). It is not clear how the production of the transgenic plants could cause the regulation of PPO activity to be altered. There is evidence however, for raised PPO activity in the process of dedifferentiation and redifferentiation of shoots from Feronia limonia (L.) Swingle (Purohit et al., 1996) and altered PPO activity in callus of Ranunculus asiaticus L. (Beruto et al., 1996). In these studies though, measurements of plants originating from tissue culture were not compared with normally grown plants so the lasting effect if any was not recorded. The similarity between the PPO activities in the TC and AS plants in our study (Table 3) suggests that the elevation could have been caused by the tissue culture process.
Whatever the cause of the elevation, the levels of PPO activity and intensity of juice color appeared to be stable for each clone. The rank order of clones was generally maintained between samplings and experiments (Table 5).
Both sense and antisense approaches to reducing PPO activity were attempted. The sense approach sometimes leads to downregulation of the target gene through cosuppression (Napoli et al., 1990). Sometimes, however, introduction of a sense construct can lead to overexpression of the target mRNA (Vaucheret et al., 1995). Despite the transgenics, in general, appearing to have higher PPO activity, some targeted manipulation was evident. In all three Set 2 experiments, plants containing the sense constructs had higher mean PPO activity than all other types of plants, transformed or controls. Overexpression appears to have been the result here. This was confirmed by northern hybridization. PPO activity in sugarcane decreases down the stem but is still measurable, particularly in the nodes of the mature stem even though below the meristem there is very little PPO gene expression (Bucheli et al., 1996). The absence of PPO gene expression in Internode 5 of the two cultivars and TC control is consistent with this result. However, in the sense transgenics PPO expression, driven by a constitutive promoter was by comparison very high. This probably explains the higher PPO activity measured in juice extracted from the whole stem. The PPO gene that was overexpressed in these experiments was a sugarcane PPO. In a previous report of PPO overexpression, a potato PPO was overexpressed in tomato, because the overexpression of a native tomato gene was unsuccessful (Li and Steffens, 2002). The only difference between the Set 1 and 2 sense plants was the additional fragment of the cloning vector in the construct of the Set 1 plants. Removing this fragment may have facilitated the overexpression.
Using the mRNA sequence of sugarcane PPO (Bucheli et al., 1996) to perform a blastx against the rice pseudomolecules (www.tigr.org; verified 12 August 2004), two genes and a pseudogene are homologous. Contig analysis using the sugarcane ESTs lodged in dbEST (www.ncbi.nlm.nih.gov; verified 8 September 2004) shows at least two genes in sugarcane. It is not clear why the antisense strategy did not work. It is possible that other PPO activities encoded by different PPO genes may have still been active. In most plants passing through the transformation process an increase in PPO activity was observed, even those not containing a PPO transgene (NAS). Consequently, it may not be possible to distinguish between a plant where PPO activity has been reduced by antisense and one where it has not when compared with the general elevation of PPO activity. Future attempts to reduce PPO activity may have to target additional sugarcane PPO genes. Although no successful use of RNAi (RNA-interference, Varsha et al., 2001) has been reported for sugarcane, this strategy could be used in a more complex silencing strategy where one or all PPO gene products are silenced.
Attempts to overexpress a spinach SPS gene in sugarcane (Grof et al., 1996) have been unsuccessful. The work reported here is the first successful attempt to overexpress either a native sugarcane or foreign gene involved in metabolism, resulting in a measurable increase in the gene product, namely PPO enzyme activity. Successful manipulation of metabolism, if combined with improvements in sugarcane transformation procedures, would be a significant step toward the directed manipulation of sugarcane metabolism for commercial purposes.
A relationship between PPO activity and juice color has been established over several experiments and sampling times. Only a weak correlation in experiments using nine varieties was found previously (Bucheli and Robinson, 1994). When the levels of PPO activity were very high (sense clones, Set 2), the color did not continue to increase. This may be due to a limited supply of substrate.
The results demonstrate that varietal differences in PPO activity can result in differences in the color of sugar crystals. This agrees with the results of experiments where PPO activity was reduced by heat (Smith, 1976) or pH (Tu, 1977), although in these latter cases the reduction in PPO activity was assumed rather than confirmed by measurement. Color in crystals can come from PPO activity, other colored compounds in the juice and compounds generated in the milling process. Here we have tried to manipulate one of these independently of the others and have shown that reduced PPO activity can lead, through lower juice color to lower crystal color.
Very high PPO activity has been reported in the growing point of sugarcane and this activity declines with increasing maturity of the stem internodes (Bucheli et al., 1996). It is apparent from the experiments that had repeated samplings, or were sampled from more mature plants (greater than 10 mo), that PPO activity was lower and that the values for the transgenics and nontransformed plants were more similar. This was very clear in the November sampling of Exp. 1, where there were no differences between the PPO activities and color measurements of the different clone types. This may have been due to a decrease in PPO activity through the stem or as more cane is produced as the crop ages, the high levels in the meristem are diluted and PPO activity measured in the juice from the whole stem is consequently lower.
In this paper, the relationship between PPO activity in juice and color of juice has been established in field grown material. In an experiment to make raw sugar from juice of high and low PPO activity, the relationship was found to follow through to the color intensity of raw sugar. Consequently, the hypothesis that lowering PPO would lead to a reduction in juice color and ultimately the color of raw sugar has been supported. As the tools to transform and control gene expression in sugarcane continue to improve, further attempts to lower PPO activity would be warranted.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication September 26, 2003.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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