Jennifer A. Thomson

An aldose reductase homolog from the resurrection plant Xerophyta viscosa Baker

Abstract. An aldose reductase homologue (ALDRXV4) was cloned from the resurrection plant Xerophyta viscosa Baker using complementation by functional sufficiency in Escherichia coli. A cDNA library constructed from X. viscosa leaves dehydrated to 85%, 37% and 5% relative water contents (RWC) was converted into an infective phagemid library. Escherichia coli (srl::Tn10) cells transformed with ds-pBluescript phagemids were selected on minimal medium plates supplemented with 1 mM isopropyl β-d-thiogalactopyranoside and 1.25 M sorbitol. Nine cDNA clones that conferred tolerance to the osmotically stressed E. coli cells were selected. The phagemid from one clone contained the ALDRXV4 insert. The E. coli cells expressing ALDRXV4 were capable of tolerating the osmotic stress, whereas control cultures were not. The ALDRXV4 insert contained an open reading frame that can code for 319 amino acids, and the predicted protein had a calculated Mr of 35,667. Amino acid sequence comparisons revealed significant similarity to several aldose reductases, with the highest similarity to aldose reductase proteins from Hordeum vulgare, Bromus inermis and Avena fatua, in the order of 66%, 65% and 65% respectively. Northern blot analysis revealed that ALDRXV4 was expressed only under dehydration conditions in X. viscosa leaves. Western blot analysis detected a protein of 36 kDa under dehydration conditions only. Aldose reductase activity levels in X. viscosa leaves increased as the leaf RWC decreased, whereas there was no significant change in aldose reductase activity in Sporobolus stafianus as the leaf RWC decreased.

The role of biotechnology for agricultural sustainability in Africa

Sub-Saharan Africa could have a shortfall of nearly 90 Mt of cereals by the year 2025 if current agricultural practices are maintained. Biotechnology is one of the ways to improve agricultural production. Insect-resistant varieties of maize and cotton suitable for the subcontinent have been identified as already having a significant impact. Virus-resistant crops are under development. These include maize resistant to the African endemic maize streak virus and cassava resistant to African cassava mosaic virus. Parasitic weeds such as Striga attack the roots of crops such as maize, millet, sorghum and upland rice. Field trials in Kenya using a variety of maize resistant to a herbicide have proven very successful. Drought-tolerant crops are also under development as are improved varieties of local African crops such as bananas, cassava, sorghum and sweet potatoes.

Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors

SummaryVirion sense gene replacement derivatives of maize streak virus (MSV) were constructed with selectable marker expression cassettes based on the bialaphos resistance gene (bar) and the CaMV 35S promoter. The effect on replication of increasing the genomic size was tested by including: (1) the 550-bp maize adh I intron and 68-bp TMV Ω RNA leader sequences upstream of the bar genes; and (2) a fusion between the bar and E. coli glutathione reductase (gor) genes. Three recombinant viral vectors ranging in size from 2.7 kb to 4.8 kb replicated efficiently in biolistically transfected cells of suspension cultured Black Mexican sweetcorn (BMS) cells. Deletions greater than 39 bp 3′ of the stemloop sequence in the LIR adversely affected replicon release. Transformed bialaphos-resistant BMS cell lines were generated with all three vectors containing the bar gene: between 38 and 60% of cell lines contained replicating viral episomes. The replicons were structurally stable, replicated to copy numbers of over 500 per haploid genome, and were detected for more than one year after introduction. We noted significant enhancement of bar gene expression, both at the protein and RNA levels, associated with the presence of episomal vector DNA. The maize adhI intron and TMV Ω RNA leader sequences did not seem to have a significant effect on bar gene expression from replicating constructs, although expression from controls was enhanced. The results suggest that MSV-based constructs would provide a useful system for long-term gene amplification in cereal cell culture systems.

Coat protein-mediated resistance in transgenic plants

SummaryThis review describes the proposed mechanism(s) of classical virus cross-protection in plants, followed by those suggested for coat protein-mediated resistance (CP-mediated resistance). Although both have common features, cross-protection is thought to be a complex response caused by the replication and expression of the entire viral genome, whereas the resistance conferred by the expression of a virus coat protein gene is more limited. The term genetically engineered cross-protection is frequently used because in many cases the phenotype of resistance mimics that of cross-protection. However, CP-mediated resistance, although a narrow term, more accurately describes the resistance that results from the expression of a virus CP gene in transgenic plants.

Cloning, sequencing and expression of a gene encoding a 73 kDa xylanase enzyme from the rumen anaerobe Butyrivibrio fibrisolvens H17c

SummaryThe cloning, expression and nucleotide sequence of a 3 kb DNA segment on pLS206 containing a xylanase gene (xynB) from Butyrivibrio fibrisolvens H17c was investigated. The open reading frame (ORF) of 1905 by encoded a xylanase of 635 amino acid residues (Mr 73156). At least 850 by at the 3′ end of the gene could be deleted without loss of xylanase activity. The deduced amino acid sequence was confirmed by purifying the enzyme and subjecting it to N-terminal amino acid sequence analysis. In Escherichia coli C600 (pLS206) cells the xylanase was localized in the cytoplasm. Its optimum pH for activity was between pH 5.4 and 6, and optimum temperature 55° C. The primary structure of the xylanase showed a significant level of identity with a cellobiohydrolase/endoglucanase of Caldocellum saccharolyticum, as well as with the xylanases of the alkaliphilic Bacillus sp. strain C-125, B. fibrisolvens strain 49, and Pseudomonas fluorescens subsp. cellulosa.

Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1.

The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) from Ruminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CelA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other organisms. Two lysozyme-type active sites were found in the amino-terminal third of the enzyme. In E. coli the cloned Cel A protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposon phoA mutagenesis experiments indicated that CelA is secreted by a mechanism other than a leader peptide.

Transcriptional regulation of an endoglucanase and a cellodextrinase gene in Ruminococcus flavefaciens FD-1

A gene which encodes a 35 kDa protein with both carboxymethylcellulase and xylanase activity was cloned from Ruminococcus flavefaciens FD-1 and the nucleotide sequence determined. The FD-1 gene, celE, and the celA gene, which encodes a cellodextrinase, were used as probes to analyse transcription in R. flavefaciens grown under different conditions. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose, although the effect was not as pronounced as induction by cellulose and was greater for the celA gene than for the celE gene.

Molecular characterization of XVT8, a stress-responsive gene from the resurrection plant Xerophyta viscosa Baker.

Xerophyta viscosa (Baker) is a monocotyledonousresurrection plant that is capable of tolerating extremes of desiccation. Uponrewatering, it rehydrates completely, assuming its full physiologicalactivities. Studies on changes in gene expression associated with dehydrationstress tolerance were conducted. A cDNA library was constructed from mRNAisolated from dehydrated X. viscosa leaves [85%,37% and 5% relative water content (RWC)].XVT8 represents one of 30 randomly selected clones thatwere differentially expressed when X. viscosa wasdehydrated. Sequence analysis of XVT8 revealed thatXVT8 exhibited 45% and 43% identity todehydrin proteins from Arabidopsis thaliana andPisum sativum respectively, at the amino acid level.XVT8 encodes a glycine -rich protein (27 kDa)which is largely hydrophilic and contains a hydrophobic segment at theC-terminus. Southern blot analysis confirmed the presence ofXVT8 in the X. viscosa genome.XVT8 transcripts accumulated in X.viscosa plants that were exposed to heat, low temperature anddehydration stresses, and to exogenous abscisic acid and ethylene. Theseresultsprovide direct evidence for the heat, low temperature, dehydration, abscisicacid and ethylene -dependent regulation of the XVT8 geneinX. viscosa.

Nucleotide sequence of thecelA gene encoding a cellodextrinase ofRuminococcus flavefaciens FD-1

SummaryThe nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) fromRuminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region inEscherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CeIA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other oganisms. Two lysozymetype active sites were found in the amino-terminal third of the enzyme. InE. coli the cloned CeIA protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposonphoA mutagenesis experiments indicated that CeIA is secreted by a mechanism other than a leader peptide.

Protocols for nuclei isolation and nuclear protein extraction from the resurrection plant Xerophyta viscosa for proteomic studies

The plant nucleus is an important subcellular organelle but the isolation of pure and enriched nuclei from plants and subsequent extraction of nuclear proteins for proteomic studies is challenging. Here, we present protocols for nuclei isolation and nuclear protein extraction from the resurrection plant, Xerophyta viscosa, and show optimization and modification of the most critical steps.