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Keywords:

  • 6-FEH;
  • Arabidopsis thaliana;
  • Beta vulgaris;
  • defence;
  • invertase;
  • Pichia expression

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

About 15% of flowering plant species synthesize fructans. Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated. This paper describes the unexpected finding that 6-FEHs also occur in plants that do not synthesize fructans. The purification, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described. Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots. Classical enzyme purification followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-flight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levan-type fructans (β-2,6) are the best substrates for the enzyme, while inulin-type fructans (β-2,1) and sucrose are poorly or not degraded. Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI), clearly different from typical high pI cell wall invertases. Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely. The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris. As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious. Like chitinases and β-glucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Fructans, a class of water-soluble, fructose-based oligo- and polysaccharides, occur as vacuolar (Wiemken et al., 1986) reserve carbohydrates in about 15% of the flowering plant species (Hendry, 1993). Dicotyledonous species store inulin-type fructans consisting of linear (β-2,1) linked fructofuranosyl units. Inulins are commercially extracted from chicory roots and used mainly for food and feed applications (Roberfroid et al., 1998). Levan-type fructans, consisting of linear (β-2,6) linked fructofuranosyl units, and branched fructan types are found in monocots (Van den Ende et al., 2002). Apart some exceptions (van Hijum et al., 2003), bacterial fructans are of the levan type. They are much longer than plant fructans and are biosynthesized by levansucrases (Han, 1990). Besides having a role as a reserve component, fructans perhaps also fulfil more specific roles in plants and could contribute to drought resistance, frost tolerance or osmoregulation (Hendry, 1993; Hincha et al., 2000; Livingston and Henson, 1998; Vergauwen et al., 2000).

Several fructan biosynthetic enzymes have been purified and cloned (Ritsema and Smeekens, 2003). Fructan breakdown is catalysed by fructan exohydrolases (FEH; G-Fn + H2[RIGHTWARDS DOUBLE ARROW] G-Fn−1 + F with n > 1) essentially transferring a fructose moiety to a water molecule as acceptor. Both 1-FEH (inulinase) and 6-FEH (levanase) can be discerned and are known to occur in bacteria, fungi and fructan plants (Hendry, 1993). Plant 1-FEH enzymes from dicots have been studied extensively (Van Laere and Van den Ende, 2002). From monocots, 1-FEH and 6-FEH iso-enzymes were characterized (Van den Ende et al., 2003 and references therein). Only recently, the first plant 1-FEHs were cloned from chicory (Van den Ende et al., 2000, 2001) and wheat (Van den Ende et al., 2003). Surprisingly, plant 1-FEHs apparently evolved from cell-wall-type and not from vacuolar invertases (Van den Ende et al., 2002). Although both endo- and exo-type 6-FEHs have been characterized and cloned from microorganisms (Han, 1990; Pereira et al., 2001), so far no 6-FEH cDNA has been cloned from any plant species.

FEHs hydrolyse fructan reserves whenever the plants need important energy supplies. Surprisingly, in fructan plants, it was found that FEH is also highly expressed in rapidly growing sink tissues lacking fructans (Van den Ende et al., 2001), suggesting an alternative function. To study this alternative function, it would be interesting to reduce the complexity by choosing a non-fructan plant as a model system. So far, the complete absence of FEH in non-fructan plants has been considered as trivial, and therefore, they were used as host plants for heterologous expression of fructosyl transferases (Caimi et al., 1996; Hellwege et al., 2000; van der Meer et al., 1994; Pilon-Smits et al., 1995; Sévenier et al., 1998).

For a long time, the weak 1-FEH activities found in non-fructan plants were neglected and considered to be the result of non-specific side reactions of invertases. However, when we tested a series of non-fructan plants, including sugar beet with levan as substrate, much higher activities were found. We remained cautious about the existence of plant-specific 6-FEHs, as plant endophytes or bacterial pathogens also could produce 6-FEHs.

This paper describes the characterization, mass mapping, cloning and heterologous expression of a cell-wall invertase-like specific 6-FEH from sugar beet roots, an economically important crop in temperate regions (400 million tons year−1). In contrast to, e.g. Arabidopsis thaliana, sugar beet storage roots are an ideal bulky starting material for protein purification purposes. Some invertases in sugar beet and other plants are induced by wounding and/or by bacterial infection (Rosenkranz et al., 2001; Sturm and Chrispeels, 1990). Extracellular invertases are key metabolic enzymes controlling plant growth and development, but they may also be considered as pathogenesis-related (PR) proteins (Roitsch et al., 2003). However, so far the physiological significance of extracellular invertases in plant–pathogen interactions is not straightforward (Sturm, 1999). They were suggested to play an indirect role by supplying extra hexoses for fuelling a cascade of further defence reactions. It is possible that cell-wall invertase-like plant 6-FEHs directly act on levans synthesized by bacterial pathogens or endophytes.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Purification of the 6-FEH enzyme from sugar beet roots

The 6-FEH enzyme was obtained from both fresh and cold-stored sugar beet roots. The purification procedure was based on (NH4)2SO4 precipitation, lectin affinity chromatography (Con A) and anion and cation exchange chromatography (Table 1). The total activity increased slightly after (NH4)2SO4 precipitation, perhaps because of the removal of an inhibitor. Although activity losses occurred after each further purification step, the purified enzyme was found to be stable when stored for 1 month at 4°C (not shown). A maximal purification of almost 800-fold was obtained and the enzyme had a very high specific activity of 12.5 U mg−1 protein, which is roughly 10 times as much as that for 1-FEHs purified from fructan plants. The amount of pure 6-FEH that could be obtained (estimated at 0.6 µg kg−1) was almost 100 times less than the yield of 1-FEH IIa from chicory (Van den Ende et al., 2001). The pure enzyme (Q 8.7 fraction) was used for enzyme characterization and for electrospray ionization (ESI)-Q-TOF MS analysis, whereas the Q 8.5 fraction was used to visualize protein components on SDS–PAGE. Three low molecular weight bands of 36, 20 and 19 kDa were found to originate from 6-FEH (Figure 1), as proven by ESI-Q-TOF MS analysis (see further Table 3). As judged by gel filtration, sugar beet 6-FEH had an apparent molecular mass of 75 kDa (not shown).

Table 1.  A typical purification of 6-FEH from 3.5 kg sugar beet roots (B. vulgaris)
Purification stepProtein (mg)Total activity (U)Recovery (%)Specific activity (U mg−1 protein)Purification (fold)
  1. Elution pHs on Mono Q are indicated.

Crude extract246.43.911000.0161
30–80% (NH4)2SO4241.54.251090.0442.8
Con A14.01.2030.70.2314.2
Mono Q (7.0)1.951.0927.90.5635.2
Mono S (4.5)0.0450.266.85.87370
Mono Q (8.5)0.0140.133.49.21581
Mono Q (8.7)0.0020.0250.6412.5788
image

Figure 1. SDS–PAGE of sugar beet 6-FEH.

Lane 1: Molecular mass marker proteins. Numbers on the left indicate their mass in kDa. Lane 2: SDS–PAGE of 3 µg of the partially purified sugar beet 6-FEH (Mono Q pH 8.5 fraction). Arrows indicate (a) 19, (b) 36 and (c) 20 kDa polypeptides, which make up the complete sugar beet 6-FEH. The 75-kDa band fragment (m) is not related to 6-FEH; peptide analysis points to an α-mannosidase.

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6-FEH properties

Sugar beet 6-FEH has a pH optimum around 5.0 (Figure 2a) and a temperature optimum around 30°C (Figure 2b). In severe contrast to chicory 1-FEH IIa (De Roover et al., 1999) and wheat 1-FEH w1 and w2 (Van den Ende et al., 2003), the enzyme is not strongly inhibited by sucrose (Ki = 134 mm; Figure 2c). For comparison, the avid sucrose inhibition (Ki of 6 mm) of chicory 1-FEH IIa is also shown (Figure 2c). Because of the low inhibition by sucrose, no inhibitory problems occurred during the kinetic analysis of the enzyme with 6-kestose as a substrate (Km of 77 mm; Figure 2d).

image

Figure 2. Characterization of 6-FEH and activity in planta.

(a) Effect of pH on the activity of purified sugar beet 6-FEH. 6-FEH was incubated for 1 h with 2 mm levan at different pH values using 100 mm sodium acetate buffer (▪) and sodium phosphate buffer (▴). Activity is expressed as a percentage of the maximal activity (i. e. 12.5 U mg−1 protein).

(b) Effect of incubation temperature on the activity of sugar beet 6-FEH. 6-FEH was incubated with 2 mm levan for 1 h at different temperatures between 0 and 60°C.

(c) Double reciprocal plots of the percentage of inhibition of sugar beet 6-FEH (▴) and chicory root 1-FEH IIa (▪) as a function of the sucrose concentration. The fitted linear regression lines and parameters are indicated. From these, Ki-values were derived.

(d) Double reciprocal plot of 6-FEH activity against the concentration of 6-kestose. The fitted linear regression line and its parameters are presented. The Km value was estimated based on these parameters.

Enzymatic activity of 6-FEH (▴), 1-FEH (●) and invertase (▪) in sugar beet storage roots (e), petioles (f) and leaf parenchyma (g) throughout the growing season. In panels (f) and (g), the invertase activity is presented on the left y-axis and the FEH activities on the right y-axis.

(h) Evolution of the percentage of root weight (▵, left y-axis) and the sucrose concentration (□, right y-axis) in the sugar beet storage roots throughout the growing season.

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6-FEH substrate specificity

Sugar beet 6-FEH is an exohydrolase using a multi-chain mechanism of hydrolysis as no products other than fructose could be detected by using levan as a substrate (not shown). No activity was found with sucrose as substrate, indicating that the enzyme is indeed an FEH and not a β-fructofuranosidase or invertase (Table 2). Of all fructans tested, the purified enzyme most efficiently hydrolysed (β-2,6) linkages: levan, DP4-12 phlein, neokestose, 6-kestose and levanbiose are the best substrates, while (β-2,1)-type fructans are poor (1-kestose) or no substrates at all (sucrose, 1,1-nystose and inulin; Table 2). Therefore, the enzyme can be designated as a real 6-FEH. Interestingly, the enzyme is able to remove the (β-2,6) glucose-bound fructosyl unit from neokestose, demonstrating that the presence of a fructose–fructose linkage is not a prerequisite for enzymatic activity.

Table 2.  Comparison of substrate specificities (1 mm) of the native sugar beet 6-FEH and the heterologously expressed sugar beet 6-FEH cDNA in P. pastoris
SubstrateDPNative activity (%)Pichia-derived activity (%)
  1. Results are shown as values relative to the activity with levan as substrate.

Sucrose200
Levanbiose26362
1-Kestose31213
6-Kestose37768
Neokestose393111
1,1-Nystose400
Phlein4–128492
Inulin>1000
LevanNd100100
NeokestinNd2539

Activities of 6-FEH, 1-FEH and invertase in sugar beet, a sucrose-storing plant

Figure 2 also shows the activity profiles of 6-FEH, 1-FEH and invertase in field-grown sugar beet storage roots (Figure 2e), petioles (Figure 2f) and leaf parenchyma (Figure 2g) throughout the growing season (June–October). Except for some of the first and the last sampling dates, the 6-FEH activity in the storage roots (Figure 2e) and petioles (Figure 2f) was rather constant throughout the growing season. As expected, the activity of invertase was very low in the sucrose-storing roots and only the very young roots showed somewhat higher invertase activities. Very high invertase activities were detected in the petioles of young plants, but these activities strongly decreased subsequently (Figure 2f). Invertase and 6-FEH activities greatly fluctuated in leaf parenchyma, with FEH activities about 20 times less than invertase activities (Figure 2g). Most probably, the observed 6-FEH activities in leaf blades and the apparent higher 6-FEH activity in young petioles (Figure 2f) can be explained as a side activity of vacuolar-type invertases. Therefore, it is unlikely that a specific 6-FEH occurs in leaf parenchyma. However, it was found that the 6-FEH/invertase ratio was much higher in the main vein tissue than in leaf parenchyma, indicating the presence of a specific 6-FEH in leaf veins. 6-FEH activities were also demonstrated in both leaves and roots of sugar beet seedlings and 1-month-old plants (not shown). Overall, in roots, petioles and leaf parenchyma, only very low 1-FEH activities were detected (Figure 2e–g).

Sugar beet is a typical sucrose-storing plant used for commercial sucrose extraction. Figure 2(h) shows that the contribution of root weight to the total plant weight first increased from 20 to 40% but remained roughly constant afterwards. The sucrose concentration in the storage roots gradually increased from 300 to 600 µmol g−1 FW (Figure 2h). Although the majority of this sucrose is stored within vacuoles, apoplastic fluid isolations demonstrated the presence of higher sucrose concentrations in the apoplast than could be explained by cellular leakage. To verify that sugar beet is a real non-fructan plant, neutral sugar beet extracts were analysed by anion exchange chromatography with pulsed amperometric detection (AEC-PAD), but no fructans could be detected in any tissue. To test for large fructans not detected by AEC-PAD, the extracts were incubated with 1-FEH and 6-FEH, but no fructose was formed. Acid hydrolysis of the neutral extracts produced equal amounts of glucose and fructose.

Cloning strategy

An RT-PCR-based strategy was used for cloning the 6-FEH cDNA (for details, see Experimental procedures). The primers used were based on both internal 6-FEH peptides generated by Q-TOF MS and conserved regions. The 3′ part was cloned with an oligo dT-based primer and the missing 5′ part was cloned by PCR from a sugar beet root cDNA library by combining vector- and 6-FEH-specific primers. After the sequencing of the 5′ and 3′ cDNA parts, final primers were used to amplify the whole cDNA by RT-PCR. The cDNA coding region is preceded by a short 53-bp 5′ untranscribed part and followed by a longer 141-bp untranslated 3′ region. The sugar beet 6-FEH cDNA contains a single open-reading frame (ORF) of 606 codons and its translated amino-acid sequence is compared with some highly related cell-wall-type invertases and FEHs in Figure 3. Remarkably, the enzyme contains a unique long C-terminal hydrophilic extension (Figure 3), which is not common among cell-wall invertase-like enzymes. The enzyme also contains a unique asparagine-rich region NNNNIN (Figure 3) that is near to the FRDP region and might participate in the formation of the active site (Song and Jacques, 1999). Comparison of the cDNA-derived amino acid sequence with the presumptive N-terminal sequence of the mature sugar beet 6-FEH enzyme (see further Table 3) demonstrates that the primary translation product has a 50 amino-acid signal peptide, which is post-translationally removed. The estimated pI of the mature polypeptide encoded by the sugar beet 6-FEH was calculated at 5.0, which is in agreement with its behaviour on anion and cation exchange columns. Furthermore, the derived polypeptide of the sugar beet 6-FEH cDNA contains nine potential glycosylation sites (N-X-S/T: see Figure 3), of which three occur in the special C-terminal extension (Figure 3). Extensive glycosylation (see also below) might explain the observed difference between the cDNA-derived Mw (62.9 kDa) and the Mw of 75 kDa (19 + 36 + 20) as estimated from SDS–PAGE and gel filtration.

image

Figure 3. Multiple alignment of sugar beet 6-FEH and some highly related translated cDNAs.

Alignment of the deduced amino acid sequences of 6-FEH, cell wall invertase and a cDNA with an unknown functionality of B. vulgaris, a cell wall invertase from A. thaliana and 1-FEH I from C. intybus. Potential glycosylation sites are underlined. The peptide regions where the cloning primers were chosen are indicated in bold and italic. The N-terminal amino-acids of the mature proteins of the sugar beet 6-FEH and chicory root 1-FEH I are indicated with an arrow. Boxed regions are unique and do not occur in other cell-wall-type invertase-like enzymes. The three carboxylic acids that are thought to be crucial for enzyme activity are indicated ($). Consensus line: asterisks (*) indicate identical residues; colons (:) indicate conserved substitutions; and periods (.) indicate semiconserved substitutions.

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Table 3.  Fragment ions detected in Q-TOF after tryptic digest of sugar beet 6-FEH, with calculated matches to theoretical digest of virtual cDNA derived protein, and confirmation of identity by tandem MS/MS sequencing
Observed massCharge stateCalculated mass (presumptive 6-FEH fragment ion)MS/MS sequence (from N- to C-terminus)
  1. Glycosylation sites NX(S/T) are presented in italic and underlined.

  2. t, native purified enzyme, no electrophoresis; a, SDS–PAGE band of 19 kDa; b, SDS–PAGE band of 36 kDa; c, SDS–PAGE band of 20 kDa; h, heterologous Pichia-expressed enzyme.

780.4 (a)2+780.32 [T1 + 2H]2+ (partial)1 DDDPYR 6
1078.6 (t,a,h)3+1078.53 [T2 + 3H]3+7 TAYHFQSPK 15
1579.8 (t,a,h)2+1579.70 [T3 + 2H]2+16 NWMNDPNGPMIYK 28
1595.8 (a)2+No match16 NWMoxNDPNGPMIYK 28 and 16 NWMNDPNGPMoxIYK 28 (T3 derived)
1287.8 (t,a,h)2+1287.71 [T5 + 2H]2+103 NYQVQNLALPK 113
2323.0 (a)3+No matchT6 N-linked glycopeptide: 114 NLSDPYLK 121 (Pen)1 (HexNAc)1 (Deo)1 (Hex)3 (HexNAc)2
1958.0 (a)3+No matchT6 N-linked glycopeptide: 114 NLSDPYLK 121 (Pen)1 (Deo)1 (Hex)2 (HexNAc)2
2976.4 (h)3+No matchT6 N-linked glycopeptide: 114 NLSDPYLK 121 (Hex)7 (Hex)3 (HexNAc)2
4336.3 (h)3+No matchT9 N-linked glycopeptide: 126 LPQNPLMAGTPTNNNNINASSFR 148 (Hex)6 (Hex)3 (HexNAc)2
3085.6 (b)3+No matchPartial T9-10 or T9-11 N-linked glycopeptide: 138 NNNNINASSFRDPSTAWQLSDGK 160 (HexNAc)2 (Pen)1 (Deo)1 (Hex)3 (HexNAc)2 or 141 NINASSFRDPSTAWQLSDGKWR 162 (HexNAc)2 (Pen)1 (Deo)1 (Hex)3 (HexNAc)2
1304.6 (b,h)2+1304.61 [T10 + 2H]2+149 DPSTAWQLSDGK 160
1647.1 (b)3+1646.79 [T10-11 + 3H]3+149 DPSTAWQLSDGKWR 162
1412.0 (b,h)2+1411.75 [T14 + 2H]2+173 GLAVLFTSDDFVK 185
1391.0 (t,b,h)2+1390.71 [T16 + 2H]2+215 SLGADTSLLGDDVK 228
1869.0 (t,h)2+1868.91 [T18 + 2H]2+233 LSLFDTQYEYYTIGR 247
667.4 (b,h)2+667.33 [T19 + 2H]2+248 YDIEK 252
1878.1 (t,b,h)2+1877.91 [T20 + 2H]2+253 DLYVPDEGSLESDLGLR 269
645.4 (b,h)2+645.29 [T21 + 2H]2+270 YDYGK 274
615.4 (b,h)2+615.31 [T22 + 2H]2+275 FYASK 279
1130.6 (t,b,h)2+1130.47 [T23 + 2H]2+280 SFFDDETNR 288
1286.7 (b)2+1286.58 [T23-24 + 2H]2+280 SFFDDETNRR 289
4117.9 (b)3+No matchT25-26 N-linked glycopeptide: 290 ILWGWVNESSTQADDIKK 307 (Hex)7 (Hex)3 (GlcNAc)2
1070.4 (t,b,h)2+1070.57 [T27 + 2H]2+308 GWSGVQAIPR 317
674.6 (b,h)2+674.41 [T28 + 2H]2+318 TVVLDK 323
946.6 (b)3+946. 56 [T28-29 + 2H]3+318 TVVLDKSGK 326
1698.2 (t)2+1697. 90 [T30 + 2H]2+327 QLVQWPLAEVDMLR 340
1370.8 (t,b,h)2+1370.72 [T31 + 2H]2+341 ENDVELPSQVIK 352
962.4 (c,h)2+962.48 [T40 + 2H]2+447 SSLNPTTDK 455
1151.6 (c,h)2+1151.58 [T45 + 2H]2+497 VYPTMAINDK 506
1167.6 (c)2+No match497 VYPTMoxAINDK 506 (T45 derived)
2626.3 (c)3+No matchT47 N-linked glycopeptide: 509 LYVFNNGTEDVKITK 520 (Pen)1 (HexNAc)1 (Man)3 (GlcNAc)2
2829.4 (c)3+No matchT47 N-linked glycopeptide: 509 LYVFNNGTEDVKITK 520 (Pen)1 (HexNAc)2 (Man)3 (GlcNAc)2
3425.8 (h)3+No matchT47 N-linked glycopeptide: 509 LYVFNNGTEDVKITK 520 (Hex)7 (Man)3 (GlcNAc)2

Homology to other glycosyl hydrolases

Similar to the other plant FEHs so far cloned, sugar beet 6-FEH is more related to cell wall invertases (dicots: 49–64% identity; monocots: 45–52% identity) than it is to vacuolar invertases and fructan biosynthetic enzymes (37–48% identity). Similarities to microbial fructan hydrolases are much lower (below 28% identity). Similarities with other known plant FEHs are as follows: chicory 1-FEH I (55% identity), chicory 1-FEH IIa and IIb (52% identity), and wheat 1-FEH w1 and w2 (47% identity).

An unrooted radial tree of some cell-wall-type glycosyl hydrolases is presented in Figure 4. Four distinct groups can be discerned: the first group (I) and the second group (II) contain mainly basic cell wall invertases from monocots and dicots, respectively. A third group (III) contains dicotyledonous and mainly basic cell-wall-type invertases, but there are two exceptions: an acid cell wall invertase from A. thaliana (CW INV5) and the sugar beet 6-FEH (arrow in Figure 4). A fourth group can be further divided in two subgroups: IVa contains dicotyledonous enzymes, harbouring the cloned 1-FEH cDNAs from chicory; and IVb harbours monocotyledonous enzymes, including the wheat 1-FEH cDNAs. Within group IV, all members have an acidic pI. Interestingly, the recently annotated rice genome resulted in six new putative invertase cDNAs (Figure 4). Four of them have a low pI and fall in group IVb, while two others appear in group I.

image

Figure 4. Unrooted tree of cell-wall invertase-like cDNAs.

Unrooted phylogenetic tree containing cell-wall invertase-like cDNA-derived amino acid sequences. Four groups can be discerned. First group (I): Zea mays INV1–INV3; two putative (indicated by ?) and one effective Oryza sativa INV; T. aestivum INV. Second group (II): Vicia faba INV1; Daucus carota INV1–INV3; Solanum tuberosum INV, INVE, INVF; Lycopersicon esculentum INV5; A. thaliana CW INV2 and INV3; Nicotiana tabacum INV. Third group (III): B. vulgaris 6-FEH (arrow) and INV; A. thaliana INV1 and putative INV and INV5; V. faba INV2; Pisum sativum INV; Chenopodium rubrum INV1; and Fragaria × ananassa INV. Fourth group (IVb): Z. mays INV4; four putative and one effective INV from O. sativa; T. aestivum 1-FEH w2; IVa: a putative INV from B. vulgaris; C. intybus 1-FEH I, IIa and IIb and a putative INV; A. thaliana fructosidase. Sequences derived from O. sativa are boxed. Iso-electric points and Accession numbers are presented between brackets. Acid iso-electric points are underlined. The scale bar indicates a distance value of 0.1. INV, cell wall invertase.

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Q-TOF analyses of 6-FEH tryptic fragments

A theoretical tryptic digest of the cDNA-derived 6-FEH protein sequence (Figure 3) yielded 53 peptides (designated T1-T53 from N- to C-terminus). Masses of ZipTip eluted tryptic 6-FEH peptides (originating from a gel-free purification, from three gel-cut bands and from Pichia-expressed 6-FEH) were determined by Q-TOF and compared (Table 3) to the masses of theoretical peptides (with the consideration of one possible missed cleavage site). Most masses detected matched – within the acceptable mass measurement error of ±0.2 Da – with one of the theoretical fragments (Table 3). Collision-induced dissociation (CID) MS/MS analysis yielded a number of sequence tags that proved the identity of the tryptic peptides (Table 3). Most of the unexplained fragments can be understood by the occurrence of oxidized methionines or by the presence of N-linked glycosyl chains. These latter fragments fit perfectly (Table 3) with four out of nine potential N-glycosylation sites (Figure 3). The other unexplained masses can be understood by the post-translational cleavage of the pre-peptide region and by the apparent double cleavage from the mature protein (6-FEH is a heterotrimer on SDS–PAGE). In this way, DDDPYR is the presumptive N-terminal sequence of the mature enzyme as no K or R is directly in front of this peptide region in the translated cDNA (Figure 3). The two other cleavage sites cannot be exactly attributed. Most likely, the first cleavage site is within or adjacent to the asparagine-rich region (cleavage between LPQNPLMAGTPT and NNNNINASSFR or between LPQNPLMAGTPTNNN and NINASSFR; Table 3). The second cleavage site should be between the T31 and T40 peptides (Table 3). In contrast to the native 6-FEH, the heterologously expressed Pichia enzyme is a monomer after SDS–PAGE and thus contains the complete glycosylated tryptic peptide LPQNPLMAGTPTNNNNINASSFR (Table 3).

Heterologous Pichia expression of sugar beet 6-FEH

The heterologously expressed sugar beet 6-FEH enzyme was incubated with various fructan substrates and sucrose (Table 2). Levan was hydrolysed to fructose and the process is linear as a function of time (not shown). Sucrose and inulin were not hydrolysed, demonstrating that the enzyme retained its 6-FEH specificity. A detailed comparison of both native and heterologous enzymes demonstrated a very similar substrate specificity, although the heterologous 6-FEH appeared to have a slightly higher affinity towards neokestose and neokestin (Table 2).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Properties and localization of plant FEHs

An elaborate purification protocol resulted finally in a small amount of pure 6-FEH enzyme. Its purity was confirmed by ESI-Q-TOF MS analysis, where all observed peaks could be explained (Table 3; Figure 1). Like the FEHs purified from fructan plants (De Roover et al., 1999; Marx et al., 1997a,b; Van den Ende et al., 2003), the sugar beet 6-FEH has negligible invertase activity (Table 2). Fungal β-fructo(furano)sidases (EC 3.2.1.80) on the contrary can degrade sucrose as well as fructans. Therefore, plant FEHs should no longer be classified under EC 3.2.1.80 but a new EC number should be created.

The apparent molecular mass of 75 kDa as estimated by SDS–PAGE (Figure 1; 19 + 36 + 20 kDa) and gel filtration is similar to 1-FEH I (72 kDa; Claessens et al., 1990) and 1-FEH II from Cichorium intybus (64 kDa; De Roover et al., 1999), a 1-FEH from Helianthus tuberosus (75 kDa; Marx et al., 1997a), a 6-FEH from Lolium perenne (69 kDa; Marx et al., 1997b), 1-FEH w1 and w2 from Triticum aestivum (70 kDa; Van den Ende et al., 2003) and a 6-FEH from Dactylis glomerata (57 kDa; Yamatoto and Mino, 1985). The apparent heterotrimeric nature of the sugar beet 6-FEH as demonstrated after SDS–PAGE is unique. Indeed, so far all reported plant FEHs and cell-wall-type invertases were characterized as monomeric enzymes. Generally, fructan biosynthetic enzymes and a number of vacuolar invertases occur as heterodimers (Van den Ende et al., 1996) that contain a large subunit (N-terminal) of about 50–55 kDa and a small subunit (C-terminal) of about 20 kDa. Interestingly, a C-terminal subunit of a similar mass of 20 kDa (besides an additional 19 kDa N-terminal one) appears in sugar beet 6-FEH. So far, it is not clear whether these polypeptides represent proteolytic artefacts or real enzyme subunits in planta. The development of specific antibodies and Western blots on rapidly boiled fresh material should resolve this issue. Overall, the physiological significance of proteolytic cleavage of fructosyltransferases into different subunits is unclear. With barley 6-SFT (Hochstrasser et al., 1998), the monomeric Pichia-expressed heterologous enzyme had the same substrate specificity as its native, heterodimeric counterpart, although the invertase side activity increased. The monomeric Pichia-derived 6-FEH from beet keeps about the same 2,6 substrate specificity as the native, heterotrimeric enzyme but, importantly, the invertase activity remained essentially zero (Table 2). It cannot be ruled out that the slightly higher affinity of the Pichia-derived 6-FEH enzyme for neokestose and neokestin is related to the absence of proteolytic cleavage or the presence of different glycosyl chains in the Pichia enzyme (Table 2).

The pH and temperature optima around 5.0 (Figure 2a,b) are similar to most other fructan enzymes known. The estimated Km of the 6-FEH towards 6-kestose (Figure 2c) is in the same range as chicory 1-FEH IIa towards 1-kestose (De Roover et al., 1999). Contrary to chicory 1-FEH IIa (Ki 6 mm; Figure 2c) or the wheat 1-FEHs (Van den Ende et al., 2003), the sugar beet 6-FEH is not avidly inhibited by sucrose (Ki 134 mm). Perhaps two major classes of plant FEHs can be distinguished based on their inhibition by sucrose.

Like vacuolar invertases, FEHs and fructan biosynthetic enzymes, sugar beet 6-FEH has a low pI. Based on vacuole isolation experiments, it is widely accepted that fructans and fructan metabolic enzymes, including FEHs, are located within the vacuoles of fructan plants (Wiemken et al., 1986). However, FEH has been reported in the apoplastic fluid after cold hardening in the fructan plant Avena sativa (Livingston and Henson, 1998), and a low pI soluble apoplastic invertase has been reported in the non-fructan plant maize (Kim et al., 2000). Therefore, FEH localization should be re-considered in both fructan and non-fructan plants.

Plant FEH genes are derived from cell-wall-type invertase genes

So far, no plant 6-FEH cDNAs had been cloned. It is ironic that the first plant 6-FEH cDNA described here (Figure 3) does not originate from a levan-accumulating plant, but from a non-fructan plant. The tryptic peptides retrieved from the purified 6-FEH covered 31% of the cDNA-derived sequence information, with an identity of 100% (Table 3). This result convincingly demonstrates the link between the cDNA and the corresponding purified enzyme. Absolute certainty about the functionality of the clone was obtained by heterologous expression of the 6-FEH cDNA in Pichia pastoris (Table 2).

Sugar beet 6-FEH is more identical to cell-wall than to vacuolar invertases but, although it has a low pI, it does not fall in the same subgroup as the plant 1-FEHs (Figure 4). Further cloning of FEHs from fructan and non-fructan plants should clarify whether groups III and IV (Figure 4) are specific for 2,6 and 2,1 types of FEH, respectively. As only a minority of the translated cDNAs listed in Figure 4 have been identified by means other than homology, it is precocious to call them cell wall invertases. Further heterologous expression (e.g. from the rice and Arabidopsis cDNAs) will show whether they are 1-FEHs, 6-FEHs, fructosidases or genuine invertases. The substantially higher homology of beet sequence to plant cell wall invertases than to bacterial and fungal FEHs is a convincing argument to refute a possible origin of enzyme and cDNA from endophytic or pathogenic microorganisms.

Functions and regulation of FEHs in fructan plants

Up to now, the proposed functions of 1-FEHs in fructan plants were straightforward: (i) to break down the endogenous fructan reserves when energy is needed (Van den Ende et al., 2001); (ii) to de-polymerize fructan to increase the osmotic pressure (e.g. Campanula flower opening; Vergauwen et al., 2000); (iii) cause a cold-induced shift from high to low degree of polymerisation (DP) fructans by partial fructan degradation (Van Laere and Van den Ende, 2002); or (iv) prevent the formation of higher 2,1-type fructans during the period of fructan biosynthesis in wheat (Van den Ende et al., 2003). It cannot be excluded that, especially low DP fructans, are important for membrane stabilization for drought or frost tolerance (Hincha et al., 2000). In contrast to chicory 1-FEH I, chicory 1-FEH IIa and wheat 1-FEH w1 and wheat 1-FEH w2 enzymes (present in high concentrations in some developmental stages or plant parts) are very sensitive to sucrose in vitro (Van den Ende et al., 2001, 2003) and might be largely inhibited in vivo by sucrose under normal circumstances. However, these enzymes would almost immediately be activated by stress-induced sugar starvation (e.g. reduced photosynthesis, defoliation, cold, drought, etc.), allowing the plants to quickly access their fructan reserves. An additional (but slower) increase in FEH activity was reached by upregulating the genes under some types of stress (Michiels, unpublished; Van den Ende et al., 2001).

Putative functions of plant 6-FEHs

A specific 6-FEH appears rather constitutively expressed during the main storage period in (Figure 2e,f) sugar beet roots (sink) and petioles (vascular system) but not in the parenchyma of source leaves. The role of 6-FEHs in non-fructan plants is not obvious and opens a completely new research area. As endogenous fructan substrates are undetectable in sugar beet, the most plausible role for a specific 6-FEH would be to degrade (and/or prevent the formation of) bacterial levans. In analogy to chitinases and β-glucanases hydrolysing cell-surface components of fungal plant pathogens, plant 6-FEHs might participate in hydrolysis of levan-containing slimes surrounding endophytic or phytopathogenic bacteria like, e.g. Pseudomonas or Erwinia.

It was demonstrated that levan solutions exclude plant cell wall polymers like pectins and cellulose. This behaviour supports the idea that levans fulfil a crucial role during the early phase of infection by creating a separating layer between bacteria and plant cell wall polymers, in a way preventing recognition of the pathogen (Hettwer et al., 1995). Further straightforward evidence for the importance of levans during the plant–pathogen interaction comes from the fact that Erwinia amylovora strains deficient in levansucrase had reduced virulence (Bereswill et al., 1997; Tharaud et al., 1994).

A constitutive 6-FEH preventing bacterial levan synthesis would allow the plant to recognize pathogens at an early stage and induce a set of PR enzymes. This would be particularly important in sink tissues of sucrose-storing plants like sugar beet (Figure 2e), carrot and sugar cane (preliminary results). It can be further hypothesized that 6-FEHs might be involved in stabilizing symbiosis between plants and fructan-producing endophytic bacteria known to occur in sugar beet taproots (Tallgren et al., 1999) and sugar cane (e.g. Gluconacetobacter diazotrophicus; Hernandez et al., 2000). In this respect, 6-FEH might function as a detoxification enzyme, as it was demonstrated with transgenic plants expressing levansucrase that levans become toxic at higher concentration in many plants (as discussed by Cairns, 2003).

It is known that some extracellular invertases, essential for supplying hexoses to sink organs in apoplasmic unloaders, are induced by sugars, wounding, phytohormones, fungal and bacterial infection and elicitors (Benhamou et al., 1991; Sinha et al., 2002; Sturm and Chrispeels, 1990; Zhang et al., 1996). They seem to be regulated by an identical set of stimuli inducing well-known defence-related genes. Therefore, extracellular invertases may be considered as PR-related enzymes (Roitsch et al., 2003), although there is no expected direct inhibiting effect of invertases on the microorganisms in vitro. The physiological relevance of the upregulation of extracellular invertases has always been understood in terms of creating a localized increase in hexoses, necessary to sustain and even induce a cascade of further defence reactions. Co-expression of extracellular invertases and specific hexose transporters has been demonstrated (Ehness and Roitsch, 1997; Fotopoulos et al., 2003; Weschke et al., 2003). It would be interesting to investigate whether a 6-FEH is also co-expressed in these systems. Indeed, co-expression of invertase, 6-FEH and hexose transporters would: (i) prevent bacterial fructan synthesis by hydrolysing most of the apoplastic sucrose, the substrate of levansucrase; (ii) allow an efficient take up of hexoses in the cell as a lot of energy is necessary for the cascade of defence reactions; and (iii) prevent the formation and/or hydrolyse the levans that still could be produced by the bacteria.

The low concentration of levan found in sugar beet transformed with a bacterial levansucrase gene (Pilon-Smits et al., 1999) might be related to the presence of 6-FEH, but the subcellular localization of both the 6-FEH and the levansucrase remains to be investigated. However, it was reported (Ritsema and Smeekens, 2003) that sugar beets harbouring two fructosyltransferases from onion (targeted to the vacuole) are able to synthesize neokestose and neokestose-based onion-type fructans. As neokestose is an excellent substrate of sugar beet 6-FEH (Table 2), this might indicate that 6-FEH cannot be present in the vacuole but rather outside the cell. This would be the preferential localization for a putative constitutive defence enzyme-like 6-FEH.

Conclusion and perspectives

Although 6-FEH or levanase activities are well-known in fructan plants, fungi and bacteria (see Introduction), the occurrence of specific 6-FEH enzymes in non-fructan plants was never demonstrated or even suspected. In this paper, we describe the purification, characterization, cloning, mass mapping and functional analysis of 6-FEH from sugar beet, a novel enzyme from an agro-economically important species. The absence of fructans in sugar beet, the clear (β-2,6) substrate specificity of the enzyme and its apparent constitutive expression in sink tissues suggest that the role of 6-FEH might be to prevent the formation of bacterial levans from sucrose. Proving this role will be a challenging task, as an understanding of the interaction between 6-FEH and levan-producing bacteria in vitro and in vivo will be necessary.

The Arabidopsis genome contains six putative cell wall invertase genes (Sherson et al., 2003). If some of these might prove to be genuine FEHs, e.g. after Pichia expression, the availability of knock-out plants should greatly help in clarifying the role of these novel enzymes. Once the function of 6-FEH in non-fructan plants has been resolved, it will be necessary to investigate whether some of the FEHs in fructan plants might have an exogenous rather than endogenous substrate.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material

Beta vulgaris L. (cv. Opus) was sown in a local field with sandy loam soil on April 26, 2002. For RT-PCR, a number of sugar beet roots were uprooted on September 30, 2002. For purification, fresh September–October roots were taken as well as roots that were stored at +1°C for various time intervals starting from November 7, 2002. For enzyme activity measurements, representative samples of field-grown sugar beet plants (roots, petioles and leaves without the major vein) were collected weekly between June 21 and October 5, and samples were stored at −80°C for later analysis. Sugar beet seedlings and young plants were also grown in a controlled growth chamber as described by De Roover et al. (2000). A number of 6-day-old seedlings and 1-month-old plants were harvested. Samples for RNA and for enzyme activity measurements were taken from both root and leaf material.

Carbohydrate analyses and enzyme activity determinations

Sugar beet tissues (stored at −80°C) were ground with mortar and pestle until a very fine powder was obtained. Subsequently, an equal amount of extraction buffer (EB; 50 mm sodium acetate pH 5.0 containing 10 mm NaHSO3, 1 mm mercaptoethanol and 0.1% (w/v) Polyclar AT; Serva, Heidelberg, Germany) was added and homogenized with mortar and pestle. Further handlings were as described by Van den Ende and Van Laere (1996). To measure the activities of 1-FEH, 6-FEH and invertase, aliquots were incubated with 3% (w/v) commercial chicory root inulin (Sigma-Aldrich, St Louis, USA), 10 mm levan and 100 mm sucrose in 50 mm sodium acetate buffer pH 5.0 for different time intervals at 30°C. Sodium azide (0.02%, w/v) was added to all buffers to prevent microbial growth. Fructose formation was determined by AEC-PAD as described by Van den Ende and Van Laere (1996). Throughout purification, aliquots were taken for activity determination with 2 mm levan as substrate. Enzymatic activity is expressed in units (U), defined as the amount of enzyme, which formed 1 µmol fructose min−1 at 30°C.

Fructan substrates

1,1-Nystose and levanbiose were prepared as described by Van den Ende et al. (2003). Low molecular weight levan (produced by immobilized Bacillus subtilis levansucrase; Iizuka et al., 1993), 6-kestose and 1-kestose were generous gifts from Dr Iizuka. Neokestose, phlein and neokestin were kindly provided by Dr Chatterton and Dr Livingston, respectively. The mean DP of levan, phlein and neokestin was estimated from the fructose/glucose ratio after mild acid hydrolysis in 60 mm HCl at 70°C for 75 min. Subsequently, molar concentrations were estimated based on this mean DP and the glucose liberation. A concentration of 1 mm of all fructan substrates were used for testing substrate specificities of both the native and the heterologous 6-FEH.

Purification of sugar beet 6-FEH

Both fresh (harvested in October) and stored (1–4 months at 4°C) sugar beet roots were used as a starting material for purification. A total of 3.5 kg roots were washed, cut into small pieces and subsequently homogenized with a Waring blender in 1.75 l EB. The homogenate was squeezed through cheese cloth. An (NH4)2SO4 precipitation step was performed as described by Van den Ende et al. (2003). The precipitate was collected and re-dissolved in 150 ml 50 mm sodium acetate buffer of pH 5.0 containing 1 mm of CaCl2, MgCl2 and MnCl2. Undissolved material was spun down for 10 min at 40 000 g and 4°C. The supernatant was applied to a Con A Sepharose column (25 mm × 100 mm) and eluted as described by Van den Ende et al. (1996). Active fractions (already free from invertase activity at this point) were pooled, adjusted to pH 7.0 with concentrated Tris and applied on a Mono Q column (Pharmacia Biotech HR 5/5, Uppsala, Sweden) equilibrated with 20 mm Tris–HCl buffer pH 7.0. Active fractions were pooled, diluted five times with 50 mm sodium acetate buffer pH 4.5 and the pH was adjusted to pH 4.5 with concentrated acetic acid. Subsequently, the enzyme mixture was loaded onto a pre-equilibrated Mono S column (Pharmacia Biotech HR 5/5, Uppsala, Sweden). The active fractions were diluted 10 times with 50 mm HEPES buffer pH 8.5. These were applied on a pre-equilibrated Mono Q column. Only the most active fraction was re-chromatographed on a next Mono Q column equilibrated with 50 mm HEPES buffer pH 8.7, resulting in a very small amount of pure protein (as proven after Q-TOF analysis). Proteins were eluted from Mono Q and S columns by using a linear gradient from 0 to 0.3 m NaCl in 30 min (flow rate 1 ml min−1) and fractions of 1 ml were collected. Whenever possible, enzymes were kept on ice and 0.02% sodium azide (w/v) was added to all buffers to prevent microbial growth. Proteins were measured by the method described by Sedmak and Grossberg (1977). SDS–PAGE was performed on 12.5% (w/v) polyacrylamide gels; staining was with Coomassie Brilliant Blue-R250 as described by Van den Ende et al. (1996). A Superdex XK 16 (Pharmacia Biotech, Uppsala, Sweden) column was used for gel filtration and reference proteins were as described by Van den Ende and Van Laere (1995).

RNA preparation, cloning and sequencing

Total RNA was isolated from field-grown sugar beet storage roots by using the Rneasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Four primers were constructed based on the conserved amino-acid sequences found in sugar beet and other cell-wall-type invertases (bold and italic in Figure 3): YRTAYHF (5′-TACAGGACNGCNTAYCAYTT-3′), NDPNG (5′-TGGATGAAYGAYCCNAAYGG-3′), WECPD (5′-GAATGTGGGARTGYCCNGA-3′) and VFNNGT (5′-GGTNCCRTTRTTRAANAC-3′). Further, four 6-FEH-based degenerated primers were designed based on the ESI-Q-TOF MS/MS internal sequences (Table 3): IIGDDVK (5′-ATHATHGGNGAYGAYGTNAA-3′), KSFFDDE (5′-AARTCTTTYTTYGAYGAYGA-3′), WSGVQAI (5′-GGAATNGCYTGNACNCCNGACCA-3′) and VQWPLAE (5′-CYTCNGCNANNGGCCAYTGNAC-3′). One-step RT-PCR was performed with primers YRTAYHF and VFNNGT (Access RT-PCR System, Promega, Madison, WI, USA) but no clear bands appeared at this stage. RT reaction was at 47°C. PCR conditions: 94°C, 3 min; followed by 35 cycles: 94°C, 40 sec; 47°C, 40 sec and 72°C, 2 min. Final extension was at 72°C, 10 min. Subsequently, nested PCR (30 cycles, annealing temperature 54°C) was performed with MWECPD and VQWPLAE. Different bands still appeared and therefore a next PCR (30 cycles, annealing temperature 50°C) with IIGDDVK and WSGVQAI was performed resulting in a single band of about 300 bp. The 300-bp band was ligated in the TOPO-TA vector and transformed to E. coli (TOPO-TA cloning kit, Invitrogen, Groningen, the Netherlands). Plasmid was extracted using the Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA). Partial sequencing resulted in the development of two specific primers: GRYDIEK (5′-GGGAGGTATGACATTGAGAAGG-3′) and RILWGW 5′-ACCCACCCCCATAGTATTCTT-3′). To obtain a longer fragment, RT-PCR was performed with primers TAYHF and VQWPLAE. Subsequently, nested PCR (30 cycles, annealing temperature 57°C) was performed with NDPNG and RILWGW. From the sequence of this 850-bp fragment, a highly specific antisense primer PVWHTE (5′-TTTCAGTGTGCCATACAGGAT-3′) was selected. The 3′ part of the cDNA was cloned by RT-PCR using GRYDIEK and an oligo dT-based primer (35 cycles, annealing temperature 59°C) followed by seminested PCR (30 cycles, annealing temperature 52°C) with KSFFDDE and oligo dT. The missing 5′ part of the cDNA was cloned by PCR on a sugar beet root cDNA library by combining the vector-specific primer M13F (5′-GTAAAACGACGGCCAG-3′) with RILWGW (30 cycles, annealing temperature 50°C), followed by a nested PCR with another vector-specific primer T7 (5′-GTAATACGACTCACTATAGGGC-3′) and PVWHTE (30 cycles, annealing temperature 56°C). After sequencing of the resulting 400-bp fragment, final primers BFINF (5′-AACAATGGGAGTTGGTTAGTGCTG-3′) and BFINR (5′-TACGCAGCAACTAATTTGGGACAC-3′) were designed from the outer 5′ and 3′ cDNA parts and used to amplify the whole cDNA by RT-PCR. First, total RNA was reverse transcribed with Avian Myeloblastosis Virus (AMV) Reverse Transcriptase XL (TaKaRa) and BFINR at 50°C during 1 h. A number of independent PCRs were performed with BFINF and BFINR as primers and proofreading Pfu DNA polymerase (Promega, Madison, WI, USA). PCR conditions: 35 cycles of 94°C, 30 sec; 64°C, 40 sec and 72°C, 5 min. After A-tailing, fragments were ligated in the TOPO-XL vector (TOPO-XL cloning kit, Invitrogen, Groningen, the Netherlands).

From a number of clones, the full-length sugar beet 6-FEH fragment was fully sequenced on both DNA strands and phylogenetic trees were constructed as described by Van den Ende et al. (2000). The sequences were deposited in the European Molecular Biology Laboratory (EMBL) sequence library (Accession number AJ508534).

Q-TOF analyses on tryptic fragments

The SDS–PAGE protein bands of 6-FEH (36, 20 and 19 kDa) exhibiting 1-FEH activity were subjected to mass spectrometric (MS) identification. The Coomassie BB stained protein bands were excised, trypsinized, extracted, de-salted and analysed on Q-TOF under reduced pressure as described earlier. A low amount of pure 6-FEH obtained from the final Mono Q 8.7 was concentrated 10 times. DDT was added to a final concentration of 10 mm, and the enzyme mixture was subsequently heated at 65°C for 1 h. After cooling, 10% (v/v) acetonitrile and 0.1 µg of trypsin (sequencing-grade-modified porcine trypsin, Promega, Madison, WI, USA) were added, and the mixture was incubated overnight at 37°C. Further analysis was performed as described by Van den Ende et al. (2001). Sequence information was derived from the MS/MS spectra with the aid of the MaxEnt 3 (de-convoluting and de-isotoping of data) and pepseq software from the micromass biolynx software package.

Heterologous Pichia expression

To construct the expression plasmid pSB6FEH, containing the mature protein part (starting from DDDPYR, see Figure 3), PCR was performed by using the primers 6FEHF (5′-GCCGGAATTCGATGATGATCCATATAGAACAGC-3′) and 6FEHR (5′-GAATGCGGCCGCGGTTGTCAGAACTCTTCTTTCTC-3′). The restriction sites of EcoRI and NotI are indicated in bold in the primers. Pfu Proofreading polymerase (Promega, Madison, WI, USA) was used, and three independent PCR reactions were performed in parallel (annealing temperature 52°C for five cycles, followed by 30 cycles at 57°C). Both the amplified fragment and the pPICZα A vector (Invitrogen, Groningen, the Netherlands) were digested with EcoRI and NotI. Subsequently, the DNA fragments were further purified by using the Ultraclean DNA Purification Kit (MOBIO, Solana Beach, CA, USA). The pPICZα A vector was cut out of a 1.1% (w/v) agarose gel by using the crystal violet visualization method (as described in the manual of the TOPO XL Cloning Kit from Invitrogen). After vector de-phosphorylation, the PCR fragment was cloned into the vector, resulting in the expression plasmid pSB6FEH, having the 6-FEH in frame behind the α-factor signal sequence of the vector. This plasmid was transformed to E. coli competent cells as described by Van den Ende et al. (2001). Cells were plated on a 2× yeast tryptone (YT) medium supplemented with zeocine as a selection marker. Positive colonies were used for vector amplification. Pichia pastoris strain X33 was subsequently transformed by using electroporation with both 20 µg of SacI linearized pSB6FEH and with empty vector (as a control). Further selection and handlings were as described by Kawakami and Yoshida (2002), except that a concentration of 2% (v/v) methanol was maintained in the Pichia expression media.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

W. Van den Ende is supported by grants from the Fund for Scientific Research (FSR, Flanders). B. De Coninck has a grant from the Belgian IWT. We thank E. Nackaerts for his technical assistance. We thank Prof. M. McGrath from the Michigan State University (USA) for providing the sugar beet cDNA library. We thank Prof. Dr J. Bennett from IRRI (Philippines) for reviewing the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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Accession number of sugar beet 6-FEH cDNA: AJ508534.