Variation of Fructan and Its Metabolizing Enzymes in Onions with Different Storage Characteristics
Article ID: 7202103
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Article ID: 7202103
Onions (Allium cepa L.) accumulate fructans, which are fructose polymers, in their bulbs as reserve carbohydrates and as a source of energy for sprouting. Onions with high fructan content and slow fructan degradation by hydrolysis are considered suitable for long-term storage. We have previously found that ‘Pole Star’ accumulates fewer fructans than ‘Kita-momiji 2000’ in their bulbs. In this study, we attempted to clarify the differences in storage characteristics, fructan content, and fructan metabolizing enzyme activities between the two cultivars during storage. Sprouting was not observed in the bulbs of ‘Kita-momiji 2000’ stored at 15 °C for up to 20 weeks, whereas it was observed in ‘Pole Star’ from around 14 weeks. The fructan content during storage showed a gradual decrease in the inner and outer scales of the bulbs in ‘Kita-momiji 2000’, whereas a rapid decrease was observed in ‘Pole Star’. In the basal plate, the fructan contents in ‘Kita-momiji 2000’ were higher than those in ‘Pole Star’ after 16 weeks of storage. Fructan metabolizing enzyme activities were low and constant in ‘Kita-momiji 2000’, whereas their activities increased in ‘Pole Star’ during storage. The low fructan content of ‘Pole Star’ was thought to be due to the high activity of fructan metabolizing enzymes, but the variation of fructan content was difficult to clearly explain using the balance of fructan hydrolase and synthase activities alone.
DP, degree of polymerization; DTT, dithiothreitol; FEH, fructan exohydrolase; Fru, fructose; FW, fresh weight; Glc, glucose; HPAEC, high performance anion exchange chromatography; KEH, 1-kestose hydrolase; NEH, nystose hydrolase; Suc, sucrose; 1-FEH, fructan 1-exohydrolase; 1-FFT, fructan:fructan 1-fructosyltransferase; 1-SST, sucrose:sucrose 1-fructosyltransferase; 6G-FFT, fructan:fructan 6G-fructosyltransferase; 6G&1-FEH, fructan 6G&1-exohydrolase; 6&1-FEH, fructan 6&1-exohydrolase; 6-FEH, fructan 6-exohydrolase.
Fructans are fructose (Fru)-based oligosaccharides and polysaccharides, which are the storage carbohydrates of onions and are beneficial to human health [1]. Onion fructans accumulate abundantly in the bulbs, which are part of the leaf sheath base [2, 3], and are a source of energy for sprouting. Fructans are classified into four categories (a)-(d) according to their linkage type, glucose (Glc) position, and the presence or absence of branching [4]: (a) inulin, a linear Fru polymer with β-(2→1) fructosyl linkages; (b) levan, linear Fru polymers with β-(2→6) fructosyl linkages; (c) graminan, a branched Fru polymer containing equal proportions or equivalent amounts of β-(2→1) and β-(2→6) fructosyl linkages; and (d) neofructans, branched fructans with a Fru polymer at the C6 position of the Glc residue. Neofructans are classified into three subgroups: (d1) neo-inulin, Glc with two β-(2→1) fructosyl chains; (d2) neo-levan, Glc with two β-(2→6) fructosyl chains; and (d3) agavin, Glc with both β-(2→1) and β-(2→6) fructosyl linkages present in the two fructosyl chains, forming a highly branched structure. Onion fructans comprise inulins with a degree of polymerization (DP) ranging from 3 to approximately 6, and neo-inulins with DP ranging from 3 to approximately DP 20 [5, 6].
Onion fructan is synthesized by two enzymes, sucrose: sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) and fructan:fructan 6G-fructosyltransferase (6G-FFT, EC 2.4.1.243) (Fig. S1; see J. Appl. Glycosci. Web site) [7, 8, 9]. 1-SST synthesizes 1-kestotriose (DP 3 of inulins) using sucrose (Suc) as the substrate [7]. The other enzyme, 6G-FFT, synthesizes 6G-kestotriose (DP 3 of neoinulins) from Suc and 1-kestotriose and 1 and 6G-kestotetraose (DP 4 of neoinulins) from two molecules of 1-kestotriose [8, 9]. The fructosyl chains of plant fructans are elongated by fructan: fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), which transfers a fructosyl residue from one 1-kestotriose to another 1-kestotriose or other fructans [10, 11, 12]. The onion 6G-FFT enzyme also exhibits 1-FFT activity, suggesting that 6G-FFT/1-FFT is responsible for elongating of the fructosyl chains of onion fructans [8, 9]. To date, no proteins showing only 1-FFT activity, nor their corresponding genes, have been identified in onions.
Plant fructans are hydrolyzed by fructan exohydrolase (FEH, EC 3.2.1.80), with four types of FEH identified: fructan 1-exohydrolase (1-FEH, EC 3.2.1.153) hydrolyzes β-(2→1) fructosyl linkages of fructan [13, 14, 15], fructan 6-exohydrolase (6-FEH, EC 3.2.1.154) hydrolyzes β-(2→6) fructosyl linkages of fructan [16, 17], fructan 6&1-exohydrolase (6&1-FEH) hydrolyzes both β-(2→1) and β-(2→6) fructosyl linkages of fructan [18], and fructan 6G&1-exohydrolase (6G&1-FEH) preferentially hydrolyzes β-(2→6) linkages between Fru and Glc in 6G-kestotriose and also hydrolyzes β-(2→1) fructosyl linkages of fructan [19]. Unlike bacterial exoinulinases, plant FEHs do not act on Suc. Starch, the general carbohydrate storage form in plants, is synthesized from ADP-glucose by starch synthase and degraded by various glucoside hydrolases and α-glucan, water dikinase [20, 21]. On the other hand, the enzymes involved in fructan synthesis and degradation (fructosyltransferases and FEHs) are both belong to the glycoside hydrolase family 32 (GH32), which includes inulinases and invertases [22]. Fructan metabolizing enzymes have evolved from invertases, and fructosyltransferases belong to the same clade as vacuolar invertases, whereas FEHs belong to the same clade as cell wall invertases [23]. Recently, we showed that onion 1-FEH belongs to the vacuolar invertase group, contrary to previous theories, and localizes to the vacuole like fructosyltransferases such as 1-SST and 6G-FFT [24]. However, how fructan metabolizing enzymes are regulated within the same cellular compartment and how they affect fructan content in onions remain poorly understood.
The fructan content in onions are related to the storage characteristics and sprouting [25, 26]. Onion cultivars with high fructan content and slow fructan hydrolysis can be stored for long periods. The levels and ratios of monosaccharides and disaccharides during storage have been identified as potential predictors of sprouting [27]. In our previous research comparing the fructan content and composition of seven Japanese onion cultivars [28], we found that ‘Kita-momiji 2000’ accumulates sufficient amounts of fructans whereas ‘Pole Star’ mainly accumulates Glc, Fru, Suc, and a small amount of fructans [28]. The difference in fructan content between these two cultivars was observed in the later stages of bulb enlargement, closer to harvest [29]; suggesting that the two cultivars have different storage characteristics and fructan metabolism during storage after harvest. Many studies on variations in the carbohydrates in onions during storage have been reported [25, 30, 31, 32]. Recently, an increase in fructans in the basal plate of onions during storage before sprouting has been reported, and the relationship between increased fructans and sprouting has been discussed. However, few studies have investigated variations in fructan metabolizing enzyme activities during storage [33]. In this study, we stored onions with different storage characteristics and examined changes in fructan content and fructan metabolizing enzyme activities.
Plant materials. Two onion cultivars, ‘Kita-momiji 2000’ (Shippo Co., Kagawa, Japan) and ‘Pole Star’ (Watanabe Seed Co., Miyagi, Japan), were harvested on August 3, 2021, at the Tohoku Agricultural Research Center, NARO, Iwate, Japan, and naturally dried in the dark at room temperature (approximately 23 °C) for 2 weeks. Subsequently, the bulbs were sent to Rakuno Gakuen University and stored in the dark in an incubator at 15 °C and 45 % relative humidity. Over a period of 20 weeks, four bulbs of the stored onions were collected every four weeks for ‘Kita-momiji 2000’ and every two weeks for ‘Pole Star’. At each sampling point, the percentage of sprouting (i.e., the percentage of bulbs with visible buds sprouting from the top relative to the total number of bulbs) was recorded. The collected bulbs were dissected into inner and outer scales and a basal plate using a stainless-steel knife (Fig. S2; see J. Appl. Glycosci. Web site). All the samples were snap-frozen immediately in liquid nitrogen and stored at −30 °C.
Saccharides. Saccharides used in the experiments were prepared as follows: crystalline 1-kestotriose (former name, 1-kestose) and 1,1-kestotetraose (former name, nystose) were prepared from Suc using Scopulariopsis brevicaulis β-fructofuranosidase [34]; 1,1,1-kestopentaose (former name, fructosylnystose [1F (1-β-D-fructofuranosyl)3 sucrose]) and 1,1,1,1-kestohexaose [1F (1-β-D-fructofuranosyl)4 sucrose] were prepared from Jerusalem artichoke tubers in our laboratory; 6G-kestotriose (former name, neokestose), 1 and 6G-kestotetraose (DP 4 of neoinulins, [1F, 6G-di-β-D-fructofuranosylsucrose], 1, 6G-kestotetraose (DP 4 of neoinulins, [6G(1-β-D-fructofuranosyl)2 sucrose]), 1, 1 and 6G-kestopentaose (DP 5 of neoinulins, [1F (1-β-D-fructofuranosyl)2-6G-β-D-fructofuranosylsucrose]), 1 and 1, 6G-kestopentaose (DP 5 of neoinulins, [1F-β-D-fructofuranosyl-6G(1 -β-D-fructofuranosyl)2 sucrose]), and 1, 1, 6G-kestopentaose (DP 5 of neoinulins, [6G(1-β-D-fructofuranosyl)3 sucrose (1, 1, 6G-kestopentaose]) were prepared from asparagus roots as described previously [35, 36, 37].
Extraction and analysis of carbohydrates. Sugars were extracted from 1-5 g of bulbs, according to the method of Ishiguro et al. [38]. The amounts of Glc, Fru, Suc, and fructans were determined using high-performance anion exchange chromatography (HPAEC). HPAEC was performed using an ICS5000 Plus chromatograph (Thermo Fisher Scientific Inc., Sunnyvale, CA, USA) equipped with a CarboPac PA-1 anion-exchange column (Thermo Fisher Scientific Inc.) and a pulsed amperometric detector, as described previously [37]. The flow rate through the column was 1.0 mL/min. Calibration curves prepared from 0, 2, 10, 20 μg/mL of 1-kestotriose were used for the quantification of DP 3 (1-kestotriose and 6G-kestotriose), and those of 1,1-kestotetraose were used for quantifying of DP 4 and higher DPs (DP ≥ 5). The oligosaccharides were identified based on the retention times of the isolated sugars [35, 36].
Preparation and enzyme activity assay of the crude enzyme. The crude enzyme was prepared from onion bulbs at 0-4 °C. Sample (1-5 g) was homogenized in 25 mL of 10 mM sodium phosphate buffer (pH 6.0) containing 2 mM dithiothreitol (DTT, Nacalai Tesque, Inc., Kyoto, Japan) for 2 min using a mixer (Panasonic Corporation, Osaka, Japan). The homogenate was filtered through a cheesecloth and centrifuged (19,000 × g for 20 min). The resulting supernatant was treated with solid ammonium sulfate (Nacalai Tesque, Inc., Kyoto, Japan) to 80 % saturation and stored overnight at 4 °C. After centrifugation at 19,000 × g for 30 min, the precipitated protein was dissolved in 10 mM sodium phosphate buffer (pH 6.0) containing 2 mM DTT. The solution was dialyzed for 2 days against the same buffer. The dialysate was centrifuged at 19,000 × g for 30 min, and the supernatant was used as the crude enzyme. The enzymatic activities of 1-FFT, 6G-FFT, and 1-SST were assayed using 1-kestotriose and Suc as the substrates. Hydrolytic activities were evaluated by quantifying Fru formation using Suc, 1-kestotriose, and 1,1-kestotetraose as substrates.
The reaction conditions for measuring the enzyme activity of the crude enzyme were as follows: a reaction mixture consisting of 25 μL of crude enzyme, 50 μL of 20 mM substrate solutions in distilled water, and 25 μL of 400 mM sodium acetate buffer (pH 5.0) was incubated at 30 °C for 4 or 21 h. The reaction was stopped by boiling the mixture for 3 min. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of reaction products per minute under aforementioned conditions. The 1-SST, 1-FFT, and 6G-FFT activities were calculated based on the quantification of 1-kestotriose, 1,1-kestotetraose, and the sum of 6G-kestotriose and 1 and 6G-kestotetraose as reaction products, respectively. Fru was used as the reaction product to calculate the hydrolytic activities of Suc (invertase activity), 1-kestotriose (1-kestose hydrolase, KEH activity), and 1,1-kestotetraose (nystose hydrolase, NEH activity). The amount of each reaction product was analyzed using HPAEC.
Statistical analysis. Sugar content and enzymatic activity are expressed as the mean ± standard error (SE) on a fresh weight (FW) basis from four onion samples. Differences between ‘Kita-momiji 2000’ and ‘Pole Star’ were analyzed using paired t-tests. Statistical differences were considered significant at P < 0.05. All analyses were performed using SPSS Version 27 (IBM Corporation, Armonk, NY, USA).
Sprouting in onions during storage. During 20 weeks of storage, no sprouting was observed in ‘Kita-momiji 2000’, whereas sprouting in ‘Pole Star’ began from 14 weeks of storage. By 20 weeks, over 70 % of the ‘Pole Star’ samples exhibited sprouting (Fig. S2; see J. Appl. Glycosci. Web site). These results align with those of previous reports [25], indicating that onions with high fructan content rarely sprout during storage and exhibit better storage characteristics than those with low fructan content. For example, onion cultivar ‘Sturon’, which contains a high amount of fructan (420-460 mg/g dry weight), showed a low fructan hydrolysis and high storage stability [25].
Fructan content in the onion bulb during storage. The total fructan content in both the inner and outer scales tended to be lower in ‘Pole Star’ than that in ‘Kita-momiji 2000’ for all the storage periods (Fig. 1). Significant differences were observed in total fructan contents in outer scales at all storage times between the two cultivars (Fig. 1B).
Total fructans (sum of fructan from DP 3 to DP 20) are shown in (A-C), low DP fructan (sum of fructan DP 3, 4, 5), and high DP fructan (sum of fructan from DP 6 to DP 20) are shown in (D-F) and (G-I), respectively. Error bars indicate standard errors (n = 4). Asterisks indicate significant differences between the mean concentrations in the two cultivars at 0, 4, 8, 12, 16, and 20 weeks of storage (t-test, P < 0.05).
The total fructan contents in the outer scales of ‘Kita-momiji 2000’ were lower than those in the inner scales, and a mild decrease was observed in both scales during storage (Fig. 1A, B). The total fructan content in the inner scales of the ‘Pole Star’ at 0 weeks was 29 mg/g FW, comparable to that of ‘Kita-momiji 2000’, but showed a decreasing trend over the next 4 weeks. In the outer scales, the total fructan content of ‘Pole Star’ was lower compared with that at 0 and 2 weeks (Fig. 1B). These results indicate that total fructan content decreases more quickly in ‘Pole Star’ than in ‘Kita-momiji 2000’. The variation in fructan content of each DP in the inner and outer scales of both cultivars is shown in Fig. 1D, E, G, H, and Fig. S3 (see J. Appl. Glycosci. Web site). High DP fructan contents in the inner and outer scales of both cultivars during storage were similar values, but the low DP fructan content of the ‘Pole Star’ were decreasing or lower. This would be the reason for the difference in total fructan content between the two cultivars.
Benkeblia et al. reported that in a storage test with the onion cultivar ‘Tenshin’, total fructan was maintained for the first 6 weeks and then decreased due to sprouting from week 8 onward [33]. Since this report was conducted under the same conditions as the current study, it was expected that fructan would decrease more rapidly in ‘Pole Star’ than in ‘Tenshin’, and more slowly in ‘Kita-momiji 2000’ than in ‘Tenshin’. Our present study demonstrated that ‘Kita-momiji 2000’, which retained high fructan content during storage, did not sprout for 20 weeks in storage. In contrast, ‘Pole Star’ with its low fructan content, showed early sprouting. These findings support the conclusion that a correlation between fructan content and shelf life at harvest exists [25, 26, 27, 30, 31, 32, 33].
In a previous study with ‘Tenshin’, the decrease in low DP fructan during storage was slow because low DP fructan was produced by the hydrolysis of high DP fructan [33]. In the inner scales of ‘Pole Star’, DP 3, 4, 5, and 6 fructans decreased rapidly until 4 weeks and then fluctuated at low levels throughout the storage (Fig. S3; see J. Appl. Glycosci. Web site). Decrease in fructans with DP 7, 8, and 9 and increase in low DP fructans were not clearly observed. On the other hand, a mild decrease in each DP fructan was observed in ‘Kita-momiji 2000’. These results suggest that the hydrolysis of fructan in the bulbs of ‘Pole Star’ is more rapid than that in ‘Kita-momiji 2000’.
In the basal plate, the total fructan contents of ‘Kita-momiji 2000’ and ‘Pole Star’ varied little during storage, but the total fructan contents in the basal plate were higher in ‘Kita-momiji 2000’ than those in ‘Pole Star’ after 16 weeks of storage (Fig. 1C). Each DP fructan content in the basal plate of ‘Pole Star’ changed similarly, while in the basal plate of ‘Kita-momiji 2000’, DP 3, 4, and 5 remained almost unchanged, but DP 6 and the higher DPs increased slightly from 8 weeks onward (Fig. 1F, I, and Fig. S4; see J. Appl. Glycosci. Web site). Increases in the high DP fructan content in the basal plate during storage have been previously reported [39, 40, 41]. Ohanenye et al. found that DP 7 and DP 8 fructans in the basal plate increased 2-6 weeks before sprouting and decreased after sprouting, suggesting that a transient increase in fructans ensures adequate energy and osmotic protection at the basal plate before sprouting [41]. During storage, fructan content in the basal plate seems to be more variable in the sprouted ‘Pole Star’ than unsprouted ‘Kita-momiji 2000’ (Fig. S4; see J. Appl. Glycosci. Web site). To clarify whether the increase or decrease in fructan content in the basal plate is related to the difference in storage stability between the two cultivars, further careful analysis with large sample size is needed.
Glc, Fru, and Suc contents in the onion bulb during storage. Glc content in ‘Kita-momiji 2000’ varied little during storage, while that of ‘Pole Star’ fluctuated in all tissues (Fig. 2A-C). Furthermore, during the first 4 weeks of storage, the Glc content in the inner scale and basal plate of ‘Pole Star’ was higher than that in ‘Kita-momiji 2000’. The slight variations in Glc content in the scales observed in ‘Kita-momiji 2000’ were similar to those of other cultivars [30, 42], and those in ‘Pole Star’ showed a tendency to decrease, especially in the inner scale and basal plate (Fig. 2A, C), suggesting a difference in Glc metabolism in the ‘Pole Star’ from the other cultivars.
Glc, Fru, and Suc are shown in (A-C), (D-F), and (G-I), respectively. Error bars indicate standard errors (n = 4). Asterisks indicate significant differences between the mean concentrations in the two cultivars at 0, 4, 8, 12, 16, and 20 weeks of storage (t-test, P < 0.05).
At the start of storage, differences in Fru content were observed in the basal plate, but no differences were observed between ‘Kita-momiji 2000’ and ‘Pole Star’ in the inner and outer scales (Fig. 2D-F). The amount of Fru in both scales and the basal plate in ‘Kita-momiji 2000’ tended to increase slowly during 20 weeks of storage, and that of ‘Pole Star’ increased to approximately 15-18 mg/g FW in the inner-outer side and 9.0 mg/g FW in the basal plate by the first 4 weeks of storage. The increase in Fru in ‘Pole Star’ was likely due to the degradation of fructan by FEH and the release of Fru. Fru content in onion bulbs gradually continues to increase during storage at 0 °C [25, 30, 42]. In this and previous studies, the temperature during storage was approximately 15 °C [33, 40], and there was a rapid increase in Fru. Thus, Fru accumulation is likely affected differently depending on the storage temperature, and the increased Fru in ‘Pole Star’ would lead sprouting because Fru is used to supply energy for respiration [43]. In all the tissues, Fru content was higher in ‘Pole Star’ than in ‘Kita-momiji 2000’ during the first half of the storage period (2-10 and 12 weeks). Significant differences were observed in Fru content between two cultivars in all tissues at almost all times after storage (Fig. 2D-F). The results of Fru content in the two cultivars suggest that the fructan hydrolase activities of ‘Pole Star’ is higher than that of ‘Kita-momiji 2000’.
Suc content in the inner and outer scales of ‘Kita-momiji 2000’ varied slightly, ranging from 11 to 13 mg/g FW, while the Suc content in the basal plate was increased slightly from 4 weeks onward (Fig. 2G-I). In contrast, a temporary increase was observed at 8 weeks in the basal plate of ‘Pole Star’. The Suc content in the inner scale of ‘Pole Star’ (9.0-15 mg/g FW) was similar to that of the ‘Kita-momiji 2000’, but the amount of Suc in the outer scale of ‘Pole Star’ (5.0-9.0 mg/g FW) was lower than that of the ‘Kita-momiji 2000’. Significant differences were observed in Suc content in the outer scales at all times after storage between the two cultivars (Fig. 2H). The amount of Suc in the entire bulb of ‘Pole Star’ was reported to be less than that of ‘Kita-momiji 2000’ [28, 29], likely due to the lower amount of Suc in the outer scales than those in the inner scales in ‘Pole Star’.
Enzymatic activities of fructan metabolic enzymes in onion bulbs during storage. The activities of fructan hydrolase (KEH and NEH) and synthase (transferase, including 1-FFT and 6G-FFT) during storage in the two cultivars are shown in Fig. 3. The invertase activity for hydrolyzing Suc and the 1-SST activity for synthesizing 1-kestotriose from Suc are shown in Fig. S5 (see J. Appl. Glycosci. Web site). Both the invertase and 1-SST activities were lower than those of the fructan hydrolase (KEH, NEH) and fructosyltransferase (6G-FFT, 1-FFT) activities, as described below.
(A) Enzymatic activities of KEH, NEH, 6G-FFT, and 1-FFT in the inner scale (a, d, g, j), outer scale (b, e, h, k), and basal plate (c, f, i, l) of ‘Kita-momiji 2000’ and ‘Pole Star’ during storage at 15 °C. KEH, NEH, 6G-FFT, and 1-FFT activities are shown in (a-c), (d-f), (g-i), and (j-l), respectively. Error bars indicate standard errors (n = 4). Asterisks indicate significant differences between the mean concentrations in the two cultivars at 0, 4, 8, 12, 16, and 20 weeks of storage (t-test, P < 0.05). (B) Fructan hydrolysis and synthesizing activity ratios in the inner and outer scale, and basal plate of ‘Kita-momiji 2000’ and ‘Pole Star’ during storage at 15 °C. The ratio was calculated using KEH activity as the fructan hydrolase enzyme and 6G-FFT activity as the fructan synthase enzyme. The dotted line is drawn at a ratio of 1.0.
In ‘Kita-momiji 2000’, the fructan hydrolase activity was not drastically changed and remained low across all the tissues during storage, while in ‘Pole Star’ an increase in activity was observed (Fig. 3A). KEH and NEH activities in ‘Pole Star’ showed a similar trend, increasing from 12 weeks onward and showing high activities (Fig. 3A, a, d). These activities in the outer scales showed a slight increase from 0 to 4 weeks, followed by a decrease, and an increase again at 20 weeks (Fig. 3A, b, e). KEH and NEH activities in the basal plate tended to be high around 12 and 14 weeks (Fig. 3A, c, f).
Onion 1-FEH, which we have previously reported [24], has 55-77 % relative activity against 1,1-kestotetraose (NEH activity), with 100 % relative activity against 1-kestotriose (KEH activity). In this study, the activity of the crude enzyme solution in each sample during storage was generally higher for KEH than for NEH, and it did not act on Suc. indicating that onion fructan is degraded by FEH [24] during storage. However, there remains the possibility of other FEH isozymes [44]; therefore, further research to identify candidate genes is necessary.
Fructan synthase activities in ‘Kita-momiji 2000’ showed low values in all the tissues during storage and fluctuated slightly in the basal plate but remained low in the inner and outer scales (Fig. 3A). In ‘Pole Star’, fructan synthase activity in the outer scale was also low and showed little variation, whereas an increase in its activity was observed in the inner scale and the basal plate (Fig. 3A). 6G-FFT and 1-FFT activities in the inner scale of ‘Pole Star’ increased from 0 to 2 weeks, similar to the fructan hydrolase activities, followed by an increase from 12 weeks onward (Fig. 3A, g, j). 6G-FFT and 1-FFT activities in the basal plate of ‘Pole Star’ increased from 12 weeks onward (Fig. 3A, i, l). Purified 6G-FFT/1-FFT native enzyme from the onions showed both 6G-FFT and 1-FFT activities, with a product ratio of approximately 2:1 when 1-kestotriose was used as the substrate [8]. The ratio of 6G-FFT activity to 1-FFT activity in each sample during storage was approximately 2, suggesting that 6G-FFT/1-FFT was expressed in the inner and outer scales and basal plates.
The ratios of fructan hydrolase to synthase activity, based on KEH and 6G-FFT activities, are shown in Fig. 3B. In the inner scales, the KEH/6G-FFT ratio gradually increased during storage in both cultivars, but no clear differences were observed between cultivars. In the outer scale and basal plate, the KEH/6G-FFT ratio of ‘Pole Star’ showed high values compared with those of ‘Kita-momiji 2000’ during the first half of the storage period, suggesting ‘Pole Star’ had higher fructan hydrolase activity than ‘Kita-momiji 2000’ for several weeks after storage. Therefore, the earlier sprouting in ‘Pole Star’ compared to ‘Kita-momiji 2000’ during storage is likely due to the high activities of fructan degrading enzymes in ‘Pole Star’, which result in lower fructan levels and the higher Glc and Fru levels, than those in ‘Kita-momiji 2000’.
Increased enzyme activities for both synthesis and degradation enzymes were observed in the inner scale and the basal plate at approximately 12 weeks, which coincided with beginning of sprouting in ‘Pole Star’. This suggests that fructan synthesis and degradation were simultaneously active in the inner and basal plates during sprouting. The ratios of hydrolase and synthase activities (Fig. 3B) and variations in fructans and Fru (Figs. 1 and 2) seem to be contradictory. For example, during the 0-4 weeks of storage in the outer scale of ‘Pole Star’, hydrolase activity was predominant, resulting in a low level of fructan and an increase in Fru. Notably, the levels of fructan in the inner scale in ‘Pole Star’ in this period decreased and Fru increased, similar to the outer scale. These results suggest that the variation in fructan content cannot be clearly explained through the balance between fructan hydrolase and synthase activities alone.
Fructans are localized in vacuoles, and we have previously shown that onion fructan synthase (6G-FFT/1-FFT, 1-SST) and the hydrolytic enzyme FEH are localized in vacuoles [24]. It is still unknown whether fructan synthases and fructan hydrolases colocalize in the same vacuole at the same time. In ‘Pole Star’, an increase in the activities of the fructan synthases was observed in the early stages of storage, but the activities of the fructan hydrolases increased even more than that of synthases, resulting in a decrease in the fructan content (Fig. 3B). The balance between fructan metabolizing enzyme activities determines the fructan contents in onion tissues, but the mechanism that controls this balance remains unclear. The increase in fructans requires not only high fructan synthase activity but also the supply of Suc. The availability of Suc, regulated by Suc transport, may have a significant impact on the balance of fructan metabolizing enzymes. Thus, investigating the transport of Suc or fructan as a substrate for fructan metabolizing enzymes from the bulb to the basal plate is necessary.
The fructan content and fructan-metabolizing enzyme activities during storage in two onion cultivars with different sprouting characteristics were compared. ‘Kita-momiji 2000’, which did not sprout under the storage conditions, showed constant and low activities of the fructan metabolizing enzymes and a slower decrease in fructan. In contrast, the early sprouting cultivar ‘Pole Star’ showed a faster decrease in fructan. Furthermore, ‘Pole Star’ showed fructan hydrolase activities exceeding fructosyltransferase activities during the first half of the storage period in the outer scale and basal plate tissues. The fructan content in ‘Pole Star’ is thought to be affected by the fluctuating activities of fructan metabolizing enzymes than in ‘Kita-momiji 2000’.
The authors declare no conflict of interests.
This work was supported by a Grant-in-Aid to Cooperative Research from Rakuno Gakuen University. Rakuno Gakuen University Dairy Science Institute, 2020. We would like to thank Editage (www.editage.jp) for English language editing.
