Wheat granule-bound starch synthase I and II are encoded by separate genes that are expressed in different tissues

Abstract

Studies of waxy mutations in wheat and other cereals have shown that null mutations in genes encoding granule-bound starch synthase I (GBSSI) result in amylose-free starch in endosperm and pollen grains, whereas starch in other tissues may contain amylose. We have isolated a cDNA from waxy wheat that encodes GBSSII, which is thought to be responsible for the elongation of amylose chains in non-storage tissues. The deduced amino acid sequences of wheat GBSSI and GBSSII were almost 66% identical, while those of wheat GBSSII and potato GBSSI were 72% identical. GBSSII was expressed in leaf, culm, and pericarp tissue, but transcripts were not detected in endosperm tissue. In contrast, GBSSI expression was high in endosperm tissue. The expression of GBSSII mRNA in pericarp tissue was similar at the midpoints of the day and night periods. The GBSSII genes were mapped to chromosomes 2AL, 2B, and 2D, whereas GBSSI genes are located on group 7 chromosomes. Gel-blot analysis indicated that genes related to GBSSII also occur in barley, rice, and maize. The possible role of GBSSII in starch synthesis is discussed.

Figure 1

Alignment of wheat GBSSII and GBSSI cDNA sequences. Sequences were aligned using the Gentyx-Mac program. Identical nucleotides are boxed. The translational start codons are indicated with solid lines, the first codons of the mature proteins with dotted lines, the stop codons with stars, and the putative polyadenylation signals with double lines.

Figure 2

Alignment of the primary structures of GBSS from several plant species. Sequences were aligned using the Genetyx-Mac program. The wheat GBSSII sequence was deduced from the cDNA. Sources of other sequences are as follows: wheat GBSSI, accession number AF163319; potato, accession number X58453; and pea, accession number X88789. The first amino acid of the mature protein is indicated with a dot. The eight sequence motifs conserved among procaryotic glycogen synthase and plant starch synthase sequences (Cao et al., 1999) are shown, and the 33 conserved residues within these motifs are indicated by asterisks.

Figure 3

N-terminal sequences of GBSSII (Nakamura et al., 1998) and GBSSIs from the A (Wx-A1), B (Wx-B1), and D (Wx-D1) genomes. Amino acids that differ between the GBSSI proteins are shown in outline. X, Undetermined residue.

Figure 4

Dendogram representation of the phylogenetic relationships between GBSS from several plant species. The sources of wheat GBSSI and GBSSII, pea, and potato sequences are as given in Figure 2. Sources of other sequences are as follows: sweet potato, accession number U44126; cassava, accession number X74160; maize, accession number X03935; sorghum, accession number U23945; rice, accession number X62134; and barley, accession number X07932.

Figure 5

Northern-blot analysis of GBSSI and GBSSII mRNA accumulation in wheat tissues. Total RNA (10 μg) was loaded in each lane. After probing with the radiolabeled insert of the GBSSII cDNA (top), the blot was stripped and reprobed with the radiolabeled insert of the GBSSI cDNA (middle). The ethidium-bromide-stained gel is shown at the bottom. CS, Chinese Spring; Pericarp, seed harvested at 3 DPA. Since endosperm development could not be detected at this time, whole seeds were used. Endosperm, Endosperm tissue removed from 20-DPA seed; Waxy Seed, whole seeds of waxy wheat harvested at 20 DPA.

Figure 6

Northern-blot analysis of GBSSI and GBSSII mRNA accumulation at the midpoints of day and night. Total (10 μg) RNA was loaded in each lane. The ethidium-bromide-stained gels are shown at the bottom. Day and night samples were harvested from the same group of Chinese Spring plants at the midpoint of the day, and 12 h later at the midpoint of the night. Pericarp, Seed harvested at approximately 0 DPA (flowering). Since endosperm development could not be detected at this stage, whole seed was used. Endosperm, Endosperm tissue removed from 15-DPA seed.

Figure 7

Southern analysis of DNA from nullisomic-tetrasomic and ditelosomic lines of Chinese Spring wheat. DNA was digested with KnpI and 15 μg was analyzed by electrophoresis. The approximate size of fragments are shown on the right. N1DT1B, Chinese Spring with no 1D chromosomes but with four copies of chromosome 1B; DT2AS, Chinese Spring with no copies of the long arm of chromosome 2A but with four copies of the short arm; DT2AL, Chinese Spring with no copies of the short arm of chromosome 2A but with four copies of the long arm; N2BT2A, Chinese Spring with no copies of chromosome 2B but with four copies of chromosome 2A; N2DT2A, Chinese Spring with no copies of chromosome 2D but with four copies of chromosome 2A.

Figure 8

Southern analysis of GBSSII and GBSSI in various plant species. DNA was digested with HindIII, and 15 μg was analyzed by electrophoresis. Lanes containing taro, pea, potato, soybean, and tomato DNA were also included on the blots, but are not shown since bands hybridizing to GBSSII or GBSSI were not detected.