A tuber-specific cDNA library of cassava (Manihot esculenta Crantz) was constructed and a full-length cDNA for granule-bound starch synthase (GBSS, also known as waxy protein), the enzyme responsible for the synthesis of amylose in reserve starch, was cloned. Sequencing of the cloned cDNA showed that it has 74% identity with potato GBSS and 60-72% identity with GBSS from other plant species. The cDNA encodes a 608 amino acid protein of which 78 amino acids form a chloroplast/amyloplast transit peptide of 8.37 kDa. The mature protein has a predicted molecular mass of 58.61 kDa (530 amino acids). Comparison of the GBSS proteins of various plant species and glycogen synthase of bacteria showed extensive identity among the mature form of plant GBSS proteins, in which the monocots and dicots form two separate branches in the evolutionary tree. From analysis of the genomic DNA of allotetraploid cassava, it is shown that GBSS is a low-copy-number gene. GBSS transcript is synthesized in a number of different organs, but most abundantly in tubers. Potato plants were transformed with the cassava GBSS cDNA in antisense orientation fused between the potato GBSS promoter and the nopaline synthase terminator. The expression of the endogenous GBSS gene in these transgenic potato plants was partially or completely inhibited. Complete inhibition of GBSS activity by the cassava antisense gene resulted in absence of GBSS protein and amylose giving rise to almost complete amylose-free potato starch. This shows that also heterologous genes can be used to achieve antisense effects in other plant species.

Branching enzyme is involved in the synthesis of amylopectin in plant reserve starch. A cDNA coding for cassava (Manihot esculenta Crantz) branching enzyme was cloned from a lambda gt11 cDNA library using a potato cDNA probe. The cloned cDNA was partially sequenced. The sequence data confirmed the identity of the clone when compared to that of potato, the homology being ca. 80% at the nucleotide level and 85% at the amino acid level. Furthermore, the cloned cassava cDNA was able to restore branching enzyme activity in a branching enzyme deficient Escherichia coli mutant. Results of the Southern analysis suggested that there is a single gene for this particular branching enzyme in the cassava genome. Study of expression patterns by northern hybridization showed that the gene is highly expressed in tubers. The transcript is detectable in stem and petiole, but not in leaves. In roots, the mRNA is hardly present. The expression levels at different stages of tuber growth are similar with exception of very young tubers in which it is relatively low. It is also shown that there is a difference in the level of branching enzyme expression between different cassava genotypes.

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Granule-bound starch synthase I (GBSS I) is responsible for the synthesis of amylose in starch granules. A heterologous cassava GBSS I gene was tested for its ability to restore amylose synthesis in amylose-free (amf ) potato mutants. For this purpose, the cassava GBSS I was equipped with different transit peptides. In addition, a hybrid containing the potato transit peptide, the Nterminal 89 amino acids of the mature potato GBSS I, and the C-terminal part of cassava GBSS I was prepared. The transgenic starches were first analysed by iodine staining. Only with the hybrid could full phenotypic complementation of the amf mutation be achieved in 13% of the plants. Most transformants showed partial complementation, but interestingly the size of the blue core was similar in all granules derived from one tuber of a given plant. The amylose content was only partially restored, up to 60% of wild-type values or potato GBSS I-complemented plants; however, the GBSS activity in these granules was similar to that found in wild-type ones. From this, and the observation that the hybrid protein (a partial potato GBSS I look-alike) performs best, it was concluded that potato and cassava GBSS I have different intrinsic properties and that the cassava enzyme is not fully adapted to the potato situation.