An Arabidopsis gene encoding a chloroplast‐targeted β‐amylase

Abstract

Summary β‐Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that β‐amylases are not involved in the metabolism of chloroplast‐localized transitory leaf starch. However, we have identified a novel β‐amylase gene, designated ct‐Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct‐Bmy encodes a precursor protein which contains a typical N‐terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic β‐amylases of higher plants. Expression of the ct‐Bmy cDNA in E. coli confirmed that the encoded protein possesses β‐amylase activity. CT‐BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT‐BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT‐BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct‐Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.

The ram1 Mutant of Arabidopsis Exhibits Severely Decreased β-Amylase Activity

“…In addition, starch is localized to plastids, whereas most ␤-amylases lack chloroplast transit peptides (Kreis et al, 1987;Monroe et al, 1991;Yoshida and Nakamura, 1991). Although chloroplast-localized ␤-amylases have been identified (Lao et al, 1999), the majority of ␤-amylase activity is extrachloroplastic (Beck and Ziegler, 1989;Nakamura et al, 1991). For example, 80% of the amylase activity present in Arabidopsis rosette leaves is located outside of chloroplasts (Lin et al, 1988b).…”

Identification of Two Plastid-Targetedβ-Amylases in Rice

“…1). In Arabidopsis, AtBAM1 and AtBAM3 belonging to subfamily II target chloroplasts (Lao et al, 1999;Sparla et al, 2006). In addition, total β-amylase activity is signifi cantly reduced in leaves of Arabidopsis bam1 and bam3 mutants, and bam1 and bam3 double mutants have a severe starch-excess phenotype (Fulton et al, 2008).…”

“…In Arabidopsis, analyses of recombinant proteins expressed in E. coli revealed that AtBAM1, AtBAM2 and AtBAM3 have measurable β-amylase activity (Lao et al, 1999;Sparla et al, 2006;Fulton et al, 2008). On the other hand, AtBAM4 lacks β-amylase activity and amino acid residues of the key active site, despite the fact that the bam4 mutant has elevated leaf starch (Fulton et al, 2008;Li et al, 2009).…”

“…1). OsBAM2, OsBAM3, OsBAM4 and OsBAM5 belong to subfamily II corresponding to AtBAM1 and AtBAM3 that encode chloroplast-targeted active enzymes in Arabidopsis (Lao et al, 1999;Sparla et al, 2006). OsBAM7 and OsBAM9 belong to subfamily III.…”

“…In the Arabidopsis genome, nine genes (BAM1−BAM9) encode the β-amylase-like proteins (Smith et al, 2004). Of these isoforms, at least BAM1, BAM2, BAM3 and BAM4 are chloroplast-targeted β-amylase (Lao et al, 1999;Sparla et al, 2006;Fulton et al, 2008). A loss of BAM3 leads to elevated levels of starch in leaves (Kaplan and Guy, 2005).…”