In vitro pullulanase activity of wheat (Triticum aestivum L.) limit-dextrinase type starch debranching enzyme is modulated by redox conditions☆
Introduction
Debranching enzymes (DBE) belong to the glycoside hydrolase family 13 (http://www.cazy.org/fam/GH13.html; Coutinho and Henrissat, 1999) and catalyze hydrolysis of α(1→6) glucosidic branch linkages of various glucan polymers present in bacteria, plants and animals. The members of the DBE family can be separated into two subclasses, isoamylase (ISA; EC 3.2.1.68) or limit dextrinase (LD; EC 3.2.1.142), based on their substrate specificity: The LD class is often referred to as R-enzyme or pullulanase in plants. ISA type enzymes readily hydrolyze α(1→6) branch linkages in amylopectin and glycogen, but are unable to debranch pullulan, a bacterial polymer of α(1→4)-linked glucose trisaccharide units joined by α(1→6)-linkages. The pullulanase/LD isoforms cleave pullulan, amylopectin and β-limit dextrins, but show no detectable activity on glycogen. All DBE belong to the α-amylase family of starch hydrolytic enzymes which are characterized by four common sequence motifs at the active site (Jespersen et al., 1993). Additional motifs distinguish the ISA and LD types of DBE (Beatty et al., 1999).
The major components of plant starches, amylose and amylopectin, together with starch-derived dextrins constitute the main substrates for DBE. Plant DBE, along with α-glucosidases, α-amylases and β-amylases are active during seed germination when starch granules are degraded to provide glycosyl units at the initial stages of seedling growth. In addition, DBEs have a role in starch biosynthesis as demonstrated by studies of DBE mutants in maize (Pan and Nelson, 1984; James et al., 1995), rice (Nakamura et al., 1996a) and Chlamydomonas reinhardtii (Mouille et al., 1996). Deficiency in ISA activity in maize sugary-1 mutants results in a higher sucrose concentration in kernels and production of a water-soluble and highly branched glucan polymer, denoted phytoglycogen, at the expense of amylopectin (James et al., 1995). Based on similar findings for sugary-1-type mutants in Chlamydomonas, it has been proposed that DBE-type enzymes are responsible for “trimming” of soluble pre-amylopectin branches to allow crystallization and packaging of the glucan polymer into starch granules (Ball et al., 1996). Another model based on studies of starch biosynthesis in Arabidopsis leaves proposes that DBEs prevent build-up of soluble α-glucans, which may yield phytoglycogen upon accumulation (Zeeman et al., 1998). A role for DBE isoforms in the initiation of starch granule formation was suggested from studies on a sugary-1 (notch) mutant in barley (Burton et al., 2002), transgenic barley expressing an LD inhibitor gene (Stahl et al., 2004) and potato tubers with reduced ISA activity (Bustos et al., 2004). In some maize and rice sugary-1 lines, both ISA and LD activities are reduced and the ratio of phytoglycogen to amylopectin in these lines can be related to the level of LD activity (Nakamura et al., 1997; Kubo et al., 1999). Analysis of LD mutants in intact and compromised ISA backgrounds in maize and Arabidopsis have provided support for involvement of both ISA and LD in starch biosynthesis and degradation (Dinges et al., 2003; Wattlebled et al., 2005).
Genes or cDNA clones encoding LD activity in cereal crops have been isolated from barley (Burton et al., 1999; Kristensen et al., 1999), rice (Nakamura et al., 1996b; Francisco et al., 1998) and maize (James et al., 1995; Beatty et al., 1999). In hexaploid wheat, three different loci on chromosome arms 7AS, 7BS and 7DS (http://wheat.pw.usda.gov/wEST/) have been shown to carry sequences with homology to LD genes. In this report we have isolated and characterized a full-length cDNA, denoted TaLD1 (Triticum aestivum Limit Dextrinase), that is expressed from one of the LD genes in T. aestivum cv. Fielder. A recombinant form of TaLD1 was expressed and purified from a bacterial system in an inactive form and thereafter refolded to regain its catalytic activities.
Section snippets
Plant material
Soft white spring wheat (T. aestivum L.cv Fielder) was grown in a growth chamber maintained at 25 °C, and 20±2 °C day and night temperatures, respectively, under a 16 h photoperiod and 93 μE m−2 s−1 light intensity. Developing kernels were collected at various days post anthesis (DPA) and stored at −80 °C until needed. The endosperm and seed coat (aleurone, testa and pericarp) tissues of 10-DPA old kernels were isolated and stored separately at −80 °C.
Mature kernels were surface-sterilized by agitation
Characterization of a wheat LD cDNA
Screening of a wheat cDNA library (Nair et al., 1997) with a 736-bp LD-specific probe resulted in six positive clones. DNA sequence analysis of the six positive clones revealed that the LD-like sequences could be divided into three groups (data not shown), representing three different LD alleles in wheat. The largest clone was designated pTaLD1 (Fig. 1A) and carried a 3300-bp insert with 92% sequence identity to a barley LD gene active in aleurone cells (Burton et al., 1999). Four in-frame ATG
Conclusions
Growing evidence demonstrates that LD-type starch debranching activity is involved in both starch biosynthesis and degradation in several plant species (Kubo et al., 1999; Dinges et al., 2003; Wattlebled et al., 2005). The present study provides further evidence for a strong conservation of LDs between wheat and other cereals, in terms of primary structure and physiological functions. Furthermore, the great sensitivity of the recombinant wheat LD protein to the chemical environment suggests the
Acknowledgments
Financial support from Canada Research Chair program, Canada Foundation for Innovation, Saskatchewan Government (Department of Industry and Research) and Natural Science and Engineering Research Council is gratefully acknowledged.
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The nucleotide sequence is deposited to GenBank with accession number EF137375.
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Present address: Laboratoire d’Ecophysiologie Moléculaire UMR 137-IRD BioSol, Université Paris XII-Val de Marne, 61 avenue du Général de Gaulle, 94010 Créteil Cedex, France.