Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta.

Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.

Fermentation and recovery of L-glutamic acid from cassava starch hydrolysate by ion-exchange resin column

Investigators were carried out with the aim of producing L-glutamic acid from Brevibacterium sp. by utilizing a locally available starchy substrate, cassava (Manihot esculenta Crantz). Initial studies were carried out in skate flasks, which showed that even though the yield was high with 85-90 DE (Dextrose Equivalent value), the maximum conversion yield (-34 per cent) was obtained by using only partially digested starch hydrolysate, i.e. 45-50 DE. Fermentations were carried out in batch mode in a 5 L fermenter, using suitably diluted cassava starch hydrolysate, using a 85-90 DE value hydrolysate. Media supplemented with nutrients resulted in an accumulation of 21 g/L glutamic acid with a fairly high (66,3 per cent) conversation yield of glucose to glutamic acid (based on glucose consumed and on 81,74 per cent theoretical conversion rate). The bioreactor conditions most conductive for maximum production were pH 7.5, temperature 30§C and an agitation of 180 rpm. When fermentation was conducted in fed-batch mode by keeping the residual reducing sugar concentration at 5(per cent) w/v, 25,0 g/L of glutamate was obtained after 40 h fermentation (16 per cent more the batch mode). Chromatographic separation by ion-exchange resin was used for the recovery and purification of glutamic acid. It was further crystallized and separated by making use of its low solubility at the isoelectric point (pH 3.2).

Molecular characterization and expression of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of cassava (Manihot esculenta Crantz).

A cDNA, mSOD1, encoding cytosolic copper/zinc superoxide dismutase (CuZnSOD) was cloned and characterized from cell cultures of cassava (Manihot esculenta Crantz) which produce a high yield of SOD. mSOD1 encodes a 152-amino acid polypeptide with a pI value of 5.84. Southern analysis using an mSOD1-specific probe indicated that a single copy of the mSOD1 gene is present in the cassava genome. The mSOD1 gene is highly expressed in cultured cells, as well as in intact stems and tuberous roots. It is expressed at a low level in leaves and petioles. Transcripts of mSOD1 were not detected in nontuberous roots. Transcriptional level of mSOD1 reaches a high level at stationary phase, and then sharply decreases during further culture. In excised cassava leaves, the mSOD1 gene responded to various stresses in different ways. The stresses tested included changes in temperature and exposure to stress-inducing chemicals. Levels of mSOD1 transcript increased dramatically a few hours after heat stress at 37 degrees C and showed a synergistic effect with wounding stress. Levels decreased in response to chilling stress at 4 degrees C and showed an antagonistic effect with wounding stress. The gene was induced by abscisic acid, ethephon, NaCl, sucrose, and methyl viologen. These results indicate that the mSOD1 gene is involved in the response to oxidative stress induced by environmental change.

Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato.

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.

Cloning, partial sequencing and expression of a cDNA coding for branching enzyme in cassava.

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.