Simultaneous silencing of asparagine synthetase (Ast)-1 and -2 limits asparagine (ASN) formation and, consequently, reduces the acrylamide-forming potential of tubers. The phenotype of silenced lines appears normal in the greenhouse, but field-grown tubers are small and cracked. Assessing the effects of silencing StAst1 and StAst2 individually, we found that yield drag was mainly linked to down-regulation of StAst2. Interestingly, tubers from untransformed scions grafted onto intragenic StAst1/2-silenced rootstock contained almost the same low ASN levels as those in the original silenced lines, indicating that ASN is mainly formed in tubers rather than being transported from leaves. This conclusion was further supported by the finding that overexpression of StAst2 caused ASN to accumulate in leaves but not tubers. Thus, ASN does not appear to be the main form of organic nitrogen transported from leaves to tubers. Because reduced ASN levels coincided with increased levels of glutamine, it appears likely that this alternative amide amino acid is mobilized to tubers, where it is converted into ASN by StAst1. Indeed, tuber-specific silencing of StAst1, but not of StAst2, was sufficient to substantially lower ASN formation in tubers. Extensive field studies demonstrated that the reduced acrylamide-forming potential achieved by tuber-specific StAst1 silencing did not affect the yield or quality of field-harvested tubers.
Asparagine synthetase (Ast) catalyses the last step in ASN formation. Plants contain multiple Ast isoenzymes, each of which functions during a specific process in development, such as the mobilization of N during seed germination, the recycling of N in vegetative organs in response to stress, the mobilization of N from source to sink and the storage of N in seeds. However, the regulation of a specific Ast-encoding gene is not necessarily conserved across plant species (Duff et al., 2011; Herrera-Rodríguez et al., 2002; Lam et al., 1998; Tsai and Coruzzi, 1991). Molecular and genetic analyses indicate that distinct Ast genes are regulated differentially by environmental stimuli (light/dark, stress), metabolic status (sugar and N levels), developmental cues and tissue/cell-type specificity (Gaufichon et al., 2010). Further studies are required to determine whether any correlation exists between phylogenetic classification and the diverse physiological roles of Ast genes during the life cycle of plants. The predominantly expressed Ast genes in potato, StAst1 and StAst2, are poorly characterized. However, as StAst1 has high sequence similarity with Arabidopsis Asn1, it may play a similar role in assimilating, transporting and storing N (Lam et al., 2003). On the other hand, StAst2 is homologous to Arabidopsis Asn2 and may thus play a role in ammonium metabolism (Wong et al., 2004).
As ASN is oxidized into a toxic compound, acrylamide, during cooking of starchy foods in a low-water environment, high tuber concentrations of ASN are undesirable. Both acrylamide and its reactive metabolite, glycidamide, are neurotoxins and probable carcinogens (Parzefall, 2008; Tareke et al., 2002), and the consumption of certain heat-processed foods, including French fries, potato chips, bread and coffee, results in an average dietary intake of 0.3–0.7 μg acrylamide/kg per day (Dybing et al., 2005). This intake may be linked to the development of certain degenerative diseases, including cancer (Tardiff et al., 2010; Tareke et al., 2002).
In an effort to reduce the acrylamide-forming potential of potatoes by minimizing tuber concentrations of ASN, we previously generated transgenic potato plants, in which StAst1 and StAst2 were simultaneously silenced (Rommens et al., 2008). These lines exhibited a ∼20-fold reduction in ASN and, when processed into French fries, contained just 5% of the acrylamide present in the control. In the light- and humidity-controlled conditions of a greenhouse, the transgenic potato plants appeared normal and had normal yields. However, we subsequently found that the StAst1/2-silenced plants did not grow well in the field, producing small, deformed tubers and fewer tubers per plant. We thus aimed to generate transgenic potato lines that had reduced levels of ASN, but grew normally in the field. By silencing StAst1 and StAst2 individually, we established that StAst1 is mainly involved in ASN formation in tubers, whereas StAst2 has a larger impact on agronomic traits in field-grown plants. Silencing of only StAst1 in tubers reduced ASN content by 80% without compromising the quality or yield of field-grown plants.
Simultaneous silencing of StAst1 and StAst2 in tubers leads to cracking
To reduce the levels of free ASN in tubers and thereby limit the precursors of the Maillard reaction, we previously generated transgenic potato lines, in which StAst1 and StAst2 were simultaneously silenced, particularly in the tubers (Rommens et al., 2008). Greenhouse trials showed that tubers of these plants contained less ASN than the control and produced less acrylamide when prepared as French fries (Rommens et al., 2008). These plants exhibited a normal phenotype in the greenhouse, producing similar yields of tubers as the control. To our surprise, upon subsequent field trials, tubers of all 23 StAst1/2-silenced lines were small and cracked, while also developing secondary growth (Figure 1a). These abnormal tubers maintained reduced levels of ASN when compared with wild type (WT) and empty vector tissue culture (TC) controls (Figure 1b). Previous studies had shown that tuber cracking in potato is generally associated with environmental stress (Hochmuth et al., 2001; Jefferies and Mackerron, 1987). However, because our control tubers did not show any signs of stress, the altered phenotype of silenced tubers was because of simultaneous silencing of StAst1 and StAst2. These unexpected results prompted us to more thoroughly analyse the role of the two ASN biosynthetic genes in potato.
StASt1 silencing reduces yield in the field
To constitutively silence the StAst1 gene alone, an inverted repeat carrying two copies of a small fragment of this gene was operably linked to the promoter of the potato ubiquitin-7 (Ubi7) gene (Garbarino et al., 1995) and cloned into pSIM1714. pSIM401, which only contains the selectable marker gene nptII, was used as empty vector control. The plants were transformed using a standard protocol (Richael et al., 2008) and rooted on kanamycin-containing medium. Plant lines positive for the presence of the transgene, as determined by PCR, were transferred to the greenhouse 1 month after rooting. RNA gel blot analysis demonstrated that StAst1 was silenced in most lines, but lines 3, 9, 11, 13, 17 and 19–21 did not contain reduced transcript levels, possibly because of position integration effects (Figure 2a).
In the greenhouse, these plants were phenotypically similar to the controls (Figure 2b,c) while also displaying similar yields. Both the leaves and tubers of StAst1-silenced plants had reduced levels of ASN. The GLN levels were higher, especially in the tubers of the silenced lines, indicating that the precursor of ASN accumulated in the absence of ASN (Figure 2c). Lines 1714-9 and 1714-13 were not silenced and served as internal controls (Figure 2c).
The tubers of greenhouse-grown StAst1-silenced lines were stored at 38 °C for 5 months to break dormancy and then planted in the field. In a five-hill trial, transgenic lines were agronomically similar to the controls (Figure 3a), but their leaves had slightly reduced ASN levels (Figure 3b). No significant change was observed in total and N content in the leaves (Figures 3c and 5d). However, the yield of tubers (Figure 6a) and the concentration of ASN in the tubers were reduced (Figure 6b), as were the total N and total protein content in the tubers on a per plant basis (Figure 6c,d).
Therefore, although the aerial and below-ground parts of StAst1-silenced lines resembled the controls, constitutive silencing of StAst1 resulted in reduced ASN levels in both the leaves and tubers, which in turn affected tuber yield and thus reduced the total N and total protein content in the tubers.
Loss of StAst2 function leads to aberrant phenotype and decreased yield in the field
StAst2 alone was constitutively silenced using the pSIM1715 construct, in which a StAst2 silencing cassette was placed under the control of the potato Ubi7 promoter. Upon transformation, lines silenced for StAst2 were identified by RNA blot analysis. The blots were also probed with a StAst1 probe to ensure that StAst1 alone was not cross-silenced (Figure 4a).
In the greenhouse, the phenotypes and yield of these plants were similar to those of the controls (Figure 4b). Neither the leaves nor the tubers of the StAst2-silenced plants exhibited any changes in ASN/GLN levels in the greenhouse (Figure 4c). Lines 1715-15 and 1715-23 were not silenced and served as internal controls (Figure 4c).
These StAst2-silenced lines and their controls were stored for 5 months and then planted in the field. Surprisingly, all StAst2-silenced lines that looked normal in the greenhouse exhibited a conditional phenotype in the field. The plants were stunted and exhibited reduced vigour, narrow leaf blades with wavy margins and a chlorophyll deficiency (Figure 5a–c). Biochemical analysis of the leaves showed a significant reduction in total N (P value <0.05) (Figure 3c). ASN levels were lower than in the controls, whereas GLN levels were higher (Figure 3b). In agreement with findings in Arabidopsis lines silenced in AtAst2 expression (Wong et al., 2004), levels were significantly (P < 0.05) elevated in the leaves of StAst2-silenced lines (Figure 5d). Thus, StAst2, like Arabidopsis AtAsn2, may be involved in detoxification in the leaves.
Although the tubers had normal morphologies, the yield per plant appeared drastically decreased in all transgenic lines containing pSIM1715 (Figure 6a). ASN levels were comparable to those in the controls (Figure 6b). Therefore, StAst2 does not contribute significantly towards tuber ASN levels. However, total N and total protein content was significantly reduced in the tubers and was even lower than in the constitutive StAst1-silenced lines (Figure 6c,d). No significant changes in level were observed in the tubers (data not shown). The decrease in the yield of StAst2-silenced lines could be due to an overall reduction in growth and a change in aerial morphology, as seen in these field-grown plants (Figure 5a). Therefore, StAst2 function is important for maintaining a normal plant phenotype, overall growth, and yield in potato.
To determine the effect of StAst2 on tuber ASN levels, we designed a construct (pSIM1716) that drives the tuber-specific silencing of StAst2. The AGP promoter was used to drive the tuber-specific silencing of StAst2. RNA blot analysis identified lines 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24 and 25 as being silenced for StAst2 (Figure 7a). Biochemical analysis of the tubers of these plants grown in the greenhouse revealed no change in tuber ASN levels compared with the controls (Figure 7b). Therefore, neither constitutive nor tuber-specific silencing of StAst2 influenced tuber ASN levels. Thus, StAst2 does not affect tuber ASN levels, and its constitutive silencing is deleterious in the field.
Increased ASN formation in leaves does not increase ASN content in the tubers
To further assess the effect of individual asparagine synthetase proteins on the ASN pool in tubers, we constructed pSIM1704, in which the StAst2 coding region is driven by the 35S promoter.
StAst2 expression was tested in the leaves of the transgenic plants to identify overexpressing lines (Figure 8a). Based on their high levels of expression, lines 1704-3, 13, 15, 18, 19, 20 and 23 were chosen for biochemical analysis. Surprisingly, we found that ASN levels were elevated up to threefold in the leaves of 35S:StAst2 plants, while the level of ASN in tubers was unaffected (Figure 8b,c). Thus, StAst2 is predominantly active in leaves. Similarly, Arabidopsis AtAsn2 was predominantly expressed in leaves and was found to be regulated by light (Wong et al., 2004). We thus propose that StAst2, like AtAsn2, is involved in detoxification in the leaves. The morphology and yield of 35S:StAst2 lines grown in the greenhouse were comparable with those of WT plants (data not shown).
A key finding of this analysis is that increased (up to threefold) ASN levels in leaves do not correlate with increased levels of ASN in tubers. Foliar ASN appears to have a limited impact on ASN levels in the potato tuber.
Contribution of long-distance transport to ASN accumulation in tubers
To reduce ASN levels to a minimum in the tuber, we needed to establish the contribution of foliar ASN to the tuber pool. ASN is the major nitrogenous compound in the phloem of several legumes (up to 30 mm) (Atkins et al., 1975), whereas in maize, ASN has been reported to represent as much as 14% of the free amino acids in phloem (Lohaus et al., 1998; Weiner et al., 1991).
We designed a construct (pSIM1256) to simultaneously silence StAst1 and StAst2 expression under the control of the AGP and GBSS promoters, which are much more active in tubers than in the aerial parts (Rommens et al., 2008). Both of these genes were silenced in line B26, which was used to study the extent of ASN transport from the leaves to the tubers.
We performed a reciprocal grafting experiment between line B26 and the WT. Whereas line B26 contained StAst1 and StAst2 transcripts in the leaves, but not in the tubers, the WT contained transcripts of both genes in the leaves and tubers. Transcripts of both genes were also detected in grafted tubers whenever the WT parent was used as stock (Table 1). Three-week-old plants intended to be used as the scion were cut two to three nodes beneath the apical meristem and were secured and sealed to the desired stock as described in Methods. New leaves that emerged from the stock were removed to ensure that all N was transported from the scion. A control grafting of WT/WT and B26/B26 was performed to study the effect of grafting itself on transcript and amino acid levels. The tubers of control B26/B26 grafts had low levels of ASN (2.8%), as did the parental B26 lines, and the WT/WT graft exhibited levels similar to those in WT tubers (100%). Whereas ASN levels in the tubers of B26/WT grafts were similar to those in WT tubers, the tubers of WT/B26 grafts had low levels of ASN (4%; Table 1). Hence, transport of ASN from leaves to tubers is limited, and ASN does not appear to be the primary amino acid that transports N to sink tissue in potato. In the grafted lines that exhibited reduced levels of ASN (i.e. B26/B26 and WT/B26), GLN levels were increased two- to threefold (Table 1). Therefore, a lack of StAst1 in tubers leads to GLN accumulation, which is the precursor of ASN (Table 1). In potato, GLN seems to be the major amino acid transported from the leaves to tubers, where it is converted to ASN by Ast.
Table 1. Relative StAs1 and StAs2 transcript levels and amino acid levels for ASN and GLN in leaves and tubers of grafted plants.* The column headings show the scion/stock genotype
ASN, asparagine; GLN, glutamine.
*Data are shown as percentage of wild type.
†100% = 0.169 mg/g FW.
‡100% = 1.374 mg/g FW.
§100% = 8.25 mg/g FW.
¶100% = 2.59 mg/g FW.
Potato quality can be improved by tuber-specific silencing of StAst1
We next silenced StAst1 alone, using two convergently oriented tuber-specific promoters from the ADP-glucose pyrophosphorylase (Agp) gene (Muller-Rober et al., 1994) and granule-bound starch synthase (Gbss) gene (Visser et al., 1991), respectively. Five silenced lines were identified by RNA blot analysis. The leaf and tuber morphology of these plants were similar to controls grown in the greenhouse. Sixty tubers from each of the selected lines were planted in three locations (three random plots per field with one row of 20 tubers per plot). No differences were observed between the controls and the StAst1-silenced lines in agronomic traits monitored such as emergence and stand counts (early-season), date of flowering, date of row closure, plant vigour, foliage colour, leaflet size, leaflet curl, incidental disease, and incidental insects (mid-season), and date of vine kill, vine maturity, and incidental disease (late-season). RNA gel blot analyses were performed to confirm that, despite the lack of observable effects (Figure 9a,b), the intragenic tubers were still silenced for StAst1 (Figure 9c). The yield of plants silenced for StAst1 was similar to that of WT plants under field conditions (P > 0.05) (Figure 9d). The tubers were also indistinguishable from the WT in terms of size profile, specific gravity by size, colour analysis and internal analysis during grading. However, StAst1-silenced tubers showed a considerable reduction in ASN levels (ranging from 60% to 80%), leading us to conclude that StAst1 is the major enzyme responsible for ASN synthesis in tubers (Figure 9e). Data from two potato varieties Russet Ranger (R) and Russet Burbank (B) showed a similar trend. We propose that StAst1 enzyme can be used as a tool to lower acrylamide levels in heat-processed potato products (Figure 9f).
In this study, we sought to limit acrylamide production in French fries by reducing the amount of ASN in potato tubers. The choice to target asparagine synthetase was clear from the start of our investigation, based on successful greenhouse trials conducted previously (Rommens et al., 2008). However, as this approach affected the quality of potatoes grown in the field, we set out to determine whether (i) one of the ASN synthetases had a greater impact on tuber ASN levels and could be fine-tuned to minimize the negative impact on the agronomic characteristics of the plant, (ii) the asparagine synthetase should be silenced constitutively or specifically in tubers, and (iii) the strategy to silence asparagine synthetase only in tubers is sufficient, or if significant amounts of ASN are transported from foliar tissues to the potato via other mechanisms. The following paragraphs discuss our findings.
StAst1/2 double mutants exhibit a synergistic phenotype
Tuber folding was only observed when both StAst1 and StAst2 were silenced in the tubers (Figures 1a and 10). As the tuber phenotypes appeared normal under field conditions when only one of the genes was silenced (Figure 10), the tuber cracking phenotype has a synergistic nature. The deleterious effect of silencing both enzymes in tubers can be attributed to the fact that ASN is critical for potato tubers. Levels of ASN that are below a certain threshold are perceived as stress and thus lead to the cracking of the tubers in the field (Hochmuth et al., 2001; Jefferies and Mackerron, 1987). Tuber cracking could be because of global alterations in ASN levels and metabolism, which is an important intermediate in C/N portioning within the plant and thus affects other metabolites. It is also possible that significant phenotypic effects on tuber could be as a result of modified tuber water relations as we could mimic tuber cracking of StAst1/StAst2 plants in greenhouse by overwatering stress.
Increased foliar ASN levels do not result in increased ASN in potato tubers
In this study, we explored the extent of ASN transport by reciprocally grafting low ASN stock with WT scions. Interestingly, there was no increase in the level of free ASN in tubers of these grafted lines (Table 1). Also, lines overexpressing StAst2 had increased StAst2 transcript and ASN levels in the leaves, but did not have increased levels of ASN in the tubers (Figure 8b,c). This result indicates that ASN is not the major amino acid that transports N from source to sink in potato. This finding differs from observations in 35S-AtAsn1 Arabidopsis lines, where sink tissues such as flowers and developing siliques exhibited higher levels of free ASN than source tissues, such as leaves and stems, despite the presence of significantly higher levels of AtAsn1 mRNA in the source tissues. This was partially attributed to enhanced ASN transport from source to sink tissues via the phloem, based on increased levels of ASN in the phloem exudates of 35S:AtAsn1 plants (Lam et al., 2003). This difference may be due to physiological differences between potato tubers and Arabidopsis siliques or may be a side effect of the constitutive promoters used.
However, our results are in agreement with the results of an aphid-feeding study that found that phloem exudates of 5-week-old potato plants contained significantly more GLN (30.1 mol%) than ASN (6.1 mol%; Karley et al., 2002). A similar observation was made in Nicotiana tabacum (tobacco), where GLN was implicated as the major N-transporting amino acid in the phloem sap (Masclaux-Daubresse et al., 2006). Furthermore, GLN is the major amino acid entering the Zea mays (maize) cob just after silking, where it is further metabolized to ASN, a process that is required for optimal kernel growth and accumulation of storage proteins (Canas et al., 2010; Seebauer et al., 2004). Phloem sap analysis confirmed that GLN is the major form of N transported in maize (Oaks, 1992). GLN concentration was considerably reduced in the gln1-3, gln1-4 and gln1-3/gln1-4 maize mutants (Martin et al., 2006). Whereas ASN accumulated in the leaves of these plants during kernel filling, it was not sufficiently transported to the developing ears and was present in the phloem sap at concentrations below those found in WT plants. Furthermore, the concentration of soluble ASN was lower in the kernels of these mutants than in those of the WT (Martin et al., 2006). These findings support the hypothesis that GLN is the major amino acid involved in N transport in potato and maize.
Loss of StAst2 function is deleterious because of its important role in ammonium detoxification
StAst2 is required for overall vigour and yield in the field. Like AtASN2, StAst2 is involved in ammonium detoxification, and it is the accumulation of in the leaves of StAst2-silenced plants that cause the severe phenotype (Figure 5). Although toxicity is a universal phenomenon in both plant and animal systems (Britto and Kronzucker, 2002), the threshold at which symptoms develop differs widely among species. Chlorosis and the overall suppression of growth have been observed in species that are sensitive to . Solanaceae has been ranked as the family that is most sensitive to toxicity (Britto and Kronzucker, 2002), with symptoms of toxicity (i.e. decreased dry weights) occurring earlier than in other plants grown in growth chambers in the presence of 8–12 mm (Cao and Tibbits, 1998).
Therefore, StAst2 appears to have a major role in detoxification in potato (Figure 5d). is generated during nitrate reduction, photorespiration and senescence. Stress factors, including drought, overwatering and infection, enhance the formation of ASN (Kanamori and Matsumoto, 1974; Lea et al., 2007; Ta and Joy, 1986; Wong et al., 2004), and accumulation is generally viewed as an index of stress in plants (Barker, 1999a,b; Britto and Kronzucker, 2002). Although the physiological significance of up-regulating the biosynthesis of ASN is not fully understood, it is likely that an inability to detoxify would, directly or indirectly, affect stress tolerance in the field. Based on phylogenetic analysis and on the observation that the expression of AtAsn1 and AtAsn2 is reciprocally regulated by light and metabolites, it has been proposed that these genes have different physiological roles in N metabolism in plants (Lam et al., 2003 and Wong et al., 2004). In Arabidopsis, light induction of AtAsn2 is dependent on the presence of in the medium (Wong et al., 2004). Accumulation of ASN in response to abiotic stresses could be a mechanism to detoxify and a means of storing N when protein synthesis is inhibited by stress conditions. ASN also accumulates when high amounts of ammonium are generated by deamination of soluble amino acids released by proteolysis during leaf senescence and thereby prevents ammonium toxicity (Herrera-Rodríguez et al., 2007).
Furthermore, this study illustrates that field conditions are vastly different from those in the greenhouse and emphasizes the importance of field trials. Whereas StAst1- and StAst2-silenced plants appeared to be normal in the greenhouse, a variety of aberrations emerged in field trials (Figure 10). Such phenotypic variations would be hard to identify in model species, for example Arabidopsis, grown under the carefully controlled conditions in a growth chamber. Thus, field trials are critical when evaluating the phenotypes of genetically modified crops.
StAst1 is the major enzyme catalysing ASN production both in the leaves and in the tubers of potato
Most ASN in the potato plant is produced by StAst1, which contributes to both foliar and tuber ASN levels (Figures 9e and 10). The majority of N/C transport in potato occurs as GLN, which is converted to ASN by StAst1 in the tuber. In agreement with results obtained for Arabidopsis (Lam et al., 2003), changes in total free ASN in tubers parallel the expression of StAst1, and not StAst2. Although StAST1 is the major enzyme in the production of ASN, it was not possible to silence this enzyme altogether in the plant. Constitutive silencing of StAST1 affects the overall yield and the total N and protein content, indicating that some basal level of ASN transport from the leaves to the tubers is required to maintain the overall tuber yield. However, tuber-specific silencing of StAst1 was sufficient to reduce levels of ASN in tubers without having a negative effect on yield in field trials (Figure 9a,b,d,e). Furthermore, this strategy was sufficient to reduce acrylamide levels by up to 70% of those in the control (Figure 9f). This study thus highlights the importance of using a tissue-specific promoter for fine-tuning the expression in a particular organ and is a key feature of the novel genetic modification strategy presented here (Figure 10).
Despite the central role of ASN in N storage in potato, most N is transported to the potato tuber as GLN. In tubers, the majority of GLN is converted to ASN by StAST1 (Figure 10). It is likely that StAST2 is involved in stress-induced ASN synthesis, although a more detailed study would enhance our understanding of this process. The present investigation shows that silencing of StAst1 in tubers decreases the levels of free ASN, while increasing GLN amounts, without affecting plant growth or tuber phenotype and can be safely used as strategy to lower acrylamide levels in French fries. The increased GLN levels did not alter the quality of potato as food because they were still within the normal range for potato (Davies, 1977; Lisinska and Leszczynski, 1989). Furthermore, GLN is a nonessential amino acid that can be synthesized by the body and does not represent a significant precursor for acrylamide (Stadler, 2005). Consumption of processed potato products contributes to approximately one-third of the average dietary exposure to acrylamide (Boettcher et al., 2005). Thus, an eventual replacement of existing potatoes by low-StAst1 varieties would lower the acrylamide intake by ∼30%.
In the light of our new understanding of the role of StAst1, it might be tempting to try and knock this gene out through methods other than genetic engineering. However, the creation of such a recessive mutant would be difficult to achieve because backcrossing triggers inbreeding depression in potato (Golmirzaie et al., 1998). Furthermore, constitutive down-regulation of StAst1 expression was shown here to negatively affect the plant’s agronomic characteristics.
A multigene silencing construct pSIM1256 containing 348-bp fragments of StAst1 and StAst2 was cloned into a basic P-DNA vector, as previously described (Rommens et al., 2004, 2008). For tuber-specific expression, the gene fragments were driven by the Gbss promoter (coordinates 1138–1823 of GENBANK accession X83220), and the Agp promoter is identical to coordinates 2183–4407 of GENBANK accession X96771 as described in Rommens et al., 2008;. RNA isolation was carried out using Plant RNA easy kit (Qiagen), and RT-PCRs were performed with the one-step RT-PCR kit (Qiagen). Amplified products were gel purified and cloned into pGEM-Teasy (Promega) for subsequent sequence analysis. For constitutive expression of StAst2, pSIM1704 the complete ORF of StAst2 was amplified using primers for RT-PCR (1704F GGA GCA TCG TGG AAA AAG AAG ACG and 1704R GAA CAA AAG AGA ACA TCC CAT CC) placed downstream of the 35S CaMV promoter. For StAst1 silencing, inverted repeat corresponding to 403 bp (coordinates 355–757 of accession DM 178284) was cloned into binary vector under the control of potato ubiquitin-7 (ubi7) gene promoter (Garbarino et al., 1995) and the ubiquitin-3 gene terminator (Garbarino and Belknap, 1994) using a standard restriction enzyme method. The primers used for RT-PCR of StAst1 are forward TCA TGT GTG GAA TTT TGG CTT TG and reverse AAA CAT CAA GTA TGG ACA GCA AGA. pSIM 1715 silencing targeted 250 bp of StAst2 (coordinates of accession DM 178281). Primers used to amplify this region were forward TGG TCT TGT CCT CAT TAT ACA GC and reverse TGG TCT TGT CCT CAT TAT ACA GC. The same region was silenced in pSIM1716 using the ubi7 promoter and ubi3 terminator. Control binary vector pSIM401 (Rommens et al., 2005) was used to produce empty vector tissue culture (TC) control plants. The binary vectors were transferred into Agrobacterium tumefaciens strain LBA4404 for plant transformation.
Plant transformation and genotyping
Stock plants were maintained in magenta boxes containing 40 mL half-strength M516 medium (PhytoTechnology, Shawnee Mission, KS) with 3% sucrose and 2 g/L gelrite. Plants were transformed using Agrobacterium as described previously (Richael et al., 2008). Transformed plants were genotyped for the presence of P-DNA, T-DNA and backbone DNA using a robust and reliable PCR method, as described in Xin et al. (2003).
Field plot design and maintenance
Field trials using select StAst1- and StAst2-silenced lines were conducted at the University of Idaho Research and Extension Center. Field trials using StAst1-silenced lines were conducted at two additional sites in Idaho and Michigan, respectively. Seeds were sown 3–4 inches deep, at 10-inch spacing within rows and 36-inch spacing between rows. Pre-plant and in-season fertilizer applications were based on soil tests and petiole analyses, respectively, following standard protocols for russet potatoes. Each plot was evaluated qualitatively, in some cases using standardized monitoring scales for differential responses to insect, disease and environmental stresses. Herbicides, insecticides and fungicides were applied as needed. Vines were removed by a flail-type mower at Parma or desiccated with 6,7-dihydrodipyrido(1,2-a:2′,1′-c)pyrazinediium dibromide (Reglone) (Syngenta, Greensboro, NC) 2 weeks prior to harvest. Tubers were harvested using a single-row mechanical harvester, and ∼40 kg of tubers/line/site (equal amounts from each plot) was scored for defects, about 2 weeks later, according to standard industry practices.
Plant growth and grafting
Plants were grown in a controlled greenhouse environment with average day temperatures of 21 ± 3 °C and night temperatures of 16 ± 3 °C and 16-h days, which were facilitated by supplemental high-pressure sodium lights. The plants were grown in SunGro Mix 1 (SunGro Horticulture, Bellevue, WA) (8 L/pot) for 3 months. Fertilizations were carried out at 2, 4 and 6 weeks after planting with All-Purpose MiracleGro 24-8-16 (Scotts Company, Marysville, OH) according to the manufacturer’s recommendations.
Plants were grafted using a technique similar to that described by others (Clayber, 1975; Marcotrigiano and Gouin, 1984). Seedlings were grown in a greenhouse in individual 1-gallon pots for about 3 weeks or until the stems were about 5 mm in diameter. The stem that was to be used as the scion was cut about two to three nodes below the apical meristem. Using a sharp scalpel, all leaves but the youngest were removed from the scion stem, and the bottom was trimmed on either side to form a ‘V’, with a greatest width of 1 mm. All leaves were removed from the stock. The stem that was to be used as the rootstock was cut widthwise to about the third node. The stem was sliced lengthwise down the centre to form a cut that was as long as the ‘V’ of the scion. The scion was then placed into the cut of the rootstock and secured with a strip of parafilm, and the graft was enclosed in a plastic bag fastened with a twist tie. After 7 days, a hole was punctured in the plastic bag to allow the scion to acclimate to a lower humidity, and the bag was removed 2 days later.
RNA isolation and RNA blot analysis
RNA was extracted from 1 g of leaf and tuber tissue using TRIZOL and Plant RNA Reagent (Invitrogen), respectively, according to the manufacturer’s protocol. For RNA blot analysis, 20 μg RNA was blotted to the nylon membrane using the standard protocol. DIG-labelled DNA probes were synthesized using StAst1 and StAst2 primers, according to the protocol from Roche. Prehybridization and hybridization were performed in DIG-Easy Hyb buffer, and washing and developing were carried out as per the protocol (Roche). Nonradioactive digoxigenin RNA gel blot hybridization was performed according to the manufacturer’s recommendations (Roche Applied Science). A 1.1-kb labelled probe for StAst2 was derived from a gene fragment amplified with the primer pair 5′-CTT GCT C AT CAA CGA TTG GCA ATA G and 5′-AGG TCG GAT CAT TTT CCA TTC TG. The primers used to produce a 1.1-kb probe for StAst1 were 5′-GGT TGA TGACTG ATG TCC CCT TTG and 5′-AGT TA G CTC CTT ATT GTG AGC TC. After exposure, the film was developed using a Konica SRX-101A developing machine.
Total ammonium, protein and nitrogen and acrylamide quantification assays
Protein and total nitrogen levels were determined according to the Dumas method as described in Official Methods of analysis of AOAC International (2005, 18th edn, Official Method 992.15, Gaithersburg, MD). The amount of ammonium/μg of protein was quantified at Covance Inc. (Madison, WI) according to protocols described in Official Methods of analysis of AOAC International (2000, 17th edn, Official Method 920.03, Gaithersburg, MD). Acrylamide levels in French Fries were measured as described previously (Rommens et al., 2008).
Determination of ASN and other amino acid levels
Asparagine and other amino acids were extracted by homogenizing 250 mg of ground freeze-dried tissue in 5 μmol sarcosine (internal standard) in 3.0 mL of a 0.03 m triethylamine HCl buffer. Next, 150 μL of 3.2% potassium hexacyanoferrate trihydrate, 150 μL of 7.2% zinc sulphate and 250 μL 0.1 N NaOH with 3.0 mL 0.03 m TEA buffer were added, with vortexing after each addition. The extract was centrifuged for 15 min at 4 °C, 2147 g, and the supernatant was transferred to a new tube. The pellet was re-suspended in 5 mL nanopure water and centrifuged. Supernatant was pooled with the first tube, and the volume was adjusted to 12.5 mL with water. The extracted free amino acids were derivatized using the EZ:faast method (Phenomenex) according to the user’s manual. Derivatized samples were analysed by liquid chromatography–mass spectrometry (LC-MS) using an Agilent 1200 series HPLC system coupled with a 6300 series ion trap. For HPLC, an EZ:faast AAA-MS column (250 × 3.0 mm) was used, and the mobile phase was 10 mm ammonium formate in water (buffer A) and 10 mm ammonium formate in methanol (buffer B), with a gradient elution of 68%–83% buffer B over a period of 0–11 min at a flow of 0.25 mL/min and a column temperature of 35 °C. The mass spectrometer was equipped with an electrospray ionization source and was operated in the positive mode with auto MSn. The source was operated using drying gas (N2; 350 °C; 10 L/min) and nebulizer gas (N2; 30 psi), and the source voltage had a scan range of 50–550 m/z. Automated MS/MS analysis was conducted using Agilent’s SmartFrag software, with a Smart ICC Target of 50 000 and a maximum accumulation time of 200 ms. Bruker’s quant analysis software was used for quantification.
Data are presented as the means of the results of at least three experiments, and the error bars shown represent the standard deviation (SD) of the mean. Significance was determined using the Student’s two-tailed t-test.
We thank Robert Chretien and Michele Krucker for plant transformations and grafting/greenhouse maintenance of plants. Craig Richael and Mike Thornton are acknowledged for managing field trials.