Factors affecting fructosyltransferases and fructan exohydrolase activities in Agave tequilana Weber var. azul

María Concepción García-Péreza and Mercedes G. Lópeza, *

a Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, CINVESTAV-Irapuato. Km 9.6 Libramiento Norte, Carretera León-Irapuato, 36821. Irapuato, Guanajuato, México.

* Corresponding author: Tel.: +52 (462) 623 9600; Fax: +52 (462) 624 5846. E-mail address:

Supplementary material

Inhibition and activation of 1-SST, 1-FFT varied over time

HgCl2 and AgNO3 clearly inhibited 1-SST and 1-FFT in a time-dependent manner: both agents inhibited 1-SST by 82% after 30 min; although inhibition by HgCl2 peaked after 2 h at 92% (Table 1). MgCl2 increased 1-SST activity by 11% after 2 h, but ultimately decreased 1-SST activity by 38% after 24 h. DNa and SDS, on the other hand, increased 1-SST activity by 22% and 75%, respectively, after 30 min. DNa increased 1-SST activity by 32% after 24 h; the increase in 1-SST activity after 24 h caused by SDS was less than 1/3 of that caused by DNa after 24 h. HgCl2 and AgNO3 strongly inhibited 1-FFT activity throughout the reaction (Table 1). HgCl2 inhibited 1-FFT by 54% after 30 min, by 84% after 12 h, and by 75% after 24 h. AgNO3 inhibited 1-FFT by 49% after 30 min and by 94% after 24 h. DNa activated at 1-FFT by 7%, 34%, and 45% after 30 min, 12 h, and 24 h, respectively.

Confirmation of the inhibition or activation of fructosyltransferases

At the beginning of the reaction (0 h) only sucrose, the substrate, was present in the reaction mixture with a retention time (tr) of 12.5 min; there were no fructans in the control reaction or in the reactions with inhibitors or activators (Fig. 5a). After 2 h and 24 h, however, kestose (tr = 16.7 min) was clearly present in the control reaction and in the reactions containing MgCl2, DNa, and SDS, but not in those containing HgCl2 and AgNO3, indicating that 1-SST was inhibited by HgCl2 and AgNO3 (Fig. 5b, c). The formation of kestose was accompanied by the release of glucose. The glucose levels increased over time in the control reaction and in the reactions containing activators; whereas they were relatively stable in the reactions containing HgCl2 and AgNO3. The nystose (tr = 21.3 min) synthesized by 1-FFT after 0, 2, and 24 h in the presence and absence of inhibitors and activators is shown in Fig. 6. At the beginning of the reaction, the substrate, kestose, was present along with small amounts of glucose and fructose (Fig. 6a). After 2 h, the control reaction and the reaction containing DNa had produced small amounts of nystose, whereas the other reactions did not (Fig. 6b). After 24 h, the reactions containing HgCl2 and AgNO3 have clearly produced much less nystose than the other reactions; AgNO3 completely inhibited nystose synthesis, while the reaction containing DNa produced even more nystose than the control reaction (Fig. 6c). HgCl2 and AgNO3 strongly inhibited 6G-FFT activity (Fig. 7). At the beginning of the reaction, the reaction mixtures contained both substrates, sucrose (tr = 11.1 min) and kestose (tr = 15.7 min), and small amounts of glucose and fructose (Fig. 7). After 2 h, none of the reactions had produced a detectable level of neokestose; however, the control reaction and those containing MgCl2, DNa, and SDS had produced small amounts of nystose (Fig. 7b). After 24 h, the control reaction and the reactions containing MgCl2, DNa, and SDS had produced neokestose; and the reactions containing HgCl2 and AgNO3 had produced only a small amount of nystose or no nystose at all, respectively (Fig. 7c). The complete inhibition of 6G-FFT activities by HgCl2 and AgNO3 is consistent with the results of the TLC analysis shown in Fig. 1c.

Chemical agents altered FEH activity

We confirmed the inhibition of FEH activity by TLC. The TLC analysis showed that in the presence of HgCl2, AgNO3, and SDS, agave fructans were slightly hydrolyzed; however, in the presence of MgCl2 and DNa the FEH activity was very similar to that in the control, presenting complete hydrolysis (Fig. 8a). The inhibitory effects of HgCl2 and AgNO3 on FEH were stronger when chicory fructans were the substrates independently of the inulin type (Fig. 8b, c). On the other hand, MgCl2 and SDS were strong activators of FEH. The smaller fructose spot compared with those corresponding to MgCl2, SDS, and the control confirmed the inhibition of FEH by HgCl2 and AgNO3. Fig. 8d presents the TLC profiles of the fructans(10%) substratesusedin this work.

Figure caption

Fig. 5The chromatograms show the synthesis of kestose by 1-SST at 0 (a), 2 (b) and 24 h (c). STD: mixture of standards, G: glucose, F: fructose, S: sucrose, K: kestose, N: nystose, DP5: kestopentaose, Ctr: control, DNa: sodium deoxycholate, SDS: sodium dodecyl sulphate

Fig. 6The chromatograms show the synthesis of nystose by 1-FFT at 0 (a), 2 (b) and 24 h (c). STD: mixture of standards, G: glucose, F: fructose, S: sucrose, K: kestose, N: nystose, DP5: kestopentaose, Ctr: control, DNa: sodium deoxycholate

Fig. 7The chromatograms show the synthesis of neokestose by 6G-FFT at 0 (a), 2 (b) and 24 h (c). STD: mixture of standards, G: glucose, F: fructose, S: sucrose, K: kestose, 6K: 6-kestose, nK: neokestose, N: nystose, DP5: kestopentaose, Ctr: control, DNa: sodium deoxycholate, SDS: sodium dodecyl sulphate

Fig. 8 TLC show fructan exohydrolase activity with three different fructans substrates at 1%: A. tequilana (AF) (a), Raftilose (RS) DP < 8 (b), Raftiline (RN) DP > 20 (c) and Fructans substrates at 10% (d). Ctr: control, STD: mixture of standards, G: glucose, F: fructose, S: sucrose, K: kestose, N: nystose, DP5: kestopentaose, Fn: fructan series without glucose

1

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 / 2 h / 12 h / 24 h / 30 min / 2 h / 12 h / 24 h
Ctr / 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 / _ / _ / _ / _
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 / _ / _ / _ / _

+: indicates percentage of activation

1

Fig. 5

Fig. 6

Fig. 7

Fig. 8

1