Isolation of a cDNA encoding a granule-bound 152-kilodalton starch-branching enzyme in wheat

Abstract

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5'-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80-100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.

Figure 1

Schematic alignment of pABEI and pRN60 cDNA. Hatched area of pABEI coding region (gray box) represents sequence encoding a putative transit peptide and horizontal arrows on the pRN60 cDNA show location of imperfect direct repeats. The four black areas within the coding region represent sequences encoding the highly conserved regions of enzymes belonging to the α-amylase family (Svensson, 1994). DNA fragments used as probes in DNA and RNA hybridizations are indicated below.

Figure 2

RNA-gel analysis of Sbe1 expression during wheat kernel development. A, Analysis of total RNA (20 μg) prepared from developing kernels harvested at different DPA. The blot was hybridized with probe 2 (Fig. 1) and estimated sizes of hybridizing RNA species are shown to the left. Migration of RNA size markers is indicated to the right. B, Same blot as above hybridized with a 25S rRNA DNA probe.

Figure 3

Isolation of cDNA corresponding to 5′ end of 4.6-kb Sbe1c transcript. A, Schematic illustration of the 4.6-kb Sbe1c transcript and product obtained from 5′-RACE analysis. Start of pRN60 sequence and location of PCR primers used in the 5′-RACE and RT-PCR reactions are indicated. B, Gel analysis of 5′-RACE products obtained in reactions with primers indicated and poly(A⁺) RNA prepared from 12-d-old wheat kernels. Arrow indicates migration of product carrying the 5′ end of the 4.6-kb Sbe1c cDNA. Migration of standard DNA fragments are indicated to the right. C, Gel analysis of RT-PCR products obtained from reactions with PCR primers BE65 and BE38.

Figure 4

Nucleotide sequence and deduced amino acid sequence of the 4.6-kb SBEIc transcript produced in the wheat endosperm. Possible polyadenylation sequence is underlined and proposed transit peptide cleavage site is indicated by a vertical arrow. Shadowed regions represent conserved sequences in enzymes belonging to the α-amylase family (Svensson, 1994). Start of pRN60 sequence and location of PCR primers used in the study are indicated.

Figure 5

Schematic illustration of SBEIc precursor encoded by 4.6-kb Sbe1c transcript. DNA sequences corresponding to exons 1 to 14 on wheat genomic Sbe1 (Båga et al., 1999; Rahman et al., 1999) are indicated. Hatched area indicates location of predicted transit peptide and domains 1 and 2 encompass SBEI-like sequences. The location of the four highly conserved regions on (βα)₈ barrels of amylolytic enzymes (Svensson, 1994) are indicated by black boxes, and their sequences are shown below. Highly conserved residues are indicated by asterisks and catalytic residues present only on domain 2 are underlined. SBEIc is aligned with the SBEI-like protein deduced from the wSBEI-D2 cDNA (Rahman et al., 1999) and the wheat 87-kD SBEIb (Repellin et al., 1997).

Figure 6

Expression analysis of Sbe1c in E. coli. A, Schematic illustration of the expression vector pQE-SBEIc carrying sequences encoding mature SBEIc with His tag (black box) added at the amino-terminal end. B, Analysis of BE activity by iodine staining and phosphorylase a stimulation assay. BE activities were determined from the BE-positive strain DH5α and the BE-deficient strain KV832, transformed with plasmids indicated. Construct pREP4-cm expresses the Lac repressor and pQE30 is a cloning vector used for construction of pQE-SBEIc. The BE activity values and ses determined by the phosphorylase a stimulation assay (Hawker et al., 1974) are expressed as μmol Glc-1-P incorporated mg protein⁻¹ min⁻¹ and were determined from three separate experiments. C, SDS-PAGE and immunoblot analysis of recombinant wheat SBEIc produced in E. coli. Total cell extracts of noninduced and IPTG-induced cultures of the BE-deficient strain, KV832, harboring pREP4-cm and plasmid indicated were analyzed. The immunoblot analysis was done with antibodies prepared against wheat 87-kD SBEI. Migration of marker proteins revealed by amido black staining is shown to the right.

Figure 7

Immunoblot analysis of starch granule-bound proteins. A, Analysis of starch granule-bound proteins by SDS-PAGE and silver staining. Migration of marker proteins (St) is shown to the left. B, Immunoblot analysis of starch granule-bound proteins using antibodies prepared against wheat 87-kD SBEI and SBEII. Migration of marker proteins revealed by amido black staining is shown to the right.

Figure 8

SDS-PAGE analysis of starch granule proteins produced in wheat endosperm. A, Analysis of granule-bound proteins produced in developing endosperm of the hexaploid wheat cv Fielder. Solid arrow indicates migration of SBEIc isoforms and open arrow shows migration of the 59-kD GBSSII present in pericarp starch (Nakamura et al., 1998). B, SDS-PAGE analysis of granule-bound proteins extracted from mature kernels of T. monococcum Tm 23 (lane 1), T. tauschii (accession no. PI 511–380) (lane 2), T. turgidum subsp. durum cv Kyle (lane 3), and T. aestivum cv Fielder (lane 4). Arrows indicate proteins recognized by SBEI antibodies and with similar migration as SBEIc.