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Factors affecting fructosyltransferases and fructan exohydrolase activities in Agave
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tequilana Weber var. azul
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María Concepción García-Péreza and Mercedes G. Lópeza, *
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del IPN, CINVESTAV-Irapuato. Km 9.6 Libramiento Norte, Carretera León-Irapuato, 36821.
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Irapuato, Guanajuato, México.
Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados
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* Corresponding author: Tel.: +52 (462) 623 9600; Fax: +52 (462) 624 5846. E-mail address:
mlopez@ira.cinvestav.mx
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Supplementary material
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Inhibition and activation of 1-SST, 1-FFT varied over time
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HgCl2 and AgNO3 clearly inhibited 1-SST and 1-FFT in a time-dependent manner: both
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agents inhibited 1-SST by 82% after 30 min; although inhibition by HgCl2 peaked after 2 h
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at 92% (Table 1). MgCl2 increased 1-SST activity by 11% after 2 h, but ultimately
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decreased 1-SST activity by 38% after 24 h. DNa and SDS, on the other hand, increased
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1-SST activity by 22% and 75%, respectively, after 30 min. DNa increased 1-SST activity
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by 32% after 24 h; the increase in 1-SST activity after 24 h caused by SDS was less than
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1/3 of that caused by DNa after 24 h. HgCl2 and AgNO3 strongly inhibited 1-FFT activity
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throughout the reaction (Table 1). HgCl2 inhibited 1-FFT by 54% after 30 min, by 84% after
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12 h, and by 75% after 24 h. AgNO3 inhibited 1-FFT by 49% after 30 min and by 94% after
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24 h. DNa activated at 1-FFT by 7%, 34%, and 45% after 30 min, 12 h, and 24 h,
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respectively.
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Confirmation of the inhibition or activation of fructosyltransferases
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At the beginning of the reaction (0 h) only sucrose, the substrate, was present in the
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reaction mixture with a retention time (tr) of 12.5 min; there were no fructans in the control
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reaction or in the reactions with inhibitors or activators (Fig. 5a). After 2 h and 24 h,
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however, kestose (tr = 16.7 min) was clearly present in the control reaction and in the
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reactions containing MgCl2, DNa, and SDS, but not in those containing HgCl2 and AgNO3,
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indicating that 1-SST was inhibited by HgCl2 and AgNO3 (Fig. 5b, c). The formation of
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kestose was accompanied by the release of glucose. The glucose levels increased over
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time in the control reaction and in the reactions containing activators; whereas they were
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relatively stable in the reactions containing HgCl2 and AgNO3. The nystose (tr = 21.3 min)
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synthesized by 1-FFT after 0, 2, and 24 h in the presence and absence of inhibitors and
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activators is shown in Fig. 6. At the beginning of the reaction, the substrate, kestose, was
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present along with small amounts of glucose and fructose (Fig. 6a). After 2 h, the control
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reaction and the reaction containing DNa had produced small amounts of nystose,
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whereas the other reactions did not (Fig. 6b). After 24 h, the reactions containing HgCl2
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and AgNO3 have clearly produced much less nystose than the other reactions; AgNO3
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completely inhibited nystose synthesis, while the reaction containing DNa produced even
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more nystose than the control reaction (Fig. 6c). HgCl2 and AgNO3 strongly inhibited 6G-
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FFT activity (Fig. 7). At the beginning of the reaction, the reaction mixtures contained both
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substrates, sucrose (tr = 11.1 min) and kestose (tr = 15.7 min), and small amounts of
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glucose and fructose (Fig. 7). After 2 h, none of the reactions had produced a detectable
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level of neokestose; however, the control reaction and those containing MgCl2, DNa, and
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SDS had produced small amounts of nystose (Fig. 7b). After 24 h, the control reaction and
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the reactions containing MgCl2, DNa, and SDS had produced neokestose; and the
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reactions containing HgCl2 and AgNO3 had produced only a small amount of nystose or no
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nystose at all, respectively (Fig. 7c). The complete inhibition of 6G-FFT activities by HgCl2
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and AgNO3 is consistent with the results of the TLC analysis shown in Fig. 1c.
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Chemical agents altered FEH activity
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We confirmed the inhibition of FEH activity by TLC. The TLC analysis showed that in the
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presence of HgCl2, AgNO3, and SDS, agave fructans were slightly hydrolyzed; however, in
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the presence of MgCl2 and DNa the FEH activity was very similar to that in the control,
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presenting complete hydrolysis (Fig. 8a). The inhibitory effects of HgCl2 and AgNO3 on
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FEH were stronger when chicory fructans were the substrates independently of the inulin
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type (Fig. 8b, c). On the other hand, MgCl2 and SDS were strong activators of FEH. The
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smaller fructose spot compared with those corresponding to MgCl2, SDS, and the control
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confirmed the inhibition of FEH by HgCl2 and AgNO3. Fig. 8d presents the TLC profiles of
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the fructans (10%) substrates used in this work.
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Figure caption
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Fig. 5 The chromatograms show the synthesis of kestose by 1-SST at 0 (a), 2 (b) and 24 h
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(c). STD: mixture of standards, G: glucose, F: fructose, S: sucrose, K: kestose, N: nystose,
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DP5: kestopentaose, Ctr: control, DNa: sodium deoxycholate, SDS: sodium dodecyl
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sulphate
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Fig. 6 The chromatograms show the synthesis of nystose by 1-FFT at 0 (a), 2 (b) and 24
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h (c). STD: mixture of standards, G: glucose, F: fructose, S: sucrose, K: kestose, N:
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nystose, DP5: kestopentaose, Ctr: control, DNa: sodium deoxycholate
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Fig. 7 The chromatograms show the synthesis of neokestose by 6G-FFT at 0 (a), 2 (b)
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and 24 h (c). STD: mixture of standards, G: glucose, F: fructose, S: sucrose, K: kestose,
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6K: 6-kestose, nK: neokestose, N: nystose, DP5: kestopentaose, Ctr: control, DNa:
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sodium deoxycholate, SDS: sodium dodecyl sulphate
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Fig. 8 TLC show fructan exohydrolase activity with three different fructans substrates at
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1%: A. tequilana (AF) (a), Raftilose (RS) DP < 8 (b), Raftiline (RN) DP > 20 (c) and
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Fructans substrates at 10% (d). Ctr: control, STD: mixture of standards, G: glucose, F:
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fructose, S: sucrose, K: kestose, N: nystose, DP5: kestopentaose, Fn: fructan series
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without glucose
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Table 1
Percentage of inhibition or activation of 1-SST and 1-FFT in protoplast extract of A. tequilana micropropagated plants
1-SST
1-FFT
30 min
2h
12 h
24 h
30 min
2h
12 h
24 h
0
0
0
0
0
0
0
0
HgCl2 82.50 +0.28
92.02 +0.76
88.98 +2.22
87.60 +2.80
53.94 +1.25
74.75 +1.11
84.47 +2.26
75.56 +1.63
AgNO3 82.86 +0.34
81.78 +0.42
74.38 +2.86
76.33 +2.39
48.99 +0.69
78.50 +0.58
92.45 +0.53
93.86 +0.42
MgCl2 0.26 +2.14
+11.62 +0.38
+7.17 +4.08
38.58 +5.93
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Ctr
DNa
+22.27 +2.23
14.91 +1.35
+8.84 +2.94
+31.89 +7.36
+7.46 +0.50
+1.99 +2.34
34.16 +2.73
+44.62 +2.59
SDS
+75.10 +2.28
48.11 +0.72
43.08 +2.61
+11.92 +4.92
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_
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+: indicates percentage of activation
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Fig. 8
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