Present address: Australian Centre for Plant Functional Genomics, Waite Campus, University of Adelaide, PMB1, Glen Osmond, SA 5064, Australia.
Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers
Article first published online: 23 JUL 2012
© 2012 Blackwell Publishing Ltd
Plant, Cell & Environment
Volume 36, Issue 1, pages 176–185, January 2013
How to Cite
MCKENZIE, M. J., CHEN, R. K. Y., HARRIS, J. C., ASHWORTH, M. J. and BRUMMELL, D. A. (2013), Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers. Plant, Cell & Environment, 36: 176–185. doi: 10.1111/j.1365-3040.2012.02565.x
- Issue published online: 3 DEC 2012
- Article first published online: 23 JUL 2012
- Accepted manuscript online: 26 JUN 2012 07:10AM EST
- Received 17 May 2012; accepted for publication 14 June 2012
- Solanum tuberosum;
- reducing sugars
Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.
In order to provide a continuous supply of material during the year after harvest, tubers of potato (Solanum tuberosum L.) are commonly stored at low temperatures (<10 °C) to reduce sprouting, weight loss and pathogenesis. However, the cold stress experienced during storage causes an imbalance between the rate of starch breakdown and the subsequent use of the generated sucrose (Suc) by the cell. This creates a serious problem for the potato-processing industry since it results in the accumulation of the reducing sugars glucose (Glc) and fructose (Fru), in a metabolic process known as cold-induced sweetening (CIS; Sowokinos 2001a).
CIS can be a rapid process, occurring in a matter of weeks if tubers are stored at temperatures as low as 4 °C. When affected tubers are fried or roasted at high temperatures, the reducing sugars react with free asparagine via the non-enzymatic Maillard reaction, resulting in a product that is darkly coloured, unpalatable and unacceptable to the consumer. More seriously, the neurotoxin and probable carcinogen acrylamide has been identified as a by-product of the Maillard reaction, and is an important consequence of the CIS process (Mottram, Wedzicha & Dodson 2002; Stadler et al. 2002). Fried potato products (French fries, potato crisps) are one of the largest contributors to dietary acrylamide exposure, which has become a significant public health concern (Halford et al. 2012; Medeiros Vinci, Mestdagh & De Meulenaer 2012). Unfortunately, despite extensive breeding efforts, no truly CIS-resistant cultivars have been released onto the market, and CIS remains one of the major issues facing the potato-processing industry.
In potato tubers, the biochemical pathway from starch breakdown to the production of Suc, and ultimately to reducing sugars, is complex (Sowokinos 2001a; Blenkinsop, Yada & Marangoni 2004). Multiple enzymes along the pathway have been investigated as possible control points for CIS, and potential roles have been established for glucan water dikinase, β-amylase, UDP glucose pyrophosphorylase, sucrose phosphate synthase and acid invertase (Richardson et al. 1990; Zrenner, Willmitzer & Sonnewald 1993; Borovkov et al. 1996; Hill et al. 1996; Zrenner, Schüler & Sonnewald 1996; Nielsen, Deiting & Stitt 1997; Reimholz et al. 1997; Lorberth et al. 1998; Sowokinos 2001b; McKenzie et al. 2005). A large proportion of the Suc produced by these enzymes ultimately enters the vacuole (Blenkinsop et al. 2004), where it is irreversibly cleaved into Glc and Fru by acid invertase.
Acid invertases are found in the vacuole and the apoplast (Roitsch & González 2004). Most of the reducing sugars accumulating during CIS are located in the vacuole, and strong correlations have been established between hexose : Suc ratio and acid invertase activity in cold-stored tubers from a range of cultivars (Richardson et al. 1990; Zrenner et al. 1996; Matsuura-Endo et al. 2004; McKenzie et al. 2005). This association has implicated vacuolar acid invertase as a key control point. Nearly complete silencing of the vacuolar invertase gene by RNAi resulted in tubers that could be stored at 4 °C for up to 180 d with minimal Maillard browning on crisping and an up to 15-fold reduction in acrylamide content (Bhaskar et al. 2010). Similar results have been reported using other cultivars (Ye et al. 2010; Liu et al. 2011; Wu et al. 2011).
A second approach to limit acid invertase action is to target its post-translational control by native invertase inhibitors (Pressey 1966). Invertase inhibitors are part of the family that includes pectin methylesterase inhibitors (Hothorn et al. 2004), and are small proteins of approximately 17 kDa that bind irreversibly to invertase, producing an inactive complex (Rausch & Greiner 2004). Overexpression of a heterologous vacuolar invertase inhibitor from tobacco resulted in potato tubers that could be stored at 4 °C for up to 6 weeks without exhibiting symptoms of CIS (Greiner et al. 1999). However, despite these results and the long-established biochemical role of invertase inhibitor proteins in controlling invertase activity, genes encoding the apoplastic and vacuolar invertase inhibitors of potato have only recently been described (Liu et al. 2010; Brummell et al. 2011).
In potato, the INH1 gene encodes an invertase inhibitor protein targeted to the apoplast and INH2, a protein localized in the vacuole (Brummell et al. 2011). Because of unusual cold-induced splicing events, multiple transcripts were detected from the INH2 gene, encoding proteins with divergent carboxyl termini. Of these, the predominant INH2α transcript encoded a highly active protein, whereas two different versions of INH2β encoded proteins with slight or no detectable activity (Brummell et al. 2011). Molecular and biochemical analyses of a potato-mapping population showed that high mRNA accumulation of INH2 was correlated with low acid invertase activity and resistance to CIS (Brummell et al. 2011). This suggests that post-translational regulation of invertase activity by the vacuolar invertase inhibitor is an important component of resistance to CIS. The aim of the current work was to confirm this endogenous mechanism for regulation of acid invertase activity and CIS resistance using two opposing methods: tuber-specific overexpression of the native potato vacuolar invertase inhibitor INH2α in a potato cultivar with poor CIS resistance, and tuber-specific silencing of this inhibitor in a potato cultivar with high CIS resistance.
MATERIALS AND METHODS
Constructs and plant transformation
RNA was purified from potato tubers using the hot borate method (Wan & Wilkins 1994) and converted to cDNA using oligo d(T)17 and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For the overexpression construct, the open reading frame of potato invertase inhibitor INH2α was amplified from cDNA by PCR using Platinum Pfx DNA polymerase (Invitrogen) with sense primer 5′-AAAAGAATTCAAATGAGAAATTTATTCCCCATA-3′ and antisense primer 5′-AAAAGGATCCTTCATAATAACATTCTAATTATGGATTTAG-3′. These primers added an EcoRI restriction site (underlined) upstream of the translation start and a BamHI site (underlined) downstream of the translation stop. The resulting band of ∼570 bp was purified by gel electrophoresis, ligated into pBluescript and sequenced.
The primary cloning vector pART7PAT3, in which the 35S promoter of pART7 (Gleave 1992) had been replaced by the tuber-specific potato patatin promoter, was a generous gift of Dr Tony Conner. The open reading frame of INH2α was ligated in the sense orientation into pART7PAT3, using EcoRI and BamHI restriction sites between the patatin promoter and the ocs terminator.
For the RNAi silencing construct, the upstream primer 5′-TAATGCTAGCCATGGTTCCACGTAGTAACGAGGTTG-3′ added overlapping NheI (underlined) and NcoI (bold) sites while the downstream primer 5′-TAATAGATCTCCAACCATTCCATCTTCTGC-3′ added a BglII site (underlined). These primers were used to PCR amplify a 430 bp fragment that consisted of the downstream 310 bp of the coding region and 109 bp of the 3′-UTR of the INH2β transcript. Of these, the first 275 bp were identical to the INH2α transcript, and it is expected that this construct would bring about post-transcriptional gene silencing of both the INH2α and β mRNAs. This INH2 fragment was ligated into the pART7PAT3 vector in the sense orientation using NcoI and BglII sites. A spacer consisting of non-plant DNA (a 125 bp BglII-BamHI fragment of the bacterial sialidase NAN gene, Kirby & Kavanagh 2002) was ligated downstream, followed by the BglII-NheI INH2 fragment in the antisense orientation using the BglII site to ligate into the BamHI site of the NAN fragment and destroy both sites.
The PAT:INH2α:ocs overexpression cassette and the PAT:INH2:INH2antisense:ocs silencing cassette were moved into the binary vector pART27 (Gleave 1992), which confers resistance to kanamycin, and transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation.
Potato cultivars ‘Karaka’ (CIS sensitive) and ‘1021/1’ (CIS resistant) were transformed with the overexpression or silencing constructs, respectively. Potato transformation was conducted using kanamycin selection as described by Barrell et al. (2002). Young seedlings were confirmed as transgenic by screening genomic DNA for the presence of the transgene and the selectable marker gene using PCR (data not shown). Independent transformants were multiplied by excising axillary buds and regenerating them into whole seedlings. Six clonal copies of each line that had been confirmed as transformed were hardened off and transferred to 16 L size plastic bags of potting mix for tuber production. Non-transformed wild type (WT) material was similarly regenerated in tissue culture medium (lacking kanamycin) and potted up for use as controls.
Plant growth and treatments
Plants were grown to maturity in a greenhouse during the southern summer and autumn of 2010 and 2011, under ambient lighting with a maximum daytime temperature of 27 °C and minimum night-time temperature of 16 °C, and the tops were allowed to senesce naturally. Tubers were harvested, then stored for 7 d in the dark at 20 °C to set skins and repair scars. Following this, a T0 control sample was taken (as described below) and the remaining tubers from each line were stored in constant temperature rooms in the dark at either 10 or 4 °C for 6 weeks. After storage treatment, tubers were thickly peeled and the central cores chopped into cubes with sides of ∼3 mm. Tissue of three tubers from each plant was combined per biological replicate, placed in two plastic vials and frozen in liquid N2. One set of tissue was stored at −80 °C until needed for RNA preparation or enzyme activity, and the other set was freeze-dried for sugar analysis.
For an initial screen of the overexpressing lines by RNA gel blot analysis, total RNA was purified from frozen, powdered tuber tissue (Wan & Wilkins 1994), and separated by electrophoresis in 1.2% agarose denaturing formaldehyde gels. RNA was blotted overnight to Hybond-XL nylon membrane (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions, and immobilized by ultraviolet irradiation. Labelled probes were synthesized from a template consisting of the entire open reading frame of INH2α using [α-32P]dATP and the Klenow fragment of DNA polymerase I. This probe would detect the sum of both INH2α and INH2β transcripts. RNA gel blots were hybridized overnight with the denatured probe in Church & Gilbert (1984) buffer at 65 °C, then washed in 1× saline-sodium citrate (SSC) at 65 °C and exposed to X-ray film (Kodak, Rochester, NY, USA).
The mRNA abundance of INH2 was too low to detect by gel blot analysis in the silenced lines in background ‘1021/1’. For quantitative real-time PCR (qPCR) analysis, 1 µg of RNA per sample was treated with DNase I (Invitrogen), then converted to cDNA using oligo d(T)18 primer, dNTPs and Superscript III reverse transcriptase (Invitrogen) following the manufacturer's instructions. qPCR was carried out using 2.5 µL of a 25-fold dilution of cDNA template, 0.5 µL of a 10 µm solution of primers specific for INH2α (Brummell et al. 2011) and the reference gene EF1α (Jung et al. 2010), and 5 µL of Light Cycler 480 SYBR green I master mix (Roche, Auckland, New Zealand) in a final reaction volume of 10 µL. PCR was performed in triplicate on a RotorGene RG-3000 (Corbett Research, Sydney, Australia) with conditions of 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 15 s at 62 °C and 15 s at 72 °C. Transcript abundance was calculated relative to the EF1α reference gene using the Relative Quantification feature of the RotorGene Series 6000 software version 1.7. Experiments were repeated using at least two RNA preparations from different biological replicates.
Invertase activity and sugar content
Total protein was extracted and acid invertase activity determined as previously described (Brummell et al. 2011). Glc, Fru and Suc contents were determined by high-performance liquid chromatography (HPLC) analysis. Freeze-dried tuber tissue was powdered, and aliquots of 100 mg were extracted with 200 µL of 62.5% (v/v) methanol at 55 °C for 1 h. Extracts were clarified by centrifugation and aliquots of 20 µL injected into a Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) fitted with a 4.6 mm × 250 mm Prevail Carbohydrate ES, 5µ, column (Grace Davison, Deerfield, IL, USA). Separation was achieved using an isocratic elution mobile phase of acetonitrile-water (3:1, v/v) flowing at 1 mL min−1, and a constant column temperature of 25 °C. Sugars were detected by evaporative light scattering with nitrogen gas. Peaks were identified by comparing retention times with sugar standards, and Glc, Fru and Suc contents were estimated against standard curves.
Fry tests and acrylamide determination
Slices 2 mm thick were cut from the equatorial region of tubers using a kitchen mandolin slicer (Bron-Coucke, Orcier, France). Slices were cooked in a deep fryer in canola oil at 180 °C for 2 min, then cooled on paper towels. The irregular nature of the colouration meant that overall colour readings could not be taken, so fried crisps were photographed to record the colour.
Acrylamide determination was carried out using a SampliQ QuEChERS AOAC kit (Agilent Technologies, Santa Clara, CA, USA) for two-stage separation, followed by liquid chromatography–tandem mass spectrometry/mass spectrometry (LC-MS/MS; Mastovska & Lehotay 2006). After deep frying, replicates of 2–3 crisps were homogenized, and 1 g aliquots were spiked with a deuterated acrylamide solution as an internal standard. Samples were held overnight in sealed vials to allow the sample and spike to equilibrate. Acetonitrile (10 mL), water (10 mL) and n-hexane (5 mL) were added, then anhydrous MgSO4 (4 g) and NaCl (0.5 g), and vials were immediately shaken vigorously for 1 min. Samples were centrifuged (10 min, 3500 g), and an aliquot of the acetonitrile phase (1 mL) was transferred for clean-up with primary secondary amine sorbent (50 mg) and anhydrous MgSO4 (150 mg). After centrifugation (5 min, 3500 g), the analyte and internal standard were separated on a reversed phase C-18 column following the manufacturer's instructions, and eluants were analysed with LC-MS/MS using a UHPLC 1200 series chromatography system (Agilent) equipped with a Linetex C18 column (mobile phase: 2.5% methanol, 1% formic acid, under isocratic flow conditions). The mass spectrometer (60410 triple-quad, Agilent) was operated in positive ion mode using multiple reaction monitoring mode (MRM). Calibration was performed using an acrylamide dilution series with a deuterated acrylamide internal standard. Acrylamide sample concentrations were automatically corrected with recovery efficiency derived from the internal standard.
Mean content of Glc, Fru and Suc in the transformed potato lines was analysed using a linear mixed model separately for each of the three different storage treatments (T0, 4 °C for 6 weeks, and 10 °C for 6 weeks). Comparisons were made between each line and WT using Fisher's least significant differences.
In order to investigate the role of the native vacuolar invertase inhibitor in controlling CIS, two potato populations were produced with the opposing aims of (1) using tuber-specific overexpression to increase the amount of vacuolar invertase inhibitor INH2α in a cultivar that is susceptible to CIS (‘Karaka’), and (2) using tuber-specific RNAi-mediated gene silencing to reduce the amount of INH2 in a cultivar that is highly resistant to CIS (‘1021/1’). The RNAi construct used would be expected to bring about post-transcriptional gene silencing of both the INH2α and β mRNAs. In both populations, tuber-specific modification of INH2 mRNA abundance was achieved by using the patatin promoter to control the expression of the transgene.
Molecular characterization of potato populations with altered INH2 mRNA abundance
An initial screen of approximately 20 independent transgenic lines from each of the two populations was conducted by analysing the reducing sugar content of tubers from each line following storage at 4 °C for 6 weeks (data not shown). Six and three lines transformed with the overexpression (OE) and silencing constructs for gene expression knock-down (KD), respectively, were chosen for further analysis.
RNA gel blot analysis was used to determine the abundance of INH2 mRNA in INH2-overexpressing tubers after three different storage treatments (T0, and after storage at 4 °C or 10 °C for 6 weeks; Fig. 1). At T0, the highest INH2 overexpression was detected in lines OE-12, OE-16 and OE-28. INH2 mRNA was detected at an abundance only slightly greater than WT in line OE-3. Lines OE-5 and OE-34 had mRNA abundance similar to, or less than, WT, respectively. INH2 mRNA abundance in these lines increased slightly following storage, even in WT, but was similar at both 4 and 10 °C, showing that transgene expression driven by the patatin promoter was not greatly affected by either storage time or temperature. An exception was line OE-16, which appeared lower after storage at 4 °C. To extend the results of the RNA gel blot, two further biological replicates were examined by qPCR analysis (Fig. 2a), which showed that line OE-12 had an INH2α mRNA abundance ∼10-fold greater than WT, lines OE-16 and OE-28 ∼4-fold greater than WT, and lines OE-3 and OE-5 ∼2-fold greater than WT.
qPCR analysis was also conducted on the INH2-silenced KD lines, and demonstrated a marked reduction in INH2α gene expression (Fig. 2b). INH2α mRNA abundance was less than 3% that of WT in all three lines analysed.
Biochemical characterization of potato populations with altered INH2 mRNA abundance
Tuber material of each line was assessed for acid invertase activity following cold storage at 4 °C for 6 weeks (Fig. 3). The lines in which the INH2α invertase inhibitor was substantially overexpressed (see Fig. 2a) had reduced acid invertase activity (Fig. 3a), with activity ranging from 66% of WT in line OE-16 to 27% of WT in line OE-12. The WT ‘Karaka’ activity was 697 nmol Glc h−1 (mg protein)−1. Lines OE-5 and OE-34 had acid invertase activity indistinguishable from WT. Conversely, tubers from lines with silenced INH2 expression had significantly increased acid invertase activity, ranging from ∼10-fold greater than WT in line KD-35 to ∼4-fold greater than WT in line KD-16 (Fig. 3b). The WT ‘1021/1’ activity was 9.6 nmol Glc h−1 (mg protein)−1.
HPLC analysis of tuber-soluble sugar content (Glc, Fru and Suc) was conducted for each transgenic line following the three storage treatments (Figs 4 & 5). In the ‘Karaka’ lines transformed with the INH2α overexpression construct, at T0 the total soluble sugar content was similar in most of the lines (Fig. 4a), except for lines OE-12 and OE-16, which had reduced contents of Glc and Fru compared with the other lines and the WT control. Following storage at a chilling temperature of 10 °C for 6 weeks (Fig. 4b), WT tubers experienced a modest rise in Glc and Fru content compared with all the transgenic lines, which maintained the sugar contents at similar amounts to those at T0. Following storage of the tubers in the cold at 4 °C for 6 weeks (Fig. 4c), there was a drastic increase in Glc and Fru content in WT tubers (cold-induced sweetening), as well as in tubers of line OE-5. Substantial increases were also seen in two of the other transgenic lines (OE-28 and OE-34). However, three lines had significantly lower contents of total reducing sugars (Glc + Fru), the most substantial being a decrease to 38% that of WT in line OE-12, and to 50% and 59% that of WT in lines OE-3 and OE-16, respectively.
In the reciprocal experiment, where INH2 mRNA accumulation was suppressed in ‘1021/1’, little difference was seen between the transformants and WT at T0 or following storage at 10 °C for 6 weeks (Fig. 5a,b), where the predominant sugar was Suc, and only traces of Glc and Fru could be detected. However, following cold storage at 4 °C for 6 weeks, the Glc and Fru content of tubers from all three INH2-silenced lines rose significantly, to concentrations two- to threefold that detected in WT tubers (Fig. 5c).
A comparison of the WT tubers in Figs 4c and 5c shows the extent of the difference in CIS between the cultivars: in CIS-susceptible ‘Karaka’, the total hexose reducing sugars were almost 900 µmol g−1 dry weight (DW), whereas in the CIS-resistant ‘1021/1’, they were only 37 µmol g−1 DW. In the transgenic lines where hexose accumulation was most strongly affected, following cold storage total reducing sugar content was decreased to 340 µmol g−1 DW in line OE-12, and increased to 100 µmol g−1 DW in line KD-35.
These trends in the accumulation of reducing sugars after cold storage were also observed when tuber-soluble sugar content was expressed as the ratio of hexoses (Glc + Fru) to Suc (Table 1). Overexpression of INH2 generally resulted in a reduced ratio of hexose : Suc relative to WT, with the greatest reduction occurring in the most strongly overexpressing line, OE-12. Conversely, silencing of INH2 mRNA expression in lines KD-12, KD-16 and KD-35 all resulted in an increased hexose : Suc ratio relative to WT. However, because of metabolic and post-translational factors, there is not always a simple correlation between the abundance of an mRNA and the abundance or activity of the corresponding protein, and a more relevant correlation may be found between acid invertase activity and hexose : Suc ratio. A strong correlation (R = 0.95) between acid invertase activity and hexose : Suc ratio existed in the transgenic populations of INH2-overexpressing and INH2-silenced lines (Fig. 6).
|Cultivar||Line||Hexose : Suc (SE)|
Fry test colour and acrylamide content of tuber slices from potato lines with altered INH2 mRNA abundance
The two best-performing INH2α-overexpressing lines were selected based on the lowest amount of reducing sugars accumulating after cold storage (lines OE-3 and OE-12, Fig. 4c). These lines, together with all three INH2-silenced lines, were analysed for colour production and acrylamide content following deep frying of slices from cold-stored tubers. These crisps were compared with corresponding WT crisps fried in the same batch.
The crisps from INH2α overexpressing lines OE-3 and OE-12 were substantially lighter than the ‘Karaka’ WT controls, and acrylamide content analysis revealed that both lines had significantly reduced acrylamide concentrations, in the case of line OE-3 to only 50% that of WT (Fig. 7a). In contrast, the crisps from lines KD-12, KD-16 and KD-35, where INH2 mRNA accumulation was suppressed, were all darker than the ‘1021/1’ WT controls and had increased acrylamide contents (Fig. 7b). Acrylamide content was twofold greater than WT in line KD-16, and as much as 5.5-fold greater than WT in line KD-35.
Previous work has shown that overexpression of a heterologous vacuolar invertase inhibitor from tobacco reduced the accumulation of hexose sugars in transgenic potato tubers during cold storage (Greiner et al. 1999). In this work, the tobacco transgene was controlled by the powerful and constitutive cauliflower mosaic virus 35S promoter. In order to minimize potential abberant plant phenotypic effects, we investigated the role of the recently characterized endogenous potato vacuolar invertase inhibitor INH2 using the potato tuber-specific patatin promoter. Up-regulating the expression of the native vacuolar invertase inhibitor INH2α in CIS-susceptible cultivar ‘Karaka’ decreased acid invertase activity (in the case of line OE-12 to only 27% that of WT) and the accumulation of reducing sugars, and consequently increased resistance to CIS (Figs 3a & 4c). Post-translational regulation of acid invertase activity was confirmed in the reciprocal experiment by silencing INH2 in three transgenic lines of ‘1021/1’, all of which showed increased acid invertase activity and the accumulation of reducing sugars, and strongly decreased resistance to CIS (Figs 3b & 5c).
In the range of independent transgenic lines in which acid invertase activity was modified by up- or down-regulating expression of the endogenous potato vacuolar invertase inhibitor, a strong correlation between acid invertase activity and hexose : Suc ratio was found (Fig. 6). There was also an approximate correlation between INH2 mRNA abundance and acid invertase activity, but the relationship was not exact. For example, line OE-3 was a weak overexpressor but acid invertase activity (and reducing sugar content) were substantially decreased. However, taken together, these findings provide direct evidence that a component of CIS resistance is due to post-translational inhibition of acid invertase activity by the vacuolar invertase inhibitor INH2.
Suppression of acid invertase activity by silencing of the major vacuolar invertase gene itself gave a very strong reduction of Glc and Fru content, with essentially no increase in these sugars after cold storage (Bhaskar et al. 2010). This implies that the activity of vacuolar invertase present in a particular line is the primary determinant of CIS. Potato possesses at least six genes encoding acid invertase, four for cell wall-localized proteins and two for vacuolar proteins (Draffehn et al. 2010; Liu et al. 2011). During cold storage at 4 °C, the breakdown of starch results in an increase in Suc content by 14–20 d, followed by a conversion of this Suc to Glc and Fru, which reach substantial concentrations by 28 d (Bagnaresi et al. 2008). The transcript abundance of the major vacuolar acid invertase (termed Pain-1, VINV or vacINV1; Zrenner et al. 1996; Bhaskar et al. 2010; Liu et al. 2011) is strongly increased by cold treatment (Zrenner et al. 1996), and by 20 d was >1000-fold higher than before cold storage and remained high until at least 28 d (Bagnaresi et al. 2008). The correlation between the accumulation of vacuolar acid invertase vacINV1 mRNA and Glc content indicates that cold induction of vacINV1 is a major cause of CIS (Bagnaresi et al. 2008; Liu et al. 2011).
Interestingly, mRNA of the vacuolar invertase inhibitor INH2 also showed induction by cold storage over the same time period in the strongly CIS-resistant cultivar ‘1021/1’, at both chilling (10 °C) and cold (4 °C) temperatures (Brummell et al. 2011). This is consistent with a component of CIS resistance being brought about by post-translational regulation of acid invertase activity by an invertase inhibitor. The presence of an invertase inhibitor may also explain discrepancies where acid invertase mRNA abundance and activity are not correlated. For example, in cultivar ‘ND860-2’ acid invertase mRNA abundance was high but activity was low, as opposed to four other cultivars where an approximate correlation was observed (Liu et al. 2011).
Acrylamide production is an unfortunate consequence of CIS in processed potato products. Acrylamide is a proven neurotoxin and suspected carcinogen, and when eaten in food is well absorbed and widely distributed to tissues, including to the foetus and in breast milk (Sorgel et al. 2002). Fried and roasted potato products are some of the largest contributors to dietary acrylamide intake, along with bread and bakery products and coffee (Friedman 2003; Halford et al. 2012; Medeiros Vinci et al. 2012). Both asparagine and reducing sugars are required for acrylamide production from the Maillard reaction, and it has been shown that in potato silencing of two of the genes responsible for asparagine production reduced the amounts of free asparagine by up to 20-fold and acrylamide by a similar amount (Rommens et al. 2008). However, it is important to develop potato cultivars that have lower contents of reducing sugars after cold storage since potato contains very high amounts of free asparagine and it is usually the content of reducing sugar that determines acrylamide formation (Amrein et al. 2003).
Overexpression of INH2 in transgenic lines of ‘Karaka’ reduced CIS, and consequently reduced the development of brown colouration and the formation of acrylamide in subsequent fry tests (Fig. 7a). The content of reducing sugars and the amount of acrylamide formed were not directly related, with line OE-3 possessing higher reducing sugar content but less acrylamide formation than line OE-12 (Figs 4c & 7a). Similarly, line KD-35 had only slightly more reducing sugar than lines KD-12 and KD-16 but more than twice the acrylamide content after frying (Figs 5c & 7b). This may be because reducing sugar content is not the only factor affecting acrylamide formation since the Maillard reaction is actually a complex series of reactions with multiple steps and components (Halford et al. 2012). Variations in growth temperature, soil conditions and post-harvest handling can also affect tuber composition (Amrein et al. 2003; Halford et al. 2012; Medeiros Vinci et al. 2012).
Correlations of INH2 gene expression with CIS phenotype (Brummell et al. 2011) and directly altering expression through overexpression and RNAi have shown that the INH2 gene potentially plays a major role in resistance to CIS. Overexpression of this gene in transgenic lines would produce potato cultivars with both reduced CIS and reduced acrylamide content after processing. Since the INH2 transgene is a copy of an endogenous sequence and its overexpression can be controlled by a potato promoter, it is now possible to develop intragenic plants in which the transgene construct (borders, promoter, transgene, terminator) is composed entirely of native DNA (Conner et al. 2007; Rommens et al. 2007). Alternatively, the development of perfect molecular markers for INH2 expression could be a useful tool in breeding efforts aimed at improving CIS resistance. Crossing lines with low invertase activity with lines showing high expression of INH2 vacuolar invertase inhibitor might result in cultivars with exceptional resistance to CIS.
We thank Tony Conner for the generous gift of the pART7PAT3 vector, Russell Genet and John Anderson for providing plant material, Ian King for care of the transgenic plants, Erin O'Donoghue for assistance with HPLC analysis, Erin McGill and Peter Grounds for assistance with acrylamide determinations, Andrew McLachlan for statistical analysis, and Simon Deroles and Kevin Davies for comments on the manuscript. This work was funded by the New Zealand Ministry for Science and Innovation (MSI) under contract number C02X0701, and by Vital Vegetables, a Trans-Tasman research project jointly funded and supported by Horticulture Australia Ltd, The New Zealand Institute for Plant & Food Research Limited, MSI, the Victorian Department of Primary Industries, Horticulture New Zealand and the Australian Vegetable and Potato Growers Federation Inc.
- 2003) Potential of acrylamide formation, sugars, and free asparagine in potatoes: a comparison of cultivars and farming systems. Journal of Agricultural and Food Chemistry 51, 5556–5560. , , , et al. (
- 2008) Heterologous microarray experiments allow the identification of the early events associated with potato tuber cold sweetening. BMC Genomics 9, 176. , , , , & (
- 2002) Alternative selectable markers for potato transformation using minimal T-DNA vectors. Plant Cell, Tissue and Organ Culture 70, 61–68. , , & (
- 2010) Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato. Plant Physiology 154, 939–948. , , , , , , , & (
- 2004) Metabolic control of low-temperature sweetening in potato tubers during postharvest storage. Horticutural Reviews 30, 317–354. , & (
- 1996) Effect of expression of UDP-glucose pyrophosphorylase ribozyme and antisense RNAs on the enzyme activity and carbohydrate composition of field-grown transgenic potato plants. Journal of Plant Physiology 147, 644–652. , , , & (
- 2011) Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants. Journal of Experimental Botany 62, 3519–3534. , , , , , & (
- 1984) Genomic sequencing. Proceedings of the National Academy of Sciences of the United States of America 81, 1991–1995. & (
- 2007) Intragenic vectors for gene transfer without foreign DNA. Euphytica 154, 341–353. , , , , , , & (
- 2010) Natural diversity of potato (Solanum tuberosum) invertases. BMC Plant Biology 10, 271. , , & (
- 2003) Chemistry, biochemistry, and safety of acrylamide. A review. Journal of Agricultural and Food Chemistry 51, 4504–4526. (
- 1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology 20, 1203–1207. (
- 1999) Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers. Nature Biotechnology 17, 708–711. , , & (
- 2012) The acrylamide problem: a plant and agronomic science issue. Journal of Experimental Botany 63, 2841–2851. , , , , & (
- 1996) The onset of sucrose accumulation in cold stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase. Plant, Cell & Environment 19, 1223–1237. , , , & (
- 2004) Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins. The Plant Cell 16, 3437–3447. , , , & (
- 2010) Expression of multiple expansin genes is associated with cell expansion in potato organs. Plant Science 179, 77–85. , , & (
- 2002) NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS. The Plant Journal 32, 391–400. & (
- 2010) Cloning and molecular characterization of putative invertase inhibitor genes and their possible contributions to cold-induced sweetening of potato tubers. Molecular Genetics and Genomics 284, 147–159. , , , , & (
- 2011) Systematic analysis of potato acid invertase genes reveals that a cold-responsive member, StvacINV1, regulates cold-induced sweetening of tubers. Molecular Genetics and Genomics 286, 109–118. , , , , , , & (
- 1998) Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nature Biotechnology 16, 473–477. , , & (
- 2005) Investigations on the role of acid invertase and UDP-glucose pyrophosphorylase in potato clones with varying resistance to cold-induced sweetening. American Journal of Potato Research 82, 231–239. , , , , & (
- 2006) Rapid sample preparation method for LC-MS/MS or GC-MS analysis of acrylamide in various food matrices. Journal of Agricultural and Food Chemistry 54, 7001–7008. & (
- 2004) Changes in sugar content and activity of vacuolar acid invertase during low-temperature storage of potato tubers from six Japanese cultivars. Journal of Plant Research 117, 131–137. , , , , & (
- 2012) Acrylamide formation in fried potato products – present and future, a critical review on mitigation strategies. Food Chemistry 133, 1138–1154. , & (
- 2002) Acrylamide is formed in the Maillard reaction. Nature 419, 448–449. , & (
- 1997) A β-amylase in potato tubers is induced by storage at low temperature. Plant Physiology 113, 503–510. , & (
- 1966) Separation and properties of potato invertase and invertase inhibitor. Archives of Biochemistry and Biophysics 113, 667–674. (
- 2004) Plant protein inhibitors of invertases. Biochimica et Biophysica Acta 1696, 253–261. & (
- 1997) Potato plants contain multiple forms of sucrose phosphate synthase, which differ in their tissue distributions, their levels during development, and their responses to low temperature. Plant, Cell & Environment 20, 291–305. , , , , , & (
- 1990) Invertase activity and its relation to hexose accumulation in potato tubers. Journal of Experimental Botany 41, 95–99. , , & (
- 2004) Function and regulation of plant invertases: sweet sensations. Trends in Plant Science 9, 606–613. & (
- 2007) The intragenic approach as a new extension to traditional plant breeding. Trends in Plant Science 12, 397–403. , , , & (
- 2008) Low-acrylamide French fries and potato chips. Plant Biotechnology Journal 6, 843–853. , , , & (
- 2002) Acrylamide: increased concentrations in homemade food and first evidence of its variable absorption from food, variable metabolism and placental and breast milk transfer in humans. Chemotherapy 48, 267–274. , , , , , & (
- 2001a) Biochemical and molecular control of cold-induced sweetening in potatoes. American Journal of Potato Research 78, 221–236. (
- 2001b) Allele and isozyme patterns of UDP-glucose pyrophosphorylase as a marker for cold-sweetening resistance in potatoes. American Journal of Potato Research 78, 57–64. (
- 2002) Acrylamide from Maillard reaction products. Nature 419, 449–450. , , , , , , & (
- 1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical Biochemistry 223, 7–12. & (
- 2011) Developing cold-chipping potato varieties by silencing the vacuolar invertase gene. Crop Science 51, 981–990. , , , , & (
- 2010) Tuber-specific silencing of the acid invertase gene substantially lowers the acrylamide-forming potential of potato. Journal of Agricultural and Food Chemistry 58, 12162–12167. , , & (
- 1993) Analysis of the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA. Planta 190, 247–252. , & (
- 1996) Soluble acid invertase determines the hexose-to-sucrose ratio in cold-stored potato tubers. Planta 198, 246–252. , & (